AN EVALUATION OF TETRACYCLINE STAIN REMOVAL BY. BLEACHING VITAL RABBIT INCISORS by Patrick A~ Fleege Submitted to the Faculty of the Graduate School in partial fulfillment of the requirements for the degree of Master of Science in Dentistr,y, Indiana University, School of Dentistry, 1974. ACKN~"fLEDGMENTS The author wishes to thank the members of his graduate committee for their help and guidance in the preparation of this manuscript: Dr. James Roche, my graduate committee chairman, who has been a friend and teacher; Dr. Arthur Klein, for his assistance with the instrumentation and measurement procedures; Dr. Simon Katz and Dr. Leonard Koerber for their assistance with the statistical analysis and interpretation of data; and Dr. David !1Ii tchell for his assistance and editorial direction. Special thanks go to llr. Dwight MacPherson for his technical genius in designing the electronic measurement system, without which this stuqy could not have been completed. Sincere appreciation is extended to the follovnng: .Dr. A. II. El-Kafrawy for selecting the ground sections to be measured and for his encouragement throughout the study. Mrs. Helen Campbell and her library staff for assistance they gave during the literature review. ?.irs. Mildred Redford and her staff for caring for the animals and help during the clinical bleaching procedure. Professor Paul Barton for his expertise in reviewing the manuscript. Mr. Richard Scott and his staff for their help with the illustrations. 1liss Shirley Shazer for her instructions on the preparation of the ground sections. My classmates, Drs. Harry Bopp, Dennis Duffield, Jo_hn Deppen, Arthur Mourino, and Frederick Sinnnons, who made the last two years an enjoyable learning experience. Miss Merry Spoelstra for her assistance in mounting the illustrations. Dr. David Hennon for proof-reading the final manuscript. The entire faculty and staff with whom the author vras acquainted, for helping make his graduate experience a rewarding one. A final expression of gratitude goes to my loving parents for making my professional career possible and for their constant encouragemento TABLE OF CONTENTS Introduction Page 1 Review of the Literature 3 3 6 8 11 13 I. The Tetracyclines A. Physical Properties B. Clinical Pharmacologic Properties c. Clinical Antimicrobial Spectrum D. Side Effects and Toxicity rr: The Effect of Tetracyclines on Teeth 17 A. Prevalence in the General Population 17 B. Intrinsic Tetracycline Stain 19 1. Color 19 2. Effects of Sunlight on Tetracycline Staining 21 3. Severity of Staining 22 c. Fluorescence: Normal Versus Tetracycline Teeth 25 D. Dental Caries Protection? 31 E. Tetracycline Induced ~aplasia of Enamel 33 F. Possible Binding Mechanisms of Tetracyclines 38 G. Tetracyclines: Uses in Research 46 III. Bleaching of Teeth 48 48 51 51 53 54 A. Non-Vital Teeth B. Vital Teeth 1. Fluorosed Teeth 2. Tetracycline-Stained Teeth c. Considerations in Bleaching Teeth IV~ Fluorescent Photography V. Television-Electronics in Dental Research Methods and Materials Results Tables and Figures Discussion Summar,y and Conclusions Appendix I References Curriculum Vitae Abstract 55 56 57 65 70 111 119 122 176 LIST OF ILLUSTRATIONS TABLE I TABLE II TABLE III TABLE TV TABLE V TABLE VI TABLE VII I TABLE VIII TABLE IX TABLE X TABLE XI TABLE XII TABLE XIII The clinical antimicrobial spectrum of tetracyclines 13 Comparison of dentin and enamel fluorescence of un bleached and bleached once maxillary rabbit incisors which received no tetracycline 70 Comparison of dentin and enamel fluorescence of un bleached and bleached once mandibular rabbit incisors which received no tetracycline 72 Comparison of dentin and enamel fluorescence of un bleached and bleached tvvice maxillary rabbit incisors which received no tetracycline 73 Comparison of dentin and enmnel fluorescence of un bleached and bleached twice mandibular rabbit incisors which received no tetracycline 75 Comparison of enamel tetracycline fluorescent in tensity of right and left maxillary rabbit incisors 76 Comparison of enamel tetracycline fluorescent in tensity of right and left mandibular rabbit incisors 77 Comparison of enamel tetracycline fluorescent in tensity of unbleached and bleached once maxillar,y rabbit incisors 78 Comparison of enamel tetracycline fluorescent in tensity of unbleached and bleached once mandibular rabbit incisors 79 Comparison of enamel tetracycline fluorescent in tensity of unbleached and bleached tvdce maxillary rabbit incisors 80 Comparison of enamel tetracycline fluorescent in tensity of unbleached and bleached twice mand- ibular rabbit incisors 82 Comparison of dentin tetracycline fluorescent in tensity of right and left maxillar,y rabbit incisors 84 Comparison of dentin tetracycline fluorescent in tensity of right and left mandibular rabbit incisors 85 TABLE XIV TABLE X!! TABLE YJ!I TABLE XVII T .ABLE XVIII TABLE XIX TABLE XX FIGURE 1 FIGURE 2 FIGUF.E 3 FIGURE 4 FIGURE 5 FIGURE 6 FIGURE 1 Comparison of dentin tetracycline fluorescent intensity of unbleached and bleached once maxillary rabbit incisors 86 Comparison of dentin tetracycline fluorescent intensity.of unbleached and bleached once mandibular rabbit incisors 87 Comparison of dentin tetracycline fluorescent intensity of unbleached and bleached twice maxillar.y rabbit incisors 88 Comparison of dentin tetracycline fluorescent intensity of unbleached and bleached twice mandibular rabbit incisors 90 Comparison of the group mean difference of tetracycline fluorescent intensity in dentin in groups of unbleached control teeth with groups of teeth bleached once or twice 92 Comparison of clinical Kodachromes taken in white and ultraviolet light vri th depth of strongest bleaching in the ground sections from those rabbits which were bleached 94 Average thickness and range of thickness of the enamel in rabbit incisors 98 Photographs of the instruments used during the bleaching of the rabbits' incisors 99 Photographs of the isolation of a mandibular rabbit incisor with the rubber dam 100 Photographs of the thin sectioning instrument used to prepare the transverse ground sections 101 A schematic block diagram of the television fluorescent intensity measurement instrumentation 102 Photographs of the television fluorescent intensity measurement instrumentation and wave form of the video signal on the oscilloscope 103 Composite photomicrograph of fluorescence from a ground section of an unstained maxillary rabbit incisor 104 Composite photomicrograph of fluorescence from a ground section of an unstained mandibular rabbit incisor 105 FIGURE 8 FIGURE 9 FIGURE 10 FIGURE 11 FIGURE 12 Diagram of transverse ground sections of maxillary and mandibular rabbit incisors with oxytetracycline stain lo6 Clinical photographs of unstained and stained, unbleached and bleached rabbit incisors 107 Photographs taken with ultraviolet light of macro scopic fluorescence in unstained and stained, un- bleached and bleached rabbit incisors 108 Photomicrographs of fluorescence from transverse ground sections of unbleached and bleached maxil- lar.y rabbit incisors with oxytetracycline 109 Photomicrographs of fluorescence from transverse ground sections of unbleached and bleached mandibular rabbit incisors with oxytetracycline 110 INTRODUCTION -1- In 1956 tetracyclines were first implicated in the intrinsic stain ing of teeth being formed at the time of administration of this family of drugs. Even though only a limited portion of the population is affect ed, this very unesthetic staining can be damaging psychologically.1' 2'3 Spencer4 suggested the use of an acrylic veneer, acid-etched to the labial surfaces of these teeth, to mask the stain, but the sole treatment for permanent improvement of this affliction is still the porcelain veneer crown. Several clinicians have attempted to bleach the tetracycline stain from the teeth. Although monthly re-bleaching was necessary, Cohen and Parkins5 reported in 1970 that in five of six patients esthetic i~prove­ ment was achieved by bleaching the tetracycline-stained. teeth with con centrated hydrogen peroxide and heat. Arens, Rich and Healey6 in 1972 reported that three of five patients showed marked esthetic improvement after bleaching their patients' teeth with concentrated hydrogen peroxide and heat. They also stated that the yellow and brown hues of tetracycline staining bleached more successfully than the gray huese No reports have been published offering evidence as to why o~ yellow and yellow-brown teeth, .as opposed to teeth stained gr~, respond to the bleaching procedure. The bleaching process is not fully understood: there is a question as to whether its effect is due to the depth of penetration of the bleaching -2- agent into the affected tooth, to a masking effect of the stain in the tooth, or to some other factor. It is hypothesized (1) that the procedure used in the clinical bleach ing of tetracycline-stained teeth alters the tetracycline in the stained tooth to a less fluorescent state throughout the crown, with no distinct boundaries in the enamel or dentin, and (2) that the strongest bleaching occurs at the dentino-enamel junction. This stuqy was designed to investigate the effectiveness of the bleach ing technique described by Cohen and Parkins) and Arens, Rich and Healey6 by measuring the tetracycline fluorescence in ground sections from rabbit incisors. The fluorescence was measured both in location and in amount by using an ultraviolet light microscope coupled to a specifically designed television electronic measurement system. REVIEW OF THE LITERATURE -3- ~ The Tetracyclines Broad spectrum antibiotics came into medical use after Duggar in 19487 isolated chlortetracycline from Streptomyces auriofaciens and it became the third major antibiotic to be discovered, after penicillin and s~reptomycin. A number of analogues of the basic molecule have been derived either from active chemical variants in the fermentation products or from synthetic chemical alterations. The eight major analogues of tetracycline are chlortetracycline (CTC), oxytetracycline (OTC), tetra cycline (TC), demethylchlortetracycline (DMTC), N-(pyrrolidinomethyl) tetracycline ( PMTC), methacycline ( ~~ITC), doxy-cycline ( DTC), and minocycline (MITC). 8 These differ in the position of chloride, hydroxyl, methyl, or other organic radicals on positions five, six and seven of the basic naphthacene ring system. Also, these radicals represent the primary basis of deriving the generic name of the various analogues.9,10 All of these analogues are on the market in the United States. OTC was introduced in 1950 after being prepared from Streptomyces rimosus.11,12 TC was introduced in 1953 after being prepared by cata~tic hydrogenation over palladium of the chlorine radical.9 D!ITC produced by a mutant of Duggar's original strain was described in 1957, 13 and became available in 1959. In 1959 an injectable form PMTC was introduced for those who could not take the drug orally.l4,15,l6 MTC and DTC were introduced in 1967.l7,lB 1ITTC came into the market in 1972 after being mentioned in 1966.lB,l9 In 1962 6-demet~l-6-deoxytetracycline was synthesized -h- chemical~ and was considered a major break-through in pharmaceutical research, although the chemical synthesis is not commercial~ advantageous.20 The structural formulas of the tetracyclines are as follows: 15,l8, 21,22,23 II 0 I ,, OK 0 chlortetracycline tetracycline C?H c.H3 c.~3 " / N I OH N-(pyrrolidinomethyl)- tetracycline c.H3 c~3 oH \.J/ I I oH I "'c- NH~ 01-\ 1J II cH 0 0 methacycline c- tJ 11.1. II 0 o:>:ytetracycline demethylchlortetracycline c- (\}H~ II () do.xycycline c- rJH~ Jl 0 minocycline -5- 'l'he trade names of the various tetracyclines are as follows: 15' 16' 18,19,21,24,25 TC-HCl Achromycin, Achromycin V, Kesso-Tetra, Rexamycin, Steclin, Sumycin, Tetrachel, Tetracyn., Panmycin, Polycycline, Cyclomycin, Bristaciclina, Hostacyclin, Omegamycin, Tetradecin, Agromicina, Sanclomycin, Purocyclina, Tetrabon, Criseociclina, Ambramicia, Bristacycline, Gyclopar, Azotrex, 1'trsteclin-F, Robi tet, SK Tetracycline, Tetracycline, QID-tet, Retet TO-Phosphate Complex--Tetrex CTC OTC DMTC PMTC MTC DTC MiTe. Aureomycin, Biomycin, Biomitsin, Acromize, Aurecina, Chrysomykeil Terra~cin, Biostate, Ryomycin, ~-Kesso-Tetra, Uri-Tet, Azopak, OXy-Tetrachel, Urobiotic 250 Declornycin, Demeclocycline Tetracycline, Syntetrin, Velacycline, Rolitetracycline, Herverin Rondomycin Vibramycin Hyclate, Vibramycin :Monohydrate, Doxy-II Minocin, Vectrin The drugs are supplied in oral and parenteral forms except for P1ITC, which is used only parenterally.16 Terramycin is supplied in opaque, yellow, hard gelatin capsules which contain either 250 mge or 125 mg. OTC-HCl and glucosamine. Terramycin Syrup (calcium oxytetra- cycline) is available as a preconstituted fruit-flavored aqueous suspension in 5 cc. equalling 125 mg. of OTC and N-acetylglucosamine. Terramycin also is in an intramuscular and an intravenous form;l9,26 however, OTC is the most toxic upon intraperitoneal injection of all the analogues.27 -6- A. ~ysical Properties As a group, the tetracycline antibiotics are amphoteric cr.ystalline compounds soluble in glycol ethers, pyridine and dilute acid and alkali, ver.y slightly soluble. in water and in lower molecular weight alcohols, and insoluble in ether -and hydrocarbons. The acid salts (used thera- peutically) are well formed crystalline compounds with high solubility in water. 28 The tetracycline analogues will vary- as to solubility and stability according to the radicals that are substituted or replaced. CTC is the most unstable of the tetracyclines ~ vitro.23,29 Of the four major analogues (CTC, OTC, TC, DMTC) DMTC has the grea·bast •resistance -.:·.to degradation b,y acids or by alkali.l0,30 PMTC was developed and marketed as more soluble than the original four and thus the best analogue for inj ec tion.l6' 31 Terranucin, the analogue which will be used in this study, is a pale yellow, amphoteric substance with a bitter taste. Elemental analysis indicates the emperical formula to be C22H22-24 N209 •H20• The antibiotic is optically active and gives positive ferric chloride, Pauly, Friedel Crafts, Fehling and Molisch tests.ll Its ultraviolet absorption spectrum is maximum at approximately 247, 275 and 353 mJl. It also shows a char- acteristic absorption in the infrared region. Terramycin and its qydro- chloride are moderately soluble in methanol, ethanol, acetone, and pro- pylene g~col. OTC hydrochloride is soluble in water but on standing precipitates to the amphoteric base. OTC is soluble in water to the extent of • 25 mg ./ml. at 25°C. Aqueous solutions of the hydrochloride -7- at pH 1.0 to 2.5 are stable for at least thirty days at 5°c and 25°c. Solutions at pH 3.0 to 9.0 show no detectable loss in potency on storage 0 . at 5 C for at least one month. In the dry state, OTC and its hydro- chloride show no detectable loss in activity on prolonged storage at 25°C; it has less than 5 percent inactivation after four months at 56°C, and no loss in potency on heating for four days at 100°C. OTC has a melting point of approximately 185°C with decomposition [a]D25 __ 196.6oc (1% in 0.1 N HCl). The pKa for OTC-HCl are 3.5, 7.6, and 9.2.11~12,32,33 The tetracycline structure contains essentially two 1,3 diketones with two of the ketones in the enol form. Such monoenols in 1,3 diketones chelate readi~ 1vith metallic ions. Drug-metal complexes of 1:1 and 2:1 have been formed vrith Fe-tt-t, Al+t-t-, Cu-ti-, Ni+•, l1'e+o~-, Co+-t-, zn+"", Ca*t-, Mn+~ They have also been shown to chelate with organic compounds like sodium sal~cylateJ sodium p-hydrobenzoates, riboflavin, and amino acids.31,34 I The complexes have formed distinctive colors: Cu ... .-ar.rl Ni'4-+ were green; Fet+tand Fe++were red; and Co-H", zn+-+', Mn-t-r, and ca-t--t-were yellow.35 It was shown that the constants for 6TC and CTC are almost identical in binding metal cations.35 It is suggested that such organo-metallic tetracycline complexes are responsible £or antimetabolic action of tetra- cyclines. Magnesium-tetracycline could compete with magnesium for a specific enzymic site in oxidative phosphorylation.36 v~••has been found to reverse the inhibitory action of CTC on the nitroreductase activity of E. ~.37 It has been shown that the tetracyclines are neutralized by certain metallic cations so that biological activities (which may not necessari~ be associated with its chelating activities) are lost.38 -8- OTC was found to affect excretion of B vitamins and their activity, and may conceivably involve association with essential metabolites ~ !!!£·34 The mutual effects of OTC and each of eighteen multivalent inorganic cations, eight multivalent anions, and five antibiotics on the growth of cells of Pseudomonas aeruginosa were studied. The toxicity of OTC was strongly reversed by salts of Fe+++, Fe~, Mg++, Mn++, and M.oOlf .39 All the tetracyclines fluoresce yellow under ultraviolet light.40 The amount of fluorescence varies with the analogue used and the admin- istered dose. DMTC and TC fluoresce the most, followed by DTC, .MTC, P~C, OTC, CTC and MITC. This is a qualitative judgement and varies among authors and differs with species and methods of measurement used.41,42,43 B. Clinical Pharmacologic Properties The tetrac.yclines are rapidly absorbed from the duodenum, ileum and stomach with little ab~orption from the colon.l8t29,31,32,44-47 Absorp- tion is increased if the analogues are taken in the fasting state.l8 Doxycycline and minocycline are least affected b.Y concurrent administra- tion with food or milk and gastric irritation is diminished if the drug is taken after meals, especially in large doses.l7,l8, 25 Tetracyclines due to chelation are absorbed poorly in the presence of ions of aluminum hydroxide gels, calcium and calcium-containing foods such as dair.y products, iron, magnesium sulfate, and sodium bicarbonate.18,25,31,48,49 Since the tetracyclines are also implicated in disturbing the absorption of some of the vitamins, the use of B complex concomitantly with the antibiotic is suggested.So Excipients such as citric acid or phosphate -9- compounds significantly increased the blood levels of the tetracyclines.; however, these elevated levels were of no real therapeutic value. 28,3l 34,5l,52 The addition of .5 percent terephatalic acid gave approximately a twofold increase in CTC serum levels.49 The greatest differences among analogues is the duration and con- centration of blood levels. Half -life in serum for 1vliTC is 11-17 hours; for DTC, 18-22 hours; MTC, 14 hours; DMTC, 12 hours; PMTC, 12 hours; arc, 9.6 hours; TC, 8.5 hours; and CTC, 5.6 hours.l3,l8,l9,53 The method of administration has a great deal to do with the .amount of tetracycline present, as 6 mg./kg. of OTC given intramuscularly was equal in blood levels to 22 or 33 mg./kg. taken orally, and 25 mg./kg. of OTC intramuscu larLY was equivalent to 6.6 mg./kg. intravenously.54,55 Although there are minor differences in the absorption of the different ~nalogues from the gastrointestinal tract therapeutic levels can be maintained by six-hourly adffii~stration, the usual dose being 250 mg.lO,l3,23,29,32,44,56,57,58 The administration of tetracycline in pregnancy creates another problem in that the fetus receives only about one-fourth of the maternal dose but due to the relative size of the fetus the dose can be very high, approaching 400 mg./kg.59,60,61 Tetracyclines given to infants, both pre mature and full term, in doses of 6 and 12 mg./kg. gave satisfactory therapeutic serum levels.55, 6l Intravenous dosage is 0.5 to 1.0 g./day for an adult and 10 to 20 mg./kg. total daily for a chilct. 32 The tetracyclines diffuse well into most body fluids and tissues.32, 62 -67 Their distribution in soft tissues is widespread with highest con- centrations occurring in the reticuloendothelial system, liver and -10- kidney.31,32,46,62-65,67 The analogues appear in the milk of lactating patients. 28,3l,43 Tetracyclines cross the placental barrier.21,26,28,31, 32,59,61,68-86 The drug is not seen in fat, 62, 63 and has poor penetration of the central nervous s.ystem.3l,32,54, 63-65,B7 Both in vitro and in vivo, dead and normally mineralized bone does not take up tetracycline.88 The drug is bound to plasma proteins and this affects the mode of excretion and serum half-life.l8, 2B,3l,B9 The di~tribution of antibiotics in the organs of the human boqy is the result of several conflicting relations: namely, resorption and excretion on the one hand; and on the other dis- tribution and rate of flow of the circulating blood and the conditions of transfer into the tissue liquor, including penetration of and accumula tion in the tissue cells.9° The tetracyclines are concentrated in the liver, kidney, and in calcif.ying tissues.31,46,63,65,~1,92 .The principal mechanism of excretion is passive glomerular filtration. TC is · concentrated in the urine to the greatest extent, and MITC is the least concentrated. It is evident that the analogue with the lowest excretion rate has the highest blood levels and half-lifee In oliguria and renal failure the half-life can be significantly prolonged, i.e. from 8 hours to 108 hours. CTC is not affected significantly by renal failure. The analogues appear in the urine in an active state. The drug is also found in the feces and this is due to incomplete absorption from the intestines as well as the concentration of the drug in the liver and excretion by w~ of bile. Thus, the tetracycline describes a circuit in the organism: intestine to blood to liver to bile to intestine. There is then about ninety percent excretion b,y the urinar,y and fecal -11- route with another ten percent deposited in bone and calcif.ying tissue.l7, 18,26~28,29,31,32,44-46,53,56,62,67,92-95 c. Clinical Antimicrobial Spectrum The mode of action or the precise mechanism by which the tetracyc lines exert their antimicrobial action is unknown. The following effects appear to have been established. First, tetracyclines are active chelat ing compounds with divalent and trivalent cations, and may thereby inter- fere with enzymes requiring such cations as cofactors. Next, there is an inhibition of protein synthesis which occurs simultaneously with an increase in the rate of nucleic acid formation within the cell. Finally, the 4rugs appear to interfere with the phosphorylation of glucose in both bacterial and mammalian cells. The greater sensitivity of bacteria relative to higher animals toward the tetracyclines remains unexplained, exqept that tetracyclines interfere with both oxidative process and protein ~nthesis in bacteria, and in man only the oxidative systems seem to be affected.28,32,36,37,38,96-100 The tetracyclines are considered to be bacteriostatic but at times appear to exert a bactericidal effect when massive intravenous doses are given.26-29 Mirror images of the analogues have no antimicrobial activit.y.20 In general, no important differences have been documented in clinical effectiveness of the analogues.9,l8, 27, 28,3l,44,56,57,lOl-l04 The differ- ences are quantitative rather than qualitative. A given organism will be resistant to all analogues if it is resistent to one.l8,56 As is true of m~ antimicrobial agents, the tetracyclines are frequently administered unnecessarily, such as for viral infections, or are -12- administered when less toxic or more efficacious antimicrobial agents are available.2 The problem of antimicrobial resistance. is two fold; on the one hand there is the development of resistant strains during the course of therapy by means of mutation and selection, and on the other there is the superimposition of strains of established resistance after suppr~s- sion of the susceptible rnicroflora. TPe tetracyclines have been used for chronic urinary infections but antimicrobial resistance precludes this use novT. The drug has been used very successfully to treat on a long-term basis cystic fibrosis,79,l04-l08 chronic bronchiti~, bronch iectasis and in low dosage for acne.l8,32,109 Plaza-RocallO suggested that bone could act as a depot for storing OTC and several authors have proposed that the bone-seeking properties of t~e drug could be used to combat osteomyelitis.lll,ll2 However, Anderson, Ferguson and Braudell3 showed that bone is no longer bacterio static 96 hours after the administration of tetracycline and that the persistence of tetracycline fluorescence bears an independent relation ship to its pharmacologic effect. Cullen and Hargadon114 made the point that selection of a drug to which the organism vras sensitive was more important than the bone-seeking properties of the tetracyclines. Frost in several articles gave strong evidence that tetracyclines had no advantage over the other antibiotics in treating osteomyelitis due to the architecture of bone.88,115 Oryl8 in his review of tetracyclines summarized the use of tetra cyclines by the following authors7,23,26,28,29,32,54,70,9L~,95,109,116 and is reproduced as a table on the following page. -13- TABLE I The clinical antimicrobial spectrum of tetracyclines Treatment of first Not treatment of first Treatment ineffective choice choice but usually or more effective Diplococcus pneumoniae (if patient is allerg ic to penicillin) Pasteurella tularensis Brucellosis Pseudomonas pseudornal- lei (meliodosis) Vibrio cholera (cholera) Dacterioides J.f.ycoplasma pneumoniae Rickettsial infections Rocky mountain spot- ted fever Typhus fever, murine Epidemic typhus Rickettsial pox Q fever Relapsing fever (Bor relia novyi and Bor relia recurrentis) Psittacosis Lymphogranuloma venerum granuloma inguinale Inclusion conjunctivitis Actinomycosis Reproduced from aryl8 effective therapy available Diplococcus pneumonia Streptococcus hemolyticus, Group A (sensitive strains) Streptococcus, Group B (sensitive strains) Anaerobic streptococci (most strains) Listeria monocytogenes Bacillus anthracis (anthrax) Erysiplothrix insidiosa E. coli (some strains) Hemophilus influenza Neisseria gonorrhoeae Shigella Hemophilus Ducr~y--chancroid Pasteurella pestis (plague) combined with streptomycin Malleomyces mallei (Glanders) Mima-Herellea (some strains) Clostridia tetani Clostridia welchii Treponema pallidum (syphilis) Endocarditis Staphylococcal in fections, especi ally bacteremia Gram-negative bac- illary bacteremias Meningitis Chronic osteomyelitis r'Jnpyerna and suppura- tive pericarditis Septic arthritis Tuberculosis leptospirosis D. Side Effects and Toxicity Acute toxicity of the tetracyclines is relatively low; the LD5o values for intravenous dosage were 170 mg./kg. in mice and 220 mg./kg. in rats for Tc.45 The oral LD50 was found to be greater than 3000 mg./kg. in both species. The LD5o intravenous dose of OTC in mice was 192 mg./kg.l2,32 Original therapeutic administrations of the analogues in humans seemed to confirm the consensus that the tetracyclines were relatively innocuous. Several early papers statad that the only side reaction -lh- were gastrointestinal upsets or superimposed infections. 29,32,55,61,70, 102 116 117 . ' ' It l.S now documented that tetracyclines are not as innocuous as once believed and a review of the major side effects will novr be sununarized. Allergic Reactions Hypersensitivity reactions due to tetracycline and its analogues are rare. A few cases of skin rash or other dermatologic manifestation of reaction to the drugs have been noted.8,18,19, 26,h4,81,118,119 There have been typical anaphylactoid reactions to OTC, D1.1TC, TC, and CTC.8,120,121 Phototoxicity The low allergenicity of the tetracyclines is contrasted by the ~igh incidence of phototoxicity. The reaction manifests itself as an exaggerated sunburn with high fever, eosinophilia, and increased blood plate~ets, and occurs only on skin exposed to rays in the 2,700 to 3,200 angstrom range. DriTC is usually incriminated but the effect has been reported with other analogues.8,18,23,26,31,81,105,106,122,123 Blood Blood dyscrasias due to tetracycline are rare. The clinical signif- icance of the delay which certain tetracyclines may produce in blood coagulation has not been evaluated fully. There have been reports of anemia, neutropenia, and eosinophilia--but again these are rare and not fully substantiated.B,lB, 26,l24,l25 Gastrointestinal Tract Perhaps the most commonly reported side effects are disturbance.s of the gastrointestinal tract. Nausea, vomiting, anorexia, and epi- -1.5- gastric burning occur on oral administration but not on intravenous administration~ .Stomatitis, ·cheilosis, vaginitis, proctitis, diarrhea and other enteric symptoms such as flatulence and bulky, loose stools may result from altered bacterial and fungal flora. There have also been oropharyngeal complaints of black hairy tongue, hoarseness, sore throat and a variety of eruptions in and around the mouth from the altered flora. DMrc and MTC probably irritate the gastric mucosa the most, and TC and DTC the least.8,18,23,28,31,32,44,.56,10l,l02,10.5,106, 116,117 Hepatic System Overdosage of intravenously administered tetracycline causes severe liver damage and in some cases death has been reported. A dose of up to two grams per day given intravenously m~ cause fatal damage of the liver, even in the case of normal kidney function. Pregnant women, children, and ~ose suffering from uropathies are most susceptible.8,18,28,36,67, 76,126,127 Urinary System DMTC may damage the kidneys of healthy adults and several other analogues have caused progressive decrease of renal function in patients suffering from renal insufficiency. A progressive decrease in maximum urinary concentration ability and an increase in dai~ urine volume was noted, as in nephrogenic diabetes, blood urea nitrogen rose and there 1·ras a decrease in creatinine clearance. These phenomena regressed upon vrithdrawal of the drug. Even moderate oral or parenteral doses of tetra cyclines given to patients with renal insufficiency have caused further -16- deterioration of their renal function.B,lB, 26,36, 128-l30 Another problem is the use of degraded tetracycline. It has been documented that outdated tetracyclines cause an adult 11Fanconi Syndrome"--renal tubular dysfunction leading to coma.8,l8)3l,l32 Incriminated is the degradation product epi- anhydrotetracycline. Central Nervous System In infants even a single dose of tetracycline may cause acute benign intracranial hypertension (bulging fontanelle). This symptom has also occurred in adults. These are all reversible when the drug is -vrithdrawn.B, 18,26,133 Teratogenic Changes A possible relationship has been reported between high doses of tetra cyclines in the first trimester and congenital cataracts in three cases.l34 In another case a women received tetracycline at 33 d~s gestation and her chi1d was born with deformed hands bilaterally •135 Tetracyclines also have inhibited grov~h and disturbed calcification in infants. The inhibition is reversible upon cessation of the drug.8,36,76,82,136-147 Tetracyclines also deposit in calcifying tissues and this produces an intrinsic stain in teeth if they are forming at the time of the administration. This subject will be discussed in greater detail in a following section. Miscellaneous Side Effects · · · · Superimposed infections, usually monilia, gram negative infections, and resistant strains of staphylococcal infections, have contributed to fatalities in debilitated patients.8,l8,ll7 Hetabolism of some patients has been disturbed by a catabolic effect and azotemia.8,l8 -17- II. ~ Effect £! Tetracyclines ~ Teeth Regna et al in 195133 reported that Terramycin complexed with calcium, but no real significance was attached to this until Andre 1 s63 excellent study demonstrated that tetracycline was deposited in the skeletons of rats. During the same year, 1956, Schwachman and Schuster106 published the first indication that the tetracyclines discolored teeth. As time passed more clinicians and researchers began to observe and report cases of tetracycline staining of both primary and permanent teeth, and a few reported hypoplasia of enamel which they attributed to the drug. During During the early 1960's heated debate raged in Lancet's Letter to the Editor column on the disputed subject of tetracycline staining and hypop1asia.l3,85,148-153 A. Prevalence in the General Population The prevalence of tetracycline staining in a population varies from country to country and with the sample taken. Witkop, Wolf and Mehaf'feyl-54 found discoloration and fluorescence caused by tetracycline in three sep- arate populations to be 21.2 percent of the children in a pediatric practice, 4.1 percent of urban children and only 1.4 percent of rural children. In children with cystic fibrosis the characteristic staining is from 36 to 83 percent.78,l05-l08,l55-157 Baker and Storeyl58 in 1970 reported that 71 percent of the teeth in children 6 to 7 years of age showed tetracycline deposits. The.y also noted that the analogues were administered with increasing frequency during 1960 to 1965, with the -18- peak year being 1962 followed by a decline in usage. A study by Brownl59 confirmed the frequency of use of these drugs. Brearly and Storeyl60 compared the results of clinical examinations in visible light with the results of using ultraviolet light to detect macroscopic tetracycline fluorescence and showed that the former provided a better diagnostic criterion for tetracycline staining. They confirmed this by microscopic examination of sample teeth under ul tr~violet light which ffi owed that there was microscopic fluorescence in the teeth with no macroscopic fluorescence. Witkop in 1958161 discussed the results of a survey of 96,471 children in which the different genetic problems of teeth were studied. He found that 15 percent of the children had some variation in color, usually a yellow which he attributed to a mutant strain of enamel. One must also distinguish tetracycline stains from the gray-brovrn color of amelogenesis imperfecta, the opalescent discoloration in dentinogen esis tmperfecta, the bluish-green stain of erythroblastosis fetalis, and the purplish-brov1n color of congenital porphyria. Extrinisic stains of black, green and orange can also affect the examiner's judgement.l61 A prevalence study is therefore influenced b.1 a number of external factors. The percentages given for tetracycline discoloration in major studies in the United states are 1.5 percent,162 2.3 percent,l63 and 3.5 percent;l64 in Belfast, Ireland, Stewart in 1968165 reported 7 per cent for permanent teeth and 15 percent for primary teethi and in Australia and nearby regions the percentages of too different studies were 3.4 percent,l66 17.8 percent,l59 20.1 percent,75 and 30 percent.l60 -19- B. Intrinsic Tetracycline stain Authors agree that tetracyclines are deposited along growth planes during odontogenesia, and unlike skeletal tissue, once the drug is fixed it is a permanent stain. Upon completion of calcification and develop ment dental tissues are protected against further staining.22,83,84,144, 146,158,163, 167-174 Since the tetracyclines cross the . placental barrier, the period. during which esthetically important tooth discoloration can occur in man ranges from the fourth month ~ utero to approximately the seventh year of life. More specifically, the prim~ dentition is affected from four months in utero to nine months post-partum and the permanent dentition after ten months of age to the fifth through the seventh year.8,18,72,81, 84,85,156,163,171,175,176 ,In published reports a critical assessment of the severity and the color ·caused qy tetracycline varies between authors due to: a) the ana logue used; b) the dosage level of the drug; c) the stage of development and the dentition (primary or permanent) under survey; d) degradation and bleaching of the drugs; e) the length of time and the manner in which the drug was administered; f) the species under survey; g) the presence and severity of any disease from which the drug was given; h) the length of time the affected teeth were exposed to daylight; i) whether the teeth were studied~ ~ ·or~ vitro; and j) examiner variation and subjectivity. 1. Color Much has been written about which tetracyclines cause the hues of yellow, bro•m, gray or combinations of these colors. Wallman and Hiltonl48 -20- in 1962 reported that the color varied, young children having yellow pigmentation and older children having brownish colors. OTC was least likely to stain and when it did, it was yellow; TC stained yellow but later turned brown. Weymanl77 in 1963 stated that CTC caused a gray- brawn stain and TC, DJITC, and OTC caused a yellow stain which tended to darken to brown. In two other reports, Weyman and Porteousl44,178 en- countered two types of staining: one stained with CTC was gray-brown and did not fluoresce in ultraviolet light, and the other stained with TC and OTC was yellow and did fluoresce. They concluded that a yellow tooth erupted yellow, and that a gray-brown tooth erupted slightly dark er than normal, but never would appear yellow. Douglas,74 Storey,l79 Madison,59 Stewart,72 and Maculay and Leistyna77 all stated that the pig- mentation was yellow and then turned to a yellowish-brown or brown. Several other authors reported cases in which the teeth were bluish- , 1 81 157 180 . brol'm ·to black. ' ' Me Intosh and Storey showed that ep~anhydro- tetracycline (ENfTC), a breakdown product of tetracycline, stained all teeth a very dark brown and that this substance was found in outdated tetracyclines or when tetracyclines were kept in moist and light con- ditions. Their results show unequivocally that different tetracyclines induce different degrees of discoloration in intact teeth. Harcourt, Johnson and Storey181 stated that the teeth are stained by the tetracyclines themselves as the color is identical with the original solution of the drug, it fluoresces at the correct wave length, and it has antibacterial action when released. Owen82 reported that in dogs the teeth stained with DMTC were very yellow, those with TC and CTC were less yellow, and those with OTC were white or dull white. Thanik and McMurchy -21- in 1966141 reported that in dogs DMTC produced a yellow-orange color, TC and CTC produced a yellow color, and OTC gave the least discoloration, a era~ yellow. ~ Effects of Sunlight ~ Tetracycline Staining Wallman and Hil ton148 showed that a TC stained tooth which was yellow turned brown on exposure to sunlight. They did this by splitting a TC stained tooth longitudinally and placing one part in sunlight and the other in the dark. They hypothesized that the brown pigmentation was due to an oxidation product of TC. Hilton111 did the same with TC stained bone. .Again the part exposed to sunlight turned brown while the part kept in darkness remained yellow. Mcintosh and Storeyl80 found that on exposure to light severely affected teeth become gr~-brown, those less affected become gray-yellow, and light yellow te.eth become gray wh~te and difficult to distinguish from normal. Brearley and Storeyl60 reported that sunlight changed wide yellow bands to dark brown and light yellow bands to gr~ on sectioned tetracycline stained teeth. Ibsen, Urist and Sognnaes24 exposed rabbit teeth stained with different analogues to sunlight. After exposure all teeth turned brown and then CTC, PMTC and OTC upon further exposure returned to normal color. D~ITC and TC were more severely stained and remained browno Bridges, Owen and Stewart182 showed that on exposure to daylight all teeth in their study began to discolor further but some more rapidly and to a greater extent than others. Coupled with this degradation was a gradual loss of fluorescent capacity of all samples. Once the color of each tooth had degraded to an observed maximum, it began to bleach. In any single tooth this bleaching occurred more slowly than the degradation. -22- There was also a differential rate of bleaching. In general, samples which were most stained before exposure to d~light discolored more rapid~ and to a greater extent than those whose initial staining was minimal. OTC showed the least initial discoloration, fluorescence and degradation. Tetracycline-L-methylene-~sine (LC) and clomocycline (CC) had the most, and TC and CTC were in between. Brearley, Stragis, and Storey 75 in another study examined 23 human dentitions which showed a difference in fluorescence and discoloration between anterior and posterior teeth, the posterior teeth being darker than the anterior teeth. They said that alterations of color occurring upon exposure to sunlight appeared to be inadequate to explain their clinical observations. ~ Severity of Staining · 1kintosh and Storeyl80 grouped the tetracyclines into two groups and in decreasing order of staining. The first group caused severe staining~ epianhydrotetracycline (EAHTC), demethychlortetracycline :(DMTC)i tetracycline (TC), and tetracycline-~ethylene-lysine (LC). The second group caused leas staining: chlortetracycline (CTC), methacycline (MTC), do~cycline (DTC), oxytetracycline (OTC), and anhydrotetracycline (AHTC). Three studies on the relative severity of staining in man are in close agreement. Wallman and Hil ton148 stated that TC stains more than OTC; Weyman177 in 1965 reported a decreasing order of staining in CTC, TC, DMTC, and OTC; and Swallow, De Haller and Young155 said that CTC stained more than OTC. In a atuqy of dogs Owens82 rated in decreasing order of staining DMTC TC, CTC, and OTC; another study in dogs by Thanik and McMurchyl4l arrived at the same order, although the colors were more yellow. Ibsen, Urist -23- and Sognnaes24 demonstrated in rabbits in decreasing order of severity D~.ITC, TC, PMTC, OTC and CTC. Tvro good studies have been performed in rats. Bridges, ~ven, and Stewartl82 found that LC, clomocycline (CC), CTC, TC, and OTC stained in decreasing intensity. The other study by· Ucintosh and Storeyl80 vras mentioned earliero Johnson in 1964183 showed that extremely high doses of tetracycline produced brown rather than yellow staining. Evidence has been presented that the total dosage of tetracycline is more important in the discolor ation of teeth than the total period of administration.81~,184 The color intensities in bone were directly dose-related.l4l Bevelander and l~akahara184 stated that in the rat the degree and extent to which dentin and enamel exhibit discoloration as a result of exposure to tetracycline are dependent upon age and dose. Brearley and Storeyl60 showed that in human dentitions the quantity of tetracyclire administered and the close- I ness of the staining to the denti~o-enamel junction determined the severity of discoloration. A single course of tetracycline given during the formation of the dentino-enamel junction could cause severe discolor- ation. They also showed that the color of the staining could be altered by the distance the light must pass through enamel and dentin to reach the stained area. This property of light produced different shades and different intensities of color with the same analogue. Moffitt et all7l stated that the intensity and severity of the dis- coloration are influenced by the dosage, duration, and the time of in- itiation of tetracycline series of antibiotics relative to the period of odontogenesis. The closer to the dentino-enamel junction, the more intense the staining. Bridges, Owens, and Storeyl82 shov;ed further that -24- with higher dosage levels darker discolorations occur. Harcourt, Johnson and Storey181 showed that coloration was correlated to dose and frequency of administration. Moffittl56 demonstrated in his thesis that the critical period for tetracycline-related discoloration in the primary dentition was the period of mineralization of the first millimeter of dentin nearest the dentino-enamel junction, which was the first 2h months of life. He also showed that the severity of tooth discoloration was dependent upon duration and total dosage of tetracycline therapy and that the staining became more intense as the period of tetracycline adminis tration began earlier in the child's life. Grossman et all67 reported that the darkening effect of one course of TC or Dl.ITC during the years of permanent incisor formation was negligible; however, with increasing frequency of tetracycline exposure the risk increased and four of their six patients with eight or more courses had noticeably dark teeth. &Tallow, DeHaller, and Young~SS disagreed stating that there was no statistical difference in discoloration among the analogues OTC, TC, CTC, and DMTC. They further stated that total dosage arrl time of tooth formation made no statistical difference in the degree of tooth discolor- ation. In two cases the dentin was entirely removed from the enamel and discoloration remained in the enamel, particularly in the cervical region.l69' l8.5 Bennett and Lavr186 and Bennett187 showed that tetracycline was incorporated into the calcifying dentin and enamel of dog teeth in a ratio of approximately nine times as much tetracycline in dentin as in enamel. Owen82 stated that he observed incorporation of tetracycline both in the dentin and enamel at the time of formation. Urist and Ibsenl88 -25- demonstrated that tetracycline was present in both enamel and dentin, with dentin having the higher incorporation of the drug. Moffi ttl56 stated that tetracycline incorporation in enamel was minimal or non- existent in all the primary teeth of his test population, yet tetracycline incorporation was always observed in dentin. Harcourt, Johnson and Storeyl81,189 said that they could not demonstrate tetracycline's pre sence in enamel by fluorescence. Weyman and Porteous83 were unable to find fluorescence of tetracycline in enamel in human teeth even though it was present in the dentin. l'leyman185 stated in a later study that the stain was found to be in the enamel and was not stain showing through from the dentin. This indicated a higher concentration of tetracycline in the enrunel than would be indicated by the amount of fluorescence. Summary Intrinsic tetracycline stain is deposited in both enamel and dentin and• imparts a yellovT hue to the bone or tooth substance. The severity of discoloration is dependent on total dosage, the time of administration in relation to tooth development, the analogue given, and the position of the drug in relation to the dentine-enamel junction. The yellow stain turns brown on exposure to sunlight end ·t.he gray hues of tetracycline staining seem to be due to the masking of the stain with varying thick- nesses of normal enamel and dentin. c. Fluorescence: Normal Versus Tetracycline Teeth Fluorescence is the luminescence which ceases within a very short time (lo-8 seconds) after the exciting radiation is removed.l90 ~~en a material fluoresces on its own, it is described as having primary fluorescence -26- or autofluorescence. A material ~nich fluoresces after being impregnated with a fluorescent dye is .referred to as having secondary fluorescence.l9l The particular radiation that excites fluorescence and the specific position of that fluorescence in the visible spectrum can be used to identif.y a substance. This is one of the important physical properties of tetracycline, and a major means of studing the distribution of this drug. Fluorescent microscopy is accomplished by illuminating the subject under study with ultraviolet light and observing the resulting fluoresc- ence. The ultraviolet light for the microscope is usually generated by filtering out all but the ultraviolet light from the full spectrum of white light.l90-l92 Benedictl93 in 1928 made the following observati~ns in human beings concerning fluorescence. He showed that the lens of the eye was the I strongest fluorescing organ, although the teeth were almost as brilliant. He noted that dentin fluoresces more brilliantly than enamel and with a bluer light. Initial dental caries did not fluoresce and the same was true if a tooth was treated with dilute acetic acid. Benedict also found that ashed enamel did not fluoresce and that the organic matrix of dentin retained appreciable fluorescence. Hals191 in 1953 wrote an excellent review of the fluorescence of teeth. In ful~ developed hard tooth tissues the primary fluorescence is strongest in the least mineralized parts. Regions vdth especially high mineralization, such as the inner zone of the enamel, do not fluor- esce. In teeth the cementum fluoresces more than dentin, which fluoresces -27- more than enamel. Carious regions do not fluoresce, and vital teeth have greater fluorescence than non-vital teeth. The fluorescence of many tissue elements is strongly labile when exposed to ultraviolet light and this is true of both secondar.y and primary fluorescence. Hals stated that investigations should be done on freshly prepared specimens only in visw of the decrease of primary fluorescence in tissues which occurs only a few minutes after death. From his own research, Hals also reported that the primary fluores- ence was blue for enamel and a brighter or whiter blue for dentin. In addition, he referred to a report by Pfl~ger194 in 1931 of experimental~ induced porphyria in teeth. After repeated injections of uroporphyria in the abdominal skin, the experimental animals were sacrificed and ground sections of the teeth were made and studied with a fluorescence micro,scope. In longtitudinal sections of the dentin luminous red lines were seen, and in cross-sections rings appeared whose relative position agreed with the time intervals between the injections. Red fluorescence could be observed in the enamel also, but unlike the fluorescence of the dentin there was no striped deposition, but rather a delicate, diffused red tinge in the enamel structure, strongest at the dentino~enamel·. junction and dimininishing toward the enamel surface where it was not observable. In ordinary light no coloring was demonstrable. The literature is replete with statements that tetracyclines will cause yellow fluorescence in tissues in which they are deposited.2,21,22, 24,63-67,74,81,82,llo,l5h,l6o,l63,167,171,174,175,181,189,195-2o5 Br 1 ear ey, Stragis and Storey75 demonstrated that clinical discoloration caused by tetracyclines fluoresced macroscopically in only 84.8 percent of the -28- cases they studied. Kutscher et a1200 found that patients exhibited fluorescence of greatest intensity when viewed with long-wave ultraviolet light at 366 nanometers and that short-wave ultraviolet light under 253.7 nanometers never gave as good a result. Antalovska and Beran173 stated that the localization of the fluorescence is not influenced by the type of tetracycline, the dose, or method of administration. Antalovskal70 in another article pointed out that the intensity of macro- fluorescence in dental tissues and in bone varies according to the amount of tetracycline retained. He felt that the amount of tetracycline incorporated could not be determined quantitatively on the basis of the yellow tetracycline fluorescence. Storeyl79 said the drug was incorporated into calc~ing dentin and into immature enamel, but appears to be removed or masked as the enamel calcifies, in contrast to its permanent retention in dentin. Harco~t, Johnson, and Storeyl8l showed in five children that the tetra- cycline stain localized in dentin. It fluoresced under ultraviolet light and this fluorescence was associated with a typical globular pattern of calcification in dentin and cementum. Enamel did not fluoresce yellow when viewed directly under the ultraviolet light microscope. Moffitt et all7l stated that tetracycline fluorescence in enamel was minimal or non-existent. Hammarstrom198 reported that the distribution of ca45 in the enamel remained unchanged during the four d~s of his investigation. The distribution of tetracycline fluorescence, howeve~, was markedly changed with time. One d~ after injection there was considerable increas~ in fluorescence in the whole thickness of the enamel. It then gradually decreased and extinction seemed to start at -29- tips of the cusps and proceeded cervically. After four days, fluorescence could be seen mainly in the cervical parts of the enamel. LOfgren, Omnell, and Nylenl99 observed that some hypoplastic defects exhibited tetracycline fluorescence while others were negative, as were the incremental bands and the normal enamel. The labelled lesions in cluded all of those caused by the previous injections of tetracycline and not the injection which corresponded to the time of the defect. These authors suggested that the fluorescent lesions labelled by the injection of tetracycline were labelled subsequent to the injection that caused the hypoplastic enamel. Nylen, Omnell, and LOfgren206 reported that only hypoplastic enamel.exhibited the yellow fluorescence of tetracycline. Harcourt196 reported on several cases of neonatal jaundice who had received large doses of tetracyclines during the first month of life. Tetracycline fluorescence could be seen in the enamel as well as the dent~ of the primary teeth. Both Hefferren et a1205 and Moffitt et al171 reported that the fluorescence of tetracycline in teeth could be seen most vividly at the cervicals of the teeth due to the intense fluorescence of the dentin which shows through the thinner enamel. They also observed that gray tetracycline-stained teeth had minimal fluorescence, if aQY, and that brown tetracycline-stained teeth showed less fluorescence than yellow tetracycline-stained teeth. Hiltonlll exposed TC stained bone to sunlight and after exposure the color turned from yellow to brown and no longer fluoresced under ultra violet light. Frost207 has sh~nn that TC labelled bone can be detected and measured after 109 months of life has intervened between labelling and measurement. He obtained his specimen from a patient who had received -30- TC nine years earlier in life and the bo~e was removed during surgery. Johnson and Mitchen43 stated that tetracycline fluorescence fades rapidly after preparation of sections. Mcleay and Walskell2 demonstrated that if the bone which contains the tetracycline is kept in the dark at minus ten degrees centigrade the specimen will maintain its fluorescence and bacteriostatic activity up to six months. Plaza-Roca11° was able to keep samples for a year and still have fluorescence by storing them in ethyl alcohol in hermetical~ sealed containers and refrigerated a temperatures normally used for storing food. Fluorescence ceased in specimens exposed for long periods to light and specimens lost fluor- escence if exposed to ultraviolet light contL~uously for thirty minutes to one hour. A few studies compared the analogues for degree of producing tetra- cycline fluorescence in teeth. In general, the more severe the dis- I coloration caused by the tetracycline, the more intense the fluorescence. Owen82 in his study with dogs showed that the decreasing order of fluor escence.was DriTC, TC, CTC, and OTC. Ibsen, Urist, and Sognnaes24 stated that DMTC, TC, and P1ITC were equal in fluorescence and all were more fluorescent than OTC, which was more fluorescent than CTCe Bridges, Owens and Stewart182 rated the analogues in decreasing order of fluorescence as follows: LC equaled CC, which fluoresced more than TC, which equaled .. CTC, and the least fluorescing analogue was OTC. Johnson21 showed in rats that fluorescence decreased from D~ITC and CTC to TC and then OTC. Hodson, 208 upon examination of ground sections of carious tetra- cycline-banded teeth under light and fluorescent microscopes, found that the yellow-brown pigmented bands and their fluorescence were eliminated by the carious process. Sections treated with dilute lactic acid and -31- ethylene diamine tetra-acetic acid (EDTA) showed first the quenching of the fluorescent bands, fo~lowed later by solution of the drug. Brearly and Storeyl60 and Harcourt196 both reported that dental caries removed the fluorescence in tetracycline-stained dentin. Summary Tetracyclines fluoresce yellow when deposited in growing tissue and subjected to ultraviolet light. Dentin fluoresces more than enamel which does not retain the fluorescence of tetracycline. Sunlight and chemical decomposition diminish the intensity of tetracycline fluorescence. The intensity of fluorescence varies with the analogues and follows the same order as their ability to cause discoloration. The decreasing order of fluorescence is as follows: LC, CC, D1~C, TC, PMTC, CTC, and OTC. D. Dental Caries Protection? Bevelander147 interpreted Wallman and Hilton148 as s~ing that the teeth of 50 children which demonstrated discoloration subsequent to tetracycline staining were highly susceptible to dental caries. Weyman and Porteousl44 reported the incidence of . dental decay as similar to that in children with normal teeth unaffected by tetracyclines. Hennonl64 felt the dental caries incidence was not altered by tetracycline incorporation. , Frankel and Hawesl63 and Frankel175 stated that no significant association between dental caries and tooth discoloration was apparent~ Anderson, Ferguson and Braude113 felt that teeth stained with tetracycline would not be resistant to dental dec~ because bone incorporated with tetra- cycline did not remain bacteriostatic for more than four d~s. Swallow, DeHaller and Young155 noted that in their relatively small population of cystic fibrosis patients with tetracycline discoloration, there was a -32- trend for a lower dental caries prevalence than a general population. Stephan et al in 1952209 ~ere the first to study CTC and other anti- biotics in relation to dental caries prevention in rats. : The antibiotics inhibited the formation of dental carious lesions. · The antibiotics effective against gram positive bacteria inhibited dental dec~ the most, although not completely. Shaw and &veeney in 1958210 demonstrated that CTC and OTC were moderately effective in reducing dental caries in the cotton rat and white rat. Larson and Zipkin in 1960211 stated that tetracycline treatment may be an effective means of reducing dental caries activity if the bacteria which are eliminated by the antibiotic are not re-introduced. Zipkin, Larson, and Rall in 1960212 and Zipkin and Larson in 1960213 conducted studies in which dental caries in rats was markedly reduced by adminis tering tetracycline. Larson and Zipkin in 1961214 and Larson, Zipkin and·Fltzgerald in 1963215 stated that the reduction of dental caries caused by tetracycline was due to an altered bacterial flora in the rats which was not as cariogenic. Another study by Grahnen and Larson216 found no significant difference in the incidence of tooth decay between premature and normal children. This fact is important when one notes that this controversy originated ri th the sample of Wallman and Hil ton.148 Summary Tetracycline incorporation does. not seem to alter the tooth's resist ance or susceptibility to dental caries. In animal experiments tetracyc line lowered the dental caries incidence by altering the cariogenic flora. This lower incidence was maintained as long as the animals were not re innoculated with the cariogenic strains of bacteria. -33- E. Tetracycline Induced Hypoplasia of Enamel Wallman and Hilton14B,l5l built a strong case that large doses of TC and OTC in premature infants were causing enamel hypoplasia. The most severe tooth changes ware found with the highest total dose per kilogram of birthweight. The average dose was 210 mg./kg. with OTC and 189 mg./kg. with TC. Wallman217 also reported a clinical observation that an infant who received TC immediately after birth had yellow teeth and the first and second primary molars were deformed with extremely sharp cusps. Witkop and Wol£218 noted a high degree of correlation with TC staining and hypo plasia. Again, the higher the dose in these children the more damaging the result; these authors estimated that 21 to 26 mg./kg. will cause hypo plasia. Brownl59 and Beckelman and Gingold) reported cases of tetracycline hypoplasia. Brearley, Stragis and Storey75 stated that 4.02 percent of those . teeth stained with tetrac.ycline also showed areas of hypoplasia. Bak~r ·and Storeyl58 proposed that the incidence of tetracycline-associated hypoplasia in teeth at risk is approximately 26 percent. Demers et a18l indicated that hypoplasia of tooth enamel and dentin could occur. DeBorgarello and Gendelman219 found under microscopic examination that enamel showing quantitative and qualitative alterations correspond ed to areas of tetracycline fluorescence. There was a significant dit- ference (P equals .001) in pigmented teeth displaying hypoplasia (23. 79%) and non-pigmented teeth showing hypoplasia (2.44%). However, many other authors disagreed. Millerl53 believes that the hypoplasia that Wallman and Hilton referred to was in fact caused by prematurity and not tetra cycline. 'Vleyman and PorteouslL4 felt that clinical hypoplasia could not -34- be related to the drug. Madison59 found no evidence of enamel hypoplasia attributable to the drug. Swallov·r, DeHaller and Youngl.55 noted that only one patient out of sixty-three had tetracycline staining and hypoplastic defects. Harcourt, Johnson and Storeyl81 noticed disturbances of dentin mineralization in the form of large interglobular areas but implied that these were not associated with tetracycline per ~ but were probably localized manifestations of chronic systemic illness·. Porter et al220 ran a statistical analysis of 41 matched sets of full-term children in which one set received TC placentally and the other did not. Although there was staining of primar,y teeth, no statist- ically significant difference in hypoplasia was found in the TC group. Martin and Barnard166 thought that any enamel opacities or hypoplasia was the result of the disease the drug was administered for and not the tetracyclines themselves. Mello84 stated that there is insufficient I evidence to show that tetracyclines are responsible for enamel hypo- plasia when given in therapeutic doses during tooth development. Frankel and Hawes163 said there was no significant association betvreen tooth discoloration caused by tetracycline and hypoplastic defects in these teeth. Grahnen and Larson216 examined 68 premature and 61 normal-term children and found a significantly higher frequency of 5,Ymmetrical enamel hypoplasia in the premature group, 2 percent to 21 percent. In the same stuqy references to Gaunt and Irving221 and to Lindquist and Rakit222 w·ere used to show that in animal experiments a deficiency of blood calcium causes severe disturbances in the calcification of teeth. An article by von Sydow223 was cited as sho·wing in the first days of life -35- premature children have lower blood calcium levels and a higher alkaline phosphatase levels in serum than full term children. Hamp22 compared premature infants and found that those premature infants who received tetracycline had a higher incidence of enamel hypoplasia than those who did not receive the drug. Storey179 took the middle of the road; although he did not deny that tetracyclines could cause hypoplasia, he wanted further evidence of a direct cause and effect between the drug and the structural abnormality. lie felt as others had that children given large quantities of the drug are usually extremely ill, which in itself may be sufficient to affect formation of teeth. The anm1er, appears to lie be ~reen the extremes, and experimental data seemed to indicate that tetra cyclines Tmen administered to individuals with low serum calcium could cause hypoplasia. Baker216 injec~ed TC 250 mg./kg. intraperitoneally into rats which were parathyroidectomized and calcium deficient; he observed a rapid and prolonged fall in serum calcium following the injection. Bevelander, Rolle and Cohlanl95 demonstrated an inhibition of calci- fication in rat teeth corresponding to tetracycline administration. Bevelander, Goldberg and Nakahara224 demonstrated that tetracyclines in sufficient concentration could del~ skeletal formation in sand dollars and that at higher concentrations they completely inhibited larval devel opment. Concentrations which inhibited skeletal formation exerted their effect specifically at the onset of skeletal differentiation. Bevelanderl46 in a later article stated that in teeth the larger the total dose re- lative to body weight, the more severe the abnormality. Johnsonl83 con firmed that high dosages (200-250 mg./kg.) result in definite areas of enamel hypoplasia. -36- Nylen, Omnell . and ~fgren2o6 found pronounced hypoplastic and hypomineralized lesions in the enamel laid d~rn during the full period of drug administration, while enamel formed before and after appeared normal. They said that the primary effect of tetracycline seems to be ameloblastic impairment leading to the formation of hypoplastic enamel and that impaired cells probably allow passage of the tetracycline since only the affected enamel is labelled vrith fluorescing tetracycline. In a later study LOfgren, Omnell . and N,ylen199 found little effect on the enamel vrhen TC and OTC were injected in low dosages. In contrast, high dosages of both TC (150 mg./kg.) and OTC (154 mg./kg.) resulted in developmental disturbances ranging from an incremental band only to one that included a gross hypoplastic lesion. A comparison between the two drugs revealed a much higher incidence of hypoplasia among the de- fects caused by TC than those due to OTC. Mcintosh and Storeyl80 found that in rats 50 mg./kg. caused dis- coloration; 100 mg.jkg. discolored and caused a decreased thickness in the enamel with some aplasia of enamel showing dentin in some areas;: and that 200 mg./kg. caused severe aplasia of enamel. They also found that the different analogues had effects of varying severity for hypoplasia, they were in decreasing order, EAHTC, D1ITC, LC, TC)CTC, OTC, 1ITC>DTC, AHTC. ftn oral dose had to be four or five times the intraperitoneal dose to produce the same effect. Saxen225 developed an iD vitrg test for cal- cification inhibition of the analogues. She found that different analogues behaved differently at different concentrations. At 1 microgram/mi. she listed in decreasing order TC I, DMTC, TC II, MTC, CTC and OTC; at five -37- micrograms/ml. DMTC, TC II, TC I, MTC, OTC, and CTC; and at twenty micro grams/ml. TC I equa2.ed TC . II, MTC, DMTC, CTC, and arc. Yen and Shaw226 indicated in their study that 4 mg./kg. of oral minocycline caused no apparent effect on dentin apposition. Walters and Sayegh42 found that 2 mg./kg. of minocycline intraperitoneally produced less fluorescence than 25 mg./kg. of OTC or TC given the same way. The initial microradiographic picture of the fluorescent areas in bone and dentin was suggestive of hypoplasia. Gr~n and Johannessen204 ex- arnined ground sections of teeth from rats which had been injected with 100 mg./kg. of OTC; the fluorescent bands in dentin corresponding to the time of injection were hypomineralized when the sections were submitted to microradiographs. Moffittl56 found that dentin microhardness was higher in non-stained (non-tetracycline and without yellow fluorescence) areas than in stained areas, (P equals 0.03). Antalovska and Beranl73 found that dental tissues are usually less mineralized in places which show tetracycline fluorescence. Brazda, Kolc and Zastaval72 found that high doses of tetracycline given during intensive formation of the enamel result in enamel hypoplasia. Ovren145 and Bennett and Lawl86 ident- ified areas of hypoplasia in the enamel of dogs who received tetracycline. As stated earlier, Thanik and Mc1vfurchyl-4l showed cessation of growth in rats given 80 mg./kg. of CTC or DMTC intraperitoneally, and arc was the most inhibiting at very high dosage. Cohlan, Bevelander and Tiamsici36 conducted a study concerning tetracycline administration to premature infants. They concluded that a forty percent depression of normal skeletal grovnh had occurred as measured by inhibition of fibula grov~h. Fibula growth inhibition was rapidly reversible after cessation -38- of the tetracycline. They also found that 40 mg./kg./d~ of TC during the tenth through the fifteenth day of gestation in rats resulted in a 26 percent reduction in expected fetal size at term. However, Johnson and ~Jitchell43 found that a study group of young growing rats with limit ed oral dosages of tetracycline apparently had no significant effect on either weight gain or femur growth after 39 days. Summary Tetracyclines do have the potential to alter growth. In high dos ages during periods of lowered serum calcium tetracyclines can increase the amount of enamel hypoplasia that would othervdse be seen in the developing teeth. F. Possible Binding Mechanisms of Tetracyclines The exact mechanism of tetracycline incorporation into mineralizing tis'sue is not clearly understood. Regena et al33 in 1951 said that OTC chelated to calcium ions; Albert35 demonstrated this effect with many metallic cations for both OTC and CTC in 1953. In 1957 and 1958 Hilch, Rall and Tobie65, 66 stated that it may be surmised, if only on the basis of the lack of persistence of fluorescent material in tissues richly supplied b.Y body fluids, as in the reticulo endothelial system, that mere availability of the administered analogue is not the sole mechanism involved in its persistence in osseous tissueo They noted that tetracycline localization in bone appeared to be limited to areas of nevr bone formation and suggested that bone fluorescence following tetracycline administration may be attributed to the binding of either the unaltered compound or metabolic derivative to calcium and/or the matrix of newly formed bone. -39- Lao, Titis and Rall in 1957227 presented evidence that tetracycline is unchanged and bound as · a loose complex to a peptide in mouse sarcoma tissue. Titus, Loo and Rall228 in the same year reported that the bind ing of tetracyclines to bone structure is dependent on pH and possibly involves metallic cations either to the inorganic structure or to the organic matrix. Hakkinen229 in 1958 confirmed that calcium ions are in volved with the binding of tetracyclines in experimentally produced metastatic tumors in rats. :Malek and Kolc23° found that maximum absorption of CTC in tumors occurred in areas affected by calcification. Plaza-RocallO noticed that aqueous solutions of OTC did not fluor esce but when they were passed through filter paper the filter paper fluoresced. This was the same fluorescence exhibited by the dry OTC and was seen where serum had oozed on the casts of patients receiving OTC. Plaza-Roca believed that the tetracyclines were linked to basic bone substance through the action of mucopolysaccharides at the time that calcium is laid down. Buyske, Eisner .. and Kelly in 1960231 noted that TC decline from bone is faster than for CTC; they attributed this to the fact that TC is not as good a chelating agent as CTC. They also demonstrated that physical contact of the antibiotic Ynth bone 'immediately produced attachment. This was revealed by removing a rat femur and stirring it for one minute in a solution containing twenty micrograms/ml. of tetracycline, and discover ing that the bone took up the tetracycline fluorescence which could not be washed off under running water. Frost and Villanueva232 said in 1960 that tetracycline was merely •'cemented11 in by further mineralization since with fresh bone in vitro -40- they were able to stain and destain bone surfaces of all types at will. Hattner and Frost233 in 1962 discovered that the fluorescence of tetra- cycline molecules is quenched in water and methanol but not in carbon tetrachloride, indicating that a dipole action is involved. They further stated that the fluorescence of tetracycline molecules fixed in mineral ized bone is in part the result of loss of hydration shell. The loss is probably related to the increasing amounts of mineral phase with in creasing time which characterizes all new bone ~ vivo; of major im portance in the fixation of tetracyclines to mineralizing bone is the steric placement of carbonyl and hydroxyl groups in the apatite lattice. Due to the fact that tetracycline-induced fluorescence localizes on~ in those areas where matrix calcification •vas observed, Milch, Tobie and Robinson234 postulated that tetracyclines bind to cal~ium of nseeded" cr,ystal nucleation sites and their immediate derivatives on collagen fibrils presumably via the oxygen atoms of the D-ring of the naphtha- cenecaboxamide nuclei. Kelly and Buyske92 demonstrated that except for metal chelate form ation, tetracycline vras chemically unaltered in the rat and the dog; therefore, no metabolic transformation had occurred. Harcourt, Johnson, and Storey in 1962181 noted that dentin stains and enamel does not appear to stain; therefore, tetracyclines must not complex to calcium cations and must be binding to organic matter rather than inorganic matter. They proposed the hypothesis that a complex of ground substance, collagen and mineral may be the mechanism by which tetracycline is bound. Davis, Little and Aherne13° suggested that TC is deposited on the organic matrix of bones and teeth-gave no factual sub stantiation for this opinion. -41- Hilton111 showed that when calcium orthophosphate was precipitated from aqueous solution which contained tetracycline hydrochloride, the tetracycline was adsorbed onto the precipitate. Ibsen and Urist235 in 1962 published the first of several articles dealing with tetracycline and binding. They showed that calcium and magnesium binding by OTC, observed spectrophotometrically, occurs in a step-wise fashion. Higher metal to ligand complexes may exist but the 1~1 and 2:1 OTC complexes are postulated to be the chief molecular species occurring in vivo. They postulated that when 1:1 or higher calcium to OTC complexes were formed it was unlikely that all coordinating positions of calcium were satisfied. Apatite surface calcium could react with OTC and permit accumulation of the fluorophore in bone. Possibly, it may be that spatial configuration of apatite surface calcium_ allovrs two or more calcium atoms to simultaneously bind one molecule of OTC, enhancing the force of attraction. Urist et al236 described their method using frog heart muscle in which the calcium complexes of arc were analyzed. In 1963 Urist and Ibsen188 stated that the binding of OTC by calcium salts in vitro depends upon the formation of a complex with calcium ions in the surface of the microcrystals of apatite. The large OTC molecule presumably occupies a cr.ystal surface position comparable to that of citrate, carbonate, and other ions. If OTC binding is a function -of calcium ions in the surfaces of crystallites, the mechanism can be that of chemisorption, which refers to electrovalent or covalent bonding; the chemisorped OTC is limited to a surface monolayer. It may also in- volve ion exchange. Thus, the bone mineral behaves like an ion exchange column in which arc may exchange for H-r, CO), or OH- or citrate:: ions. -42- Urist and McLean237 in 1963 outlined three possible binding mechanisms for tetracyclines. These .were (A) in the cr.ystal surfaces as complexes with calcium, (B) as complexes with collagen or (C) in complexes that share calcium ions with polysaccharides in newly mineralized tissue. Diagrammatically they are: A B c Collagen Collagen ' Collagen Polysaccharide OTC Polysaccharide • \ . .Apatite Polys ac char ide OTC l 0 l arc Apatite Apatite HYPothesis (A) was supported as the best of these, since evidence was presented that OTC is bound by apatite crystals in aqueous solution and by inorganic bone. These authors contend that all substances which bind OTC have calcium ions (gastric mucosa, scar tissue, cornea) and that the small cr,ystal size, high reactivity, and large amounts of fluid in fast-grmT.Lng bone predispose it to OTC uptake. Older bone is inert due to the large crystal size and the denseness of matrix which prevents free diffusion of the large OTC molecule. I~othesis (B) is difficult to prove since collagen binds only relatively small quantities in vitro and it cannot be divested of all mucopolysaccharide and metal ions that form complexes with tetracycline. Although (C) is a possibility, ·direct chemical evidence is lacking. In 1964 Ibsen and Urist36 admitted in another article that tetra- cyclines form complexes with organic as well as inorganic compounds, but the inorganic compounds have the characteristics of chelates and appear ·to be more stable. Bone-bound tetracycline shows polarized fluorescence; -43- fluorogens in solution do not emit polarized fluorescence~ while organized crystals of fluorophores do, this should rule out binding to po~sac­ charides. Soft tissue bindinr, does occur and is due almost certainly to tetracycline complexes with organic molecules stabilized by metal ions or perhaps by lipids. The authors hypothesized that such stabilization may be due to partial dehydration of the tetracycline molecule--breaking intramolecular hydrates and freeing reactive centers. Finerman and Milch238 in 1963 presented evidence which agreed with Ibsen and Urist in that tetracycline binds to the calcium ion in tissue. They stated that this was because decalcified bone would not take up tetracycline and deproteinized would (in vitro) and concluded that tetra cycline probably interacts, primarily if not exclusively with calcium ions, at least in hydroxyapatite seeded nucleation sites on collagen fibrils. However, Deleu239 stated in the same year that CTC was attaching to organip tissue as CTC localized in the crystalloid membrane of the eye. Prochazka et al240 in 1964 showed that the lasting tetracycline fluorescence in the islets of Langerhans in the human pancreas was due to the formation of a complex of CTC vdth insulin mitigated by a bivalent cation of zinc. Ovrenl45 in 1964 stated that if binding of tetracycline antibiotics is a function of the chelating ability, the strongest chelating agent should give the deepest coloring. His experiments seemed to support this premise. Johnson in 1964183 found that the tetracycljne distribution in dentin is apparently related to the distribution of the mineral phase and follows well known features of dentinogenesis. In enamel there appears to be an initial distribution related to the oreanic phase and a secondar.y distri- -44- bution related to the mineral phase during maturation of the preformed matrix. TC uptake by enamel is greatest where the mineral content is lowest, since it diffuses rapidly through the highly permeable immature matrix. This fact per ~ does not indicate whether the drug is associat ed with the mineral or organic phase or both. The fading during pro- gressive mineralization may be due to removal of the drug from the matrix, along with large amounts of water, protein, and mucopolysaccharides, which occurs at this stage. However, it could equally well be due to the masking of the fluorescence by additional influx of apatite crystal- lites. Probably two distinct processes are involved and that tetracyclines chelate both the inorganic and organic substances. Zastava et al in 196490 confirmed other reports that the TC molecule is mediated by the calcium cation, but fixation through other cations is also possible. ~lvaney, Beck and Qureshi91 in 1964 found that OTC binds only to reactive crystals and through aging the crystals of apatite no longer readi~ accept OTC in urinary calculi. Bevelander in 1964146 and Bevelander and Nakahara in 1965174 were unable to exclude the possibility that the fluorophore may combine with the organic matrix of dentin and enamel as well as the mineral componento Epker241 presented cases in 1966 in which the zone of mineralization in dentin was precisely labeled by tetracycline. He used this as evidence that tetracyclines combine with the mineralizing phase of dentin and not with the organic matrix of the dentinoid per ~· Eger, Gattow and Kammerer242 gave evidence on how tetracyclines de- range mineralization and osteogenesis. They presented this in a step-wise -45- fashion. A) Tetracycline molecules are incorporated into crystallite surfaces of apatite and octacalcium orthophosphate. B) The calcium ions on the surface are saturated as regards to coordination number and growth of the crystallites is blocked. Uoreover, under certain conditions the formation of cr,ystal nuclei is prevented by the presence of fully complexed calcium ions. C) The hydrolysis of octacalcium orthophosphate into apatite is impeded or even stopped by chelation of the crystal surface. The surface of these partial tetracycline--calcium ion complexes is hydrophobic and for reason of lattice dynamics their dissociation in aqueous solvents is meager. Octacalcium orthophosphate must therefore accumulate in tetracycline-labeled bone and this ·would expJa in why such bone yield more pyrophosphate on heating to 325°C than normal bone. Bennett and Law186 and Bennett187 found supporting evidence for the theory that tetracycline combines Ydth the surface calcium ions of the apatit~ crystal when their analysis of enamel and dentin showed that tetracycline bound to dentin and enamel in a ratio of 9 to l. Hammarstroml98 in 1967 shovred that shortly after injection, both tetracycline and ca45 accumulated in a superficial zone in newly deposited enamel matrix. In addition, ca45 was taken up throughout the whole thickness of other areas of enamel with no corresponding ac cumulation of tetracycline. This uptake of Ca45 was localized occlusal- ly of the superficial zone. Between these two areas there was a super- ficial zone of increased fluorescence and autoradiographic blackening. Both substances accumulated in the developing enamel, but there were great differences in their dist:r·ibutions. The distribution of ca45 supports -46- the theory of two staGes in the mineralization, and tetracycline seemed mainly associated with the primar.y stage. A physicist, Kallmann, 243 in 1968 considered Terramycin and hypo thesized that the yellow fluorescence of tetracycline from ultraviolet light in living tissue is a process of surface adsorption of the drug. Subsequent tissue growth locks the drug into bone or other living tissue. Kawasaki244 in 1972 presented evidence that the binding of tetra cycline occurs in both the organic and inorganic phase of dentin. He injected piglets with TC, tetracycline was deposited in the dentin, and the ~ ~ fluorescence could be regenerated after fixation and decal cification or after removal of the organic matrix by placing the speci mens in a solution containing tetracycline. Sununary The exact mechanism of tetracycline deposition and fixation in bone and teeth still remains unclear and additional research is necessary. The mechanism which seems to be the most feasible is a chemisorption of the tetracycline molecule to the surface of the apatite crystal mediated by calcium cations. G~ Tetracyclines : Uses in Research The fluorescence of tetracyclines has been used to great advantage in many divergent areas of research as a vital dye. Initially the pharmacology of the tetracyclines themselves was stud ied. Andra,63 Milch, Rall and Tobie,66 Helander and B~ttiger,64 and Bottiger67 traced the distribution of tetracyclin~ by means of fluorescence. Frost207,245 used tetracycline labelling to study the thickness of osteoid mineralization per d~ and the half-life of some human bones. -47- Boyne,246,138 Boyne and Kruger247 developed the use 6f tetracycline as a vital dye in dental surgery for study of the healing of fractures, for characterization and correlation of various osseous repair responses of the traumatized host in various anatomical loci, for bone graft mater- ials, and for surgical correction of malocclusions. Gregg and Avery248 used TC as a label for vital bone in the alveolous to determine growth of the alveolar bone. Cleau, Perkins and Gildal37 and Yen and Shaw226 found that tetra- cycline was an excellent vital stain for demonstrating calcifying bone or tooth structure. Tobie and Beye96 used tetracycline to locate and visualize migrating and subcutaneous filarial worms. Johnson21 and Hennonl64 proposed that tetracyclines could be used to compare the sealing capabilities of various restorative materials in I dentistry. Mulvaney, Beck and Qureshi91 proposed tttagging" urinary calculi by administering a tetracycline and examining the ring-groYrth to determine the history of the stone. Perhaps the most exciting developments in the late 1950•s was the belief by maQY authors that tetracyclines preferentially deposited in malignant tumors. Rall et al, 2h9 Hakkinen and Hartiala2.SO and Mcleay and Walskell2 all noted this and thought that the drug could be used to detect and locate malignant tissue. Berk and Kantor251,252 and Klinger and Katz253 even devised a method of pretreatment for patients 1vith sus- pected gastric cancer and reported a high degree of correlation with those ulcers that fluoresced and their malignancy. -48- However, Ackerman254 1vrote that studies in man and experimental animals demonstrated that tetracycline localization in malignant tissues was erratic and undependable. Tetracycline was often seen in non cancerous, necrotic, inflamed and calcific lesions. Therefore, he warn ed that these limitations should be realized when using the drug for cancer diagnosis. Mustakelliol52 stated that tetracycline fluorescence is not an indicator of malignancy but merely an expression of stromal reaction favoring calcification. Malek255 provided an overview of the use of this group of drugs for research in areas other than antimicrobial. He stated that the fix- ation of tetracycline is not a specific property of some cells, tissues or pathological conditions. It can be found in very divergent patholog ical conditions--lipid necrosis in pancreatitis, in tumors, in retention of burnt skin and in damaged myocardial muscle. summary The use of tetracycline in research other than antimicrobial is generally confined to its use as a vi·t,al dye to determine hard tissue growth and developments III. Bleaching of Teeth A. Non-Vital Teeth Pearson256 in 1958 described his method for bleaching a stained non vital endodontically treated tooth. After placing a rubber dam on the tooth in question, he desiccated and cleaned it with a 1:2 mixture of 95 percent ethyl alcohol and chloroform. The tooth was then pumiced. A -49- bleaching solution of 75 percent ether and 25 percent hydrogen peroxide was placed on the tooth. This mixture was activated by a photo-flood lamp. The author claimed that the actinic radiation plus heat was neces- sary for a good bleach--heat alone was not enough. The lamp was a number two photo-flood placed about twenty inches from the tooth. The treatment usually lasted about twenty minutes. Then, after the final bleach, he sealed both the enamel and dentin with self-curing acrylic monomer to prevent extrinsic stains from entering the tooth. Spasser257 in 1961 described another method for bleaching non-vital teeth. He used a solvent to remove oil residue after the pulp chamber of the endodontically treated and sealed tooth had been cleaned. Then a creamy mix of sodium perborate and water was placed in the chamber and sealed in the tooth for about four days. This procedure vras repeated three or four times until the proper shade of tooth color was obtained. I Spasser could not predict how long the tooth would maintain the new shade since the stability of the bleached tooth shade was determined qy external enamel cracks and the integrity of the marginal seal of the restoration. Nutting and Poe258 introduced an innovative approach with the nwalking bleachtt technique. This method bleached endodontically treated teeth by sealing in a paste of sodium perborate and superoxol (35 per- cent hydrogen peroxide). This paste was periodically replaced until the desired tooth color was obtained. These authors felt that their proced- ure was clinically effective, simpler and less time consuming than the heating methods. In 1967 }rutting and Poe259 restated their method of sealing sodium perborate and superoxol in the pulp chamber of endodontically treated -50- teeth and after a successful bleach, restoring the pulp chamber with silicate cement. Also, they mentioned that sodium perborate monohydrate (Amosan) could be used. Serene260 in 1973 referred to the use of 30 percent hydrogen peroxide and sodium perborate mixed as a thick paste and sealed into the pulp chamber. He believed that it was extremely important to seal the root canal filling at the base of the pulp chamber with a mixture of zinc oxide eugenol to prevent the bleaching agent from entering the root canal. He also advocated a final seal after the bleaching agent was removed by placing restorations of silicate cement covered vdth gold foil. Stewart26l in 1965 isolated the discolored tooth with rubber dam. Cotton pellets saturated with 35 percent hydrogen peroxide were inserted into the clean pulp chamber and the pellets warmed by_ applying an endo dontic drying point until the hydrogen peroxide began to nboil.u ' Caldvre11262 described the use of a comparatively new instrument* as a heat source for bleaching discolored teeth or preventing discolora tion after endodontic treatment. The technique was as follows: 1) Plug the heating instrument into a 110 volt s.ystem and allow 3 to 4 minutes for the instrument to reach working temperature • . 2) Isolate the tooth or teeth with a rubber dam. 3) Remove all restorations from the tooth or teeth to be bleached and root canal filling material to a point 2 mm. apical to the gingival margin. 4) Flush the tooth with a desired irri gating solution and dry thoroughly. .5) Place saturated cotton pellets of 35 percent hydrogen peroxide in the cavities, in the pulp chamber, and over the crovm of the tooth. · 6) Have the patient warm the 35 percent * Bleaching Tool, Fluor-Ted Co., Inc., Davis, California -51- hydrogen peroxide by holding the heating instrument against the cotton pellets on the tooth with .slight pressure. If the heat becomes uncom fortable for the patient, they are instructed to remove the instrument for a few minutes. The hydrogen peroxide is replenished as the cotton pellets dry. Most appointments will last approximately thirty to forty five minutes. B,y heating concentrated hydrogen peroxide to 165°F, the bleaching rate of the drug will be increased about 200 times. B. Vi tal Teeth 1. Fluorosed Teeth Mcinnes263 in 1966 described the following method which he had used for twenty years to improve the esthetics of fluorosed teeth. 1) The teeth were cleaned Yli th pumice and isolated Yd th a rubber dam. 2) The teeth were wetted for 15 to 30 minutes with a solution of 5 parts 30 percent· hydrogen peroxide, 5 parts 36 percent hydrochloric acid, and 1 part ether. 3) The solution was neutralized with a mixture of baking soda and distilled water. 4) The dam was removed and the teeth polished with cuttlefish discs and moistened pumice. Bailey and Christen264 in 1968 stated that endemic dental fluorosis is a common clinical entity, especially in patients living in rural areas of southwestern United States. As a safe, practical method of stain re moval, th~ used a solution of 5 parts 30 percent hydrogen peroxide, 5 parts 36 percent hydrochloric acid, and 1 part anesthetic ether, along with light discing of the tooth surfaces. Fourteen patients were treated 1vith marked success in every case and no adverse sequelae. The two -52- authors cautioned that only teeth with a smooth, marbled appearance should be chosen for the bleaching procedure; deep hypoplastic areas were contraindicated, as were teeth with paper-white areas or tetracycline stained teeth. Bailey and Christen265 conducted a test under in vitro conditions to determine whether the previously outlined technique by Mcinnes destroys excessive amounts of tooth structure. A standarized bleaching process by Mcinnes was applied for 20 minutes to the labial surfaces of 27 extracted permanent maxillary anterior teeth with endemic dental fluorosis and to 27 control teeth. The enamel thickness on the labial of each tooth was measured before and after treatment. The authors found that the enamel removed was constant for both fluorosed and normal teeth; it was less than 20 percent, approximately 0.1 mm. for each twenty minute treatment, for 92 percent of the speclinens. There was no statistically signif~cant difference betvreen fluorosed and non-fluorosed teeth in terms of thickness of labial enamel. Colon in 1973266 also used the Mcinnes technique for removal of severe endemic dental fluorosis: he used the same solution for two 15 minute appointments one week apart. He had results comparable to those of others using this technique. Bouschor267 in 1973 used superoxol and heat to bleach teeth with fluorosis. He isolated the teeth with rubber dam and vrarmed a mixture of 30 percent hydrogen peroxide and ether held against the teeth in saturated cotton rolls. The solution over each tooth was warmed for three or four seconds rotating from tooth to tooth for 20 to 30 minutes. Four such treatments were usually required. -53- 2. Tetracycline-Stained Teeth Cohen and Parkins in 19705 achieved significant esthetic improve ment in five of six patients with tetracycline staining of their teeth by bleaching these teeth Ydth 30 percent hydrogen peroxide heated to 88°F with a hand-held heating instrument for thirty minutes. Each patient was given eight treatments, generally at one week intervals, and was sub sequently evaluated at monthly visits. · The authors observed that the thinness of enamel of the lateral incisors as compared to the central incisors and cuspids accounted for the most dramatic shade improvement of the lateral incisors. They also stated that after the rubber dam was removed the teeth appeared chalky white for one hour. The long term results are still under observation and to be reported on. Arens, Rich and Healey in 19726 and Rich268 reported the results of five P,atients having tetracycline-stained teeth who were bleached -vrith 35 percent hydrogen peroxide and heat. The study demonstrated that a good esthetic result can be achieved by bleaching tetracycline-stained teeth. Bleaching was accomplished by heating 35 percent hydrogen peroxide saturated in cotton rolls and held against the teeth with a hand-held instrument at lOOF less than the highest temperature at vvhich discomfort was first illicited in the patient's tooth. Five patients from 7 to 16 years of age were treated. Yellow and yellow-brown stains were more easily and completely removed than was gray stain. The incisal one-half of the teeth bleached better than the cervical half. Vitality tests be fore and after showed no measurable change in the teeth. v~nen treatment was completed, the teeth no longer fluoresced under ultraviolet light. -54- Of the five patients, three showed a marked improvement and two a slight improvement. The patients were treated for three 20 minute sessions at weekly intervals. C. Considerations in Bleaching Teeth Nyborg and Brannstrom in 1968269 histologically exrunined teeth after the application of a heating instrument at 1)0°C for 30 seconds and reported very little cellular infiltration in 6 pulps and none in 14. Mumford in 196627° showed that when teeth are isolated by rubber dam the enamel becomes vrhiter, but regains its normal appearance when re-e.xposed to saliva. This was thought to be due to the loss of moisture from the enamel and subsequent re-L~bibing of moisture from the saliva. Another consideration in the bleaching of teeth is the effect of the heating instrument on the enamel and the additional staining that may oc'cur if the instrument causes cracks in the enamel. Peultier, Frank and Klewansky271 in 1967 found that sudden changes of temperature applied during a varied time period was the major factor in production of enamel cracks. Variations from normal to cold (21°C) were more harmful than from normal to warm (60°C). Alternating between 21°C to 60°C produced enamel cracks more easily in younger age groups. The authors also showed that a hot needle used in cautery as well as ethyl chloride produced enamel cracks. Wainwright and Lemoine272 in 19)0, using urea radioactively labeled, and Bartelstone in 1950273 and 1951, 274 using I-131, found that a diffuse penetration of enamel occurs vdthout the necessity of following lrunellae or cracks. Penetration of the isotopes through enamel into dentin was -55- more frequent near the gingival line and near the occlusal fissures. In several teeth the radioactive material was traced continuously from the grossly intact non-carious enamel and dentin to the pulp. In summary, the literature on bleaching teeth is empirically direct- ed. Further investigation is needed of the exact mechanism involved in altering tooth color. TV. l?luorescent Photography Fluorescent photography of livine subjects in color is a powerful diagnostic tool. Many pathologic conditions appear differently under ultraviolet light. Thus, the pathological state can be easily differ entiated from the normal. 275 Two methods have been used to obtain fluorescent photographs of subjects. The first, uses a continuously emitting source of long~wave ultrayiolet light and very long exposure times. Adams 276 used a camera ratio of 1:1 with an F-stop of 8, a Kodak 2B barrier filter, Kodachrome II film and a three minute exposure to obtain his fluorescent photograph. The second method which is more acceptable to the patient is to use a Wratten 47-A or a Corning Glass filter 5970 over a 200 watt-second flash source for generating flash ultraviolet light. Hi-Speed Ektachrome film is used with an F-stop of 2.8 to 8. Barrier filters are often used to enhance the contrast. The flash fluorescent photographs have the ad..:.·· vantage of being more reproducible and require a fraction of the exposure time.79,190,205,275,277 -56- V. Television-Electronics in Dental Research Using the television microscope, Klein,278 described a technique and instrumentation for intraoral clinical investigation. His initial work was follow·ed by several other investigations. 279-284 In 1967 Klein and MacPherson285 described electronic equipment which could measure distances, in microns, on the intraoral radiograph. The distances are measured 1vith a series of calibrated dots which are superimposed on a television scan line. Further investigation by these authors286 in photographing stored television microscope images led to sophisticated instrumentation that could store and measure a series of video images. A later report by Klein287 dealt with intraoral microscopy, linear- density measurements of radiographs, and subtraction radiography. The use of television-electronics in dental research is in its infancy. I Time should prove it an invaluable adjunct for the dental investigator. METHODS .Al'ID MATERIALS -57- Twenty-two male New Zealand white rabbits, each weighing three to four pounds, were selected for the investigation. The animals were caged individually in a room maintained at 70°F vvith 12 hours light and 12 hours darkness. The cages were cleaned every three days and once a week all cages were sterilized. Every three days the rabbits were weighed to the nearest tenth of a pound. Their diet consisted of Purina Rabbit Chow* given ad libitum. The rabbits were divided into two groups a~d assigned an identifi cation number by using a random numbers table. 288 To ensure that the animals would remain in their assigned group, the right ear of each rabbit w•s tattooed with the identification numbero Seven rabbits re- ceived distilled drinking water and a subcutaneous injection of 0.10 mlo of isotonic saline per pound of body weight every three days. Fifteen rabbits received 5.0 mg. of oxytetracycline** per pound of body weight in divided doses twice a d~ in their drinking water and 5.0 mgo of oxytetracycline*** subcutaneously every three days. The rabbits were maintained on this regimen for eight weeks and all incisor teeth were checked with an ultraviolet lamp~H} ever,y two weeks for evidence of tetracycline fluorescence. Administration of oxytetracycline was dis- continued the day before the clinical bleaching of the incisors, and thereafter all animals were given distilled drinking water. *Purina Rabbit Chow, Ralston Purina Co., St. Louis, Missouri **Terramycin Pediatric Syrup lot No. 1Y493, Pfizer Laboratories, New York, N.Y. ***Terr~cin Intramuscular lot No. 8Y911, Pfizer Laboratories, New York, N.Y. *~H~BlakR~ B-100 A, Ultra Violet Corporation, San Gabrial, California -58- Clinical Bleaching Of the 22 rabbits used in the stuqy, three control animals which were not to receive the drug accidentally received OTC and were destroy ed. The remaining 19 animals were randomized into five groups as follows: Three rabbits with tetracycline-stained teeth were not bleached and were used to determine if incisors in the same arch stained equal~, and could be used as their own controls for bleaqhed and unbleached stained teeth. Four rabbits without tetracycline staining would be bleached to determine if the bleaching process affected the teeth; two of these would be bleached once, the other two twice. Twelve rabbits with tetracycline stained incisors would be bleached; four would be bleached once, the re maining eight would be bleached twice. In all cases of bleaching, o~ one incisor in each arch was bleached and the other incisor acted as the unbleached control. The rabbits fasted 24 hours to prevent regurgitation of stomach contents during anesthesia. The animals were injected into an ear vein with 15.0 mg. of Nembutal* per pound of body weight. The incisor teeth were photographed with a Minolta SRT-101 camera using a 100 mm. short mount lens on an automatic bellows, a Tiffon 81-A filter and a Braun F-111 strobe-light. Kodachrome X film at 1/60 of a second and an F stop of 32 was used. A fluorescent photograph was then taken using the same basic camera system, but it was modified slightly by placing a Kodak Wratten 2B barrier filter in front of the lens system. The incisors were illuminated in the darkened room with only long-wave ultraviolet *Nembutal Sodium Solution, AbbottLaboratories, Chicago, Illinois -59- light from a 100 watt mercury vapor lamp.* The camera, light source, and rabbits were kept in a copstant relationship and using an F-stop of 5.6, a one-second exposure was made. All slides were taken at a 1:1 image to film ratio. A pre-operative and post-operative slide in white and ultra- violet light was taken during each bleacho A one-day post-operative slide in white light was taken after the final bleach and before the animal was sacrificed. The incisors were given a fiv.e second per tooth dental prophylaxis using flour of pumice moistened vdth water and applied vdth a rubber cup and slow speed handpiece. An attempt was made to match the four incisors vnth a shade guide.** An extra-heavy rubber dam was applied first to the left maxillar,y incisor, then to the mandibular right incisor, and each was bleached for 10 minutes. The dam was held in place by an Ash number nine clarn.p and a frame constructed of 0.040 stainless steel wire spot- l welded together (Figures 1 and 2)o Thirty percent hydrogen peroxide~~~ was held on the isolated tooth with saturated cotton pellets and a stop-watch was used to time the ap- plication of heat to them. Intermittent application of heat was sup-. plied by alternating the heating element**** for 30 seconds on the labial surface and 10 seconds on the lingual surface for a total time of 10 minutes. The temperature of the heating element was maintained at ll0°F and gauged periodically with a thermometero *Blak Ray B-100 A, Ultra Violet Corporation, San Gabrial, California **Trubyte Biotone Shade Guide, Dentsply International, Inc., York, Pa. **Sevriton Simplified Shade Guide, Claudius Ash Co., London, England *Y.-1Hiydrogen Peroxide 30% C.P., J. T. Baker Chemical Co., Philipsburg, N. J. ~P~Indiana University-Union Broach Bleaching Instrument, Union Broach Co., Long IsJa nd City, New York -60- Twenty-four hours after the bleaching treatments were completed, the animals were sacrificed by guillotine and the clinical crowns of the in- cisors were removed at the level of the gingiva with a high-speed carbide bur. The lingual of each incisor was scored for later orientation of the ground section on the microscopic glass slide. Throughout the preparation of the ground sections the specimens were stored in a laboratory drm~er of a darkened laboratory maintained at 60°Fo Preparation of Ground Sections The teeth were coded and placed in separate vials of 10 percent formalin for 24 hours. Next the specimens were dehydrated on successive days in alcohol in concentrations of 70, 80, 95, 100, and 100 percent etnyl alcohol. The teeth were then placed in styrene for 24 hours, fol- lowed by a d~ in an equal mixture of Bio-Plastic* and styrene. The next day the teeth were embedded in Bio-Plastic vdth their code number and allowed to cure for one week. The specimen blocks were then removed from their molds and polished with wet pumice and a cloth wheel on a lathe. The polished blocks were mounted on acrylic sectioning jigs. Serial transverse and approximately plano-parallel ground sections were prepared on a thin sectioning machine~~ (Figure 3) at 100 ±) ·microns, and checked on a stage micrometer.~L~ The teeth were arbitrarily divided into thirds representing incisal, middle and gingival thirds of the tooth. Each third produced about 4 ground sections. The ground sections Mlard's Bio-Plastic, Vfard's Natural Science Establishment, Inc., Rochester New York -~illings-Hamco Model GH-1, Hamco Machines, Inc., Rochester, New York ***Federal Products Corp. Model P-31, Providence, Rhode Island. -61- from the contralateral teeth for a specific third were mounted in pairs on the sa~e 1 inch by 3 inch glass slide with Histoclad* and cover-slip- ped with a number one Corning cover glass. All slides were stored in a light tight box at 40°F in a refrigerator until they were examined. An indepen~ent investigator selected the most ideal ground section from the incisal, middle and gingival third of each tooth for measure- ment. His selection was founded on identifying the ground section in each third closest to being plano-parallel and 100 microns thicko These sections were then coded to prevent identification of bleached and un- bleached specimens before measurement. This eliminated bias on measuring the fluorescent intensity of the specimens; however, it meant that the sections selected were not necessarily from identical positions in that third of the pair of teeth comparedo Fluorescent Intensity and Linear Measurements All electronic linear and amplitude measurements of tetracycline fluorescent intensity were completed in the Television and Electronic Dental Research Laboratory of Indiana University School of Dentistry. The television microscope measurement instrumentation was developed by Klein and MacPherson. 285,286 An ultraviolet light microscope-l* was coupled to a closed-circuit color television system with scan-line measurement circuitry (Figures 4 and 5). The microscope was adjusted for fluorescent microscopy using a planapo 4/0.16 objective mounted in *Histoclad, a low fluorescing synthetic mounting medium, Clay Adams, Parsippy, New Jersey **Zeiss Large Universal Research Microscope, Carl Zeiss, West Germany -62- a deflector FL assembly with a 53 insert barrier filter. The light source was a special purpose illuminator with a HB0-200 watt super pres- sure mercury lamp with BG-12 and BG-38 exciter filters. The lamp's power supply was stablized vdth transformers supplying a constant line voltage of 105 volts. This arrangement provided a televised image of 2.5 mm. of the tooth specimen under measurement. The color television camera* coupled to the microscope provided video signals from the red channel only. This channel provided the best video signal of the fluorescent image, but the signal was still weak and required amplification with a video distribution amplifier. The amplified signals were passed through a narrow pass filter to reduce the electronic noise caused by insufficient light from the fluorescing image. The pro- cessed signal was then applied to a calibrated differential comparator (Tektronic type ~ plug in unit) mounted in the oscilloscope.~· The com parison voltage control readout was utilized to position the video signal's wave-form at a reference level on the oscilloscope graticule and provide a digital reading of the amplitude of fluorescent intensity. The linear measurement system identified the position and distance of each fluorescing band of tetracycline from the outer enamel surface. The measurement system enabled the operator to select, identif.y, and illuminate any one of the 525 lines of scan of the televised fluorescent image, and generate a marker dot that was moved along the identified and illuminated line by a vernier control. *Shibaden Model HV-1100 U, Shibaden Company, Chicago, Illinois. **Tektronix Model RM 35 A, Tektronix, Inc., Portland, Oregon. -63- Calibration of the instrmnentation was accomplished by focusing the microscope assembly on a Bausch and Lomb glass stage micrometer slide ruled O.l/0.01 mm. The micrometer image was centered and focused with the viewing camera. The line selector was positioned through the micro meter ruling with the marker dot superimposed over the ruling. The linear measurement unit has scale factor calibration controls, these were ad justed to provide a digital display readout of 0.000 to 2.500 mm. - 0.005 mm. The measurement of the tetracycline fluorescent intensity was made by passing first one then the other of the unidentified and previously selected ground sections of each pair of incisors through the center point of the ultraviolet light. The intensity of each band of fluorescing tetracycline was measured individually starting from the outer enamel surface and ending on a tangent to the pulp chamber. The pulp canal was not included .. in the measurement to eliminate its intense autofluor escence from distorting the results (Figures 6 and 7). The maxillary incisor was measured by centering the scan line through the central groove of the labial surface. The mandibular incisor was measured by aligning the scan line perpendicular to the mid-point of the labial surface (Figure 3). Photomicrographs for publication were taken of selected sections with a Leitz ultraviolet light microscope with BG-12, BG-38 exciter filters and a 53 insert barrier filter. A Pentax camera and microscope adapter 1vith Hi-Speed Ektachrome (ASA 165) were used. Exposure time was between 5 and 60 seconds. -64- Analysis of Data The raw data were adjusted by computer to calculate the individual intensity of each band of tetracycline in a ground section. Intensity of fluorescence equaled the peak amplitude of the fluorescence minus the peak amplitude of the electronic noise and the base of the wave form (Figure 5). The numerical unit assigned equaled 1/1000 of the total oscilloscope height; since the signal was amplified and the oscilloscope was modified, no absolute unit (volts, lumens) could be assigned these values. The mean of 6 to 17 individual intensities from the tetracycline bands in a tooth was used as the fluorescent intensity of the tooth. This value for each tooth was then used to obtain a mean for a given group of teeth. The diffeFence in intensity of right and left incisor or unbleached and bleached incisor was obtained for each fluorescent band of the paired teeth and mean differences for the paired teeth and for a group of paired teeth were calculated. Using these means, t-test comparisons for statistical significance were computed: A) between each pair of teeth in the same arch of each rabbit; B) between all teeth that were unbleached compared to all that were bleached in the entire group; and C) between all teeth of the control group, all teeth that had been bleached once and all teeth that had been bleached twice. Observations made during a review of the Kodachrome slides taken during the clinical bleaching were then correlated with the measurement data. RESULTS -65- At the time of clinical bleaching, a shade guide was used for co~ parison with the rabbits' incisors. The rabbits 1vith no oxytetracycline exhibited a shade of approximately 61 on a Trubyte Biotone shade guide; those rabqits who had received oxytetracycline exhibited a color approx imating shade 69, yet the teeth exhibited a yellow hue not present in this or the Sevriton shade guide. This verified that the teeth of rabbits which had received oxytetracycline were different in color from those rabbits which had not received the drug and that tetracycline staining had been induced. Kodachromes of the rabbits taken in white and ultra violet light during the clinical bleaching (Figures 9. and 10) show that the maxillar,y incisors bleached clinically more successfully, as their shade became closer to shade 61 after the bleaching than the mandibular incisors. Table XIX compares the clinical Kodachromes vnth the depth of bleaching measured in the ground sections. The strongest bleaching occurred in the incisal one-third in both maxillar,r and mandibular incisors and was approximately 250 to 350 mic rons into the dentin in the maxillar.y and 150 to 200 microns into the dentin in the mandibular incisors. l~ important observation is that the tetracycline fluorescence was not entirely removed by the bleaching pro cess (Figures 11 and 12). The tetracycline fluorescence appeared micro scopically to be little changed by the bleach, except that its ability to fluoresce was slightly reduced and the bands exhibited less contrast than in the unbleached specimens. Tetracycline-stained teeth which were -66- not bleached exhibited a gradual increase of tetracycline fluorescent .. intensity from the outer enamel surface to the pulp canal. This pattern of fluorescent intensity was altered in about one-half of the cases sur- veyed, with the last three or four bands demonstrating a subtle decrease in fluorescent intensity. A graphic demonstration of these observations is detailed in the portion of the computer print-out reproduced from Appendix II that follows: RABBIT 12 MAXILLARY MIDDLE THIIID--T•'{O BLEACHES* I-1 I-2 I-3 D-1 D-2 (unbleached) (bleached) (unbl.-bl.) (depth I-1) (depth I-2) 233 103 130 72 52~E- 257 94 163 104 83 260 100 160 134 110 254 126 128 160 124 260 139 121 186 162 294 153 lhl 210 192 310 180 130 237 222 317 197 120 260 248~,} 307 212 95 287 269 306 . 219 87 312 292 320 242 78 336 315 300 247 53 364 345 267 237 30 388 372 257 213 44 400 400 N = 14 *The computer print-out demonstrates the fluorescent intensity of un bleached (I-1) and bleached (I-2) dentin. Each line represents a band of tetracycline. I-3 is the difference (I-1 - I-2) and represents the loss of intensity per band. D-1 corresponds to I-1 and D-2 corresponds to I-2 and is the depth of the leading edge of the tetracycline band from the outer enamel surface; the distance is in microns. The unit for intensity is 1/1000 of the oscilliscope height. **At the point of the line one can see that the greatest bleaching stopped and was betvreen 52 to 260 microns. The intensity of tetracycline fluorescence gradually changed from the first band to the last band in column I-1 and I-2. Refer to Appendix I for additional examples •. -67- The data in Table II through Table V demonstrate that there was no statistically significant difference between bleached and unbleached non- stained teeth. The bleaching process did not alter the inherent fluoresc- ence of the enamel and dentin. In one case (mandibular middle third after one bleach--Table III) vmere there was a significant difference at the {o.o5 level, the fluorescence was more intense in the bleached than in the unbleached teeth. The intensity of the unstained teeth was very low and no peaks of fluorescence could be seen on the oscilloscope above the peak of the electronic noise. To achieve a value for the fluorescence of unstained teeth, the peak value of the electronic noise was used as the peak of fluorescence of the unstained teeth. The enamel of tetracycline-stained teeth .fluoresced only sliehtly above the values obtained for unstained teeth, averaging about 10 units of fluorescent intensity (Tables VI to XI). This is in agreement with current knowle~ge _ that fluorescence of tetracycline is not seen to aqy great extent in the enamel. The difference in the intensity of tetra- cycline fluorescence of enamel as opposed to the fluorescence of tetra- cycline in dentin was highly significant. This significance is demon- strated by computing the t-test value from the mean intensities of the enamel and dentin of the unbleached teeth in the incisal one-third (Table X and Table XVI). If the t value is greater than 3.05, there is signifi- cance at the .01 level when there are 12 degrees of freedom. The computation of the t value is as follows; Intensity Standard Degrees of t-test Error Freodon value Enamel 5.57 2.54 12 8.85 Dentin 268 29.6 -68- Therefore, a highly significant difference is present between the degree of fluorescence of dentin and the degree of fluorescence of enamel in tetracycline teeth. Since the fluorescence of tetracycline in enamel is minimal, the presence or absence of tetracycline in enamel cannot be accurate~ determined by its fluorescence. Tables VIII through XI show that there was some effect of the bleaching procedure on the tetracycline in enamel, with an average de crease in fluorescent intensity of about 2 units after one bleach and about 7 units after two bleaches. This was statistically significant in only one group of animals (Table X--maxillary middle third); in this group the bleached enamel fluoresced ·with a significantly lower intensity (P <. 0.05) than the unbleached enamel. Tables XII and XIII show that there was great variation between in cisors in the same arch. In a comparison o! 36 pairs of teeth, 18 showed a significant ctifference: in 13 cases the right incisor showed a higher intensity of fluorescence, and in five cases the left showed a higher intensity. This finding invalidated the usa of tetracycline-stained in cisors of individual rabbits to act as their own controls for unbleached and bleached teeth. In comparing groups of teeth (Tables XII through XVIII), there was an insignificant statistical difference betvreen right ru1d left rabbit in cisors which were stained with tetracycline and not bleached. There was a tendency for reduced tetracycline fluorescence, with an average lowering of about 45 units of intensity after one bleach and a statistically significant loss of tetracycline fluorescence in the maxillary incisors -69- ( P < 0.001) and in the incisal one-third of the mandibular incisors (P (0.005) after two bleaches. Therefore, groups of teeth can be compar ed and there was an effect when the tetracycline-stained incisors were bleached twice. TABLES AND FIGURES TABLE II Comparison of dentin and enamel fluorescence of unbleached and bleached once maxillar.y rabbit incisors which received no tetraa,ycline. The incisal, middle and gingival thirds of the teeth were measured. Fluorescent Intensities statistical significance Rabbit Nu.rnber of Difference o£ Means.* Unbleached SE Bleached SE Difference SE t p Incisal Third Rabbit 1-section A 4o.o** 45.0 -5.00 Rabbit 1-section B 45.0 4o.o 5.00 Rabbit 1-section C 6o.o 40.0 14.0 Rabbit 1-section D 72.0 62.0 10.0 Rabbit 4-section A 73.0 63.0 10.0 Rabbit 4-section B 52.0 42.0 10.0 Rabbit 4-section C 90.0 44.0 46.0 Mean 61 .. 7 6.69 48.7 3.62 12.8 5.98 1.71 NS Middle Third Rabbit 1-section A 62.0 90.0 -28.0 Rabbit 1-section B 8.5.0 68.0 17.0 Rabbit 1-section C 72.0 71.0 1.00 Rabbit 1-section D 72.0 72.0 o.oo Rabbit 4-section A 78.0 41.0 37.0 Rabbit 4-section B 87.0 81.0 6.00 Rabbit 4-section C 90.0 79.0 11.0 Rabbit 4-section D 54.0 78.0 -24.0 Mean 75&0 4e44 72.5 5.11 2.50 7.47 .361 NS *Obtained by means of a t-test. When difference is not significant the letter NS are used instead o! P, **Each unit represents 1/1000 of total height of oscilloscope. I -.J cr TABLE II (continued) Comparison of dentin and enamel fluorescnece of unbleached and bleached once maxillar,y rabbit incisors which received no tetracycline. The incisal) middle and gingival thirds of the teeth were measured. Rabbit Number Unbleached Gingival Third 45.0~ Rabbit 1-section A Rabbit 1-section B 65.0 Rabbit 1-section C 79.0 Rabbit 1-section D 98.0 Rabbit 4-section A 79.0 Rabbit 4-section B 75.0 Rabbit 4-section C 87.0 Rabbit 4-section D 64.0 Mean 74.0 Fluorescent Intensities SE Bleached SE 90.0 85.0 78.0 65.0 76.0 86.0 93.0 86.0 5o70 82.4 .3;.18 Difference -45.0 -20.0 1.00 33.0 3.00 -11.0 -6.00 -22.0 ::n.38 SE 8.02 Statistical significance of Difference of Means.* t p 1.29 NS *Obtained bY means of a t-test. When difference is not significant the letters NS are used instead of P. **Each unit represents 1/1000 of total height of the oscilloscope. I -J J-1 I TABLE III Comparison of dentin and enamel fluorescence of unbleached and bleached once mandibular rabbit incisors which received no tetracycline. The incisal, middle and gingival thirds of the teeth were measured. Fluorescent Intensities Statistical significance Rabbit Number of Difference of Means* Unbleached SE Bleached SE Difference SE t p -Incisal Third Rabbit 1-section A 82.0** 87.0 -5.00 Rabbit 1-section B 85.0 77.0 8.oo Rabbit 1-section C 98.0 8.5.0 13.0 Rabbit 4-section A .53.0 97.0 -44.0 Rabbit 4-section B 64.0 78.0 -14.0 Rabbit 4-section C 74.0 72.0 2.00 Mean 76.0 6.53 82.7 3.64 -5.00 8.65 .896 NS Middle Third I -.J Rabbit 1-section A 71.0 85.0 -14.0 1\.) I Rabbit 1-section B 86.0 98.0 -12.0 Rabbit 1-section C Bo.o 78.0 2.00 Rabbit 4-section A 70.0 88.0 -18.0 Rabbit 4-section B 57.0 77.0 -20.0 Rabbit 4-section C 63.0 88.0 -25.0 Mean 71.2 4.35 85.7 3.15 -14.5 3.79 2.70 <·05 Gingival Third Rabbit 1-section A 89.0 92.0 -3.00 Rabbit 1-section B 105 77.0 28.0 Rabbit 1-section C 76.0 83.0 -7.00 Rabbit 4-section A 80.0 84.0 -4.00 Rabbit 4-section B · 96.0 82.0 14.0 Rabbit 4-section C 77 .o 83o0 -6.00 Mean 87.2 4.76 8).;" 1.98 j.67 5.80 .715 NS *Obtained by means of a t-test. \Vhen difference is not significant the letters NS are used instead of P. ~Each unit represents 1/1000 of the total height of the oscilloscope. TABLE IV Comparison of dentin and enamel fluorescence of unbleached and bleached twice maxi11ar.y rabbit incisors which received no tetracycline. The incisal, middle and gingival thirds of the teeth were measured. Fluorescent Intensities Statistical Significance Rabbit Number of Difference of Means* Unbleached SE Bleached SE Difference SE t p Incisal Third Rabbit 3-section A 98.o** 90.0 8.oo Rabbit 3-section B 105 103 2.00 Rabbit 3-section C 102 113 -11.0 Rabbit 21-section A 107 110 -3.00 Rabbit 21-section B 105 125 -20.0 Rabbit 21-section C 111 118 -1.00 Rabbit 21-section D 119 111 8.oo Mean 107 1 2.55 llQ 0 4.22 -3.21f 3.89 .608 NS Middle Third Rabbit 3-section A 109 114 -5.00 Rabbit 3-section B 80.0 115 -35.0 Rabbit 3-section C 101 110 -9.00 Rabbit 3-section D 98.0 1c6 -8.00 Rabbit 21-section A lll 117 -6.00 Rabbit 21-section B 111 102 9.00 Rabbit 21-section C 105 100 5.00 Rabbit 21-section D 107 122 -15.0 Mean 103 3.64 nr 2.70 -8.50 4.72 1.76 NS *Obtained bY means of a t-test. When difference is not significant the letters NS are used instead of P. **Each unit represents 1/1000 of total ~eight of the oscilloscope. I -.J \..!.) • TABLE IV (continued) Comparison of dentin and enamel fluorescence of unbleached and bleached twice maxi11ar.y rabbit incisors which received no tetracycline. The incisal, middle and gingival thirds of the teeth were measured. Rabbit Number Unbleached Gingival Third Rabbit 3-section A 94.0~ Rabbit 3-section B 91.0 Rabbit 3-section C 88.0 Rabbit 21-section A 87.0 Rabbit 21-section B 112 Rabbit 21-section C 119 Rabbit 21-section D 120 Mean 102 .. Fluorescent Intensities SE Bleached SE -· 86.0 102 108 92.0 137 123 119 5.60 llO 6.81 Difference B.oo -9.00 -20.0 -5.00 -25.0 -4.00 1 .. 00 -7.71 SE 4.35 Statistical Significance of Difference of Means* t p .901 NS *Obtaliied by means of a t-test. When difference is not significant the letters NS are used instead of P. **Each unit represents 1/1000 of total h~ight of the oscilloscope. TABLE V Comparison of dentin and enamel fluorescence of unbleached and bleached twice mandibular rabbit incisors which received no tetracycline. The incisal, middle and gingival thirds of the teeth were measured. Fluorescent Iritensities StatistiCal Significance Rabbit Number of Difference of Means* Unbleached SE Bleached SE Difference SE t p Incisal 'Ihird Rabbit 3-section A 118** 88.0 30.0 Rabbit 3-section B 105 91.0 8.oo Rabbit 21-section A 88.0 107 -19.0 Rabbit 21-section B 86.0 95.0 -9.00 Rabbit 21-section C 93.0 88.0 s.oo Mean 98.0 5.99 95.0 3.51 3.00 8.32 .432 NS Middle Third Rabbit 3-section A 100 97.0 3.00 ~ Rabbit 3-section B 98.0 98.0 o.oo \.n Rabbit 3-section C 10.5 10.5 o.oo I Rabbit 21-section A 101 98.0 3.00 Rabbit 21-section B 105 91.0 8.00 Rabbit 21-section C 100 97.0 3.00 Mean 102 1.39 98.5 1.37 ~.83 1.40 1 • .54 NS Gingival Third Rabbit 3-section A 101 98.0 3.00 Rabbit 3-section B 112 96.0 16.0 Rabbit 3-section C 107 110 -3.00 Rabbit 3-section D 102 113 -11.0 Rabbit 21-section A 88.0 114 -26.0 Rabbit 21-section B 91.0 109 -18.0 Rabbit 21-section C 104 92.0 12.0 Rabbit 21-section D 100 79.0 21.0 Mean 1o1 2.79 lOl 4.34 -6.15 5.94 o.oo NS *Obtained bY means of a t-test. · When difference is not . significant the letters NS are used instead of P. **Each unit represents 1/1000 of the total height of the oscilloscope. TABLE VI Comparison o~ enamel .tetracycline fluorescent intensity of right and left maxillar,y rabbit incisors. The incisal, ~ddle and gingival thirds of the teeth were measured. Fluorescent Intensit~es Statistical Significance Rabbit Number of Dilference of Means* Right SE Left SE Difference SE + p v Incisal Third - Rabbit lOA** 28.o*** 27.0 1.00 Rabbit lOB 47.0 34.0 13.0 Rabbit 13A . 27.0 21.0 6.00 Rabbit 13B 4.00 10.0 -6.00 Rabbit l5A 17.0 o.oo 17.0 Rabbit 15B. 10.0 3.00 7.00 Mean 22.2 6.27 15.8 5.57 b.33 3.36 .763 NS Middle Third I -.J Rabbit lOA· -10.0 27.0 -31.0 "' ' Rabbit lOB 25.0 68.0 -43.0 Rabbit 13A 26.0 n.o 15.0 Rabbit 13B 20.0 19.0 1.00 Rabbit 15A 3.00 1.00 -1.00 Rabbit l5B -9.00 35.0 -44.0 Mean 9.11 6.80 27.3 9.30 -18.2 10.6 1.57. NS Gingival Third Rabbit lOA 48.0 90.0 42.0 Rabbit lOB 56.0 46.0 -10.0 Rabbit 13A 31.0 20.0 17.0 Rabbit 13B 17.0 9.00 8.00 Rabbit 15A 43.0 14.0 29.0 Rabbit 1.5B 1.00 22.0 -21.0 Mean j:;.7 8.46 33.5 12.4 16.8 9.65 .013 NS *Obtained by means of a t-test. When difference is not significant the letters NS are used instead of P. **A and B represent different sections from the same animal in that third. ***Each unit represents 1/1000 of the total height ·of the oscilloscope. TABLE VII Comparison of enamel tetracycline fluorescent intensity of right and left mandibular rabbit incisors. The incisal, middle and gingival thirds of the teeth ~ra measured. Fluorescent Iritensities Statistical Significance Rabbit of Difference of Means* Ri~ht SE left SE Difference: t p Incisal !hird Rabbit lOA"** 4~oo*** -12.0 16.0 Rabbit lOB -9.00 o.oo -9.00 Rabbit 13A . 15.0 1.00 a.oo Rabbit 13B 21.0 1.00 20.0 Rabbit 15A 11.0 -3.00 14.0 Rabbit 15B 11.0 4.00 7.00 Mean 8.83 4.23 -0.$0 2.69 ~-33 4.18 1.86 NS Middle Third I -.J Rabbit lOA 8.00 2.00 6.00 -.J I Rabbit lOB ll.O 3.00 a.oo Rabbit l3A 6.00 17.0 -11.0 Rabbit 13B 24.0 19.0 5.00 Rabbit 15A 13.0 o.oo 13.0 Rabbit 15B 17.0 2.00 15.0 Mean 13.2 . Q 2.68 7.17 3ct46 6.05 3.76 1.38. NS Gingival Third Rabbit lOA -8.00 -2.00 -6.00 Rabbit lOB 10.0 6.oo 4.00 Rabbit 13A 9.00 22.0 -13.0 Rabbit 13B 18o0 B.oo 10.0 Rabbit 15A 5.00 o.oo 5.00 Rabbit 15B -13.0 18.0 -31.0 Mean 3.50 4.79 8.67 3.92 -5.17 6.18 .835 NS *Obtained bY means of a t-test. ~~en difference is not significant the letters NS are used instead of P. **A and B represent different sections from the same animal in that third. ***Each unit represents 1/1000 of the total height of the oscilloscope. TABLE VIII Comparison of enamel tetracycline fluorescent intensity of unbleached and bleached once maxillar,y rabbit incisors. The incisal, middle and gingival thirds of the teeth were measured. Fluorescent Intensities Statistical Sigriificance Rabbit Nllllilier of Difference of Means* Unbleached SE Bleached SE Difference SE t p Incisal Third Rabbit 6 1o.o** -15.0 25.0 Rabbit 8 17.0 11.0 6.00 Rabbit 9 2.00 8.00 -6.00 Rabbit 11 9.00 11.0 -2.00 Mean 9.50 3.10 ). 75 6.29 5.60 6.28 .788 NS Middle Third Rabbit 6 5.00 o.oo 5.00 Rabbit 8 40.0 5.00 35.0 Rabbit 9 -9.00 18.0 .' -27.0 Rabbit 11 8.oo 16.0 -8.00 Mean 11.0 10.4 9.75 4.20 1.25 12.8 .111 NS Gingival Third Rabbit 6 15.0 1.00 14.0 Rabbit 8 16.0 8.oo a.oo Rabbit 9 16.0 27.0 -11.0 Rabbit 11 34.0 14.0 20.0 Mean 2o.2 4.59 12.5 5.52 7.75 6.71 1.07 NS *Obtained by means of a t-test. When difference is not significant the letters NS are used instead of P. **Each unit represents 1/1000 of total height of the oscilloscope. I -J co ' TABLE IX Comparison of enamel tetrac,ycline fluorescent intensit,y of unbleached and bleached once mandibular rabbit incisors. The incisal, middle : and gingival thirds of the teeth were measured. Fluorescent Intensities Statistical Significance Rabbit Number of Difference of Means* Unbleached SE Bleached SE Difference SE t p Incisal Third - Rabbit 6 31.0** -s.oo · 36.0 Rabbit 8 3.00 41.0 -38.0 Rabbit 9 -5.00 8.00 -13.0 Rabbit 11 -1.00 -11.0 10.0 Mean ?.oo 8.16 8.25 11.6 -1.25 JS.B .oaa NS Middle Third Rabbit 6 o.oo o.oo o.oo Rabbit 8 5.00 o.oo s.oo Rabbit 9 8.00 5.00 3.00 Rabbit 11 1.00 24.0 -17.0 Mean s.cxr· 1.78 7.25 5.10 -2.25 5.02 .371 NS Gingival Third Rabbit 6 -5.00 8.oo -13.0 Rabbit 8 5.00 12.0 -7.00 Rabbit 9 13.0 -10.0 23.0 Rabbit 11 35.0 36.0 -1.00 Mean 12.0 8.5o 11.5 9.46 o.5o 7.89 .039 NS *Obtained b.Y means of a t-test. V~en difference is not significant the letters NS are used instead of P. **Each unit represents 1/1000 of totaL height of the oscilloscope. I -J '0 ' TABLE X Comparison of enamel tetrac,ycline fluorescent intensity of unbleached and bleached twice maxillar.y rabbit incisors. The incisal, middle and gingival thirds of the teeth were measured. Fluorescent Intensities Statistical Significance Rabbit Number of Difference of Means* Unbleached SE Bleached SE Difference SE t p Incisal Third Rabbit 7 2.00** 2.00 o.oo Rabbit 12 10.0 o.oo 10.0 Rabbit 16 10.0 9.00 1.00 Rabbit 17 12.0 10.0 2.00 Rabbit 18 -6.00 -11.0 5.00 Rabbit 19 2.00 -14.0 16.0 Rabbit 20 9.00 -6.00 15.0 Mean 5.57 2.54 -1.43 3.53 7.06 2.46 1,63 NS Middle Third Rabbit 7 5.00 7.00 -2.00 Rabbit 12 12.0 -3.00 15.0 Rabbit 16 18.0 6.00 12.0 Rabbit 17 19.0 13.0 6.00 Rabbit 18 19.0 4.00 15.0 Rabbit 19 15.0 -5.00 20.0 Rabbit 20 13.0 -5.00 18.0 Mean 14.4 1.90 2.43 2.;62 12.0 2.89 2.41 (.05 *Obtained by means of a t-test. When difference is not significant the letters NS are used instead of P. **Each number represents 1/1000 of the total height of the oscilloscope. 6, 0 1 TABLE X (continued) Comparison of enamel tetracycline fluorescent intensity of unbleached and bleached twice 1naxillary rabbit incisors. The incisal, middle and gingival thirds of the teeth were measured. Rabbit Number Unbleached Gingival Third 17.0** Rabbit 7 Rabbit 12 19.0 Rabbit 16 15.0 Rabbit 17 38.0 Rabbit 18 9.00 Rabbit 19 10.0 Rabbit 20 7.00 Mean 10.4 Fluorescent Intensities SE Bleached . SE Difference -16.0 33.0 10.0 9.00 4.00 11.0 22.0 16.0 -16.0 25.0 9.00 1.00 7.00 o.oo 3o96 2.86 5.31 13.6 .. SE 4.59 Statistical Significance of Difference of Means* t p 2.04 NS *Obtained by means of a t-test. When difference is not significant the letters NS are used instead of Po **Each number represents 1/1000 of the total height of the oscilloscope . I o:> .. TABLE XI Comparison of enamel tetracycline fluorescent intensity of unbleached and bleached twice mandibular rabbit incisors. The incisal, middle and gingival thirds of the teeth were measured. ·Fluorescent Intensities Statistical Significance Rabbit Number of Difference of Means* Unbleached SE Bleached SE Difference SE t p Incisal Third Rabbit 7 1.oo** 1o.o -9.00 Rabbit 12 9.00 3.00 6.00 Rabbit 14 62.0 -5.00 67.0·. Rabbit 16 2.00 -6.00 8.oo Rabbit 17 21.0 -3.00 24.0 Rabbit 18 6.00 o.oo 6.00 Rabbit 19 -2.00 -7.00 5.00 Rabbit 20 -6.00 -16.0 10o0 Mean 11.6 1•1S -3.00 2.71 14.6 8.12 1.77 NS Middle Third Rabbit 7 8.00 2.00 6.00 Rabbit 12 6.00 13.0 -7.00 Rabbit 14 1.00 5.00 -4.00 Rabbit 16 15.0 8.oo 7.00 Rabbit 17 33.0 10.0 23.0 Rabbit 18 3.00 o.oo 3.00 Rabbit 19 20.0 o.oo 20.0 Rabbit 20 -2.00 -10.0 8.oo Mean Io.5 4.11 3.5b 2.55 7oOO 3.68 1.44· NS *Dbtainea by means of a t-test. When difference is not significant the letters NS are used instead of P. «*Each unit represents 1/1000 of the total height of the oscilloscope. I o:> 1'\) I TABLE XI (continued) Comparison of enamel tetracycline fluorescent intensity of unbleached and bleached twice mandibular rabbit incisors. The incisal, middle and gingival thirds of the teeth were measured. Fluorescent Intensities Statistical Significance Rabbit Number of Difference of Means* Unbleached SE Bleached · SE Difference SE t p Gingival Third 6.oo** Rabbit 7 22.0 -16.0 Rabbit 12 13.0 -5.00 18.0 Rabbit 14 28.0 22.0 6.00 Rabbit 16 48.0 14.0 34.0 Rabbit 17 30.0 15.0 15.0 Rabbit 18 11.0 5.00 6.00 Rabbit 19 -3.00 15.0 -18.0 Rabbit 20 -11.0 -2.00 -9.00 Mean 15.2 6.79 10.8 3e64 4.5o 6.38 .571 NS *Obtained b.1 means of a t-test. Vfuen difference is not significant the letters NS are used instead of P. **Each unit represents 1/1000 of the total height of the oscilloscope. I CX> w ' TABLE XII Comparison of dentin tetracycline fluorescent intensity of right and left maxillar.y rabbit incisors. From six to seventeen bands were measured in the incisal, middle and gingival thirds of the teeth. Fluorescent Intensities Statistical Significance Rabbit Number Right Left Difference of Difference of Means* Mean SE Mean SE Mean t p Incisal Third Rabbit lOA** 187*** 11.6 196 3.24 -8.89 12.7 0.75 NS Rabbit lOB 223 3.28 209 2.71 13.8 4.78 3.29 .6 237 29 302 319 291 240 51 330 348 263 229 34 357 374 - -. . RABBIT 13A N= 14 MEM ~ I 1::: 239 • 643 MEAN I 2= 203·929 MEAN DI F= 3 5 ·- 714 3 . . - - SD.l~ 37· 029 3 SD2~ 27· 2974 SD3= 21·949 . SE1= 9·89 65 SE2= 1· 29 554 SE3= 5·8 6611 .... I 1 I2 I 3. Dl D2 172 84 88 28 21 207 91 . 116 50 48 202 .153 49 69 71 199 .167 32 92 94 218 18 0 38 J18 122 222 2.05 17 .148 152 254 192 62 173 18 1 228. 212 16 204 213 25.3 211 42 236 242 251 215 36 263 274 254 2!14 10 292 303 290 2.18 72 320 333 260 214 46 342 361 . . - . RABBIT 13R N= 13 -- MEAN I 1= . 231• 538 .. MEAN !2= 18 3· 538 MEAN DIF= 48 SDJ~ 32~ 1989 . SD2~ 48.7743 SD3= 30· 2297- SEl= 8 ·93037 SE2= 13·5276 SE3= 8• 3842 . -131- - - . I 1 I2 13 Dl D2 - 205. .174 31 .12.2 112 191 177 14 161 148 222 203 19 195 18 5 216 221 -s 227 218 23.0 192 38 263 252 2ea 200 28 304 290 239 204 35 335 324 22.5 19.6 29 368 354 241 213 28 400 386 200 .18 7 21 429 414 220 200 20 454 440 203 .183 20 470 462 202 184 18 493 482 -- RABBIT 15A N= 13 , - .... - . - MEAN ~ I 1= 21 7• 692 MEAL\J I a~ 194·923 MEAN DI F= 22.~ 7.692. ~ -- . . SD_1?. 15· 1788 SD2= 13.8411 SD3= 10·9784 SE 1= 4. 209'8 6 SE2= 3· 8 388 4 SE3= 3· 0446 7 I 1 12 I3 Dl D2 . . - 223 198 25 .142 152 23!1 208 a6 18 1 184 259 .18 4 75 216 220 24.0 l92 I.B 250 261 281 185 96 286 292 236 180 56 320 324 240 210 30 352 357 200 190 10 378 388 233 209 24 400 414 205 .177 00 432 441 20/i 190 14 454 464 194 19 7 -3 473 .te2 . . R.ABBIT 1511 N= 12 MEAN_ I 1= 229·003 MEAN I 2= 19 3· 333 MEAN DI F= - 35. .~ 75 -- SD.l~ 25• 6531 SD2~ 11·27_61 SD3~ 28.3007 SE1c: 7·40542 SE2= 3.25514 SE3= 8 • 169 72 I 1 18 1 19 6 215 231 234 212 198 216 207 RABBIT lOA . . MEAN Il= 210 SD_l= 16.8>523 SEl= 5e61743 I 1 192 199 216 231 221 224 223 233 220 221 206 RABBIT lOB· -132- MANDTI3ULAR INCISAL THTIID . STAINED, NO BLEACH 12 13 216 -35 210 -14 215 0 238 -7 239 -s 231 -19 225 -27 206 10 176 31 N= 9 MEAN I 2= 217• 333 SD2= 19.49 36 SD3= 19·8809 SE2= 6. 49 78 6 SE3= 6· 6269 7 !2 I3 209 .. 17 219 -20 228 -12 243 -12 237 -16 233 -9 220 3 227 6 202 18 197 24 206 0 Na 1 1 Dl 96 131 167 19 7 236 272 304 334 373 MEHN D1 113 148 183 222 262 292 328 364 398 426 452 MEAN I 1= 2 1 6. 9 09 MEAN 12= 220·091 -3. 18 18 2 SD1=: 12.8 41 SD2:= 15· 0695 SD3= 14· 6548 SE;l= 3e8717 SE2= 4e54364 SE3= 4· 418 59 D2 142 178 218 256 286 328 354 369 396 DI F=-7• 33333 D2 129 164 198 232 268 306 340 370 405 433 455 MEAN DI Fu -133- I 1 12 13 Dl D2 80 176 -96 36 43 190 18 3 7 50 63 222 248 -26 66 84 273 237 36 88 100 254 352 -98 120 138 292 281 11 148 174 294 274 20 18 0 215 325 299 26 214 258 289 260 29 251 292 316 284 32 290 330 303 276 27 331 368 297 264 33 331 398 311 251 60 404 421 R.ABBI 'T 13A N= 13 ME.AN I 1= 265.077 MEAN I 2= 260• 38 5 MEAN DI F= 4. 69231 S D .1 = 6 7 • 58 6 3 SD2?= 45·8 322 SD3= 49. 189 1 . SEt= 18· 7451 SE2= 12· 7116 SE3= 13· 6426 I 1 I I2 13 Dl D2 120 71 49 36 41 195 171 24 52 53 265 19~ 71 72 70 272 215 57 98 93 256 218 38 130 120 290 237 . 53 168 149 291 258 33 202 182 311 293 18 237 220 290 241 49 280 254 298 241 57 317 292 292 257 35 362 329 263 245 18 402 367 2 .74 251 23 439 396 319 216 103 ll67 422 RABBIT 13:S N= 14 MEAN I 1= 266.857 MEAN I 2= 222 MEAN DI F• 44. 8 57 1 SDl= 51· 7893 SD2= 52·7447 SD3= 23· 369 5 SEl= 13.8413 SE2= 14·0966 SE3= 6· 24575 -134-. I 1 I2 I3 Dl D2 136 184 -48 (!fJ 86 172 209 -37 98 125 19 7 255 -58 126 154 212 254 -42 158 192 209 230 -21 196 226 188 221 -33 247 262 189 203 •14 272 300 164 192 -28 308 328 201 207 -6 362 366 176 19 6 -20 404 400 206 218 -12 452 436 205 208 -3 497 468 223 219 4 528 500 RABBIT 15A N= 13 ME.AN I 1= 190·615 MEAN I2= 215·077 MEAN DI F= -24.4615 SD1~ 23· 6239 SD2= 21.5076 SD3= 18. 568 SE1= 6·5521 .SE2= 5·9 6514 SE3= s. 1498 3 I 1 I2 !3 D1 D2 152 176 -24 52 104 .167 190 -23 83 138 1911 230 -36 112 171 193 223 -30 136 204 201 211 -10 170 238 283 202 8 1 210 277 282 191 91 244 317 174 162 12 276 355 188 195 -7 320 394 173 172 1 364 432 194 179 15 408 468 _19 1 191 0 452 498 19 7 190 7 495 513 205 166 39 536 534 RABBIT 15R N= 14 MEAN I 1= 199·571 MEAN I 2= 191·266 MEAN DI F= 8·28571 SD1= 38 • 0176 SD2= 20· 1511 SD3= 38. 5016 SEt= 10·1606 SE2= 5· 38 56 SE3= 10· 29 I 1 l99 199 220 272 248 250 258 264 250 238 2.03 218 218 203 228 RABBIT lOA - - -135- MANDIBULAR MIDDLE THIRD STAINED, NO BLEACH .. I2 13 16 1 18 218 -19 2J&8 -28 310 -38 287 -39 260 •10 260 -2 255 9 241 9 225 13 202 1 192 26 207 11 206 -3 203 25 -- N= 15• MEAN I 1= 231•2 · MEAN I 2= 233 SDJ= 24•8458 ' ~D2?= 36·8 549 SD3= 21· 1329 SE1= 6· 41516 SE2= 9· 5159 SE3= s. 45649 I 1 ~ I2 I3 - r 189 182 7 224 211 13 245 2LIS -3 265 259 6 264 257 7 283 240 43 271 222 49 263 226 37 263 235 28 253 220 33 209 217 -8 2.19 200 19 210 192 18 RABBIT 10B N= 13 - . Dl 68 92 120 149 186 220 256 287 315 350 384 411 440 460 479 MEA'J DI F=-1•8 Dl 78 100 142 174 206 252 284 314 346 378 413 433 462 MEAN I lza 242·923 MEAI.\J I 2= 223• 769 19·1538 SDl~ 29e31~ SD2= 23·9 31 SD3= 1 7· 7569 SEl= 8 • 12992 SE2= 6· 63726 SE3= 4.92~8 D2 66 87 116 149 184 220 254 280 310 344 375 403 428 452 472 D2 81 116 145 175 212 248 281 312 342 374 396 424 443 MEAN DI F= -136- I 1 I2 I3 D1 D2 250 19 1 59 49 39 250 19 1 59 68 56 302 236 66 86 79 316 260 56 113 103 310 255 55 143 127 368 304 64 172 156 275 295 -20 203 18 Ll 383 309 74 245 217 366 311 55 278 252 371 307 64 311 289 385 305 80 348 322 333 276 55 388 357 313 302 11 418 389 RABBIT 13A N= 13 MEAN . I 1= 324• 7e:J MEAN I 2= 272• 615 ME.AN DI F= 52~_ 1538 . SDl= 47• 78 (52 SD2?7 43.3389 SD3= 27·015 SEl= 13• 2535 SE2= 12·02 SE3= 7· 49 26 I 1 I -2 13 Dl D2 162 215 - 53 37 43 264 215 49 51 62 253 246 7 84 84 262. 262 0 93 106 301 245 56 123 133 312 281 31 148 164 344 273 71 18 0 19 6 336 271 65 217 230 343 270 73 252 270 330 267 63 296 308 332 271 61 334 344 332 228 104 372 377 298 230 68 409 412 RABBIT 1JB N= 13 MEAN I 1 = 29 7 • 615 MEAN 12= 251·846 MEA'J DI F= 45.7692 .. S D .1 ~ 5 1• 718 SD2= 23·3375 SD3= 40· 669 7 SEl= 14.344 SE2= 6. 4726 7 SE3= ll e 2797 -13·7- I 1 12 13 D1 D2 163 153 10 42 59 200 190 10 62 84 19 1 216 -25 82 109 211 224 -13 106 131 200 236 -36 134 161 202 241 -39 161 191 237 243 -6 198 224 202 206 -4 232 257 .229 .247 -18 268 293 235 233 2 309 333 269 271 -2 348 367 ~ .3 261 22 390 405 311 262 49 434 438 279 307 -28 478 474 257 291 -34 516 509 272 253 19 546 535 RABBIT 15A N= 16 MEAN_ I 1= 233·813 MEP.N I 2= 239• 625 MEA\J DI F= -5~8125 SD1~ 41• 1229 SD2~ 37· 7039 SD3= 24· 2768 SEl= 10·2807. SE2= 9· 42597 SE3:: 6· 0692 I 1 I2 I3 Dl D2 157 164 -7 48 87 .149 167 -18 72 113 167 207 -40 99 141 204 223 -19 125 174 205 215 -10 152 214 19 6 212 -16 184 249 204 19 3 1 1 220 285 ~06 184 22 258 320 194 190 4 294 3S4 221 18 6 35 330 395 174 194 -20 369 426 212 185 27 410 456 213 211 2 444 Ll87 225 201 24 475 511 246 258 -12 502 528 R.ABBIT 15B N= 15 MEAN I 1= 198· 2 MEAN I 2= 199. 333 MEAN DI F= -1.13333 SD.l~ 26• 4662 SD2= 23·4114 SD3= 21·3303 SEl= 6.8 3353 SE2= 6. 0448 1 SE3= 5·50746 -138- MANDIBULAR GINGIVAL 'rHmD STAINED, NO BLEACH I 1 I2 I3 D1 D2 . .100 .60 40 38 35 .160 142 .18 57 46 114 .157 17 73 61 2.10 164 46 98 84 217 192 25 127 113 257 224 33 163 142 227 245 -18 192 170 263 231 32 224 204 255 233 .22 258 238 237 19 7 40 292 270 215 204 11 324 300 220 218 2 354 330 2.15 199 16 381 358 2.15 210 5 403 389 215 185 30 422 416 181 179 2 434 448 220 18 0 40 448 461 -- RABBIT 10A N= 17 .. MEAN 11~ 210· 647 MEAN I 2= 189 · -412 MEAN DI F= 21~. 2353 - . - SD_l~ 39 • .6389 . 'SD2~ 43· 6134 SD3:=; 1 7 ~ 1739 SEt= 9 • 6138 5 SE2= 10· 5778 SE3= 4 e l6527 . . I 1 12 13 Dl D2 -. . 152 149 3 40 36 210 168 42 52 60 222 203 19 77 82 246 23.1 _15 101 104 240 241 -1 131 133 287 253 34 160 165 257 26.1 -4 229 225 238 231 7 260 259 258 218 40 292 288 285 201 84 321 315 245 191 48 348 340 260 211 49 374 366 241 19 7 44 399 389 250 222 28 414 416 2~0 204 56 434 438 231 201 30 451 456 RABBIT lOR N= 16 MEAN I l:a 242• 625 MEAN I 2= 21lo 75 MEAN DI Fa 30·8 75 SDl~ 31· 36 SD2!= 28 ·8941 SD3= 23$ 728 7 SEt= 7·84 SE2= 7·22351 SE3:2 5·9 3217 -139- I 1 12 13 Dl D2 147 155 -8 LIB ll4 200 239 -39 64 56 232 250 -18 82 75 255 287 -32 100 99 293 282 11 132 124 279 317 -38 161 152 306 300 6 190 18 1 325 350 -25 228 211 33e 334 -2 256 250 362 346 16 290 287 334 372 -38 326 324 - RABBIT 13A N= 11 MEAN _ I _1= 278• 636 MEAN I 2= 29 3·8 18 ME.AN DI F= - 15._ 18 18 - - - - SDJ~ 64·9712 SD2= 62· 0996 SD3= 20·8 03 SEl= 19. 5895 SE2= 18. 7237 SE3= 6· 27233 . I 1 I2 I3 Dl D2 211 84 _127 44 43 200 193 15 62 54 248 206 42 86 74 252 233 19 112 95 298 265 33 141 123 285 249 36 168 148 322 292 30 200 179 320 301 19 233 213 339 299 40 269 246 348 333 15 303 278 304 306 -2 339 312 RABBIT 13B N= 1 1 MEAN I 1= 28 5 MEAN I 2= 251 MEAN DI F= 34 SDl~ 48 ·8 59. SD2:= 70· 7361 SD3= 33·5231 SE1= 14. 7315 SE2= 21·3277 SE3= 10·1076 -140- I 1 12 I3 D1 D2 158 18 5 -27 36 36 - 177 200 -23 544 54 162 21P -28 72 72 190 213 -23 92 93 2.28 263 -35 114 120 219 233 -14 146 150 223 280 -57 18 0 18 2 226 240 -14 214 214 262 281 -19 250 249 279 304 -25 289 283 279 300 -29 324 316 253 312 -59 360 354 252 275 -23 399 390 244 292 -48 436 428 239 298 -59 470 460 263 290 -27 506 492 -- . RABBIT .15A N= 16 MEAN I 1= 229 • 625 MEAN I 2= 261• 5 MEAN DI F= - 31~. 8 75 SD1= 36~ 7784 SD2= 41·9 635 SD3= 15·3444 SEl= 9•1946 SE2= 1 o. 4909 SE3= 3·8361 I 1 I2 I3 Dl D2 241 168 73 42 36 262 165 97 52 58 268 18 _1 87 68 78 284 16 1 103 88 98 274 189 85 112 124 306 211 95 138 151 - 29A 200 94 170 184 311 219 92 204 220 "327 220 107 234 253 316 252 64 274 .283 298 261 37 300 318 327 291 36 340 351 332 297 35 380 386 284 293 -9 421 425 329 298 31 456 466 RABBIT 15R N= 15 MEAN I 1= 29 6·8 67 MEAN I 2= 228• 4 MEA\1 DI F• 68.4667 SD1= 27~ 7305 SD2= 49·4568 SD3= 34.463 SEt= 7· 15999 SE2= 12·7(;f)7 SE3= 8 ·898 3 I 1 -. 126 146 18 6 .16.6 .1.51 141 HABBIT 6 -141- MAXILLARY INCISAL THIRD STAINED, 1 BLEACH I2 I3 148 -22 143 3 .148 38 153 13 154 -3 126 15 .. N= 6 Dl 286 321 360 392 422 454 MEAN I 1:::: 152· 667 MEAl\J I 2= 145· 333 7~ .33333 .. SDJ~ 20e8966 SD2~ 1 o~ 2w 1 SD3= 20· 00 65 SEl= 8 • 53099 SE2= 4· 19 259 SE3= 8. 20027 I 1 12 13 Dl 374 286 88 168 352 23~ 116 220 354 259 95 264 394 272 122 312 372 254 118 357 3LU~ 222 120 38LI 3!17 247 100 394 314 244 70 408 .. RABBIT ·a: N= 8 .. . . MEtW I 1= 356. 125 ME.Al\l I 2= 252·5 10.3· 625. SD.l~ 24. 1569 SD2~ 20~ 18 49 SD3= 18· 6236 SEl= 8 • 54074 SE2= 7·13643 SE3= 6. 58 444 D2 296 326 354 382 406 438 ME.AN DI F= D2 224 270 304 340 378 410 429 440 MEAN DI F= -142- I 1 12 13 Dl D2 354 291 63 166 104 309 284 25 199 148 334 326 8 230 174 330 314 16 254 201 318 280 38 278 226 267 238 29 304 253 280 278 2 334 281 277 265 12 354 304 240 233 7 376 325 265 239 26 392 340 ~ -- RABBIT 9 N= 10 MEAN I 1= 297· 4 MEAN I 2= 27i1·8 MEAN DI F= 22_._ 6 . . ~ . .. - . . - SDJ.~ .3P• 746. SD2:=: 31·6607 SD3= 18·1488 SEl= 11· 6201 SE2= 1 0~ 012 SE3= s. 73914 I 1 12 13 Dl D2 - - 318 235 63 208 89 344 241 103 239 118 375 310 65 268 153 373 336 37 288 176 356 342 14 317 205 356 329 27 344 241 304 329 -25 365 274 316 353 -37 382 300 -- RABBIT 11 N= 8 . . MEAt\1 ~ I 1= 342·· 75 MEAN I 2= 309· 375 MEAN DI F= 3.3.~375 SD.l:=; 27·0911 SD2~ 45· 1538 SD3~ 49· 4251 S E 1 = 9 • 5 78 1 6 SE2= 16· 1764 SE3= 17·4744 . - I 1 - - 166 .178 180 l70 .161 _190 16~ 189 .173 145 RABBIT 6: -143- MAXILLARY MIDDLE TH.ffiD STAINED, 1 BLEACH 12 13 - l22 44 _130 48 122 58 .143 27 171 10 .153 37 140 24 144 45 .126 47 146 - 1 -. N= 10 MEAN _ I 1= 173· 6 MEAN I 2= 139 · 7 -33._9- - .. SD.l~ 13 · .3~33 SD2~ 15· 3988 SD3= lf~ . 62 17 SEt= 4 e 21953 SE2= 4 · 8 69 52 SE3= 5e888 69 I 1 I2 I3 366 276 90 334 29A 40 366 31 1 55 377 309 ffi 315 305 70 395 279 116 383 252 13 1 ~00 199 209 360 225 135 RABBIT 8' N= 9 Dl 232 268 300 330 362 392 4 19 436 456 476 Dl 92 134 176 210 247 2137 320 352 379 MEAN I 1= 373· 778 MEAi\J I 2= 272· 222 - 10.1· 556 .. SD.l~ 21 . 2472 SD2= 39 . 474 SD3~ 52· 38 1 1 SEl= 7· 00 241 SE2= 13· 158 SE3= 17· 4604 D2 231 264 292 324 352 380 406 412 422 434 MEAN DI F= D2 174 217 249 284 324 360 389 414 432 ME.AN DI F= -144- I 1 12 13 Dl D2 - 335 262 73 99 61 320 278 42 132 88 361 307 54 164 116 361 302 59 191 144 342 304 38 221 174 304 293 11 252 204 3.07 310 -3 274 231 317 324 -7. 300 252 287 318 -31 322 278 276 322 -46 340 300 213 268 -ss 358 324 e29 264 -35 368 3Lt7 302 250 52 382 363 272 237 35 392 374 RABBIT ~ N= 14 MEAN I 1= 30 1·8 57 MEAN I 2= 288· 5 MERN DI F= 13· 35 71 - . - · SD.l~ 4!h 09 75 , SD2~ 28.3759 SD3= 42.8515 SEl= 11·7856 SE2= 7· 58 378 SE3= 11.4525 I 1 I2 I3 Dl D2 - . - 343 169 .174 156 9 362 253 109 190 29 396 218 _118 218 54 423 316 107 244 80 414 321 93 270 116 404 330 74 296 149 378 316 62 321 186 384 397 -13 347 220 300 339 -31 368 246 285 397 -112 382 278 - .. - RABBIT 11 N= 10 - I 2= 311· 6 MEAN_ I 1= 369· 7 ME:m MEP.t-J DI F:s 58_ •. 1 . - SDl?. AS• 58 03 SD2~ 6.7• 2478 SD3= 8 5• 2558 SE1= 14· 4138 SE2= 21·2656 SE3= 26• 9 602 . I 1 195 ~.07 212 218 229 .19.9 211 240 226 2 .16 212 232 RABBIT 6 -145- MAXILLARY GINGIVAL TH:rnD STAINED, 1 BLEACH !2 !3 178 17 .179. 28 199 13 210 6 221 8 208 -9 204 .7 224 16 212 14 213 3 198 14 212 20 .. N= 12 Dl 118 154 18 7 212 242 270 300 326 354 380 406 428 MEAN I 1= 216· 417 MEAN I 2= 204•_8 33 1.1 .• _58 33 .. SD.l~ 13·3856 SD2= 14~4715 SD3= 9. 27811 SE1= 3·8 6409 ·SE2= 4·17756 SE3= 2· 678 36 -. I 1 I2 I3 Dl 296 258 36 24 308 264 44 56 375 305 70 247 348 282 66 87 381 307 74 121 399 318 81 157 390 325 65 19 3 365 315 50 229 ~00 336 72 256 401 319 62 . 291 396 275 121 322 358 256 102 353 346 248 98 380 RABBIT 8' N= 13 MEAN I 1= 367 MEAN I 2= . 29 2• 9 2 3 MEAN SDJ~ 35.2373 SD2= 30·1426 SD3= 23· 48 57 SEl= 9. 77307 SE2= 8 • 36006 SE3= 6·51375 D2 178 214 238 270 294 322 348 372 398 423 439 448 ME.AN DI F= D2 104 140 324 174 209 246 286 322 352 381 400 430 445 DI F= 74· 0769 -146- -. I 1 I2 I3 Dl D2 - . . .167 .154 13 12 33 173 164 9 100 66 - 190 . 177 . ·13 126 91 192 180 . 12 122 122 195 174 21 18 3 156 166 .162 4 210 18 6 177 157 20 234 212 _18 3 164 19 260 234 171 160 11 284 262 .1.7~ .147 25 308 290 151 133 18 336 312 l4P 1.32 8 357 336 154 114 40 376 352 194 114 80 398 368 · - RABBIT .9 N= 14 . - . - - . - '"" ) MEAN _ I 1= 173·2.14 MEAN I 2= 152· 28 6 MEAN DI Fe - 20_._928 6 _ SD.l~ 16·9 032 SD2:; 21e5672 SD3= 19·1812 SE1= 4• 51758 SE2= 5·76407 SE3= 5· 12639 I 1 I2 13 Dl D2 378 247 .131 116 153 386 285 101 150 189 4~2 342 100 174 226 445 361 84 201 253 423 349 74 228 283 414 333 81 257 319 405 321 84 285 344 . 473 387 86 320 367 373 270 103 348 386 ·- RABBIT 11 N= 9 . 321· 667 MEAN . I 1= 415.444 MEA~ I 2= ME.AN DI F= 9 3~_ 7778 . . . SD.l~ .33• 78 28 SD2= 45• 68 1 SD3~ 1 7· 203 SEl= 11· 2609 SE2= 15· 227 SE3= 5· 73435 -147- MMIDIBULAR INCISAL THlliD STAI!·JED, 1 BlEACH - I 1 12 I3 Dl 65 .. 53 32 44 114 100 6 66 140 .115 25 89 .1.36 .13.1 5 116 .1 75 .161 .14 143 .167 154 13 172 185 .156 29 208 161 .164 -3 244 175 155 20 277 . 175 153 22 300 .1.5.7 153 4 338 .171 .15.2 19 368 151 .138 13 394 .143 .136 7 421 l!!!J 140 4 446 141 127 14 464 . . -- RABBIT 6 N= 16 MEAN I 1= 151·25 . . MEAN I 2= 137· 25 SDl~ 25· 7!>.69 SD2~ 27· 7405 SD3~ 9·93311 . SEl= 6· 44173 SE2= 6·9 3512 SE3= 2 ~ 48 328 I 1 I2 I3 Dl 73 47 26 45 87 46 41 62 9!&. . 6.7 27 87 lll 71 40 114 1.16 78 38 141 113 71 42 180 .1.31 84 47 220 .115 90 25 262 .122 76 46 304 .128 78 50 350 .148. 75 73 398 141 65 76 442 122 55 67 474 . . . . RABBIT 8 N= 13 - ... -- ... .. . 69.4615 MEJ\N I.l= 115· 462 -- - MEAN I 2= SD.l:=: 21· 00.64 SD2= 13·3767 SD3= 16·9 066 SEl= 5·82613 SE2= 3· 71003 SE3= 4· (69 05 D2 39 55 78 103 128 162 191 224 259 292 338 354 384 410 434 456 MEA\J DI F= 1-4 D2 45 72 104 132 162 206 240 274 318 361 402 434 454 ME.AN DI F= 46 -148- . . I 1 I2 13 Dl D2 -. 155 110 45 42 36 210 2.14 - 4 58 52 237 212 25 82 74 295 2.59 36 110 100 279 312 - 33 143 128 321 335 - 14 182 162 300 345 - 45 222 196 30.1 309 - 8 258 230 301 3 _10 - 9 306 266 279 319 - 40 352 300 245 285 - 40 401 300 255 308 - 53 438 38 1 RABBIT 9 N= 12 MEAN I 1::;: 264. 8 33 MEA\1 I 2= 276· 5 MEAN DI F= ~ 1_1_._ 6667- . . - SDl~ 47• 3418 SD2:= _67· 700 SD3=: 32. 7423 SEt= 13· 6664 SE2= 19 .. 5632 SE3= 9 . 45 19 I 1 I2 1 3 D1 D2 - 234 .140 9~ 106 147 2!19 1JO 19 136 182 310 2_14 9 6 162 2 13 306 2 1 0 96 194 247 295 190 105 227 285 267 221 4 6 259 3 15 257 194 63 289 349 328 20~ 122 314 369 244 169 75 342 394 265 212 53 363 4 16 .. - RABBIT 11 N= 10 - MEAN _ I 1= 275 · 5 ME.AN I 2= 192· 6 MEAN DIF= . 8 .2.•.9 . - . . - .. .. S D .1 ~ .31 • 9 69 6 SD2~ 25!t 7475 SD3= 24· 0668 SEt= 10· 1097 SE2= 8 . 14207 SE3= 7· 61059 I 1 JJS 6LI 79. Jl .100 .1 0 .1 .124 132 123 100 124 90 .144 .100 96 RABBIT 6 -149- MANDIBULAR MIDDLE THffiD STAINED, 1 BLEACH 12 13 33 15 83 -19 .71! - 5 .103 -32 115 -15 13~ - 35 .151 -27 154 -22 141 -18 .145 - 37 142 - 18 .142 - 52 124 20 100 0 130 -34 . . - N= 15 MEAN_l1~ 100·267 MEAN I 2= 118 · 2 . -1_7_._9 333 - - -. SDl~ 26.8 633 SD2::; 34~ 0613 SD3::: 20· 267.7 SE1= 6e936C6 SE2= 8. 79459 SE3= s. 23311 - - I 1 I I2 13 23, 62 - 39 113 SA 59 lOS 71 34 13.2 12 60 158 92_ 66 .163 .109 5ll .17!1 .110 64 _172 .123 49 169 106 63 186 88 96 209 99 110 205 .1 02 103 .179. 100 79 151 88 63 117 93 24 . - . RABBIT 8 N= 15 - . Dl 43 66 86 118 148 184 215 250 281 313 342 371 395 416 434 D! 44 55 73 94 126 157 19 3 234 270 311 352 394 432 464 il84 . - . ... - MEAA . . ll~ 150·4 MEAN I 2= 9 1• 2667 - 59.~_1333_ --. . . - SDl~ A 7• 2316 SD2:= 19· 2594 SD3~ 35·9997 SE1= 12·1952 SE2= 4e9 7275 SE3= 9 · 29509 D2 38 Lt;) 74 102 130 154 190 228 266 300 331 363 394 428 450 M EAI.'J DI F= D2 39 55 73 102 133 167 196 232 272 300 344 384 416 448 468 MEAN DI F=- -1.50- I 1 12 I3 D1 D2 - 235 .1 36 99 57 38 253 179 74 81 60 305 223 82 111 82 321 297 24 142 106 323 336 -1 3 176 134 361 364 - 3 218 165 340 350 - 10 262 201 365 347 18 314 233 363 337 46 369 270 386 332 54 4 13 305 367 338 29 448 346 -· RABBIT 9 N= 1 1 - . MEAN _I 1= 330· 8 18 MERN I 2= 294.455 MEAN DI F= 3.6.•. 3636 _ . . - ·- SD.l?: 50· 3007 SD2~ 78 · 1503 SD3= .38 . 08 75 SEl= 15. 1662 SE2= 23e 5632 SE3= 11~ 4838 I 1 . 12 1 3 Dl D2 224 j 7 1 53 52 10 1 ~76 16 7 89 72 130 334 237 9 7 93 157 367 28 1 80 118 168 386 26 1 125 144 218 393 267 12 6 17 4 255 3 .64 274 90 208 286 4J(> ;3~~ 82 240 309 333 271 62 270 339 ~02 305 91 . 296 366 432 3 15 1 17 320 383 " .. 33.4 282 52 346 402 388 3 10 78 368 41 6 - .. - RABBIT 11 N= 13 . - ... - - . I 2= 269 · 308 MEAN I 1=:.: 35 7. 615 ME.AN MEAN DI Fz:a - 88 _~ 3077- . . - - .. SDl::: 57• 9 203 SD2= ~7· 6609 SD3~ 24· 5234 SE1= 16· 0642 SE2= 13· 2188 SE3= 6 · 8 0157 -1.51- MANDIBULAR GINGIVAL THIRD STAINED, 1 BLEACH I 1 12 I3 Dl D2 60 30 30 38 45 - 105 53 52 51 64 100 . 58 50 76 84 .130 ~6 6!1 96 112 .131 100 31 .126 150 .15.5 93 62 158 18 6 .151 128 · 23 18 7 223 142 108 34 220 259 .140 J03 3_7 255 295 .145 94 51 284 326 l4p 99 47 320 364 .151 93 58 350 394 117 100 17 378 422 -- RABBIT 6 N= 13 - MEAN I 1~ 129· 308 MEAN I 2= 8 6• 538 5 MEAN DI F= 42_._ 7~2 . .. .. - SDl~ 26~ 465 SD2?. 26·8 72 SD3= 15· 1171 . SE1= 7· 34000 SE2= 7e45294 SE3= 4· 19272 I 1 · I 2 13 Dl D2 - 180 - 183 -3 32 60 245 • 18 3 62. 45 84 266 209 59 62 100 292 239 53 68 138 329 252 77 117 168 356 280 16 150 203 374 275 99 184 242 4.05 273 _132 222 278 418 292 126 258 314 465 292 173 295 3~ .475 3.12 .163 338 379 ~69 313 156 380 407 445 306 139 418 440 372 289 83 448 466 -- - RABBIT 8 N= 14 MEAN I 1= 363.786 MEAN I 2= 264. 143 MEP.N DI F= 99.•. 6429 . SDl~ 9 1• 388 6 SD2~ 44e7521 SD3= 50·3764 SE1= 24~4246 SE2= 11·9605 SE3= 13·· 4637 -1.52- I 1 12 I3 Dl D2 224 3 .7 18 7 38 28 276 119 157 62 42 292 134 158 83 62 335 227 .100 11 5 86 36a 255 107 .147 11 2 363 275 88 18 4 142 354 305 49 224 1 72 345 323 22 268 206 363 303 60 306 242 335 3o.g 3 1 354 274 321 .. 307 14. 402 3 16 32 1 280 41 438 352 . . RABBIT 9 N= 12 . -- . . - MEAA .. I 1~ 324· 25 MEAN I 2:: 239 · 00 3 MEAN DI F= 8 s .•. 16.67 . --- .. . . . - .. SDl?: !12· 0 .111 SD2~ 92· 4234 SD3= .58 . 559 7 SEl= 12· 1276 SE2= 26 .. 68 03 SE3= 16· 9 047 . I 1 1 2 1 3 Dl D2 206 227 - 2 1 3 2 44 2.64 286 - 22 48 60 310 . 343 - 3.3 63 76 308 369 - 6 1 83 "1 02 334 . 363 - 29 108 128 358 42.2 - .64 133 158 373 /8.9 -1 26 .158 189 ~36 470 - 34 188 218 34.0 351 - .1 7 220 247 3.61 4 0 1 -40 244 274 3!l7 ~2 1 - 74 273 300 313 359 - 46 300 327 298 340 - 42 32 1 355 288 34!1 - 56 346 380 271 379 •1 02 369 399 28~ 324 -40 387 4 16 291 316 - 25 399 4 26 . -. RABBIT 11 N= 17 . MEAN_Il= 316· 94 1 MEAN I 2= 365· 88 2 MEAN DI Fa - 48 ~. 9412 .. SD.1~ ~1.523 p SD2~ 65 · 1353 SD3= 29 9 575 1 SEl= 12· 16 63 SE2= 15e 7976 SE3= 7e 173 . I 1 32.7 3~ 353 342 3!-13 297 RABBIT 7 -153- MAXILLARY INCISAL THffiD .STAINED 2 BLEACHES I2 !3 . - - .146 .18 1 .146 .18 3 160 .19 3 _156 18 6 .147 .19 6 139 158 -·- N= 6 MEAN I .1= 331·833 - MEAN I 2= 149 SD.l?: 19· 6_002 SD2:=: 7· _6!1199 SD3= 13· 4672 SEl= 8·0017{! SE2= 3 · 1198 3 SE3= 5· 18 798 - I 1 I2 I3 225 88 137 21 .7 . 95 . .122 219 .109 110 206 .109 99 . 2~2 135 107 248 .156 92 a1o _172 98 PBO 188 92 257 .168 (:IJ 257 .166 91 .. 284 . 195 89 260 1_65 95 235 165 70 -. RABBIT 12 N= 13 Dl 281 320 353 392 424 444 MEAN Dl 85 119 152 182 209 234 239 2131 311 334 361 382 402 -.... - . - I 2= 148 . 538 MEAN . I 1= 246.300 MEAN - 9 .7 ... 7@2 - .. SDl~ 24• 5098 SD2?. 37· 1565 SD3= 16 . 6152 SEl= 6· 79 78 SE2= 10·3053 SE3= s. 1629 3 - D2 2€8 302 353 368 398 412 DI F= 18 2. 8 33 D2 97 132 160 188 215 248 273 303 329 352 379 404 426 MEAN DI F= -154- - I 1 I2 I3 Dl D2 - ~ 357 222 .135 267 234 351 232 125 302 272 332 24!4 88 336 306 321 23 .1 96 372 342 269 .169 100 400 372 273 172 101 432 398 .. RABBIT 16 N• 6 MEAN It= 319·167 M.EAN I 2= 211·667 MEAN DI F= 10.7· 5 . . . -. SD.l~ 39. 33.6.6 SD2= 32· 6599 SD3= 18.2948 SEt= 16· 0591 SE2= 13 .. 3333 SE3= 7· 4688 2 I 1 I2 I3 Dl D2 - - 462 327 .135 178 160 312 408 -96 198 223 401 326 75 250 228 415 32.3 92 284 260 404 361 43 314 294 3J6 31_1 65 348 326 356 269 87 377 357 317 306 11 394 376 - . - RABBIT 1 ,,. N= 8 MEAN I 1= 38 o. 375 ME.AN I 2= 328·875 MEAN DI F= 5.1~_5 . -- SDl~ 50·9199 SD2~ 40·9998 SD3~ 69· 771 1 SEt= 18 ~ 0029 SE2= 14· 49 56 SE3= 24 .. 6678 I 1 12 I3 Dl D2 - - 267 120 147 .103 94 266 119 147 149 140 230 226 2 . . 18 3 185 230 1.13 117 ·185 18 3 242 l50 92 220 224 2.gs 153 96 256 263 ~75 169 106 298 303. 28~ 183 106 334 339 107 80 27 372 389 RABBIT 18 N= 9 MEAN I 1= 239.333 -· MEAL\J I 2= 146 MEAN DI F= 9 3· 3333 SD_1~ 53·6004 " SD2?. 44• 0511 SD3~ 49· 2341 SEl= 17·8 668 SE2= 14~ 68 37 SE3= 16· 4114 - -155- I 1 !2 I3 Dl D2 .173 31 136 42 20 .18 7 91. 96 62 47 174 .121 53 86 72 .175 124 51 114 100 182 138 51 .143 136 eo3 167 36 178 167 324 252 72 202 204 222 185 37 231 233 212 .174 38 268 271 232 _175 57 292 303 203 .165 38 318 331 182 .16~ 16 346 358 .1.95 }83 12 374 380 18 7 90 97 394 403 -- RABBIT 19 N= 14 . --- -. - MEAN I 1= 204.143 MEAN I 2= 147· 714 MEAN DI Fa - 56.~ 428 6 . . ·- SD.l~ 38 .a 1e3 SD2~ .52· 7161 SD3= 33·899 7 SE!c 10·3891 SE2= 14· 009 SE3= 9·06006 I 1 12 I3 Dl D2 194 51 .137 88 146 eoo g.l 159 120 177 211 48 163 146 200 .1_6.2 32 .1 30 171 231 181 42 139 aot 262 .18 4 ~0 _164 235 294 164 9 155 264 320 173 · 8 _1 65 288 346 .1A6 2 144 318 372 113 6 107 349 394 103 _6 97 375 420 .106 .12 94 410 434 112 12 100 436 452 - - . RP..BBI T 20 N= 13 - I 2= 22• 6923 MEAN I .1~ 157· 615 MEAN MEAN DI F= . 1.3.'J• ~ 23 . . . . ·- SDl? .38 • 038 4 SD2=7 18. J9 SD3= 27. 0199 SE1:: to. 55 SE2= 5· 21141 SE3= 7· 49398 I 1 - 356 346 386 397 399 378 331 331 . - RABBIT -7 . - ..... -1.56- MAXILLARY MIDDLE THIRD STAIUED, 2 BLEACHES I2 I3 172 .184 172 174 227 _159 252 145 297 102 310 68 323 8 327 4 -- N= 8 MEAN I 1=. 365• 5 -- MEA~ I 2= 260 72· 214 SDl~ B8~1374 . SD2~ 64· 3295 SD3= SEl= 9e948 00 SE2= 22~ 7439 SE3= 25e5315 I 1 12 I3 233 103 . . 130 257 .94 163 260 100 160 254 126 128 260 l39 .121 294 153 141 3.10 .18 0 130 317 19.7 120 307 · 2.1.a 95 3.06 219 87 320 242 78 300 247 53 267 237 30 257 213 44 - - RABBIT 12 N= 14 . . D1 217 254 288 320 351 382 410 410 MEA'J Dl 72 104 134 160 18 6 210 237 260 287 312 336 364 388 400 . - -- - . . MEAN I 1= 281·571 MEAN I 2= 175·8 57 . 10.5· 714 - SDl= 28 • 7528 SD2::: 55· 688 5 SD3= 42· 1900 SEl= 1· 68 452 SE2= 14.88 34 SE3= 11~276 D2 172 202 232 262 292 340 362 389 DI F= 105· 5 D2 52 83 110 134 162 192 222 248 269 292 315 345 372 400 MEAN DI Fzz -157- I 1 12 13 D1 D2 - 375. 275 100 182 164 393 294 99 210 19 6 408 290 _118 242 228 .g3o 296 .134 274 255 385 270 115 300 289 ~03 289 1.14 343 328 366 255 111 377 362 366 2.78 88 400 391 312 219 93 426 413 RABBIT 16 N= 9 MEAN I 1= 382 MEAN I 2= 274 MEPJ.'J PI F=_ 108 SD_l~ 33.·5634 SD2:=: 24,-454 SD3~ 14·3178 SEl= 11· 18 78 SE2= 8·15135 SE3= 4. 77261 .. I 1 I 2. 13 Dl D2 413 3.02 .1.11 76 118 432 318 _114 114 156 Lj82 34!1 138 152 190 475 341 134 185 217 476 345 .131 218 247 446 328 .118 244 278 470 325 145 276 303 ~66 327 141 302 333 384 223 .161 334 360 397 237 160 364 378 340 263 77 384 390 . - RABBIT 17 N= 1 1 ·- . -... - - - -. MEAN I 1=_434·818 __ MEAN I 2= 304·8 18 MEAl\J DI F= 130 SDl~ ~6· 3548 SD2? 43· 126. SD3~ 24·2033 ' SEl= 13·9 765 SE2= 13· 18 39 SE3= 7·29757 · - -158- I 1 - 221 239 233 2.74 310 276 3..20 118 279 278 238 231 RABBIT 18 12 88 . 107 113 .144 158 176 165 58 117 212 214 180 .. N= 12 MEAN Il= 251·917 1 02_._58 3 . . SDl:=: 52• 39 79 SD2?. 48 • 7019 SE1= 15~126 SE2= 14~059 I 1 201 223 2~ 228 254 353 243 232 253 2.53 216 .223 195 204 RABBIT 19 - I2 - 95 130 .155 176 238 .18 1 164 210 .210 178 201 192 214 210 -· - N= 14 MEAN_ I 1= 236• 78 6 52_._9 28 6 - - SD.l::: 38·5909 SD2?. 37•1936 SEl= 10·3139 SE2= 9•9404 13 139 .132 120 130 .152 100 155 60 102 66 24 51 D1 57 90 126 163 198 238 272 304 336 362 381 393 MEAN I 2= 149 • 333 SD3~ 43• 09 7 1 SE3= 12·441 I3 106 93 73 52 _16 172 59 29 43 15 11 31 •19 -6 . . Dl 33 57 78 .104 135 .162 193 225 252 279 300 321 344 362 ME.AN I 2= 18 3• 8 57 SD3= 49 • 599 1 SE3= 13.-2559 D2 42 74 100 144 162 227 260 292 326 353 382 404 MEAN DI F= 'D2 36 58 83 110 138 168 195 226 251 274 294 316 338 360 MEAN DI F= -159- .. I 1 I2 13 Dl D2 - 192 16. 116 40 100 224 _ 151 73 62 132 199 . 88 . 111 83 160 230 70 160 107 16 1 221 88 .133 136 200 233 67 16.6 .164 220 217 56 161 189 262 222 60 162 214 273 .19 6 36 .160 243 299 18 6 27 159 270 324 .187 34 153 294 353 172 44 _128 316 380 1 .65 -4 169 334 406 .15.2 IJ1 105 348 428 158 34 124 368 448 - · - RABBIT 20 N= 15 4 - • - • • - 58 . 2667 MEAN I 1=? 19 6·933 MEA'J I 2= MEPM DI F:a 138·667. - - --SDl~ 26~94P3 SD2= 35· 62 SD3= 28 · 4421 SEt= 6·9559 6 SE2= 9· 19 703 SE3= 7e 34372 -160- MAXILLARY GINGIVAL THIRD STAINED, I 1 12 325 .1 ~9 339 l1!J 342 192 354 209 364 23!1 360 261 329 2~~ 365 299 35_6 294 371 303 _307 269 321 249 - . - RABBIT 7 N= 12 .. --- - - . MEAN I 1~ 344·417 1_0.2· 8 33 -- SDl~ 20.•.~07 SD2~ 50·7336 SE1= 5e891 SE2= 14·· 6455 -. I 1 I2 274 112 300 124 300 _147 300 149 313 .168 325 118 375 210 366 222 366 232 3~0 247 398 283 363 275 339 278 337 e73 34!1 276 271 257 225 218 -- RABBIT 12 N= 17 . - ... . MEAN I 1= 327• 294 1.12. 64 7 - - -. . -. SD.1:7 44•/$} 12 SD2~ .58 • 0323 SE 1= 1 0• 79 07 SE2= 14·· 0749 2 BLEACHES 13 D1 .176 143 165 17~ .150 203 145 234 130 264 99 295 63 332 66 358 62 384 68 407 38 416 72 425 .. MEAN I 2= 241· 58 3 .. SD3?: 47·627 SE3= 13·7487 13 Dl .162 23 17.6 54 _161 79 151 104 .145 .135 147 160 165 186 .144 200 134 234 _1 _13 261 115 289 86 317 61 342 64 369 {>6 395 14 412 7 425 MEAN I 2= 214• 647 .. SD3= 53• 1 Je 5 SE3= 12•8906 D2 126 160 168 218 247 262 312 346 370 404 424 446 MEA\J DI F= D2 23 54 80 108 140 168 ~ 19 7 223 250 278 307 332 356 378 396 414 429 M E.AN DI F= -161- I 1 I2 13 Dl D2 - 359 ·ooo 79 96 103 403 289 .114 122 129 397 280 117 .149 156 397 297 100 176 18 7 400 286 122 210 217 392 283 109 2JB 251 381 306 75 284 283 450 364 66. 314 314 4 .12 311 101 349 346 393 328 65 379 366 366 262 104 412 420 312 233 79 436 440 324 237 87 456 456 RABBIT 16 N= 13 ME.AN I 1= 38 4. 154 MEAN I 2= 288 •9 23 MEP£..1 DI F= 95 .• _2300 -- SDl?-: 36·8 5 75 SD2;: 35.0701 SD3= 17·9 544 SEt= 1 o. 2224 SE2= 9e726(/} SE3= 4·9 79 64 I 1 !2 !3 Dl D2 228 207 2 1 19 44 3!13 245 98 44 80 4 .16 317 99 74 114 ~17 329 88 104 148 445 351 88 .140 18 3 415 339 76 179 216 438 344 94 218 246 449 383 66 245 275 ~.28 344 84 280 300 ~3 40~ 79 314 342 446 331 115 344 370 420 301 119 369 388 389 359 30 390 400 RABBIT 11 N= 13 - -- - - - MEAN DI F= 8 1· 3077 MEAN I 1= 409 MEAN I 2= 327· 69 2 SDJ= 63• 724 7 SD2?: 52·9 345 SD3= 28· 7848 .. ~- 7·98 346 SEl= 1 7· 674 SE2= 14~ €814 SE3= -162- I 1 12 I3 Dl D2 209 .110 96. 19 37 273 _152 .12 1 49 64 ~9 173 126 77 97 338 193 145 100 140 328_ 232 96 145 173 - 3~1 7g _ 269 18 0 210 297 197 100 214 248 137 2 15 - 16 246 280 349 225 124 284 300 371 220 .15 1 314 336 361 203 158 345 362 325 _163 162 368 381 27_7 167 _1.1 0 384 393 274 163 11 1 396 400 -· - RABBIT 18 N= 14 . - .. - - MEAN I 1= 298 · 28 6 MEAN I 2= 177· 5 MEJ:W DI F= - 120· 78 6 . . SDl:: .63• 8 5 37 SD2~ 45. 2323 SD3= 72· 3 11 5 SEl= 1 7· 0656 SE2= 12· 0888 SE3= 19 ·· 326 1 I 1 I2 I3 Dl D2 1"75 150 25 22 42 230 21 0 . 20 44 64 2~3 3 13 - so 67 69 373 23_1 142 66 .114 284 222 62 11 2 148 .2.68 270 -2 148 177 29 .. 6 278 16 176 205 312 239 73 200 234 281 269 12 228 260 301 234 _73 254 286 259 276 ml7 283 306 303 240 63 3 11 329 305 245 60 337 352 28.7 249 ~ 36 1 373 315 202 11 3 378 388 RABBIT ·19 N= 15 . - - .. - I 2= 24 1· 8 67 MEAN DI F= MEAN I 1= . 28 3· 8 67 __ MEAN 4 2 S D.l .~ ~~· 1~00. SD2~ 38 . 498 4 SD3~ 49 ~ 478 7 - SE1= 11 · 3971 SE2= 9 • 94023 SE3= 12 .. 7753 - -163- I 1 !2 I 3 D1 D2 - - · - 82 96 -J4 20 5 7 .172 8.2_ 90 4 5 82 168 112 56 68 104 156 62 76 .8 4 122 177 .11 5 62 100 150 1.69 11 6 53 137 172 .167 _11 5 52 168 190 165 1 12 53 193 20 7 163 87 9 6 220 230 207 65 142 246 254 .170 45 125 278 002 158 60 98 - 306 304 170 53 11 7 332 334 _133 3_6 9 7 360 364 127 2 1 106 38 4 392 -- - RABBIT 20 N= 15 . . .. -.... . - - MEAN I 1::: 160· 4 MEA!.'J I 2= 79 · 8 MEPl-1 DI F= 8 0· 6 . . . . 38 · 6 11 2 .. SD.l::: 28 . 6576 . SD2?; 31 , 7382 SD3~ SEl= 7· 399 36 SE2= 8 · 19477 SE3= 9 · 96938 I 1 225 275 338 337 350 322 307 308 325 3JO 295 306 - . RABBIT 7 - -164- MANDIBULAR INCISAL THIRD ST!UNED, 2 BLEACHES . . I2 I3 82 143 95 . 18 0 .143 195 167 .170 177 173 195 127 192 115 212 96 225 100 206 .104 18 2 113 200 106 -· - N= 12 MEAt\l I 1= 308· 167 -- MEP.1.\1 I 2= 173 35· 4884 SD.l::: 33· 2288 . SD2= !-15· 0111 SD3~ SEl= 9· 59232 SE2= 12. 9936 SE3= 1 a.· 2446 I 1 I2 I3 - 106 50 56 .176 86 90 207 .132 .75 - 264 147 11 7 286 18 0 .1 0_6 346 215 131 334 .196 136 346 219 127 3.65 220 145 396 23 .6 _1_60 346 232 .1 14 345 173 172 3€8 188 18 0 -- RABBIT 12 N= 13 Dl 94 122 146 176 206 242 272 304 338 364 406 424 . MEAN Dl 35 52 7 1 94 .124 152 179 210 2LI8 287 325 368 408 - -- . - I 2= 175· 077 296·646 MEAN MEA'J I 1= - 123· 76;}- 36.5517 SD.l~ 8 6· 9 529 SD2~ 57· 3272 SD3= SEt= 24.1164 SE2= 15~ 899 7 SE3= 10· 1376 D2 83 114 143 172 200 234 268 298 328 353 382 404 DI F= 135· 167 D2 36 6 1 86 .116 149 184 220 261 305 343 380 417 450 MEAN DI F= -165- I 1 !2 13 Di D2 - -. - 289 .104 18 5 63 38 360 .100 260 60 56 3.75 .119 25 .6 .106 82 411 150 261 134 102 418 .156 260 166 130 444 197 247 198 163 428 198 230 234 195 473 220 253 268 230 ~46 219. 227 312 264 455 217 238 344 303 400 199 229 380 346 427 187 240 418 380 4~.6 190 256 448 408 442 203 239 474 436 409 19 5 214 500 468 . - RABBIT 14 N= 15 -' - 177.067 ME~ 11= 416•733 MEA\1 I 2= MEAN DI F•. - 2.39.· 667 -- SDJ:: ~5·8 68 5 SD2~ ~0·98 (:f) SD3::: 20·8 144 SEl= 11·8432 SE2= 1 0~ 58 28 SE3m 5.37425 I 1 I2 I3 D! D2 243 37 206 90 36 215 _77 198 115 47 295 .1 02 .193 142 63 270 123 147 172 86 213 153 _120 208 .114 298 166 132 245 l42 291 239 52 282 168 330 233 97 314 200 295 232 63 352 245 RABBIT 16 N= 9 MEAN I 1= 28 5· 556 MEAN I 2= 151· 333 MEJ\N DI F:s 13_4. 222. - .. .. SD.l:: 24.1356 SD2= 73·241 SD3~ 57· 29 7 SEl= 8 • 0452 SE2= 24·4137 SE3= 19.099 -166- I 1 I2 13 Dl D2 . 220 _116 104 40 36 2~ 173 95. 60 53 311 210 10 1 85 72 338 199 139 110 100 357 263 94 . 142 130 354 245 109 174 163 351 276 75 208 19 6 348 326 22 240 228 327 352 -25 278 264 388 361 2 .7 320 303 330 289 41 362 348 3.13 2~B 71 398 383 3.13 219 94 . 432 422 318 129 189 457 456 RABBIT 17· N= 14 -. - "' - .. - . MEAN I .l= _ ·324 MEAN I 2= 242· 8 57 MEAN DI F= 8 1· 1429 SD.l:=: .41 ~ 1 ~57 SD2:= 75· 4115 SD3~ 53· 0919 SEt= 11i002 SE2= 20· 1546 SE3= 14~ 1894 I 1 I2 13 Dl D2 49 .11 38 41 48 .79 23 56 58 64 82 30 59 60 92 9.7 30 67 106 150 77 5!& 23 .135 184 77 41 36 166 221 .77 37 40 202 253 60 45 35 234 290 61 53 8 267 330 67 35 32 300 378 66 49 17 334 416 ia .. RABBIT N= 11 . - . . MEA'I I 2::s 37· 09 09 MEa\1 . I 1= 74e4545 MEA\l DI F= - 31~. 3_636. - .. - - SD.l?= 13,2164 SD2::= 13! 2019 SD3= 17· 9 459 SE1= 3e98~9 SE2= 3·98 053 SE3= So 41009 -167- I 1 I2 I3 Dl D2 113 33 80 33 62 190 35 155 50 85 213 47 1 66 64 112 215 82 133 87 144 2!1~ 99 14.7 114 178 291 100 19 1 146 214 403 124 279 176 254 298 187 111 212 283 e74 .120 154 254 327 300 119 181 288 365 301 122 18 5 324 398 292 111 _181 363 420 2811 125 159 401 430 -· - RABBIT 19 N= 13 ~ MEAN I 1;:: 263· 538 MEAN I 2= 1 00· 308 MEAN DI F= . - 1~.3· 231 . . SD.l_=: _70• 2.58 ~ SD2.=- 42·8007 SD3= 46· 8 066 SEt= 19· 48 62 SE2= 11 · 8708 SE3= 12··98 18 I 1 12 13 D1 D2 25 64 - 39 35 36 42 44 - 2 19 54 54 36 18 64 82 92 62 30 88 104 .96 p7 . 29 114 132 8 7 . . 100 - .13 .143 161 1.01 JOO 1 172 194 91 95 - 4 196 228 89 9.3 - - 4 232 254 9.0 . . 103 • 13 266 291 101 .121 - 20 292 320 .108 .104 4 326 362 90 105 - 15 360 398 61 6!1 3 392 431 94 91 3 417 460 73 88 -15 442 476 RABBIT 20 N= 16 MEAN I 1= 8 2• 5 MEAN I 2= 8 4• 8 125 . . MEAN DI F=- 2• 3125 Spl~ 22.8852 SD2?. 23· 68 75 SD3= 1 7· 8 128 SE1= 5e72131 SE2= 5·9 218 7 SE3s 4o4532 -168- MANDIBULAR MIDDLE THmD STAINED, 2 BrEACHES I 1 !2 13 Dl - 203 151 52 84 ~27 203 24 112 287 222 65 140 003 276 5 167 30 .1 298 3 194 292 30.6 - .14 228 283 3.01 - 18 263 29.3 315 -22 294 32.1 337 -1.6 325 299 350 -51 352 28~ 337 -53 378 289 342 -53 405 264 295 - 31 422 RABBIT 7 N= 13 MEA'J I 1= .278 • 9 23 MEP.N I 2= 28 7· 308 -8 .!t .• 38 4.62 .. - SD1:=: 31 ~ 542g SD2?: .60· 1226 SD3= 37 · 7106 SEl= 8 • 748 23 SE2= 16· 675 SE3= 1 a.· 459 I 1 12 13 D1 150 .152 - 2 35 260 . 196 64 47 296 254 42 68 375 298 77 88 350 31.0 40 117 399 351 48 149 447 379 68 178 384 345 39 213 39.~ 322 65 248 417 357 60 285 388 358 30 323 RABBIT 12 N= 11 ~ - . - - ME:m I 1::: 350e909 MEAN I 2= 302· 636 /.18 !t.2 727_ -. SD.l?: 8 s. 1275 SD2~ 72·9277 SD3~ 22· 213 SEl= 25· 6669 SE2= 21 .. 988 5 SE3= 6· (f) 1Lt3 D2 54 78 102 124 148 178 206 235 268 298 324 354 368 MEP!N DI F= D2 35 50 71 94 123 .154 188 223 257 289 324 MEttN DI F:a -169- I 1 I2 I3 Dl D2 289 7'1 212 44 40 305 91. 214 62 60 296 .133 1~3 83 8 1 338 .126 212 100 106 362 .164 198 .136 134 . 406 . 184 . 222 163 166 37.4. 237 137 198 198 38 .1 226 .155 230 236 409 238 .171 262 272 400 239 .169 296 309 419 254 165 334 344 408 24~ .i64 362 378 ~14 231 183 392 406 418 2!!0 .178 428 438 405 218 18 7 458 466 4~ 232 216 lJS4 490 392 208 184 506 506 RABBIT 14 N= · 17 ; - -- MEAN I .1= 38 o. 706 MEAN I 2= 19 6· 588 ME.AN DI F= . 184· 118 - S D.l~ .41• 2212 SD2.=7 57·0987 SD3= 24· 778 7 SEl= 11· 4528 SE2= 13.-8485 SE3= 6. 009 72 . I 1 I2 I3 Dl D2 ~ 169 ~3 . 106 38 30 168. 123 65 52 42 201 .145 56 73 58 219. 182 31 93 80 2Q1 232 -31 118 107 227 260 -33 148 132 198 267 -fB 18 0 164 267 285 -18 210 194 273 e79 -6 248 228 263 300 -45 286 266 282 337 -55 326 296 261 355 -94 360 338 256 299 -43 394 374 308 308 0 428 414 283 318 -35 458 442 237 330 -93 486 469 -· RABBIT 16 N= 16 - MEAN . I 1= 239.563 MEAN I 2= 255· 688 MEA\1 DI F= -16~_125 . -- SD.l~ !!0•3716 SD2~ 84· .7567 SD3=7 56·9127 SE1= 1 o. 0929 SE2= 21.- 189 2 SE3= 14 .. 226 2 -170- I 1 I2 13 Dl D2 290 94 . .19~ 37 38 342 173 169 56 64 434 207 227 .79 87 ~36 . 23~ 202 100 114 463 251 212 136 139 q56 308 150 166 178 397 32.5 1a 200 206 43l 261 170 234 244 38~ 306 83 266 282 387 255 132 302 322 453 232 221 338 357 432 230 202 382 393 393 234 159 418 441 324 245 79 448 473 .. - RABBIT 17, N= 14 . . . - .. ~ - .. -· MEAN 11::: 402· 071 MEAN I 2= 239. 643 MEA.\J DI F= 162· 429 . - .. . . S D 1::: 52· 701_6 SD2~ 58.2734 SD3= 53· 3172 . SEt= 14. 00 51 SE2= 15·5742 SE3= 14~249 6 I 1 I2 !3 D1 D2 - - 45 55 -10 38 36 _7_7 84 -7 55 66 13 17 -4 88 94 8 6 . . 106 -20 113 124 _121 .114 1 147 156 .125 .112 .13 18 5 19 6 _1.02 .112 -10 216 227 1.15 .103 12 254 261 .1.1.5 .1.17 -2 288 294 .113 _110 3 321 335 131 1.15 16 366 378 1 .1.7 .117 0 407 420 121 119 2 447 458 107 108 -1 481 488 -- -. RABBIT 18 N= 14 .. . . -.. - - MEAN I 1= 99 • 1429 MEAN I 2= 99·2143 MEA'J DI F= - -7~ .. 1428 6E~2 -- 10.0036 SD.l~ 33· 67 SD2?: 29·113 SD3= - SEl= 8 ·998 (;£J SE2= 7· 78 077 SE3= 2· 67357 ...;171- ~ I 1 !2 13 Dl D2 ~ 198 27 17 1 35 33 266 51 217 53 54 2_72 67 205 73 73 315 92 223 96 98 3·15 127 188 .121 128 3.32 J..27 205 153 16 1 31.0 - 127 .183 189 188 321 2 18 ·.103 228 222 326 .135 .19 1 259 256 286 .12.6 .160 300 296 28 .7 _131 _1 56 342 334 .308 1.14 .194 376 369 2.54 9 1 .1 ~3 4 16 4 00 287 93 19 4 444 424 . ~ RABBIT 19 N= 14 - MEAN I 1= 291 · 357 . . MEAN I 2= 109 MEA'\/ DI F= 18 2· 35 7 S D.l.?. 35· 7031 SD2?. !15· 6138 SD3~ 30· 698 6 SEl= 9 · 54205 SE2= 12il900 SE3= 8 . 20453 . rt I2 I 3 D! D2 56 20 3 6 34 34 6.4 34 30 52 l9 78 58 20 72 7 1 88 58 30 9 4 93 102 83 19 120 120 1.01 7.6 25 146 148 118 99 19 179 175 127 95 . 32 200 202 123 l02 2 1 23/J 234 96 11 3 -1 5 266 26 1 9._6 96 0 300 290 116 9 4 22 330 332 92 110 -18 366 364 96 86 10 398 400 98 122 - 24 426 428 .. -. RABBIT .20 N= 15 . .. - . MEAN I 2= 8 3· 066 7 MEAN I 1= 9 6e8 667 MEA\1 DI F• 13.~.8 . . . S D.l::: 20. l 0 64 SD2~ 29 · 1289 SD3= 19 . 199 SE1= 5a 19145 SE2= 7. 52106 SE3= 4. 957 15 I 1 158 215 236 270 284 307 308 323 331 357 356 363 341 389. 391 357 333 RABBIT «· -172- MANDIBULAR GINGIVAL THmD STAINED, 2 BLEACHES !2 13 - 157 _1 229 -14 231 5 273 -3 294 -10 301 6 318 -lO 335 -12 351 -20 345 12 324 32 325 38 284 51 348 41 315 16 342 15 305 28 N= 17 Dl 42 62 87 109 134 160 190 220 250 281 310 334 360 384 407 428 446 - - MEAN I 1~ 312·882 MEA'l I 2= 298 0 647 l!l~. 2353 -- SDl~ J>3• 24~4 SD2==: 51·8 024 SD3= 27· 2638 SEl= 15· 339 SE2= 12· 5639 SE3= 6.61245 I 1 12 !3 D! - . - 211 95 - 122 35 a79 153 126 50 300 204 96 72 359 255 J04 98 39.9 257 142 124 372 332 40 156 3~1 364 -3 187 390 331 59 219 361 342 45 2Le 379 341 38 278 -. - . RABBIT 12 N= 10 -- - MEAN_ I 1= 344· 3 MEAN I 2= 267e 4 - 7_6_._9 . - SP.l:=: 59 • 4438 SD27: 9 1· 6651 SD3?= 47· 58 49 SEt= 18. 79 78 SE2= 28e98 7 SE3= 15o0477 D2 38 52 70 92 114 140 165 19 2 226 256 284 312 334 358 384 402 424 MEJW DI F= D2 36 50 70 93 .118 145 178 211 242 276 MEAN DI F= -173- I 1 I2 13 Dl D2 232 203 36 38 50 213 263 10 57 70 314 306 8 79 85 322 328 - .6 104 113 370 37 1 -l 124 138 374 38 4 -.1 0 151 172 385 4 03 -18 162 205 390 385 5 218 236 395 380 15 250 269 ~0 .1 355 46 284 303 ~02 326 74 309 342 392 308 8 4 347 378 399 3 .10 89 , 383 413 400 293 107 4 13 4 47 -- - RABBIT 14 N= 14 MEAN I 1::: 361 ·143 MEAN I 2= 329 · 78 6 ME~ DI F= 3.1 ._35 71 . . . - . . -· SD_l:: 52· 90.Pl SD2~ 55. 1434 SD3~ 4 .1• 53 15 SEl= 14· 139 7 SE2= 1 4~ 7377 SE3= 11 ~ 0998 I 1 I2 I 3 Dl D2 110 AB 62 30 3 4 251 165 86 45 ~ 278 190 88 63 64 329 252 77 87 8 4 313 275 38 110 11 0 394 29 1 103 137 133 356 3A2 14 170 160 344 310 34 207 19 6 373 32 1 52 237 228 363 384 - 2.1 272 262 381 402 - 2 1 300 298 407 369 38 346 334 o929 39 9 30 387 370 363 349 14 417 408 38.5 365 20 Jt47 440 331 350 -19 478 4 7 2 .. - . . RABBIT 16 N= 16 . - MEAI.\1 I 2= 300· 75 MEAN_ I 1= 337· 938 MEAN DIF= 3_7_._ 18 75_ · - . . SD.l::: '16· 1485 SD2::! 9 6· 70ffi SD3= 39 . 22.45 SEt= 19 . 03 7 1 SE2= 24~ 1767 SE3= 9 · 80613 ·- -174- I 1 12 I3 D1 D2 ... . - 257 134 .123 36 36 357 232 125 50 48 385 28 .7 98 72 67 425 32 1 104 96 93 445 365 80 123 118 416 390 28 158 149 449 358 9 1 192 18 2 ~1 .1 4 12 -1 . 229 218 385 335 50 266 257 430 34.6 84 303 293 472 431 4 1 343 328 ~69 412 57 379 372 3.50 38 1 - 3 1 420 404 311 307 10 452 434 329 276 53 480 462 .. - RABBIT 17 N= 15 . - . - . - . - MEAN .. I 1= 39 3· 267 MEA\J I 2= 332· 467 MEAN DI F::: 6.0.~8 - . . . . - .. SDJ.::: .61 • 4346 SD2~ 78 · 1225 SD3:= 45· 9 39 7. SE1= 15· 8 623 SE2= 20· 171 1 SE3= 11 · 8 616 - I 1 12 13 D1 D2 ~ 8.7 ~ .132 -45 33 34 143 198 - 55 49 48 9.8 . 222 -1 24 67 68 _191 212 - 8 1_ 90 92 20.7 ~ 16 - 2 11 119 114 264 32 1 - 57 .148 148 275 290 -1 5 18 1 162 245 350 - 105 216 2 18 30A 298 6 252 249 231 ~8 - 67 292 280 30.7 343 - 36 334 318 29 1 263 28 369 352 267 304 - 37 401 388 324 273 5 1 428 420 250 248 2 452 443 RABBIT 18 N= 15 ~ --- - ... MEAN I 2= . 28 2 MEA\J DI F=- 49 . 7333 MEAN I 1= . 232• 267 __ SDl~ 13· 9464 SD2::: f>7 • 758 SD3= 65· 18 38 SEl= 19 · 0929 SE2= 17· 49 5 SE3= 1 6 ~8 304 - -17$- I 1 12 I3 D1 D2 .1.28 123 5_ 44 L&4 157 -95 . 112 65 62 178 337 -159 90 85 185 176 9 120 116 194 206 -12 150 145 J97 176 21 184 18 2 193 158 35 220 215 .198 156 42 256 246 2.00 .148 60 288 284 216 167 49 324 320 232 162 70 355 356 -- RABBIT 19: N= 1 1 . - . -- - - - MEAN . I 1::= 189 e 636 MEAN I 2= 168.545 MEAN DI F= - 21 .• _09 09 . . - . . SD.l::: 28 • . 2888 SD2:=: 69 · 2566 SD3~ 66·9296 SEl= 8 • 529 39 SE2= 20 .. 88 16 SE3= 20 •. 7831 I 1 12 13 Dl D2 12 .63 . - 44 34 43 63 1.04 - 41 43 53 64 127 - 63 6 .1 73 £6 129 - 6 1 8 1 92 8.7 13~ - 47 100 118 106 .139 -31 128 143 111 163 - 52 154 172 118 153 - 35 18 2 194 103 1 4 0 - 37 200 226 .106 .1 39 - 33 241 258 .130 167 - 37 264 282 .1.15 143 - 28 29 1 314 .1.42 .153 - 11 32~ 340 147 178 - 3 1 357 372 .. - RABBIT 20 N= 14 . - .... - MEAN ! 2= 138 ME.AI.\J DI F=-39• 3571 MEAN I 1= . 98 • 6429 . . SD1::: 35~1319 SD2=: 28 . 4821 SD3= 13· 6247 SEl= 9 • 339 38 SE2= 7· 61217 SE3= 3 e 64135 I 1 !2 13 Dl D2 -TIMEs 3• 12 SECS• REFERENCES -176- 1. Mitchell, D. F.: Dental discoloration and tetracyclines. J Indianap Dist Dent Soc 17:21, 1962o 2. Keitel, H. G., and Soentgen, M. L.: Dental staining and anti microbial therapy. Canad Med Assoc J 93:129, 1965 (Letter to the editor). 3G Beckelman, J. H., and Gingold, N. L.: Developmental dental defects associated with systemic tetracycline therapy: Review of literature and case report. NY J Dent 34:377, 1964~ . 4. Spencer, D. E.: ! ~conservative method of treating tetracycline stained teeth. J Dent Child 39:443, 1972. 5o Cohen, s., and Parkins, F. M.: Bleaching tetrac,ycline-stained vital teeth. Oral SUrg 29:465, 1970. 6. Arens, D. E.; Rich, J. J.; and Healey, H. J.t A practical method of bleaching tetracycline-stained teeth. Oral Surg 34:812, 1972. 7. Duggar; B. M.: Aureomycin: A product of the continuing · search for new antibiotics. Ann NY Acad Sci 51:177, 1948o Be Manten, A.: Antibiotic Drugs: Tetracyclines. In Meyler, L., and Herxheimer, A., ed.: Side Effects of Drugs, Volume VII. Amsterdam, Excerpta Medica, 1972, pp.356-36J. 9e Boothe, J. H.; MOrton, J.; Petisi, J. P.; Wilkinson, R. G.; and Williams, J. H.: Tetracycline. J Am Chem Soc 75:4621, 1953. 10~ Ross, S • .; Puig, J. R.; and Zaremba, E. A.: Absorption of demethyl chlortetracycline in children: Some preliminary observations .. Antibiot Ann 7:388, 1959-1960~ 11. Regna, p. P., and Solomons, I. A.; The chemical properties of Terramycin. AJm N Y Acad Sci 53:229 1 1950. 12o Finl~, A. c.; Hobby, G. L.; P'an, S. Y~; Hegna, P. P.; Routien, J. B.· Seeley, D. B.; Shull, G. M.; Sob1n, B. A.; Solomana, I. A.; Vinso~, J. w.; and Kane, Je H.: Terramycin: A new antibiotic~ Science 111:85, 1950. 13. Kunin, c. M., and Finland, :M. ~ Demethylchlortetracycline: A new tetracycline antibiotic that yields greater and more sustained antibacterial activity. New Eng J Med 259:999, 1958. -177- 14. Wagner, Yf. H. ; Schmidt, W. ; Bauer, F.; and Dittmar, W.: Mode of application of therapeutic effect of tetracyclines in animal experiments. Antibiot Ann 7:358, 1959-1960. 15. Kaplan, M. A.; Albright, H.; and Buckwalter, F. H.: A new tetr4l. cycline antibiotic for parenteral use. Antibiot Ann 7:365, 1959-1960. 16. Council on Drugs:· Rolitetracycline: An antibiotic for parenteral use. J~JA 187:141, 1964. 17. Schach von Wittenau, M., and Chianini, Josephine: A pharmacokin etic analysis of the absorption and elimination of doxycycline in man. Chemo Therap 13:249, 1968. 18. Ory, E. M.: The tetracyclines. Med Clin North Am 54:1173, 1970. 19. Rtyscians' Desk Reference, 28th ed., Ocadell, N. J., Medical Economics Company, 1974, PP• 301-332. 20. Tetracycline-type antibiotic s.ynthesized. Am Druggist 146:33, September 1962. 21o Johnson, R. H.: The Effects of Tetrac.ycline on Teeth and Bones. Master's Thesis, Indiana University School of Dentistr.y, 196u, pp. 3-72. 22 • Hamp, s, E.: The tetracyclines and their effect on teeth: A clinical stuqy~ Scand J Dent Res 75:33, 1967. 23. Tod~•s Drugs--Broad spectrum antibiotics. Brit Med J 534lt 1331, 1963. 24. Ibsen, K. H.; Urist, M. R.; and Sognnaes, R. R.: tetracyclines with respect to staining of teethe 1965. Differences among J Pediat 67:459, 25. Hussar, D. A.: Interactions involving drugs used in dental practice. J J.Jn Dent J.psoc 87:349, 1973. 26. Pfizer Laboratories: Terramycin drug insert. New York, Pfizer Manufacturing, 1971. 27. Kiser, J. s.; DeMello, G. Co; Eve, V.; Lindh, H.; Malone, L.; Popken, F.; Schurr, A.; and Waters, M. K.; A comparative evalu~­ tion of chlortetracycline, oxytetracycline, and tetracycline in experimental infections. Antibiot Ann 1:56, 1953-1954o 28. Kunin, c. M.: '!be tetracyclineso Pediat Clin North kn 8:1001, 196lo 29. Welch, H. ·; Hendricks, F. D.; Price, C. W.; and.Randall, W. A.s Comparative studies on TerramYcin and Aureomyc~n: Antibacterial spectrum, serum concentrations and urinar,y excretion. J Am Fhar.m Assoc 39:185, 1950. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. -178- McCormick, J. R. 0.; Stolander, N. 0.; Hirsch, U.; Jensen, E. R.; and Doerschuk, A. P.: A new fam~ of antibiotics: The demethy chlortetracyclines. J Am Chem Soc 79:4561, 1957. Kunin, C. M., and Finland, M. : Clinical .pharmacology of the tetracycline antibiotics. Clin Pharmacol Ther 2:51, 1961. Musselman, M. M.: Terramycin. Antibiotics Monographs No. 6, New York, Medical Encyclopedia, Inc., 1956. . Regna, P. P.; Solomons, I. A.; Murai, K.; Timreck, A. E.; Brunings, K. J.; and Lazier, w. A.: The isolation and general properties of Terramycin and Terr~cin Salts. J Am Chern Soc 73:4211, 1951. . . Higuchi, T., and Bolton, s.: · The solubility and complexing prop~rties of oxytetracycline and tetracycline III: Interactions in aqueous solution with model compounds, biochemicals, metals, chelates, and hexametaphosphate. J Am Fharm Assoc, Scient Ed 48:557, 1959. Albert, A.: Avidity of Terramycin and Aureomycin for metallic cations. Nature 172:201, 1953. Ibsen, K. H., and Urist, M. R.: The biochemistr.y and physiology of the tetracyclines: With special reference to mineralized tissues. Clin Orthop 32:143, 1964. · Saz, A. K. ~ and Martinez, L. M.: Enzymatic basis of resistance to Aureomyc·in. J Bact 79:527, 1960. Weinberg, E. n.: The mutual effects of antimicrobial compounds and metallic cations. Bact Rev 21:46, 1957. Weinberg, E. D.: The reversal of the toxicity of oxytetracycline (Terramycin) by multivalent cations. J Inf Dis 95:291, 1954o Hasler, J. F., and Mitchell, D. F.: Comparison of the fluorescent properties of a new tetracycline. J Dent Res 47:1188, 1968. Mitchell D. F., and Fahmy, H.: A test of seven agents for vital dye and intrinsic dental staining activity • J Oral Therap !harm· 4:378, 1968o Walters, p., and S~egh, F. s.: Tetracycline hypoplasia in hard tissues. J Dent Res (I AD R Abstract No. 856) 53:268, 1974o Johnson, R. H., and Mitchell, D. F.s The effects of tetracyclines on teeth and bones. J Dent Res 45:86, 1966. Putnam, L. E. J Hendricks, F. D.; and Welch, H.: Tetracycline·, a new antibiotic. Antibiot Ann 1:88, 1953-1954. -179- 45. Cunningham, R. W.; Hines, L. R.; Stokey, E. H.; Vessey, R. E.; and Yuda, N. N.: Pharmacology of tetracycline. Antibiot Ann ls 63, 19.53-1954. 46. Walsh, H.t Absorption, excretion, and distribution of Terr~cin. Ann N Y Acad Sci 53:2.53, 1950. 47. Bevelander, G., and Rolle, Gloria K.~ Maternal transmission of tetracycline to the skeleton of the developing embryo. J Dent Rea 39:6.57, 1960 (Abstract). 48o Barr, W. H.; Adir, J.; and Garrettson, L.: Decrease of tetracycline absorption in man by sodium bicarbonate. Clin Pharmacol Ther 12 :· 779, 1971. 49. Stokstad, E. t. R.J Pensack, J. M.; and Huhtanen, c. N.: The effect of calcium salts on chlortetracycline absorption. Antibiot Ann 5o. 51. 53. 54. 55. 56. 57. 7:·879, 1959-1960. Andrews, n. Jeanne: pediatric practice. The use of tetracycline with vitamins in Antibiot Ann 3:280, l955-1956o Sweeney, W. M.;- Hardy, S. M.; Dornbush; A .. C.; and Ruegsegger,. J. M.: Absorption of tetracycline in human beings as affected by certain excipients. Antibiot Med 42642, 1957. Kunin, c. M.; Jones, w. F., and Finland, M.: Enhancement of tetracycii~e blood levels. New Eng J Med 2.59:147, 1958o Kunin, C. M. ; Dornbush, A. C.; and Finland, M. : The distribution and excretion of four tetracycline analogues in normal young men~ Antibiot Ann 7:386, 1959-1960. 0 'Regan, c., and Schwarzer, S.: Intramuscular Terramycin: Lab oratory and clinical studies in children. J .. Pediat 44tl72, 1954. Yadav, v.; Ziegra, s.; Smith, r.; and Keitel, H. GG: Studies of premature infants receiving oxytetracyclineo Am J Dis Child 10.5:253, 1963. Finland, M.; Purcell, E. M.; Wright, S. S.; Love, B. D.; Mou, T~ W.; and Kass, E. H.t Clinical and laborator,y observations of a new antibiotic, tetracyclineo JAMA 154:561, 19.54o Hirsch, H. A., and Finland, M.: Antibacterial activity of serum of normal subjects after oral doses of demethylchlortetrac.yc1ine, chlortetracycline, and oxytetracyclinee New Eng J Med 260:1099, 1959. Naranjo, R.t Blood level studies with new, preconstituted intra muscular solution of oxytetracycline. Antibiot Ann 7:843, 1959-1960. -180- 59. Madison, J. F.: Tetracycline pigmentation of teeth. Arch Dermatol 88:58, 1963. 60. Febles-Alfonzo, D., and Batthyany, C.: An experimental stu~ of oxytetracycline: Local genital absorption and diffusion, and placental transmission to the blood of the umbilical cord and to the amniotic fluid. Antibiot Ann 7:846, 1959-1960o 61. Gibbons, R. J., and Reichelderfer, T. E.: Transplacental trans mission of demathychlortetracycline and toxicity studies in pre mature and full term, newly born infants. Antibiot Mad 7:618, 1960. 62. Kelly, R. G.; Kanegis, L. A.; and ~ske~ D. A.: The metabolism and tissue distribution of radioisotopically labeled demethyl chlortetracycline. J Pharm Exper Therap 134:320, 1961. 63. Andre', T.: Studies on the distribution of tritium-labeled di hydrostreptomycin and tetracycline in the boqy. Acta Radiolog, Suppl. 142, 1956. 64. Helander, s., and BOttiger, L. E.r On the distribution of Terr~cin in different tissues. Acta Mad Scand 147:71, 1953o 65. Milch, R. A.; Rall, D. P.; and Tobie, J. E.: Fluorescence of tetra cycline antibiotics in bone. J Bone Joint Surg 40-A:897, 1958. I 66. Milch, R. A.; Rall, D. P.: and Tobie, J. E.: Bone localization of the tetracyclines. J Nat Cancer Inst 19:87, 1951~ 67. -~ttiger, L. E.: On the distribution of tetracycline in the boqy. Antibiot Chemother 5:?32, 1955. 68. Charles, D.: Placental transmission of antibioticse J Obstet Gynaec Brit Emp 61:750, 1954o 70o 71. 13. Swallow, J. N.: Discoloration of primary dentition after maternal tetracycline ingestion in pregnancy. Lancet 2:611, 1964. Posner, A. c.; Prigot, A.; and Konicoff, N. G.: Further observations on the use of tetracycline hydrochloride in prop~laxis and treat ment of obstetric infections. Antibiot Ann 2:594, 1954-1955o Gal~, K. A., and Mostehy, M. R.: Tetracycline staining with evidence of placental transfer. Egypt Dent J 16:297, 1970. stewart, n. J.: The effects of tetracyclines upon the dentition. Brit J Derm 76:374, 1964. AntoQY, J. R.: Effect on deciduous and permanent teeth of tetra cycline deposition in utero. Post Grad Med 48:165, 1970. 74. 15 •. 76. 77. 78. 79. 8o. 81e 82. -181- Douglas, A. c.: The deposition of tetracycline in human nails and teetht A complication of long term treatment. Brit J Dis Chest 57:44, 1963. Brearley, L. J.J stragis, A. A.; and Storey, E.: Tetracycline induced tooth changes: Part I, Prevalence in pre-school child- ren. Med J Aust 2:653, 1968. . Cohlan, S. Q.; Bevelander, G.; Bross, S.: Effect of tetracycline on bone growth in the premature infant. Antimicrob .Age11:ts Chemother 340, 1961. Macaulay, J. c., and Leistyna, J. A.: Preliminary observation on prenatal administration of demethylchlortetracycline. Pediatrics 34:423, 1964o CUttita, J. A.; Kutscher, A. H.; Zegarelli, E. V.; and Denning, Carolyn R.t Discoloration of the teeth due to antibiotics of the tetracycline family. N Y J Dent 35:89, 1965. Owen, L. N.: Fluorescence of tetraqyclines in bone tumors, normal bone, and teeth. Nature (Lond) 190:500, 1961. Kutscher, A. H.; Zegarelli, E. V.; Tovell, H. H. M.; and Hochberg, B.: Discoloration of teeth induced by tetracycline: Administered ante partum. JAMA 184:586, 1963~ l Demers, P.; ·Fraser, D.; Goldbloom, R. B.; Haworth, J. C.; LaRochelle, Jean; MacLean, R.; and Murray, T. K.: Effects of tetracyclines on skeletal growth and dentition. Canad Mad Assoc J 99:849, 1968o Owen, L. N.: The effects of administering tetracyclines to young dogs with particular reference to localization of the drugs in the teeth. Arch Oral Biol 8:715, 1963. Weyman, Joan, and Porteous, J. R.: Tetracycline discoloration bands in human teeth. Brit Dent J 115:499, 1963. Mello, H. s.: The mechanism of tetracycline staining in primary and permanent teeth. J Dent Child 34:478, 1967 • 85. Rendle-Short, J. T.: Tetracycline in teeth and bone. Lancet 1: 1188, 1962 (Letter to the editor). 86. Kline, A. H.; Blattner, R. J.; and .Lunin, M.: Transplacental effect of tetracyclines on teeth. JAMA 188:178, 1964~ Dowling, H. F.; Lepper, M. H.; Caldwell, E. R.; Whelton, R. ~; and Brickhouse, R. L.: The concentration of Aureomycin in urine and cerebrospinal, pleural and ascitic fluids after oral and intra venous administration. J Clin Invest 28:983, 1949e 88. 90. 91. 92. 93o 94. 95. 96. 97. 98. 99. 100. -182- Frost, H. M; Villanueva, A. R.; and Roth, H.: P.yogenic osteo myelitis: Diffusion in live and dead bone with particular refer ence to the tetracycline antibiotics& Henry Ford Hospit Med Bull 8:255, 1960. Wozniak, Lillian A.: Studies on binding of tetracyclines by dog and human plasma. Proc Soc Exper Biol Med 105:430, 1960. Zastava, V.; Malek, P.; Zak, F.; and Kolc, J.: On protracted fixation of tetracycline antibiotics in the tissues. In Herold M., and Gabriel, z., ed.: Antibiotics: Advances in Research ' Production and Clinical Use, (Proceedings of the Congress on ' Antibiotics held in Prague, 15-19 June, 1964). London, Butterworths, 1966, p. 218~ Mulvaney, W. P.; Beck, C. Yl.; and Qureshi, M. A.: Deposition of tetracyclines in urinary calculi. J MlA 190:1074, 1964. Kelly, R. G., and Buyske, D. A.t Metabolism of tetracycline in the rat and the dog. J Phar.macol Exp Ther 130:~~' 1960. Zaslow, J.; Cohn, E. M.; and Ball, W.: The excretion and con centration of o~tetracycline in bile following intramuscular administration of the drug. Antibiot Ann 2:964, 1954-1955. Eisner, H. J., and Wulf, R. J.: The metabolic fate of chlor tetracycline and some comparisons with other tetracyclines. J Pharmacol Eip Ther 142:122, 1963. Prat, V.; Hatala, M.; and Benesova, D.: Experimental Colibacillary p,yelonephritis and effect of tetracycline antibiotics. In Herold, M., and Gabriel, z.,· ed.: Antibiotics: Advances in Research, Production and Clinical Use, (Proceedings of the Congress on Antibiotics held in Prague, 15-19 June, 1964). London, Butterworths, 1966, p. 118. Tobie, J. E., and Beye, H. K.: Fluorescence of tetracyclines in filarial worms. Proc Soc Exp Biol Mad 104:137, 1960. Wood, w. B., Jr., and Archer, G. w.: Mechanisms of action of antimicrobial drugs. Pediat Clin North Am 8:969, 1961. Gale, E. F., and Folkes, Joan P.: The assimilation of amino acids by bacteria. Biochem J 53:493, l953G Brody, T. M., and Bain, J. A.: ~e effect of Aureomycin and Terr ~cin on oxidative phosphory1at~on. J Pharmacol Exp Ther 103:338, 1951 (Abstract). Du~ H. G. and Showacre, J. L.: Selection localization or· tetra~ycline' in mitochondria of living cells. Science 133:196, 1961. -183- 101. Perry, D. M; Hall, G. A.; and Kirby, W. M. M.; Demethylchlor tetracycline: A clinical and laboratory appraisal. Antibiot Ann 7:409, 1959-1960. 102. Garrod, L. P., and Waterworth, Pamela M.: The relative merits of the four tetracyclin~s. Antibiot Ann 7:440, 1959-1960. 103. Dowling, H. F.: Tetracycline. Antibiotics Monographs No. 3 New York, Medical Encyclopedia, Inc., 1955. . ' 104. Schwachman, H.; Silverman, B. K.; Patterson, P. R.; and Zheutlin L. J.: Antibiotics in treatment of pancreatic fibrosis, with , emphasis on Terramycin. JAMA 149:1101, 1952. 105. Schwachman, H.; Fekete, E.; Kulczycki, L. L.; and Foley, G. E.t The effect of long-term antibiotic therapy in patients with cystic fibrosis of the pancreas. Antibiot Ann 6:692, 1958-1959. Io6. Schvrachman, H., and Schuster, A.: The tetracyclines! Applied pharmacology. Pediat C1in North Am 3:295, 1956$ 107. Zegare11i, E. V.; Denning, Carolyn, R.; Kutscher, A. H.; Tuoti, F.; and Sant' Agnese, P. A.: Discoloration of the teeth in patients with cystic fibrosis of the pancreas. N Y St Dent J 27:237, 1961. 108. Zegarelli, E. V.; Kutscher, A. H.; Derming, Carolyn R.; Saporito, R. ; Slaughte_r, T. W. ; and Fahn, B. • Coloration of teeth in patients with cystic fibrosis of the pancreas, part II. Oral Surg 15:929, 1962·. 109. Hobby, Gladys 1.; Lenert, Tulita F.; Pikula, Daria; Kiseluk, Mary; and Mudders, M. Elizabeth: The antimicrobial action of Terramycin. Ann NY Acad Sci 53:266, 1950. 110. Plaza-Roca, J.: The accumulation of oxytetracycline in osteo genic zones as measured by observation of fluorescence. Antibiot Ann 1 :·850, 1959-1960. lll. Hilton, H. B.: Skeletal pigmentation due to tetracycline~ J Clin Path 15:112, 1962" 112. 113. Mc1eay, J. F., and Walske, B. R.: Tetracycline fluorescence in bone lesions. J Bone Joint SUrg 42-A:940, 1960. Anderson R. L.; Ferguson, A· B.; and Braude, A. I.: Bacter iostasis' of tetracycline deposited in bone. Surg Gynec Obstet 108:65, 1959. Cullen, c. H., and Hargadon, E. J.: Penicillin versus tetracycline in the treatment of childhood osteomyelitis. Acta Paediatr Scand 51:189, 1962o -184- 115. Frost, H. M.: P.yogenic osteo~elitiss The normal bone spaces as a bacterial reservoir. Henr.y Ford Hospit Med Bull 8:263, 1960. 116. Collins, H. S.; Paire, T. F.; and Finland, M.: Clinical studies with Aureomycin. Ann N Y Acad Sci 51:231, 1948. 117. Womack, C. R.; Jackson, G. G.; Gocke, T. M.; Kass, E. H.· Haight T. H.; and Finland, M.: Ter:rrueycin therapy of urinary t~act in-, fections. Arch Intern Med 89:240, 1952. . 118. Minkin, W. ; Cohen, H. J.; and Frank, S. B. t Fixed-drug eruption due to tetracycline: Report of a case. Arch Derm 100:749, 1969. 119. Copperman, I. J.: Hypersensitivity to tetracycline. Lancet 2:610, 1967. 120. Furey, W. W., and Tan, C.~ Anaphylatic shock due to oral demethyl chlortetracycline. Ann Intern Med 70:357, 1969. 121. Coles, R. B.; Phillips, J.; and Nuttal, J. B.: Anaphylactoid reaction to deme~lchlortetracycline. Brit Med J 2:313, 1967. 122. Tromovitch, T. A.,. and Jacobs, P. H.: Photosensitivity to oxy tetraeycline. Ann Int Med 58:529, 1963. 123. Schorr, W. F., and Monash, S. :· Photo-irradiation studies of two tetrac.yclines. Arch Dermat 88:440, 1963. 124. Kishore, B.; Bansal, 0. P•; Hazra, D. K.; and Seth, H. C.: Aplastic anemia associated with oxytetracycline therapy. Indian J Mad Sci 23:137, 1969. 125. Schwindt, W. D., and Kisken, W.: Post operative hemorrhage and the tetrac.yclines. Am J Surg 113:837, 1967. 126. IJ.oyd-Still, J. D.; Grand, R. J. t and· Vawter, G. F.: Tetracycline hepatotoxicity in the differential diagnosis of postoperative jaundiceo J Pedia~ ~4:366, 1974~ . 127. Cohn, E. M.; ·zas1ow, J.; Dickens, Janet; and Tuman, J. J.: The effect of oxytetracycline on the function and structure of the liver. Antibiot Ann 2:966, 1954-1955. 128. Edwards, o. M.; Huskisson, E. C.J and T~lor, R. T.: Azotaemia aggravated by tetracycline. Brit Med J 1:26, 1970o 129. Roth, H.; Becker, K. L.J Shalhoub, R. J.; and Katz, S.: Nephro toxicity of demethylchlortetracycline hydrochloride: A prospective etu~. Arch Intern Med 120:433, 1967. 130. 131. 132. 133. 134. 135. 136. -185- Davies, Pamela A.; Little t _ K. ; and Aherne, Yl. : Tetracyclines and yellow teeth. Lancet 1:7 4J, 1962 (Let tars to the editor) e Gross, J. M.: Fanconi Syndrome (adult type) developing secondary to the ingestion of outdated tetrac.ycline. Ann Int Mad 58=523, 1963o Frimpter, G. W.; Timpanelli, A. E.; Eisenmenger, W¥ J~; Stein, H. S.; and Ehrlich, L. I.: Reversible "Fanconi Syndrome" caused by degraded tetracycline. JNaA 184:111, 1963. Koch-Weser, J., and Gilmore, E. B.: Benign intracranial hyperten sion in an adult after tetracycline therapy. JAMA 200:345, 1967. Harley, J. D.; Farrar, J. F.; Gr~, J. B.; and Dunlop, I. c.~ Aromatic drugs and congenital cataracts. Lancet 1:472, 1964. Carter, M. P., and Wilson, F.: Tetracycline and congenital limb abnormalities. Brit Mad J 2:407, 1962. Cohlan, s. Q.; Bavelander, G.; and Tiamsic, T.: Growth inhibition of prematures receiving tetracycline. J Dis Child 105:453, 1963. 137. Cleau, J. F.; Perkins, R. E.; and Gilda, J. E.:· Bone marking agents for the longitudinal study of growth in animalsc Arch Oral Biol 138. 139. 140. 141. 142. ~. 9:627, 1969. Boyne, P. J,t Fluorescence microscopy of bone healing following mandibular ridge resection. Oral SUrg 16:749, 1963 o Yen, P. K-J.; Shaw, J. H.; and Hong, Y-C.: Effects of some stain ing agents on dentin apposition in young rabbits. J Dent Res 50:1666, 1971e Bevelander, G.; Nakahara, H.; and Rolle, Gloria K.~ Inhibition of skeletal formation in the chick embryo following administration of tetrac.ycline. Nature (Lond) 184:728, 1959. Thanik, D. D., and McMurcby, K. A.: Comparison of four tetracycline drugs at four different dose levels and their effect on animal and bone growth. Int Assoc Dent Res No. 466, 1966 (Abstract). Yen, P. K-J., and Shaw, J. H.: Preliminary study of inhibitory effects of tetracyclines on membranous bone growth in Rhesus monk~s. J Dent Res 51:16$1, 1972· · Bevelander, G.; Nakahara, H.; and Rolle, Gloria K.i The effect of tetracycline on the development of the skeletal a,ystem of the chick embr,yo. Develop Biol 2:298, 1960. Weyman, Joan, and Porteous, J. T.: Tetracycline staining of teethr A report of clinical material. J Dent Res 42:-1111, 1963 (Abstraet)e -186- 145. Owen, L. N.: Aspects of the localization of tetracyclines in tumors, bones and teeth in the dog. In Herold, M., and Gabriel z., ed.: Antibiotics: Advances in Research, Production and , Clinical Use, (Proceedings of the Congress on Antibiotics held in Prague, 15-19 June, 1964). London, Butterworths, 1966, p. 334. 146~ Bevelander, G.: The effect of tetracycline on mineralization and growth. In Staple, P. H., ed.: Advances in Oral Biology Vole 1. New York, Academic Press, 1964, p. 205. 147. Bevelander, G.: Letters to the editor, Brit Mad J 1:54, 1963e 148. Wallman, I. S., and Hilton, H. G.: Teeth pigmented by tetracycline. Lancet 1:827, 1962. · 149.. Porteous, J. R., and Weyman, Joan: Tetracyclines and yellow teeth Lancet 1:861, 1962.(Letter to the editor). . 150. Forti, G., and Benincori, C.: Doxycycline and the teeth. Lancet 1:782, 1969 (Letters to the editor). 151. Wallman, I. s., and Hilton, H. B. t Prematurity, tetracycline and oxytetracycline in tooth development. Lancet 2:720, 1962 (Letters to the editor) • 152. Mustakallio, K. K.: Tetrac,yclines and deposition of calcium~ Lancet 2:721, 1962 (Letters to the editor). 153 • Miller, J. : ·Tetracycline in teeth and bone. Lancet 2: 1072, 1962 (Letter to the editor). 155. 156. 157. Witkop, C. J.; Wol.f, R. 0.; aoo Mehaffey, H. H.: The frequency o£ discolored teeth showing yellow fluorescence under ultraviolet light. J Oral Therp Rlarm 2:81, 1965. Swallow, J,. N.; DeHaller, Jacqueline; and Young, Winifred F.: Side-effects to antibiotics in cystic fibrosis: Dental changes in relation to antibiotic administration. Arch Dis Child 42:311, 1967. Moffitt, J. M.: The Effect of Tetracycline-Type Drugs and Cystic Fibrosis on the Enamel and Dentin Microhardness in the Primary Dentition. Master's Thesis Northwestern Universit.y, 1971. Zegarel1i, E. V.; Denning, c. R.; Kutscher, H. H.; Tuotic F.; and Sant' Agnese, p. A.s Tooth discoloration in cystic fibrosis. Pediatrics 26:1050, 1960. Baker, K. L., and Storey, E.: Tetracycline-induced tooth changes, Part III incidence in extracted first permanent molar teeth. Med J Aust 1:109, 1970. -187- 159. Brown, J. P.: Effects of tetracycline antibiotics on teeth of children in the Qympie region of Queensland. J Dent Res 51: 834, 1972 (Abstract). 160. Brearley, L. J., and Storey, E.: Tetracycline induced tooth changes. Med J Aust 2:714, 1968. 161. Witkop, c. J.: Genetics and dentistr.y. Eugenics Quart 5:15, 1958. 162. 163. 164. 165. 168. 170. 17lo 172. Krasny, R. M.: The Incidence of Tetracycline Stained Teeth. Master's Thesis, Indiana University School of Dentistr.y, 1964. Frankel, M. A., and Hawes, R. R.: Tetracycline antibiotics and tooth discoloration. J Oral Therp Pharm 1:147, 1964. Hennon, D· K.: Dental aspects of tetracycline therapy, Literature review and results of a prevalence-survey. J Indiana Dent A.ssoc 44:484, 1965. Stewart, D. J.: Tetracyclines: Their prevalence in children's teeth. Brit Dent J 124:318, 1968. Martin, N. D., arrl Barnard, P. D.: The prevalence of tetracycline staining in erupted teeth. Med J Aust 1:1286, 1969. Grossman, E. R.; Walchek, A.; Freedman, Helen; and Flannagan, Cynthia~. Tetracyclines and permanent teeth: The .relationship between dose and tooth color. Pediatrics 47:567, 1971. Porteous, J. R., and Weyman, Joan: Tetracycline staining of teeth: A report of post mortem material. J Dent Res 42:1112, 1963 (Abstract). Weyman, Joan: Microscopic appearances of tetrac.ycline deposition in human dentin. J Dent Res 47:742, 1968. Antalovska, z.: taws of tetracycline antibiotic deposition in rat incisors. J Dent Res 45:1430, 1966o Moffitt, J. M.; Cooley, R. 0.; Olsen, N. H.; and Hefferren, J. J.s Prediction of tetracycline-induced tooth discoloration. J Am Dent Assoc 88;547, 1974. Bra~da D.· Kolc, J.; and Zastava, V.: The deposition of tetra cyclin~ in'hard dental tissues. In Herold, M., and Gabriel, z., ed.: Antibiotics: Advances in Research, Production and Clinical Use, (Proceedings of the Congress on Antibiotics held in Prague, 15-19 June, 1964). London, Butterworths, 1966, P• 391. -188- 173. Antalovska', z., and Beran, J.: The fluorescence of tetracycline antibiotics (TA) in the hard dental tissues in an experiment. In Herold, M., and Gabriel, z., ed.t Antibiotics: Advances in Research, Production and Clinical Use, (Proceedings of the Congress on Antibiotics held in Prague, 15-19 June, 1964). London, Butterworths, 1966, p. 392. 174o Bevelander, G., and Nakahara, H.; Correlation between tetracycline binding and mineralization in dentin and enamel. Ant Rec 153:141 1965. , 175o Frankel, M. A.: Tetracycline antibiotics and tooth discoloration. J Dent Child 37!29, 1970. 176. Winter, G. B.: Pediatrics and the dental surgeon. Brit Dent J 124:310, 1968. 177. Weyman, Joan: The clinical appearances of tetracycline staining of the teeth. Brit Dent J 118:289, 1965. 178. Yfeyman, Joan, and Porteous, J. R.: Discoloration of teeth possibly due to administration of tetracyclines:· A preliminar.y report. Brit Dent J 113:51, 1962. 179. Storey, E.: Tetracycline antibiotics and their effects on calcified and non-calcified tissues. Aust Ann Med 12:325, 1963o 180. · Mcintosh, H.~., and Storey, E.: Tetracycline-induced tooth changes~ Part IV, discoloration and hypoplasia induced b.1 tetracycline analogueso Med J Aust 1:114, 1970. 181. Harcourt, J. K.; Johnson, N. W.; and Storey, E.: In vivo incorporation of tetrac,ycline in the teeth of man. Arch Oral Biol 7:431, 1962. 182. Bridges, J. B.; Owens, P. D. A.; and Stewart, D. J.: Tetracyclines and teeth: An experimental investigation into five types in the rat. Brit Dent J 126:306, 1969. 183. Johnson, N. w.: The distribution and stability of tetracyclines in dental tissues. In Herold, M., and Gabriel, z., ed.: Anti biotics: Advances in Research, Production and Clinical Use, (Proceedings of the Congress on Antibiotics held in Prague, 15-19 June, 1964). !Dndon, Butterworths, 1966, P• 378e 184. Bevelander, G., and Nakahara, H.: The effect o£ diverse amounts of tetracycline on .fluorescence and coloration of teeth. J Pediatr 68:114, 1966. 185. Weyman, Joant Enamel discoloration by tetracyclineQ J Dent Child 34:109, 1967o -189- 186. Bennett, I. c., and Law, D. B.: Incorporation of tetracycline in developing enamel and dentin in dogs. J Dent Child 34:93, 1967. 187. Bennett, I. C.: Measurement of tetracycline incorporated in enamel and dentin. J Oral Therp Pharm 3:232, 1966. 188. Urist, M. R., and Ibsen, K. H.: Chemical reactivity of minera1ized tissue with oxytetracycline. Arch Path 76:484, 1963. 189. Harcourt, J. K.; Johnson, N. Vl.J and Storey, E.: Incorporation of tetra~ycline antibiotics in bone, teeth, and egg shells. J Dent Res 41:511, 1962 (Abstract). . 190. Kodak Publication No. }~27: Ultraviolet and Fluorescence Photo graphy. Rochester, Eastman Kodak Company, 1972, p. 1.4. 191. Hals, E., Fluorescence microscopy of developing and adult teeth. Scand J Dent Res 61:1, 1953. 192. Frost, H. M.s An economical microfluorescence set up for detection of tetracyclines in bone. Henr.y Ford Hospit Med Bull 8:197, 1960. 193. Benedict, H. C.: A note on the fluorescence of teeth in ultra violet rays. Science 67:442, 1928. 194. Pfl~ger, H. I.: Untersuchung der Experimentell Erzeugten Prophyrie der Zahne in Luminescenzmikroskop. Vierteljahschr f Zahnheilk 47: 202, 1931, as cited in Hals, E.: Fluorescence microscopy of devel oping and adult teeth. Scand J Dent Res 61:16, 1953. 195. Bevelander, G.; Rolle, G. K.; and Cohlan, s. Q.: The effect o~ the administration of tetracycline on the development of teeth. J Dent Res 40:1020, 1961. 196~ Harcourt, J. K. r Tetracyclines :to human teeth. A.ust Dent J 8:518, 1961. 197. 198o 199. Stewart, D. J.t Redeposition in calcified tissues of tetracycline mobilized during bone remodeling in the rat. J Dent Res 50:613, 19 71 (Abstract)~ HammarstrSm, L.: Different localization of tetracycline and s~ u1 taneously injected radiocalcium in developing enamel. Calc Tiss Res 1:229, 1967. LOfgren C.-G.; Omnell, K.-A.; and Nylen, M. U.: Effect of intra periton~al injections of tetrac.ycline nydrochloride and oxytetra-. cycline on forming enamel of rat incisors. Calc Tiss Res 2:145, 1968. 200. 201. 202. 203. 204. 205. 2o6. 207. 208. 209. 210. 2llo- -190- Kutscher, A. H.; Zegarelli, E. V.; Fahn, B. S.; Denning, Carolyn R.; and Douglas, R. N.; Tetracycline discoloration of teetht Diagnosis of long-wave and short-wave ultra-violet light. Oral Surg 23:91, 1967. Boyne, P. J., and Miller, C. W. : A study of tooth development b.r tetracycline-induced fluorescence. J Dent Res 40:1079, 1961 ( Annotation) • .Antalovska, A.; .Lonska, V.; and Pruchova, I. : Participation of vital dental pulp in the distribution of tetrac.ycline in dental tissues. J.Dent Res 47:8o6, 1968. Bhatia, H. L., and Sognnaes, R. F.: Tetracycline discoloration and labelling of teeth and bones: A review. J South Calif' Dent Assoe 31:215, 1963. Gr~n, P., and Johannessen, L. B.: Fluorescence of tetracycline antibiotics in dentin. Acta Odontol Scand 19:79, 1961. Hefferren, J. J.; Cooley, R. 0.; Hall, J. B.; Olsen, N. H.; and Lyon, H. W.: Use of ultra-violet illumination in oral diagnosiso J ~~ Dent Assoc 82:1353, 197lo 0 ,. Nylen, H. U.; Onnell, K.-A.; and LOfgren, C.-G.t Fine.structure of tetracycline-induced hypoplastic and hypomineralized defects in rat incisor enamel. J Dent Res 43:850, 1964 (Abstract). Frost, H. M.: ·Human osteoblastic activity--Part II: Measurement of the biological half-life of bones with the aid of tetracyclines. Henr,r Ford Hospit Med Bull 8:255~ 1960. Hodson, J. J.: The "quenching" of tetracycline fluorescence in caries of the dentin. J Dent Res 42:1102, 1963 (Abstract)o Stephan, R. M.; Fitzgerald, R. J.; McClure, F. J.; Harris, Mo Rachel; and Jordan, H.: The comparative effects of Penicillin, Bacitracin, Chloromycetin, Aureomrcin, and Streptomycin on experimental dental caries and on certain oral bacteria in the rat. J Dent Res 31:421, 1952. Shaw, J. H., and Sweeney, E. A.: Comparative studies on the in fluence of· antibiotic substances in dental caries in the l'fhite rat and the cotton rato J Dent Res 36:349, 1958. Larson, Rachel H., and Zipkin, I.: Effect of tetracycline on transmission of dental caries in rats. J Dent Res 39:725, 1960 (Abstract). 212. 213. 214. 215. 216. 217. 218. 219. 220. 221. 222 .. 223. 224. -191- Zipkin, I.; Larson, Rachel H.; and Rall, D. P.: Reduced caries in offspring of rats receiving tetracycline during various pre natal and post-partum periods. Proc Soc Exp Biol Med 104:158, 1960. Zip~in, !., and Larson, Rachel H.: Reduced caries activity in offspring of rats rec~iving tetracycline. J Dent Res 39:724, 1960 (Abstract). Larson, R. H., and Zipkin, I.: Effect of tetracycline on the transmission of dental caries in rats. J Dent Res 40:264, 1961. Larson, R. H.; Zipkin, I.; and Fitzgerald, R. J.: Effect of de hydroacetic acid and tetracycline on caries activity and .its transmission in the rat. J Dent Res 42~95, 1963. Grahnen, H., and Larson, P. G.: Enamel defects in the deciduous dentition of prematurely born children. Odont Rev 9:193, 1958. Wallman, I.: ·Tetracyclines and infant's teeth. Med J Aust 2:532, 1961. Witkop, c. J ., and Wolf, R. o. :· Hypoplasia and intrinsic staining of enamel following tetracycline therapy. J.AMA 185tl008, 1963. deBorgarello, L. T., and Gendelman, H.: Morpho structural alter ations in enamel and dentin in human deciduous teeth caused by incorporation of tetracyclines. J Dent Res 51:1223, 1972 (Abstract). l Porter, P. J.; SWeaney, E. A.; Golan, H.; and Kass, E. H.: Con trolled stu~ of the effect of prenatal tetracycline on primar,y dentition. Antimicrob Agents Chemother 5:668, 1965e Grant, W. E., and Irving, J o P.: The influence of dietary calcium and phosphorus upon tooth formation. J ~siol 99:18, 1941. Cited in Grahnen, H., and Larson, P. G.: Enamel defects in the deciduous dentition of prematurely born children. Odont Rev 9:201, 1958. Lindquist, B.: Rakit, Spasmofili Och Forandringar: Det Permanenta Bettet (Diskuggionsin Lagg). Nord Mad 49:581, 1953. Cited in . Grahnen, H., and Larson, p. · Go: Enamel defects in the deciduous dentition of prematurely born children. Odont Rev 9:201, 1958. Von Sydow, G. : Study of development of rickets in premat~e in fants. Acta Paediatr Suppl. 2, 33:7, 1946. Cited in Grahnen, H., and Larson, p. G.: Enamel defects in the deciduous dentition of prematurely born children. Odont Rev 9:201, 1958. Bevelander, G.; Goldberg, L.; and Nakahara, H.: The effect of tetracycline on skeletal development in the larval sand dollar (Echinarachnius parma). Arch Oral Biol 2:127, 1960o 225. 226. 227. 228. 229. 230. 231. 232. 233. 234. 235. 237. -192- Saxen, Laurit Drug induced teratogenesis in vitro: Inhibition ofL~alcification b,y different tetracyclines. Science 153:1384, l9uo. Yen, P. K.-J., and Shaw, J. H.: Minocycline-a new tetracycline its influence on calcified tissues of young monkeys. J Dent Res (I AD R Abstract No. 519) 53:184, 1974. Loo, T. I.; Titus, E. D.; and Rall, D. P.: Nature of fluorophore localizing in tetracycline-treated mouse tumor. Science 126:253 1957. , Titus, E. D.; Loo, T. L.; and Rall, D. P.: Identification of the bone fluorophore in tetracycline-treated rabbits. Antibiot Ann 5:949, 1957-1958. ~ . Hakkinen, I. P. T.: The fluorescence of tetracycline in rats treated with dihydrotachysterol. Act Physiol Scand 42:282, 1958. Malek, P., and Kolc, J.: Penetration of chlortetracycline into tissue affected by pathological changes. Antibiot Chemotherap 10:488, 1960. Buyske, D. A.; Eisner, H. J.; and Kelly, R. G.: Concentration and persistence of tetracycline and chlortetracycline in bone. J Pharm and Exper Ther 130:150, 1960. Frost, H. M., ·and Villanueva, A. R.: Tetracycline staining of newly forming bone and · mineralizing cartilage in vivo. Stain Technol 35:135, 1960. Hattner, R., and Frost, H. M.: Fluorescence of tetracyclines in bone: Absorption maximum, hydration shell, and polarization effects. J SUrg Res 2:262, 1962. Milch, R. A.; Tobie, J. E.; and Robinson, R. A.: A microscopic study of tetracycline localization in skeletal neoplasms. J Histochem Cytochem 9:261, 1961. Ibsen, K. H., and Urist, M. R.: Complexes of calcium and magnesium with oxytetracycline. Proc Soc Exp Biol Med 109:797, 1962. Urist, M. R.; Speer, D. P.; Kolin, A.; and McLean, F. C.: Calcium oxytetracycline complexes: New apparatus for frog heart method of estimation of calcium ion concentration. Proc Soc Exp Biol Med 109:801, 1962. Urist, M. R., and McLean, F. c.: Recent advances in physiology of bone: Part I. J Bone Joint Surg 45-A:l305, 1963. -193- 238. Finerman, G. A. M., and Milch, R. A.: In vitro binding of tetra cyclines to calcium. Nature 198:486, 19'03. 239. Deleu, J~: Tetracycline localization in the early stages of isogenous bone graft. Nature 198:194, 1963. 240. Prochazka, P.; Malek, P.; Rokos, J.; Zastava, V.; and Kolc, J.t Localization and fixation of chlortetracycline in the pancreas. In Herold, M., and Gabriel, z., ed.: Antibiotics: Advances in Research, Production and Clinical Use, (Proceedings of the Congress on Antibiotics held in Prague, 15-19 June, 1964). London, Butterworths, 1966, p. 224. 241. Epker, B. N.: Tetracycline in human dentin: Deposition in the zone of mineralization. J Dent Res 45:1825, 1966. Eger, W.; Gattaw, G.; and tammerer, H.: tion and osteogenesis by tetracyclines. 1967. Derangement of mineraliza Gar Mad Mont~ 12:175, 243. Kallmann, H. P.: A physicist looks at biology. In Philip ~rson ed.: The Biology of the Mouth. Publication of the American Association for the Advancement of Science No. 89. Washington D. C., 1968, p. 130. 244e Kawasaki, K. : In vivo and in vitro experiments on the uptake of tetracycline f?Y · tootll'germs.-J Dent Res 51:1247, 1972 (Abstract). 245. Frost, H. M.: Lamellar osteoid mineralized per day in man. Henry., Ford Hospit Med Bull 8:267, 1960. 246. Boyne, P. J.: The use of tetracycline in studies of bone healing. In staple, P. H., ad.: Advances in Oral Biology Vol. III. New York, Academic Press, 1968, p.·121. 247. Boyne, P. J., and Kruger, G. 0.: Fluorescence microscopy of alveolar bone repair. Oral SUrg 15:265, 1962. 248& Gregg, J. M., and Aver,r, J. K.: Studies of alveolar bone growth and tooth eruption using tetracycline-induced fluorescence. J Oral Therp Pharm 1:268, 1964. 249 o Rail, n. p.; I.J:Jo, T. L.; Lane, M.; and Kelly, Margaret G.: Appear ance and persistence of fluorescent material in tumor tissue after tetracycline administration. J Nat Cancer Inst 19:79, 1957. 250. H~inen, r., and Hartiala, K.: Fluorescence of tetracycline in experimental ulcers and regenerating tissue injuries. Ann Med Exp Biol Fenn 37:l15, 1959. 251. 253. 254. 255. 256. 259. 260. 261. -194- Berk, J. E., and Kantor, s. M.: Demethylchlortetracycline induced fluorescence of gastric sediment. J~~ 179:997, 1962. ~rk,·J. E., and Kantor, S.M.: The identification of gastric cancer by means of fluorescence of gastric sediment induced b,y demethylchlortetracycline. Clin Res 9:237, 1961 (Abstract) Klinger, Jaime, and Katz, R.: Tetracycline fluorescence in the diagnosis of gastric carcinoma: Preliminar.y report. Gastroenterology 41:29, 196lo Ackerman, N. B.: Limitations in the use of tetracycline antibiotics for tumor diagnosis. In Herold, M., and Gabriel, z., ed.: Antibiotics: Advances in Research, Production and Clinical Use, Proceedings of the Congress on Antibiotics held in Prague, 15-19 June, 1964). London, Butterworths, 1966, p. 321. Malek, P.: The theory and practice of the diagnostic and physio logical uses of the te~racycline antibiotics. In Herold, M., and Gabriel, z., ed.t Antibiotics: Advances in Research, Production and Clinical Use, (Proceedings of the Congress on Antibiotics held in Prague, 15-19 June, 1964). London, Butterworths, 1966, p. 298. Pearson, H. H.: Bleaching the discolored pulpless tooth. J AlD Dent Ass~ 56:64, 1958. Spasser, H. F. : A simple bleaching technique using sodium per borate. N ,Y St Dent J 27!332, 1961. Nutting, E. B., and Poe, G. S.: A new combination for bleaching teeth. J South Calif Assoc 31:289, 1963. Nutting, E. B., and Poe, G. S.: Chemical bleaching of discolored endodontically treated teeth. Dent Clin North Am, November, 1967, P• 655. Serene, T. p., and Snyder, D. E. : Bleaching technique ( pulpless anterior technique ) • J South Calif Dent As soc 41: 30, 1913. Stewart, G. G.: Bleaching discolored pulpless teeth, J Am Dent Assoc 70:325, 1965. 262. Caldwell, c. P.: Bleaching vital or non-vital teeth. J Calif' Dent Assoc 42:234, 1966. 263. Mcirmes, J. :· Removing brown stain from teeth. Ariz Dent J 12:13, May 1966CI 264. Bailey, R. w., and Christen, A. G. : Bleaching of vi tal teeth with endemic dental fluorosis. Oral Surg 26:871, 1968. -195- 265o Bailey, R. w., and Christen, A. G.: Effects of a bleaching technic on the labial enamel of human teeth stained with endemic dental fluorosis. J Dent Res 49:168, 1970. 266. Colon, P. G.: Improving the appearance of severely fluorosed teeth. J Am Bent Assoc 86:1329, 1973. 267e Bouschor, C. F., and Dorman, H. 1.: Bleaching fluoride stained teeth. Tex Dent J 91:6, June 1973. 268. Rich, J. J.t Bleaching tetracycline-stained vital teeth. Dent Stud 33:83, 1972e 269. t{vborg, H., and Brannstrom, M.: Pulp reaction to heat. J Prosth Dent 19:605, 1968o 270. Mumford, J. M.: Drying of enamel under rubber dam. Brit Dent J 121:178, 1966. 271. Peultier, P. N.; Frank, R. M.; and Klewansky, P.: Effet Des Chocs Themiques Sur L'email Humaino Schweiz Mschr Zahnheilk 77:190, 1967 (Abstracted in English). 272. Waimvright, w. w., and Lemoine, F. A.: Rapid diffuse penetration of intact enamel and dentin by carbon-6-14-labeled urea. J Am. Dent Assoc 41:135, 1950. \ 273. Bartelstone, H .. J.: In vivo study of the uptake of radioactive iodine by human teeth (III). J Dent Res 29:684, 1950. 274. Bartelstone, H. J.: Radioactive penetration through intake with uptake qy bloodstream and thyroid glando J Dent Res 30:728, 1951. 275. Gibson, D. A.~ Fluorescence photography of living subjects: With electronic flash illumination. Mad Radiogr Photogr 38:114, 1962. 276. Adams,. c. p.: Dental Fhotography. Bristol, John Wright and Sons, LTD., ~968, P• 32. 279. 280o Bergenholts, A., and Welander, U.: Ultraviolet autofluorescence: An aid in oral diagnosiso Dent Radiogr Fhotogr 43:83, 1970. Klein, A. I.: The television microscope in dental research. Dent Progr 3:274, 1963. Horowitz, B. A. s The Intraoral Television Micromeasurement o:f Cavit.y Margin Deteriorationo Master's Thesis, Indiana University School of Dentistry, 1966 o Kerkhove, B. C.; Herman, So C.~ . Klein, A. I.; and McDonald, R. E. t A clinical and television dens1tometric evaluation of the indirect pulp capping technique. J Dent Child 34:192, 1967G -196- 281. deAguiar, A. E.: Spongy Bone Architecture of Edentulous Mandibules: A television Radiographic Evaluation. Master's Thesis, Indiana University School of Dentistry, 1966. 282. Traubman, L. t .. A Critical Clinical and Television Radiographic Evaluation of Indirect Pulp Capping. Master's Thesis, Indiana University School of Dentistry, 1967. 283. Geller, J. S.; Klein, A. I.; and !~Donald, R. E.: Association between dentinal sclerosis and pulpal floor thickness: Television Radiographic evaluation. J Am Dent Assoc 83:118, 1971e 284. Jones, M. D.: Television micromeasurement of vent and non-vent cast crown marginal adaption. Dent Clin North Amer 15:663, July 1971. 285. Klein, A. ~·; and MacPherson, D.t Television measurement pulse. generator-mixer. Med Electron Biol Engin 5:267, 1967~ 286. Klein, A. I., and MacPherson, D.: "Intra-oral television micro measurement instrumentation. Med Electron Biol Engin 5:259, 1967. 287. Klein, A. I.: Clinical television reasearch instrumentation. J Am Dent Assoc 74:1210, 1967. 288. Edwards, A. L.: Experimental Design in Ps.ychological Research . New York, H?lt, Rinehart, and Winston, Inc., 1968, PPo 390-394. CURRICULUM VITAE Patrick A. Fleege August 9, 1944 Born in Hazel Green, Wisconsin, to Herbert and Elizabeth Fleege 1966 1970 B. S. cum laude - Seattle University Seattle, Washington Do D. S. with honor - University of Washington School of Dentistry Seattle, Washington 1970 - 1972 Captain in the United States Army Dental Corps - Fort Carson, Colorado 1972 - 1974 Graduate pedodontic student, Indiana University School of Dentistr.y, Indianapolis, Indiana Professional Societies American Dental Association (Student Member) American Society of Dentistr.y for Children (Student Member) American Academy of Pedodontics (Student Member) American Society of Dental flnesthesiology Associate Member Chicago Dental Society Board Eligible American Board of Pedodontics Honor Societies Alpha Epsilon Delta Omicron Kappa Upsilon ABSTRACT AN EVALUATION OF TETRACYCLINE STAIN REUOV AL BY BLEACHING VTIAL RABBIT INCISORS Patrick A. Fleege 1157 22nd Avenue East Seattle, Washington This stuqy evaluated the effectiveness of bleaching tetracycline- stained teeth by measuring the loss of fluorescent intensity from teeth that were bleached. Nineteen male New Zealand white rabbits, with 58 incisors stained with oxytetracycline and 16 incisors as unstained controls, were usedo Three rab- bits were sacrificed to determine whether the tetracycline stain was comparable between incisors in the same jawo Of the remaining 16 animals, 6 were bleach- ed once and 10 were bleached twice. One maxillar,y and one mandibular incisor were bleached in each jaw with 30 percent hydrogen peroxide and heat for ten minutes per tooth; the other incisors were protected with a rubber damo The animals were sacrificed 24 hours after the last bleach. The fluorescent in- , tensity of 374 select -ground sections 100 i 5 microns thick from the incisa~, middle and gingival thirds of the teeth were measured with an ultraviolet light microscope coupled to a television electronic measurement systemo These measurements were statistically analyzed b.Y t-test, and observations correlated~ The dentin of tetracycline-stained maxillary incisors which were bleached twice and the dentin in the incisal one-third of the mandibular incisors which were bleached twice had a significantly (P ~ 0.001; P~o005) lower -tetracycline : fluorescent intensity than the dentin of unbleached tetracycline-stained teetho The greatest loss of fluorescent intensity of tetracycline occurred in dentin closest to the dentino-enamel junction and varied from about 150 to 350 microns from the outer enamel surface. Clinical Kodachromes indicate that the loss of tetracycline pigment is associated with the loss of tetracycline fluorescence; The ground sections showed that the tetracycline fluorescence was never total~ removed by two bleaches.