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Item Advanced biomaterials for periodontal tissue regeneration(Wiley, 2022) Daghrery, Arwa; Bottino, Marco C.; Biomedical and Applied Sciences, School of DentistryThe periodontium is a suitable target for regenerative intervention, since it does not functionally restore itself after disease. Importantly, the limited regeneration capacity of the periodontium could be improved with the development of novel biomaterials and therapeutic strategies. Of note, the regenerative potential of the periodontium depends not only on its tissue‐specific architecture and function, but also on its ability to reconstruct distinct tissues and tissue interfaces, suggesting that the advancement of tissue engineering approaches can ultimately offer new perspectives to promote the organized reconstruction of soft and hard periodontal tissues. Here, we discuss material‐based, biologically active cues, and the application of innovative biofabrication technologies to regenerate the multiple tissues that comprise the periodontium.Item Contribution of Porphyromonas gingivalis lipopolysaccharide to experimental periodontitis in relation to aging(Springer, 2021) Akkaoui, Juliet; Yamada, Chiaki; Duarte, Carolina; Ho, Anny; Vardar-Sengul, Saynur; Kawai, Toshihisa; Movila, Alexandru; Biomedical and Applied Sciences, School of DentistryAging is associated with increased prevalence and severity of pathogenic outcomes of periodontal disease, including soft tissue degeneration and bone loss around the teeth. Although lipopolysaccharide (LPS) derived from the key periodontal pathogen Porphyromonas gingivalis (Pg) plays an important role in the promotion of inflammation and osteoclastogenesis via toll-like receptor (TLR)4 signaling, its pathophysiological role in age-associated periodontitis remains unclear. This study investigated the possible effects of Pg-LPS on RANKL-primed osteoclastogenesis and ligature-induced periodontitis in relation to aging using young (2 months old) and aged (24 months old) mice. To the best of our knowledge, our results indicated that expression of TLR4 was significantly diminished on the surface of osteoclast precursors isolated from aged mice compared with that of young mice. Furthermore, our data demonstrated that the TLR4 antagonist (TAK242) dramatically decreased the numbers of tartrate-resistant acid phosphatase positive (TRAP+) osteoclasts differentiated from RANKL-primed young osteoclast precursors (OCPs) compared with those isolated from aged mice in response to Pg-LPS. In addition, using a ligature-induced periodontitis mouse model, we demonstrated that Pg-LPS elevated (1) secretion of senescence-associated secretory phenotype (SASP) markers, including the pro-inflammatory cytokines TNF-α, IL-6, and IL-1β, as well as osteoclastogenic RANKL, and (2) the number of OCPs and TRAP+ osteoclasts in the periodontal lesion induced in young mice. In contrast, Pg-LPS had little, or no, effect on the promotion of periodontitis inflammation induced in aged mice. Altogether, these results indicated that periodontal disease in older mice occurs in a manner independent of canonical signaling elicited by the Pg-LPS/TLR4 axis.Item Self-assembling peptide-laden electrospun scaffolds for guided mineralized tissue regeneration(Elsevier, 2022) de Souza Araújo, Isaac J.; Ferreira, Jessica A.; Daghrery, Arwa; Ribeiro, Juliana S.; Castilho, Miguel; Puppin-Rontani, Regina M.; Bottino, Marco C.; Biomedical and Applied Sciences, School of DentistryObjectives: Electrospun scaffolds are a versatile biomaterial platform to mimic fibrillar structure of native tissues extracellular matrix, and facilitate the incorporation of biomolecules for regenerative therapies. Self-assembling peptide P11-4 has emerged as a promising strategy to induce mineralization; however, P11-4 application has been mostly addressed for early caries lesions repair on dental enamel. Here, to investigate P11-4's efficacy on bone regeneration, polymeric electrospun scaffolds were developed, and then distinct concentrations of P11-4 were physically adsorbed on the scaffolds. Methods: P11-4-laden and pristine (P11-4-free) electrospun scaffolds were immersed in simulated body fluid and mineral precipitation identified by SEM. Functional groups and crystalline phases were analyzed by FTIR and XRD, respectively. Cytocompatibility, mineralization, and gene expression assays were conducted using stem cells from human exfoliated deciduous teeth. To investigate P11-4-laden scaffolds potential to induce in vivo mineralization, an established rat calvaria critical-size defect model was used. Results: We successfully synthesized nanofibrous (∼ 500 nm fiber diameter) scaffolds and observed that functionalization with P11-4 did not affect the fibers' diameter. SEM images indicated mineral precipitation, while FTIR and XRD confirmed apatite-like formation and crystallization for P11-4-laden scaffolds. In addition, P11-4-laden scaffolds were cytocompatible, highly stimulated cell-mediated mineral deposition, and upregulated the expression of mineralization-related genes compared to pristine scaffolds. P11-4-laden scaffolds led to enhanced in vivo bone regeneration after 8 weeks compared to pristine PCL. Significance: Electrospun scaffolds functionalized with P11-4 are a promising strategy for inducing mineralized tissues regeneration in the craniomaxillofacial complex.Item Photocrosslinkable methacrylated gelatin hydrogel as a cell-friendly injectable delivery system for chlorhexidine in regenerative endodontics(Elsevier, 2022) Ribeiro, Juliana S.; Sanz, Carolina K.; Münchow, Eliseu A.; Kalra, Nikhil; Dubey, Nileshkumar; Suárez, Carlos Enrique C.; Fenno, J. Christopher; Lund, Rafael G.; Bottino, Marco C.; Biomedical and Applied Sciences, School of DentistryObjectives: This work sought to formulate photocrosslinkable chlorhexidine (CHX)-laden methacrylated gelatin (CHX/GelMA) hydrogels with broad spectrum of action against endodontic pathogens as a clinically viable cell-friendly disinfection therapy prior to regenerative endodontics procedures. Methods: CHX/GelMA hydrogel formulations were successfully synthesized using CHX concentrations between 0.12 % and 5 % w/v. Hydrogel microstructure was evaluated by scanning electron microscopy (SEM). Swelling and enzymatic degradation were assessed to determine microenvironmental effects. Compression test was performed to investigate the influence of CHX incorporation on the hydrogels' biomechanics. The antimicrobial and anti-biofilm potential of the formulated hydrogels were assessed using agar diffusion assays and a microcosms biofilm model, respectively. The cytocompatibility was evaluated by exposing stem cells from human exfoliated deciduous teeth (SHEDs) to hydrogel extracts (i.e., leachable byproducts obtained from overtime hydrogel incubation in phosphate buffer saline). The data were analyzed using One- and Two-way ANOVA and Tukey's test (α = 0.05). Results: CHX/GelMA hydrogels were effectively prepared. NMR spectroscopy confirmed the incorporation of CHX into GelMA. The addition of CHX did not change the micromorphology (pore size) nor the swelling profile (p > 0.05). CHX incorporation reduced the degradation rate of the hydrogels (p < 0.001); whereas, it contributed to increased compressive modulus (p < 0.05). Regarding the antimicrobial properties, the incorporation of CHX showed a statistically significant decrease in the number of bacteria colonies at 0.12 % and 0.5 % concentration (p < 0.001) and completely inhibited the growth of biofilm at concentration levels 1 %, 2 %, and 5 %. Meanwhile, the addition of CHX, regardless of the concentration, did not lead to cell toxicity, as cell viability values were above 70 %. Significance: The addition of CHX into GelMA showed significant antimicrobial action against the pathogens tested, even at low concentrations, with the potential to be used as a cell-friendly injectable drug delivery system for root canal disinfection prior to regenerative endodontics.Item Three-Dimensional Printing of Clinical Scale and Personalized Calcium Phosphate Scaffolds for Alveolar Bone Reconstruction(Elsevier, 2022) Anderson, Margaret; Dubey, Nileshkumar; Bogie, Kath; Cao, Chen; Li, Junying; Lerchbacker, Joseph; Mendonça, Gustavo; Kauffman, Frederic; Bottino, Marco C.; Kaigler, Darnell; Biomedical and Applied Sciences, School of DentistryObjective: Alveolar bone defects can be highly variable in their morphology and, as the defect size increases, they become more challenging to treat with currently available therapeutics and biomaterials. This investigation sought to devise a protocol for fabricating customized clinical scale and patient-specific, bioceramic scaffolds for reconstruction of large alveolar bone defects. Methods: Two types of calcium phosphate (CaP)-based bioceramic scaffolds (alginate/β-TCP and hydroxyapatite/α-TCP, hereafter referred to as hybrid CaP and Osteoink™, respectively) were designed, 3D printed, and their biocompatibility with alveolar bone marrow stem cells and mechanical properties were determined. Following scaffold optimization, a workflow was developed to use cone beam computed tomographic (CBCT) imaging to design and 3D print, defect-specific bioceramic scaffolds for clinical-scale bone defects. Results: Osteoink™ scaffolds had the highest compressive strength when compared to hybrid CaP with different infill orientation. In cell culture medium, hybrid CaP degradation resulted in decreased pH (6.3) and toxicity to stem cells; however, OsteoInk™ scaffolds maintained a stable pH (7.2) in culture and passed the ISO standard for cytotoxicity. Finally, a clinically feasible laboratory workflow was developed and evaluated using CBCT imaging to engineer customized and defect-specific CaP scaffolds using OsteoInk™. It was determined that printed scaffolds had a high degree of accuracy to fit the respective clinical defects for which they were designed (0.27 mm morphological deviation of printed scaffolds from digital design). Significance: From patient to patient, large alveolar bone defects are difficult to treat due to high variability in their complex morphologies and architecture. Our findings shows that Osteoink™ is a biocompatible material for 3D printing of clinically acceptable, patient-specific scaffolds with precision-fit for use in alveolar bone reconstructive procedures. Collectively, emerging digital technologies including CBCT imaging, 3D surgical planning, and (bio)printing can be integrated to address this unmet clinical challenge.Item Silica infiltration on translucent zirconia restorations: effects on the antagonist wear and survivability(Elsevier, 2022) Martins Alves, Larissa Marcia; da Silva Rodrigues, Camila; de Carvalho Ramos, Nathalia; Buizastrow, Jeff; Bastos Campos, Tiago Moreira; Bottino, Marco Antonio; Zhang, Yu; de Melo, Renata Marques; Biomedical and Applied Sciences, School of DentistryObjective: To assess potential antagonist wear and survival probability of silica-infiltrated zirconia compared to glass-graded, glazed, and polished zirconia. Methods: Table top restorations made of 3Y-TZP (3Y), 5Y-PSZ (5Y), and lithium disilicate (LD) were bonded onto epoxy resin preparations. Each zirconia was divided into five groups according to the surface treatment: polishing; glaze; polishing-glaze; glass infiltration; and silica infiltration. The LD restorations received a glaze layer. Specimens were subjected to sliding fatigue wear using a steatite antagonist (1.25 ×106 cycles, 200 N). The presence of cracks, fractures, and/or debonding was checked every one/third of the total number of cycles was completed. Roughness, microstructural, Scanning electron microscopy, wear and residual stress analyses were conducted. Kaplan-Meier, Mantel-Cox (log-rank) and ANOVA tests were performed for statistical analyses. Results: The survival probability was different among the groups. Silica infiltration and polishing-glaze led to lower volume loss than glaze and glass-infiltration. Difference was observed for roughness among the zirconia and surface treatment, while lithium disilicate presented similar roughness compared to both glazed zirconia. Scanning electron microscopy revealed the removal of the surface treatment after sliding fatigue wear in all groups. Compressive stress was detected on 3Y surfaces, while tensile stress was observed on 5Y. Significance: 3Y and 5Y zirconia behaved similarly regarding antagonist wear, presenting higher antagonist wear than the glass ceramic. Silica-infiltrated and polished-glazed zirconia produced lower antagonist volume loss than glazed and glass-infiltrated zirconia. Silica-infiltrated 3Y and lithium disilicate restorations were the only groups to show survival probabilities lower than 85%.Item Platform Technologies for Regenerative Endodontics from Multifunctional Biomaterials to Tooth-on-a-Chip Strategies(Springer, 2021) Soares, Diana G.; Bordini, Ester A. F.; Swanson, W. Benton; de Souza Costa, Carlos A.; Bottino, Marco C.; Biomedical and Applied Sciences, School of DentistryObjectives: The aim of this review is to highlight recent progress in the field of biomaterials-mediated dental pulp tissue engineering. Specifically, we aim to underscore the critical design criteria of biomaterial platforms that are advantageous for pulp tissue engineering, discuss models for preclinical evaluation, and present new and innovative multifunctional strategies that hold promise for clinical translation. Materials and methods: The current article is a comprehensive overview of recent progress over the last 5 years. In detail, we surveyed the literature in regenerative pulp biology, including novel biologic and biomaterials approaches, and those that combined multiple strategies, towards more clinically relevant models. PubMed searches were performed using the keywords: "regenerative dentistry," "dental pulp regeneration," "regenerative endodontics," and "dental pulp therapy." Results: Significant contributions to the field of regenerative dentistry have been made in the last 5 years, as evidenced by a significant body of publications. We chose exemplary studies that we believe are progressive towards clinically translatable solutions. We close this review with an outlook towards the future of pulp regeneration strategies and their clinical translation. Conclusions: Current clinical treatments lack functional and predictable pulp regeneration and are more focused on the treatment of the consequences of pulp exposure, rather than the restoration of healthy dental pulp. Clinical relevance: Clinically, there is great demand for bioinspired biomaterial strategies that are safe, efficacious, and easy to use, and clinicians are eager for their clinical translation. In particular, we place emphasis on strategies that combine favorable angiogenesis, mineralization, and functional tissue formation, while limiting immune reaction, risk of microbial infection, and pulp necrosis.Item Nicotine is a potent extracellular polysaccharide inducer in Fusobacterium nucleatum biofilms(SciELO, 2023) Matos, Adaias Oliveira; Barão, Valentim Adelino Ricardo; Gregory, Richard Lee; Biomedical and Applied Sciences, School of DentistryAim: The purpose of this in vitro study was to analyze the influence of nicotine on the extracellular polysaccharides in Fusobacterium nucleatum biofilm. Methods: F. nucleatum (ATCC 10953) biofilms supplemented with different concentrations of nicotine (0, 0.5, 1, 2, 4, and 8 mg/mL) were grown in two different BHI broth conditions [no sucrose and 1% sucrose]. Extracellular polysaccharides assay, pH measurements, and a spectrophotometric assay were performed. Data were submitted for ANOVA and Tukey honestly significant difference analyses (HSD) tests (α =.05). Results: Extracellular polysaccharides synthesis was influenced by an interaction between nicotine concentrations and growth medium solution containing sucrose (P<.05). The pH values declined in the sucrose-exposed biofilm were greater than in the group exposed only to nicotine (P<.05). The biofilm exposed to sucrose and nicotine had a higher total biofilm growth (P<.05) than the nicotine-treated biofilm without sucrose. Conclusions: Regardless of sucrose exposure, biofilms exposed to different nicotine concentrations influenced the amount of extracellular polysaccharides.Item Effect of phenylmethylsulfonyl fluoride, a protease inhibitor, on enamel surface remineralization(SciELO, 2023) Peres, Paulo Edelvar Corrêa; Fu, Jean; Zero, Domenick T.; Cury, Jaime Aparecido; Biomedical and Applied Sciences, School of DentistryPhenylmethylsulfonyl fluoride (PMSF) is a protease inhibitor widely used in research, but fluoride is released during its action and this knowledge has been neglected in dental research. Aim to evaluate if fluoride released by salivary protease action on PMSF affects enamel remineralization and fluoride uptake. Methods Groups of 10 enamel slabs, with caries-like lesions and known surface hardness (SH), were subjected to one of the following treatment groups: Stimulated human saliva (SHS), negative control; SHS containing 1.0 μg F/mL (NaF), positive control; and SHS containing 10, 50 or 100 µM PMSF. The slabs were subjected to a pH-cycling regimen consisting of 22 h/day in each treatment solution and 2 h/day in a demineralizing solution. After 12 days, SH was again measured to calculate the percentage of surface hardness recovery (%SHR), followed by enamel fluoride uptake determination. The time-related fluoride release from 100.0 µM PMSF by SHS action was also determined. Data were analyzed by ANOVA followed by Newman-Keuls test. Results The release of fluoride from PMSF by SHS was rapid, reaching a maximum value after 10 min. Fluoride released from PMSF was more effective in enhancing %SHR and increasing fluoride uptake in enamel compared with SHS alone (p < 0.05); furthermore, it was equivalent to the positive control (p > 0.05). Conclusion In conclusion, fluoride released by saliva from PMSF is available to react with enamel and needs to be taken into account in research using this protease inhibitor.Item Glycyrrhizin mitigates inflammatory bone loss and promotes expression of senescence-protective sirtuins in an aging mouse model of periprosthetic osteolysis(Elsevier, 2021) Yamada, Chiaki; Ho, Anny; Akkaoui, Juliet; Garcia, Christopher; Duarte, Carolina; Movila, Alexandru; Biomedical and Applied Sciences, School of DentistryAlthough periprosthetic osteolysis induced by wear debris particles is significantly elevated in senior (65+ years old) patients, most of the published pre-clinical studies were performed using young (less than three-month old) mice indicating the critical need to employ experimental models of particle-induced osteolysis involving mice with advanced age. Emerging evidence indicates that currently available antiresorptive bone therapies have serious age-dependent side effects. However, a resurgence of healthcare interest has occurred in glycyrrhizin (GLY), a natural extract from the licorice roots, as alternative sources of drugs for treating inflammatory bone lytic diseases and prevention of cellular senescence. This study investigated the effects of GLY on inflammatory bone loss as well as expression patterns of senescence-associated secretory phenotype and senescence-protective markers using an experimental calvarium osteolytic model induced in aged (twenty-four-month-old) mice by polymethylmethacrylate (PMMA) particles. Our results indicate that local treatment with GLY significantly diminished the size of inflammatory osteolytic lesions in aged mice via the number of CXCR4+OCPs and Tartrate-resistant acid phosphatase positive (TRAP+) osteoclasts. Furthermore, GLY dramatically decreased the amounts of senescence-associated secretory phenotype markers, including pro-inflammatory macrophage migration inhibitory factor (MIF) chemokine, and cathepsins B and K in the bone lesions of aged mice. By contrast, GLY significantly elevated expression patterns of senescence-protective markers, including homeostatic stromal derived factor-1 (SDF-1) chemokine, and sirtuin-1, and sirtuin-6, in the PMMA particle-induced calvarial lesions of aged mice. Collectively, these data suggest that GLY can be used for the development of novel therapies to control bone loss and tissue aging in senior patients with periprosthetic osteolysis.