Browsing by Author "Holguin, Nilsson"

Now showing 1 - 7 of 7
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    Aging aggravates intervertebral disc degeneration by regulating transcription factors toward chondrogenesis
    (Wiley, 2020-02) Silva, Matthew J.; Holguin, Nilsson; Anatomy and Cell Biology, School of Medicine
    Osterix is a critical transcription factor of mesenchymal stem cell fate, where its loss or loss of Wnt signaling diverts differentiation to a chondrocytic lineage. Intervertebral disc (IVD) degeneration activates the differentiation of prehypertrophic chondrocyte-like cells and inactivates Wnt signaling, but its interactive role with osterix is unclear. First, compared to young-adult (5 mo), mechanical compression of old (18 mo) IVD induced greater IVD degeneration. Aging (5 vs 12 mo) and/or compression reduced the transcription of osterix and notochordal marker T by 40-75%. Compression elevated the transcription of hypertrophic chondrocyte marker MMP13 and pre-osterix transcription factor RUNX2, but less so in 12 mo IVD. Next, using an Ai9/td reporter and immunohistochemical staining, annulus fibrosus and nucleus pulposus cells of young-adult IVD expressed osterix, but aging and compression reduced its expression. Lastly, in vivo LRP5-deficiency in osterix-expressing cells inactivated Wnt signaling in the nucleus pulposus by 95%, degenerated the IVD to levels similar to aging and compression, reduced the biomechanical properties by 45-70%, and reduced the transcription of osterix, notochordal markers and chondrocytic markers by 60-80%. Overall, these data indicate that age-related inactivation of Wnt signaling in osterix-expressing cells may limit regeneration by depleting the progenitors and attenuating the expansion of chondrocyte-like cells.
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    Factors influencing cartilage wear in an accelerated in vitro test: collagen fiber orientation, anatomic location, cartilage composition, and photo-chemical crosslinking
    (2018) Hossain, M. Jayed; Wagner, Diane; Jones, Alan; Holguin, Nilsson
    Articular cartilage (AC) is a strong but flexible connective tissue that covers and protects the end of the long bones. Although cartilage has excellent friction and wear properties that allow smooth joint function during daily activities, these properties are not fully understood. Many material properties of cartilage are anisotropic and vary with anatomic location and the composition of the tissue, but whether this is also true for cartilage friction and wear has not been previously determined. Furthermore, cartilage disease and injury are major health concerns that affect millions of people, but there are few available treatments to prevent the progression of cartilage degeneration. Collagen crosslinking may be a potential treatment to reduce cartilage wear and slow or prevent the progression of cartilage disease. The objectives of this thesis were to investigate the relationships between the friction/wear characteristics of cartilage and the orientation of the preferred fiber direction, the anatomic location of the tissue, the composition of the tissue, and exogenous photochemical crosslinking. In the superficial zone, AC has preferential fiber direction which leads to anisotropic material behavior. Therefore, we hypothesized that AC will show anisotropic behavior between longitudinal and transverse direction in an accelerated, in vitro wear test on bovine cartilage in terms of friction and wear. This hypothesis was proven by the quantification of glycosaminoglycans released from the tissue during the wear test, which showed that more glycosaminoglycans were released when the wear direction was transverse to the direction of the fibers. However, the hydroxyproline released from the tissue during the wear test was not significantly different between the two directions, nor was the coefficient of friction. The material properties of AC can also vary with anatomic location, perhaps due to differences in how the tissue is loaded in vivo. We hypothesized that cartilage from a higher load bearing site will give better wear resistance than cartilage from lower load bearing regions. However, no differences in friction or wear were observed between the different anatomic locations on the bovine femoral condyles. The concentration of collagen, glycosaminoglycans, cells and water in the tissue was also quantified, but no significant differences in tissue composition were found among the locations that were tested. Although wear did not vary with anatomic location, variation in the wear measurements were relatively high. One potential source of variation is the composition of the cartilage. To determine whether cartilage composition influences friction and wear, a correlation analysis was conducted. An accelerated, in vitro wear test was conducted on cartilage from bovine femoral condyles, and the tissue adjacent to the wear test specimens was analyzed for collagen, glycosaminoglycan, cell, and water content. Because wear occurs on the cartilage surface, the superficial zone of the cartilage might play an important role in wear test. Therefore, composition of the adjacent cartilage was determined in both the superficial zone and the full thickness of the tissue. A significant negative correlation was found between wear and collagen content in the full thickness of the tissue, and between the initial coefficient of friction and the collagen content in the superficial zone. This correlation suggests that variation in the collagen content in the full thickness of the cartilage partially explains differences in amount of wear between specimens. The wear resistance of cartilage can be improved with exogenous crosslinking agents, but the use of photochemical crosslinking to improve wear resistance is not well understood. Two photochemical crosslinking protocols were analyzed to improve the wear resistance of the cartilage by using chloro-aluminum phthalocyanine tetrasulfonic acid (CASPc) and 670nm laser light. The cartilage treated with the two crosslinking protocols had lower wear than the non-treated group without changing the friction properties of the cartilage.
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    Global Deletion of Sost Increases Intervertebral Disc Hydration But May Trigger Chondrogenesis
    (2020-05) Kroon, Tori; Holguin, Nilsson; Wallace, Joseph; Wagner, Diane
    Intervertebral discs (IVD) degenerate earlier than many other musculoskeletal tissues and will continue to degenerate with aging. IVD degeneration affects up to 80 percent of the adult population and is a major contributing factor to low back pain. Anti-sclerostin antibody is an FDA-approved treatment for osteoporosis in postmenopausal women at high-risk for fracture and, as a systemic stimulant of the Wnt/LRP5/β-Catenin signaling pathway, may impact the IVD. Stabilization of β-Catenin in the IVD increases Wnt signaling and is anabolic to the extracellular matrix (ECM), while deletion of β-catenin or LRP5 decreases Wnt signaling and is catabolic to the ECM. Here, we hypothesized that a reduction of Sost would stimulate ECM anabolism. Lumbar and caudal (tail) IVD and vertebrae of Sost KO and WT (wildtype) mice (n=8 each) were harvested at 16 weeks of age and tested by MRI, histology, immunohistochemistry, Western Blot, qPCR, and microCT. Compared to WT, Sost KO reduced sclerostin protein and Sost gene expression. Next, Sost KO increased the hydration of the IVD and the proteoglycan stain in the nucleus pulposus and decreased the expression of genes associated with IVD degeneration, e.g., heat shock proteins. However, deletion of Sost was compensated by less unphosphorylated (active) β-Catenin protein in the cell nucleus, upregulation of Wnt signaling inhibitors Dkk1 and sFRP4, and catabolic ECM gene expression. Consequently, notochordal and early chondrocyte-like cells (CLCs) were replaced by mature CLCs. Overall, Sost deletion increased hydration and proteoglycan protein content, but activated a compensatory suppression of Wnt signaling that may trigger chondrogenesis and may potentially be iatrogenic to the IVD in the long-term.
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    In-Vivo Nucleus Pulposus-Specific Regulation of Adult Murine Intervertebral Disc Degeneration via Wnt/Beta-Catenin Signaling
    (Nature Publishing Group, 2018-07-25) Holguin, Nilsson; Silva, Matthew J.; Mechanical and Energy Engineering, School of Engineering and Technology
    B-Catenin, transcription factor of Wnt signaling, is promoted in patients with intervertebral disc (IVD) degeneration, but Wnt signaling decreases with aging. We hypothesize that IVD degeneration is associated with decreased Wnt signaling despite more b-Catenin. Chronic compression of tail IVDs of young-adult and aged Wnt-reporter (TOPGAL) animals initiated an age-related cascade of degenerative-like changes, which included reduced Wnt ligand expression and Wnt signaling in nucleus pulposus cells, despite elevation of b-Catenin protein and gene expression. To determine the effect of upregulated and downregulated Wnt signaling in adult discs, b-Catenin in the nucleus pulposus was stabilized (Shh-CreErT2/b-Cateninfl(Ex3)/fl(Ex3), cACT) or knocked out (Shh-CreErT2/b-Cateninfl/fl, cKO). cACT discs had promoted expression of Wnt-targets and -ligands, brachyury, extracellular matrix production and 34% greater compressive stiffness than WT (b-Cateninfl(Ex3)/fl(Ex3)) discs, but 50% less tensile stiffness. By contrast, knockout reversed the cACT phenotype: less protein expression of b-catenin in the nucleus pulposus, less expression of brachyury, heightened expression of extracellular matrix breakdown and 46% less compressive stiffness than wild-type (b-Cateninfl/fl,WT) discs. These data suggest that intervertebral disc degeneration is associated with loss of Wnt signaling and that the concomitant increase in b-catenin is a regenerative response, potentially offering a therapeutic approach to degeneration.
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    The Quantification of Force Distribution of a Vibrational Device for Accelerating Tooth Movement
    (2019-08) Akbari, Amin; Chen, Jie; Wagner, Diane; Holguin, Nilsson
    One of the most common concern among patients who need orthodontic treatment is treatment duration. The ability to accelerate orthodontic tooth movements would be bene cial to reduce the undesired side-effects of prolonged treatment. Methods have been used in conjugate with common orthodontic appliances to shorten the treatment. One of them is to use vibrational force (VF), which is non-invasive. The VF stimulates bone modeling and remodeling, which is essential to tooth movement. However, commercial devices used in the clinic failed to deliver consistent outcomes. The effects of the VF highly depend on its intensity the tooth receives. There must be a range of stimulation that optimizes the ffeects. The stimulation outside the range either have no effects or creates damages, which adversely affects the orthodontic treatment. Since these devices have generic mouthpiece and teeth are in di erent heights, hence some teeth cannot get force stimulation and others may be overloaded. The current designs also do not have ability to adjust the level of VF intensity that individual tooth needs, as in some cases orthodontists are required to move a tooth faster than others or even slower, which needs the device to be personalized. There- fore, the primary cause of inconsistent clinical outcomes is the inadequate design of the mouthpiece of the current device. The goal of this study is to design a better vibratory device that not only guarantees VF delivery but also enables orthodontists to control the level of VF on the individual tooth, which meets the patient's treat- ment needs. This is a preliminary study to understand the effects of different design parameters affecting the VF distribution on teeth. A nite element model, which consists of human upper and lower jaws in their occlusal positions and a mouthpiece, was created. The VF was from a vibratory source with a peak load of 0.3N and speci ed frequencies (30 and 120 Hz). The element size was determined through a convergence test and the model was validated experimentally. Results showed that the VF distribution among the teeth relies on the material property of the mouthpiece. The distribution is uneven, meaning some teeth bearing much more load than others. This means, with the current device design, teeth would be a ected with di erent level of force stimulation, which results in di erent clinical outcomes consequently. Dynamic load (VF) changes the force distribution on the teeth comparing to the dis- tribution from a static load. Frequency does not affect the peak load. Finally, the study demonstrated that the level of VF stimulation can be adjusted by introducing clearance or interference between the teeth and mouthpiece. It is feasible to control the level of the VF intensity for individual tooth based on treatment requirement.
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    Raloxifene Stimulates Estrogen Signaling to Protect Against Age- and Sex-Related Intervertebral Disc Degeneration in Mice
    (Frontiers Media, 2022-08-11) Bhadouria, Neharika; Berman, Alycia G.; Wallace, Joseph M.; Holguin, Nilsson; Mechanical and Energy Engineering, Purdue School of Engineering and Technology
    Estrogen agonist raloxifene is an FDA-approved treatment of osteoporosis in postmenopausal women, which may also be a promising prophylactic for painful intervertebral disc (IVD) degeneration. Here, we hypothesized that 1) aging and biological sex contribute to IVD degeneration by reducing estrogen signaling and that 2) raloxifene stimulates estrogen signaling to protect against age- and sex-related IVD degeneration in mice. 2.5-month-old (male and female) and 22.5-month-old (female) C57Bl/6J mice were subcutaneously injected with raloxifene hydrochloride 5x/week for 6 weeks (n = 7–9/grp). Next, female mice were ovariectomized (OVX) or sham operated at 4 months of age and tissues harvested at 6 months (n = 5–6/grp). Advanced aging and OVX increased IVD degeneration score, weakened IVD strength, reduced estrogen receptor-α (ER-α) protein expression, and increased neurotransmitter substance P (SP) expression. Similar to aging and compared with male IVDs, female IVDs were more degenerated, mechanically less viscoelastic, and expressed less ER-α protein, but unlike the effect induced by aging or OVX, IVD mechanical force was greater in females than in males. Therapeutically, systemic injection of raloxifene promoted ER-α protein to quell these dysregulations by enlarging IVD height, alleviating IVD degeneration score, increasing the strength and viscoelastic properties of the IVD, and reducing IVD cell expression of SP in young-adult and old female mice. Transcriptionally, injection of raloxifene upregulated the gene expression of ER-α and extracellular matrix-related anabolism in young-adult and old IVD. In vertebra, advanced aging and OVX reduced trabecular BV/TV, whereas injection of raloxifene increased trabecular BV/TV in young-adult and old female mice, but not in young-adult male mice. In vertebra, advanced aging, OVX, and biological sex (females > males) increased the number of SP-expressing osteocytes, whereas injection of raloxifene reduced the number of SP-expressing osteocytes in young-adult female and male mice and old female mice. Overall, injection of estrogen agonist raloxifene in mice normalized dysregulation of IVD structure, IVD mechanics, and pain-related SP expression in IVD cells and osteocytes induced by aging and biological sex. These data suggest that, in addition to bone loss, raloxifene may relieve painful IVD degeneration in postmenopausal women induced by advanced age, biological sex, and estrogen depletion.
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    Suppression of sost/sclerostin and dikkopf-1 augment intervertebral disc structure in mice
    (Wiley, 2022) Kroon, Tori; Bhadouria, Neharika; Niziolek, Paul; Edwards, Daniel; Clinkenbeard, Erica L.; Robling, Alexander; Holguin, Nilsson; Biomedical Engineering, School of Engineering and Technology
    Intervertebral disc (IVD) degeneration is a leading cause of low back pain, characterized by accelerated extracellular matrix breakdown and IVD height loss but there is no approved pharmacological therapeutic. Deletion of Wnt ligand competitor Lrp5 induces IVD degeneration, suggesting that Wnt signaling is essential for IVD homeostasis. Therefore, the IVD may respond to neutralization of Wnt ligand competitors sost(gene)/sclerostin(protein) and/or dikkopf-1 (dkk1). Anti-sclerostin antibody (scl-Ab) is an FDA-approved bone therapeutic that activates Wnt signaling. We (1) determined if pharmacological neutralization of sclerostin, dkk1 or their combination would stimulate Wnt signaling and augment IVD structure and (2) determined the prolonged adaptation of the IVD to global, persistent deletion of sost. Nine-week-old C57Bl/6J female mice (n = 6-7/grp) were subcutaneously injected 2x/wk for 5.5 wk with scl-Ab (25 mg/kg), dkk1-Ab (25 mg/kg), 3:1 scl-Ab/dkk1-Ab (18.75:6.25 mg/kg) or vehicle (veh). Separately, IVD of sost KO and wildtype (WT) mice (n = 8/grp) were harvested at 16 weeks of age. First, compared to vehicle, injection of scl-Ab, dkk1-Ab and 3:1 scl-Ab/dkk1-Ab similarly increased lumbar IVD height and β-catenin gene expression. Despite these similarities, only injection of scl-Ab alone strengthened IVD mechanical properties and decreased heat shock protein gene expressions. Genetically and compared to WT, sost KO enlarged IVD height, increased proteoglycan staining and imbibed IVD hydration. Notably, persistent deletion of sost was compensated by upregulation of dkk1, which consequently reduced the cell nuclear fraction for Wnt signaling co-transcription factor β-catenin in the IVD. Lastly, RNA-sequencing pathway analysis confirmed the compensatory suppression of Wnt signaling and revealed a reduction of cellular stress-related pathways. Together, suppression of sost/sclerostin or dkk1 each augmented IVD structure by stimulating Wnt signaling, but scl-Ab outperformed dkk1-Ab in strengthening the IVD. Ultimately, postmenopausal women prescribed scl-Ab injections to prevent vertebral fracture may also benefit from a restoration of IVD height and health.