Design of Alginate/Gelatin Hydrogels for Biomedical Applications: Fine-Tuning Osteogenesis in Dental Pulp Stem Cells While Preserving Other Cell Behaviors.

Alginate/gelatin (Alg-Gel) hydrogels have been used experimentally, associated with mesenchymal stromal/stem cells (MSCs), to guide bone tissue formation. One of the main challenges for clinical application is optimizing Alg-Gel stiffness to guide osteogenesis. In this study, we investigated how Alg-Gel stiffness could modulate the dental pulp stem cell (DPSC) attachment, morphology, proliferation, and osteogenic differentiation, identifying the optimal conditions to uncouple osteogenesis from the other cell behaviors. An array of Alg-Gel hydrogels was prepared by casting different percentages of alginate and gelatin cross-linked with 2% CaCl2. We have selected two hydrogels: one with a stiffness of 11 ± 1 kPa, referred to as "low-stiffness hydrogel", formed by 2% alginate and 8% gelatin, and the other with a stiffness of 55 ± 3 kPa, referred to as "high-stiffness hydrogel", formed by 8% alginate and 12% gelatin. Hydrogel analyses showed that the average swelling rates were 20 ± 3% for the low-stiffness hydrogels and 35 ± 2% for the high-stiffness hydrogels. The degradation percentage was 47 ± 5% and 18 ± 2% for the low- and high-stiffness hydrogels, respectively. Both hydrogel types showed homogeneous surface shape and protein (Alg-Gel) interaction with CaCl2 as assessed by physicochemical characterization. Cell culture showed good adhesion of the DPSCs to the hydrogels and proliferation. Furthermore, better osteogenic activity, determined by ALP activity and ARS staining, was obtained with high-stiffness hydrogels (8% alginate and 12% gelatin). In summary, this study confirms the possibility of characterizing and optimizing the stiffness of Alg-Gel gel to guide osteogenesis in vitro without altering the other cellular properties of DPSCs.

Barriers and Enablers to Implementing Teledentistry From the Perspective of Dental Health Care Professionals: Protocol for a Systematic Quantitative, Qualitative, and Mixed Studies Review.

BACKGROUND: There is growing literature on the potential of digital technologies for improving access to, ensuring continuity and quality of health care, and to strengthen health systems. Some studies have reported the cost-effectiveness of teledentistry, its reliability for remote dental screening, diagnosis, consultation, and treatment planning. Nonetheless, current evidence suggests that teledentistry implementation faces many challenges and is not yet adopted by dental health care providers (DHCPs). Developing strategies to improve teledentistry adoption requires an understanding of the factors that promote or hinder its successful implementation. OBJECTIVE: This systematic review aims to identify and synthetize barriers and enablers to implementing teledentistry as perceived by DHCPs in their clinical practices, using the Theoretical Domains Framework (TDF) and the Capacity, Opportunity, and Motivation Behavior (COM-B) model. METHODS: This protocol follows the PRISMA-P (Preferred Reporting Items for Systematic Reviews and Meta-Analyses Extension for Protocols) checklist. Literature will be searched in the following databases: PubMed, Cochrane Library, Web of Science, CINAHL, Embase, and PsycINFO. We will perform additional searches on Google, Google Scholar, and ProQuest Dissertations & Theses Global, screen the references of the included studies to capture additional relevant studies, and contact the authors of studies if we need more details. We will consider studies using qualitative, quantitative, and mixed methods. There will be no restrictions on the publication date and dental setting. We will include studies published in French, English, and Portuguese. Two independent reviewers will select the study, extract data, and assess methodological quality using the Mixed Methods Appraisal Tool's checklist. Data analysis will include a descriptive and a thematic content analysis. We will synthetize and categorize the barriers and enablers using the TDF and COM-B model and present a narrative synthesis of our results using tables, figures, and quotes. RESULTS: By March 2023, the literature search has retrieved 7355 publications. We will identify the range of barriers and enablers to implementing teledentistry through DHCPs' perspectives. Considering the critical need for theory-based implementation interventions to improve the use of evidence-informed practices, we will synthesize the factors influencing the adoption of teledentistry based on the TDF domains and the 3 essential conditions predicting behavior change in accordance with the COM-B model. As needed, we will include additional determinants if not included in the TDF. We will conduct some subgroups analyses if studies are sufficient. We expect to complete the review by July 2024. CONCLUSIONS: This review will provide some insights on the determinants of teledentistry implementation as perceived by DHCPs in dental settings. These findings will cater to patients, families, DHCPs, researchers, academic and professional decision-makers, and policy makers. The results of the systematic review could be used to develop theory-led interventions in improving teledentistry implementation. TRIAL REGISTRATION: PROSPERO CRD42021293376; https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=293376. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): PRR1-10.2196/44218.

Porphyromonas gingivalis under palmitate-induced obesogenic microenvironment modulates the inflammatory transcriptional signature of macrophage-like cells.

Metabolic diseases and low-grade chronic inflammation are interconnected: obese persons are at higher risk of developing periodontitis. However, the molecular mechanisms involved in the development and progression of periodontitis in an obesogenic microenvironment in response to periodontopathogens are still lacking. This study aims to investigate the combined effects of palmitate and Porphyromonas gingivalis on the secretion of pro-inflammatory cytokines and on transcriptional landscape modifications in macrophage-like cells. U937 macrophage-like cells were treated with palmitate and stimulated with P. gingivalis for 24h. Cytokines IL-1ß, TNF-α and IL-6 were measured by ELISA in the culture medium and cell extracted RNA was submitted to a microarray analysis followed by Gene Ontology analyses. P. gingivalis, in presence of palmitate, potentiated IL-1ß and TNF-α secretion in comparison to palmitate alone. Gene Ontology analyses also revealed that the combination palmitate-P. gingivalis potentiated the number of gene molecular functions implicated in the regulation of immune and inflammatory pathways compared to macrophages treated with palmitate alone. Our results provide the first comprehensive mapping of gene interconnections between palmitate and P. gingivalis during inflammatory responses in macrophage-like cells. These data highlight the importance of considering systemic conditions, specifically obesogenic microenvironment, in the management of periodontal disease in obese patients.

Multi-Omics Data Integration Reveals Key Variables Contributing to Subgingival Microbiome Dysbiosis-Induced Inflammatory Response in a Hyperglycemic Microenvironment.

Subgingival microbiome dysbiosis promotes the development of periodontitis, an irreversible chronic inflammatory disease associated with metabolic diseases. However, studies regarding the effects of a hyperglycemic microenvironment on host-microbiome interactions and host inflammatory response during periodontitis are still scarce. Here, we investigated the impacts of a hyperglycemic microenvironment on the inflammatory response and transcriptome of a gingival coculture model stimulated with dysbiotic subgingival microbiomes. HGF-1 cells overlaid with U937 macrophage-like cells were stimulated with subgingival microbiomes collected from four healthy donors and four patients with periodontitis. Pro-inflammatory cytokines and matrix metalloproteinases were measured while the coculture RNA was submitted to a microarray analysis. Subgingival microbiomes were submitted to 16s rRNA gene sequencing. Data were analyzed using an advanced multi-omics bioinformatic data integration model. Our results show that the genes krt76, krt27, pnma5, mansc4, rab41, thoc6, tm6sf2, and znf506 as well as the pro-inflammatory cytokines IL-1ß, GM-CSF, FGF2, IL-10, the metalloproteinases MMP3 and MMP8, and bacteria from the ASV 105, ASV 211, ASV 299, Prevotella, Campylobacter and Fretibacterium genera are key intercorrelated variables contributing to periodontitis-induced inflammatory response in a hyperglycemic microenvironment. In conclusion, our multi-omics integration analysis unveiled the complex interrelationships involved in the regulation of periodontal inflammation in response to a hyperglycemic microenvironment.

Anethole induces anti-oral cancer activity by triggering apoptosis, autophagy and oxidative stress and by modulation of multiple signaling pathways.

Oral cancer is one of the major public health problems. The aim of this study was to evaluate the effects of anethole, 1-methoxy-4-[(E)-1-propenyl]-benzene, on growth and apoptosis of oral tumor cells, and to identify the signaling pathways involved in its interaction with these cancer cells. Cancer gingival cells (Ca9-22) were treated with different concentrations of anethole. Cell proliferation and cytotoxic effects were measured by MTT and LDH assays. Cell death, autophagy and oxidative stress markers were assessed by flow cytometry while cell migration was determined by a healing capacity assay. The effect of anethole on apoptotic and pro-carcinogenic signaling pathways proteins was assessed by immunoblotting. Our results showed that anethole selectively and in a dose-dependent manner decreases the cell proliferation rate, and conversely induces toxicity and apoptosis in oral cancer cells. This killing effect was mediated mainly through NF-κB, MAPKinases, Wnt, caspase 3, 9 and PARP1 pathways. Anethole showed an ability to induce autophagy, decrease reactive oxygen species (ROS) production and increased intracellular glutathione (GSH) activity. Finally, anethole treatment inhibits the expression of oncogenes (cyclin D1) and up-regulated cyclin-dependent kinase inhibitor (p21WAF1), increases the expression of p53 gene, but inhibits the epithelial-mesenchymal transition markers. These results indicate that anethole could be a potential molecule for the therapy of oral cancer.

The curcumin analog (PAC) suppressed cell survival and induced apoptosis and autophagy in oral cancer cells.

PAC (3,5-Bis (4-hydroxy-3-methoxybenzylidene)-N-methyl-4-piperidone), a novel bioactive curcumin analog, has been reported to have anticancer properties against various tumors. However, the anti-cancer effects of PAC on oral cavity squamous cell carcinoma were not studied yet. Our aim is to investigate the anti-oral cancer properties of PAC in vitro, and determine the molecular mechanisms underlying these effects. Viability assays including MTT and LDH were conducted to measure cell proliferation. Flow cytometry-based cytotoxicity assay was performed to detect autophagic cell death and oxidative stress markers. Western blotting was used for measuring protein expression/activation in apoptotic, autophagic and pro-carcinogenic cellular signaling pathways. We demonstrated that PAC preferentially and, in a dose, -dependent way kills oral cancer cells, but was not toxic to normal human gingival cells. PAC destabilizes cell-cycle distributions, inhibits the expression of oncogenes (cyclin D1) and that of cyclin-dependent kinase inhibitor (p21WAF1) is upregulated, increases the expression of p53 gene, and inhibits epithelial-mesenchymal transition markers in oral cancer cells. The PAC effect involve various signaling pathways including NF-κB, MAPK, Wnt, caspase-3/9 and PARP1. Finally, PAC demonstrated ability to induce autophagy, decrease production of reactive oxygen species, increase intracellular glutathione (GSH) activity, and reduce mitochondrial membrane potential in oral cancer cells. In conclusion, PAC inhibits the proliferation and increases the apoptosis and autophagy and oxidative stress of oral cancer cells. These effects involve ERK1/2, p38/JNK, NF-κB and Wnt cellular signaling pathways. Overall, our study suggests the potential use of PAC to treat oral cancer.

Biochemical parameters and oxidative stress markers in Tunisian patients with periodontal disease.

BACKGROUND: Oxidative stress is involved in many diseases including diabetes and cancer. Numbers of studies have suggested its involvement in the pathogenesis of periodontal diseases. The aim of this study was to evaluate the levels of biochemical parameters and oxidative stress markers in plasma of healthy and chronic periodontitis patients. METHODS: One hundred thirty subjects were divided into two groups; patients (mean age = 42 ± 13.6 y.o) and control (mean age = 44.8 ± 12.6 y.o). Patients and healthy subjects were free from any infection, coronary or heart disease, diabetes or liver failure. Total cholesterol, LDLc, HDLc, Triglycerides (TG), creatinine, uric acid (UA), glucose and urea levels as well as the activities of enzymatic antioxidants such as catalase, glutathione reductase (GR) and total antioxidant capacity (TAOC), were measured in plasma samples using colorimetric assays. Statistical differences between groups were determined by Student's t-test and p ≤ 0.05 was considered as significant. RESULTS: Periodontitis patients exhibited significant decrease in the activities of catalase, TAOC, GR and TG, cholesterol, LDLc, glucose, HDLc, uric acid levels in plasma samples in comparison with healthy subjects. However, no statistically significant differences in the levels of creatinine and urea were observed between the two groups. CONCLUSION: The reduction of plasma antioxidant activities (Catalase, TAOC, GR) may have a role in the pathogenesis of periodontal diseases. Our findings suggest a decrease in the host capacity to control the damage caused by oxidative stress. Therefore, therapeutic strategies, aiming at modulating the oxidative stress could be considered as potential tools for the prevention or treatment of periodontal diseases and their potential systemic effects on the general health.

Piezocorticision-assisted orthodontics: Efficiency, safety, and long-term evaluation of the inflammatory process.

OBJECTIVES: The aim of this work was to compare the duration of treatment between orthodontic treatment combined with piezocorticision (OT-PC) and conventional orthodontic treatment (COT), as well as to evaluate the safety, inflammatory process, periodontal health, and soft tissue healing in the OT-PC group. METHODS: Twelve patients were included in each group, and their treatment times were compared for preliminary bracket alignment (PBA) and for overall treatment. In the OT-PC group, the inflammatory process was evaluated by quantifying cytokines in the gingival crevicular fluid. A calibrated examiner measured the probing depth (PD), the distance between the gingival margin and the cementoenamel junction (GM-CEJ), and the clinical attachment level (CAL), before and after OT-PC. The presence of gingival scars was evaluated. Bone and root injuries were assessed with the use of cone-beam computed tomography. RESULTS: The treatment time was significantly reduced in the OT-PC group for PBA in both maxilla (45%; P = 0.001) and mandible (31%; P = 0.023), as well as for overall treatment (52%; P < 0.0001). Although not statistically significant, several inflammatory mediators demonstrated peaks at 3-5 and 16 weeks. There were not significant changes in PD and GM-CEJ after OT-PC treatment, unlike CAL (0.09 ± 0.12 mm; P = 0.024); 47.5% of piezocorticisions caused gingival scars. Only one patient showed no scars. Four cortical bones did not heal completely, and 2 roots had piezoelectric injuries. CONCLUSION: OT-PC was effective at reducing the orthodontic treatment time.

Dental caries are positively associated with periodontal disease severity.

OBJECTIVES: To examine associations between periodontal disease severity and clinical and microbiological measures of caries in adults. MATERIALS AND METHODS: A cross-sectional study of 94 healthy adults ((mean ± SD) 55.4 ± 13.0 years) was conducted. Data were collected by means of questionnaire and a clinical examination that included the Decayed, Missing, Filled teeth Surfaces (DMFS) index, probing depth (PD), clinical attachment level (CAL), and gingival bleeding and plaque scores. Supra- and subgingival plaque samples were collected to assess the presence of Streptococcus mutans and six periodontal pathogens. Participants were subsequently categorized using Center for Disease Control and Prevention/American Academy of Periodontology (CDC-AAP) definitions and tertiles of percentage of sites with CAL ≥ 3mm. RESULTS: Significant positive associations were found between the periodontal disease severity (CDC-AAP) and the DMFS (aOR = 1.03; 95% CI 1.01-1.05) and DS indices (aOR = 1.18; 95% CI 1.05-1.32) as well as between the tertiles of percentage of sites with CAL ≥ 3 mm and DMFS (aOR = 1.03; 95% CI 1.00-1.05) and DS indices (aOR = 1.12; 95% CI 1.00-1.25). A significant positive association was also found between oral levels of F. nucleatum and S. mutans (aOR = 6.03; 95% CI 1.55-23.45). CONCLUSIONS: A small but positive association was found between clinical measures of caries and periodontal disease severity. Further research is warranted to examine the association between these two common oral diseases. CLINICAL RELEVANCE: Periodontal diseases and caries are the two most common oral diseases. There was a positive association between clinical and microbiological markers of both diseases. Therefore, strategies in oral health education should involve both caries and periodontitis prevention.

Use of platelet-rich fibrin in regenerative dentistry: a systematic review.

OBJECTIVES: Research across many fields of medicine now points towards the clinical advantages of combining regenerative procedures with platelet-rich fibrin (PRF). This systematic review aimed to gather the extensive number of articles published to date on PRF in the dental field to better understand the clinical procedures where PRF may be utilized to enhance tissue/bone formation. MATERIALS AND METHODS: Manuscripts were searched systematically until May 2016 and separated into the following categories: intrabony and furcation defect regeneration, extraction socket management, sinus lifting procedures, gingival recession treatment, and guided bone regeneration (GBR) including horizontal/vertical bone augmentation procedures. Only human randomized clinical trials were included for assessment. RESULTS: In total, 35 articles were selected and divided accordingly (kappa = 0.94). Overall, the use of PRF has been most investigated in periodontology for the treatment of periodontal intrabony defects and gingival recessions where the majority of studies have demonstrated favorable results in soft tissue management and repair. Little to no randomized clinical trials were found for extraction socket management although PRF has been shown to significantly decrease by tenfold dry sockets of third molars. Very little to no data was available directly investigating the effects of PRF on new bone formation in GBR, horizontal/vertical bone augmentation procedures, treatment of peri-implantitis, and sinus lifting procedures. CONCLUSIONS: Much investigation now supports the use of PRF for periodontal and soft tissue repair. Despite this, there remains a lack of well-conducted studies demonstrating convincingly the role of PRF during hard tissue bone regeneration. Future human randomized clinical studies evaluating the use of PRF on bone formation thus remain necessary. CLINICAL RELEVANCE: PRF was shown to improve soft tissue generation and limit dimensional changes post-extraction, with little available data to date supporting its use in GBR.

Osteogenic gene array of osteoblasts cultured on a novel osteoinductive biphasic calcium phosphate bone grafting material.

OBJECTIVES: Recently, novel biphasic calcium phosphate (BCP) scaffolds have emerged as a new class of bone grafts with osteoinductive potential demonstrating the ability to form ectopic bone in extra-skeletal sites. The aim of the present study was to perform an osteogenic gene array to target possible genes responsible for eliciting the changes in cell expression responsible for inducing osteoblast differentiation. MATERIALS AND METHODS: Human MG63 osteoblast-like cells were seeded for 24 h on tissue culture plastic or osteoinductive BCP particles and analyzed for upregulated genes using an osteogenesis super-array. Osteoblast-related genes including those transcribed during bone mineralization, bone metabolism, cell growth and differentiation, as well as gene products representing extracellular matrix molecules, transcription factors, and cell adhesion molecules were investigated. RESULTS: An upregulation of genes transcribing biglycan (1.7-fold), bone morphogenetic proteins 1, 2, 4, 6, and 7 (1.5-2.1-fold), various collagen isoforms including 1a1, 1a2, 2a1, and 5a1 (1.73-2.72-fold), colony stimulating factor 2 (2.59-fold), fibroblast growth factor receptor 2 (1.79-fold), fibronectin (2.56-fold), integrin alpha 1, 2, and 3 (1.82-2.24-fold), SOX9 (2.75-fold), transforming growth factor beta receptor 2 (1.72-fold), vitamin D (1.89-fold), and vascular endothelial growth factor A and B (2.00, 1.75-fold) were all significantly (p < 0.05) increased on BCP particles when compared to control tissue culture plastic. CONCLUSION: In summary, a number of activated genes were involved in bone formation following osteoblast attachment to BCP particles. The involvement of key chondrogenic genes hints that bone grafts capable of spontaneously inducing ectopic bone formation may implicate endochondral ossification. Further investigations in the triggered pathways involved in the process of ectopic bone formation are necessary to understand the key inductive properties of these novel osteoinductive BCP particles. CLINICAL RELEVANCE: Novel osteoinductive BCP particles demonstrate a wide range of significant increases over several key molecules implicated in osteogenesis that may be implicated in their ability to form ectopic bone formation.

Periodontal Disease and Adverse Pregnancy Outcomes: A Prospective Study in a Low-Risk Population.

BACKGROUND: Periodontal disease has been associated with systemic inflammation and adverse pregnancy outcomes, including preeclampsia and preterm birth. OBJECTIVE: To examine the relationship between periodontal disease in early pregnancy and the risk of amniotic inflammation, preterm birth, and preeclampsia. METHODS: We performed a prospective cohort study of women undergoing amniocentesis for fetal karyotype between 15 and 24 weeks' gestation. Participants underwent periodontal examination by a certified dentist, and a sample of amniotic fluid was collected. Periodontal disease was defined as the presence of one or more sites with probing depths ≥ 4 mm and ≥ 10% bleeding on probing. Matrix metalloproteinase-8 and interleukin-6 concentrations in the amniotic fluid were measured. Medical charts were reviewed for perinatal outcomes. Univariate and multivariate logistic regression analyses were used to assess the association between periodontal disease and adverse pregnancy outcomes. RESULTS: We recruited 273 women at a median gestational age of 16 weeks (range 15 to 24), and 258 (95%) agreed to undergo periodontal examination. Periodontal disease was observed in 117 of the participants (45%). We observed no significant association between periodontal disease and preterm birth (relative risk [RR] 2.27; 95% CI 0.74 to 6.96) or spontaneous preterm birth (RR 0.90; 95% CI 0.20 to 4.11). However, women with periodontal disease were more likely to develop preeclampsia, and this association remained significant after adjustment for potential confounders (adjusted RR 5.89; 95% CI 1.24 to 28.05). Periodontal disease was not associated with significant differences in the intra-amniotic concentration of matrix metalloproteinase-8 (13.0 ± 46.6 vs 5.7 ± 10.4 ng/mL, P = 0.098) or interleukin-6 (3.3 ± 20.3 vs 1.0 ± 1.6 ng/mL, P = 0.23), although a non-significant trend was observed. CONCLUSION: Periodontal disease is associated with preeclampsia but not with spontaneous preterm birth. The current study cannot exclude an association between periodontal disease and intra-amniotic inflammation.

Gene array of PDL cells exposed to Osteogain in combination with a bone grafting material.

OBJECTIVES: The aim of the present study was to investigate the effects of Osteogain, a new formulation of enamel matrix derivative (EMD) in combination with a grafting material on a wide variety of genes for cytokines, transcription factors and extracellular matrix proteins involved in osteoblast differentiation. MATERIALS AND METHODS: Primary human periodontal ligament (PDL) cells were seeded on natural bone mineral (NBM) particles coated with Osteogain for 24 h and analyzed for regulated gene expression using a human osteogenesis gene super-array kit. Osteoblast-related genes include those transcribed during bone mineralization, ossification, bone metabolism, cell growth and differentiation as well as gene products representing extracellular matrix molecules, transcription factors and cell adhesion molecules. RESULTS: Osteogain significantly upregulated the expression of over 20 of the 100 genes examined including bone morphogenetic protein 2 (BMP2), TGFß1, fibroblast growth factor (FGF), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) as well as some of their associated receptors. Osteogain also promoted gene expression of a number of osteoblast differentiation markers including collagen1α2 and alkaline phosphatase as well as cell adhesion molecules including fibronectin and a variety of integrin binding proteins. Interestingly, Osteogain promoted calcitonin receptor 55-fold and also promoted annexin A5 gene expression over 12-fold. CONCLUSION: The present study demonstrates that Osteogain is capable of either upregulating or downregulating the expression of a wide variety of genes including those for growth factors and cytokines when combined with a bone grafting material. CLINICAL RELEVANCE: The results from the present study demonstrate the large and potent effect of addition of Osteogain in combination to a bone grafting material over a wide variety of genes supporting osteogenesis.

Osteoinductive potential of 4 commonly employed bone grafts.

OBJECTIVES: Guided bone regeneration (GBR) aims to predictably restore missing bone that has been lost due to trauma, periodontal disease or a variety of systemic conditions. Critical to this procedure is the ability of a bone grafting material to predictably serve as a 3-dimensional scaffold capable of inducing cell and bone tissue in-growth at the material surface. Although all bone grafts are osteoconductive to bone-forming osteoblasts, only a small number of commercially available bone grafts with FDA approval are osteoinductive including demineralized freeze-dried bone allographs (DFDBA) and scaffolds containing bone morphogenetic proteins (BMPs). Recently, a class of synthetic bone grafts fabricated from biphasic calcium phosphate (BCP) sintered at a low temperature have been shown to form ectopic bone formation in non-skeletal sites without the use of growth factors. Therefore, the present study aimed to compare the osteoinductive potential of this group of synthetic BCP alloplasts with autografts, allografts and xenografts. MATERIALS AND METHODS: In the present study, 4 types of bone grafting materials including autogenous bone harvested with a bone mill, DFDBA (LifeNet, USA), a xenograft derived from bovine bone mineral (NBM, BioOss, Geistlich, Switzerland) and a novel synthetic biphasic calcium phosphate (BCP, Straumman, Switzerland) were implanted into intramuscular pouches of 24 rats and analysed histologically for their ability to form ectopic bone formation around grafting particles. A semi-quantitative osteoinductive score was used to quantify the osteoinductive ability of each bone graft. RESULTS: The results from the present study reveal that (1) autogenous bone resorbed rapidly in vivo, (2) the xenograft showed no potential to form ectopic bone formation and (3) both DFDBA and BCP were able to stimulate ectopic bone formation. CONCLUSION: These studies demonstrate that these newly developed synthetic bone grafts have potential for inducing ectopic bone formation similar to DFDBA. Future clinical testing is necessary to reveal their bone-inducing properties in clinical scenarios including GBR procedures and in combination with implant dentistry. CLINICAL RELEVANCE: Novel BCP scaffolds are able to induce ectopic bone formation without the use of osteoinductive growth factors such as BMP2 and thus demonstrate a large clinical possibility to further enhance bone formation for a variety of clinical procedures.

Osteoinductive potential of a novel biphasic calcium phosphate bone graft in comparison with autographs, xenografts, and DFDBA.

OBJECTIVES: Since the original description of osteoinduction in the early 20th century, the study and development of innovative biomaterials has emerged. Recently, novel synthetic bone grafts have been reported with potential to form ectopic bone in vivo. However, their full characterization in comparison with other leading bone grafts has not been investigated. The aim of this study was to determine the osteoinductive potential of bone grafts by comparing autogenous bone grafts, demineralized freeze-dried bone allografts (DFDBA), a commonly utilized natural bone mineral (NBM) from bovine origin (Bio-Oss), and a newly developed biphasic calcium phosphate (BCP). MATERIALS AND METHODS: Grafts were compared in vitro for their ability to stimulate bone marrow stromal cell (BMSC) migration, proliferation, and differentiation as assessed by quantitative real-time PCR for genes coding for bone markers including Runx2, collagen I, and osteocalcin. Furthermore, bone grafts were implanted in the calf muscle of 12 beagle dogs to determine their potential to form ectopic bone in vivo. RESULTS: The in vitro results demonstrate that both autografts and DFDBA show potential for cell recruitment, whereas only autografts and BCP demonstrated the ability to differentiate BMSCs toward the osteoblast lineage. The in vivo ectopic bone model demonstrated that while NBM particles were not osteoinductive and autogenous bone grafts were resorbed quickly in vivo, ectopic bone formation was reported in DFDBA and in synthetic BCP grafts. CONCLUSION: The modifications in nanotopography and chemical composition of the newly developed BCP bone grafts significantly promoted ectopic bone formation confirming their osteoinductive potential. In conclusion, the results from this study provide evidence that synthetic bone grafts not only serve as a three-dimensional scaffold but are also able to promote osteoinduction.

Effect of Enamel Matrix Derivative Liquid on Osteoblast and Periodontal Ligament Cell Proliferation and Differentiation.

BACKGROUND: Enamel matrix derivatives (EMDs) have been used clinically for more than a decade for the regeneration of periodontal tissues. The aim of the present study is to analyze the effect on cell growth of EMDs in a gel carrier in comparison to EMDs in a liquid carrier. EMDs in a liquid carrier have been shown to adsorb better to bone graft materials. METHODS: Primary human osteoblasts and periodontal ligament (PDL) cells were exposed to EMDs in both gel and liquid carriers and compared for their ability to induce cell proliferation and differentiation. Alizarin red staining and real-time polymerase chain reaction for expression of genes encoding collagen 1, osteocalcin, and runt-related transcription factor 2, as well as bone morphogenetic protein 2 (BMP2), transforming growth factor (TGF)-ß1, and interleukin (IL)-1ß, were assessed. RESULTS: EMDs in both carriers significantly increased cell proliferation of both osteoblasts and PDL cells in a similar manner. Both formulations also significantly upregulated the expression of genes encoding BMP2 and TGF-ß1 as well as decreased the expression of IL-1ß. EMDs in the liquid carrier further retained similar differentiation potential of both osteoblasts and PDL cells by demonstrating increased collagen and osteocalcin gene expression and significantly higher alizarin red staining. CONCLUSIONS: The results from the present study indicate that the new formulation of EMDs in a liquid carrier is equally as potent as EMDs in a gel carrier in inducing osteoblast and PDL activity. Future study combining EMDs in a liquid carrier with bone grafting materials is required to further evaluate its potential for combination therapies.

Bone grafting material in combination with Osteogain for bone repair: a rat histomorphometric study.

OBJECTIVES: Enamel matrix derivative (EMD) has been successfully used for the regeneration of periodontal tissues including new cementum, periodontal ligament, and alveolar bone. Combination of EMD with bone grafting materials has however generated variable clinical results. Recently, we have demonstrated that a new formulation of EMD in a liquid carrier system (Osteogain®) has improved physicochemical properties for the adsorption of EMD to a bone grafting material. The aim of the present study was to investigate the regenerative potential of Osteogain®, in combination with a bone graft, on new bone formation in a rat femur defect model. MATERIALS AND METHODS: Fifty-four critically sized femur defects (3 mm in diameter) were created bilaterally in 27 rats and treated following the group allocation: (1) drilled unfilled control, (2) a natural bone mineral (NBM), and (3) NBM + Osteogain®. All defects were histologically analyzed at 2, 4, and 8 weeks after surgical intervention. Micro-CT analysis, hematoxylin and eosin (H&E) staining, and Safranin O staining were performed to quantify new bone formation. RESULTS: Significantly more new bone formation was observed in defects treated with NBM + Osteogain® at both 4 and 8 weeks when compared to NBM alone and the control unfilled defects (P < 0.05). Histologically, the formation of more mature mineralized bone with the presence of osteocytes were found more commonly in defects treated with Osteogain® + NBM at 8 weeks post-healing when compared to NBM alone. CONCLUSIONS: The present study demonstrate that Osteogain® in combination with a bone grafting material improves the speed and quality of new bone formation in rat osseous defects. CLINICAL RELEVANCE: Future clinical research are now warranted to fully characterize the benefits of Osteogain®, a new formulation of enamel matrix proteins delivered in liquid formation when used in combination with a bone grafting material.

Comparison of the capacity of enamel matrix derivative gel and enamel matrix derivative in liquid formulation to adsorb to bone grafting materials.

BACKGROUND: The use of an enamel matrix derivative (EMD) has been shown to enhance periodontal regeneration (e.g., formation of root cementum, periodontal ligament, and alveolar bone). However, in certain clinical situations, the use of EMD alone may not be sufficient to prevent flap collapse or provide sufficient stability of the blood clot. Data from clinical and preclinical studies have demonstrated controversial results after application of EMD combined with different types of bone grafting materials in periodontal regenerative procedures. The aim of the present study is to investigate the adsorption properties of enamel matrix proteins to bone grafts after surface coating with either EMD (as a liquid formulation) or EMD (as a gel formulation). METHODS: Three different types of grafting materials, including a natural bone mineral (NBM), demineralized freeze-dried bone allograft (DFDBA), or a calcium phosphate (CaP), were coated with either EMD liquid or EMD gel. Samples were analyzed by scanning electron microscopy or transmission electron microscopy (TEM) using an immunostaining assay with gold-conjugated anti-EMD antibody. Total protein adsorption to bone grafting material was quantified using an enzyme-linked immunosorbent assay (ELISA) kit for amelogenin. RESULTS: The adsorption of amelogenin to the surface of grafting material varied substantially based on the carrier system used. EMD gel adsorbed less protein to the surface of grafting particles, which easily dissociated from the graft surface after phosphate-buffered saline rinsing. Analyses by TEM revealed that adsorption of amelogenin proteins were significantly farther from the grafting material surface, likely a result of the thick polyglycolic acid gel carrier. ELISA protein quantification assay demonstrated that the combination of EMD liquid + NBM and EMD liquid + DFDBA adsorbed higher amounts of amelogenin than all other treatment modalities. Furthermore, amelogenin proteins delivered by EMD liquid were able to penetrate the porous surface structure of NBM and DFDBA and adsorb to the interior of bone grafting particles. Grafting materials coated with EMD gel adsorbed more frequently to the exterior of grafting particles with little interior penetration. CONCLUSIONS: The present study demonstrates a large variability of adsorbed amelogenin to the surface of bone grafting materials when enamel matrix proteins were delivered in either a liquid formulation or gel carrier. Furthermore, differences in amelogenin adsorption were observed among NBM, DFDBA, and biphasic CaP particles. Thus, the potential for a liquid carrier system for EMD, used to coat EMD, may be advantageous for better surface coating.

Effect of bone graft density on in vitro cell behavior with enamel matrix derivative.

OBJECTIVES: Bone replacement grafting materials play an important role in regenerative dentistry. Despite a large array of tested bone-grafting materials, little information is available comparing the effects of bone graft density on in vitro cell behavior. Therefore, the aim of the present study is to compare the effects of cells seeded on bone grafts at low and high density in vitro for osteoblast adhesion, proliferation, and differentiation. MATERIALS AND METHODS: The response of osteoblasts to the presence of a growth factor (enamel matrix derivative, (EMD)) in combination with low (8 mg per well) or high (100 mg per well) bone grafts (BG; natural bone mineral, Bio-Oss®) density, was studied and compared for osteoblast cell adhesion, proliferation, and differentiation as assessed by real-time PCR. Standard tissue culture plastic was used as a control with and without EMD. RESULTS: The present study demonstrates that in vitro testing of bone-grafting materials is largely influenced by bone graft seeding density. Osteoblast adhesion was up to 50 % lower when cells were seeded on high-density BG when compared to low-density BG and control tissue culture plastic. Furthermore, proliferation was affected in a similar manner whereby cell proliferation on high-density BG (100 mg/well) was significantly increased when compared to that on low-density BG (8 mg/well). In contrast, cell differentiation was significantly increased on high-density BG as assessed by real-time PCR for markers collagen 1 (Col 1), alkaline phosphatase (ALP), and osteocalcin (OC) as well as alizarin red staining. The effects of EMD on osteoblast adhesion, proliferation, and differentiation further demonstrated that the bone graft seeding density largely controls in vitro results. EMD significantly increased cell attachment only on high-density BG, whereas EMD was able to further stimulate cell proliferation and differentiation of osteoblasts on control culture plastic and low-density BG when compared to high-density BG. CONCLUSION: The results from the present study demonstrate that the in vitro conditions largely influence cell behavior of osteoblasts seeded on bone grafts and in vitro testing. CLINICAL RELEVANCE: These results also illustrate the necessity for careful selection of bone graft seeding density to optimize in vitro testing and provide the clinician with a more accurate description of the osteopromotive potential of bone grafts.

Silver-zeolite combined to polyphenol-rich extracts of Ascophyllum nodosum: potential active role in prevention of periodontal diseases.

The purpose of this study was to evaluate various biological effects of silver-zeolite and a polyphenol-rich extract of A. nodosum (ASCOP) to prevent and/or treat biofilm-related oral diseases. Porphyromonas gingivalis and Streptococcus gordonii contribute to the biofilm formation associated with chronic periodontitis. In this study, we evaluated in vitro antibacterial and anti-biofilm effects of silver-zeolite (Ag-zeolite) combined to ASCOP on P. gingivalis and S. gordonii growth and biofilm formation capacity. We also studied the anti-inflammatory and antioxidant capacities of ASCOP in cell culture models. While Ag-zeolite combined with ASCOP was ineffective against the growth of S. gordonii, it showed a strong bactericidal effect on P. gingivalis growth. Ag-zeolite combined with ASCOP was able to completely inhibit S. gordonii monospecies biofilm formation as well as to reduce the formation of a bi-species S. gordonii/P. gingivalis biofilm. ASCOP alone was ineffective towards the growth and/or biofilm formation of S. gordonii and P. gingivalis while it significantly reduced the secretion of inflammatory cytokines (TNFα and IL-6) by LPS-stimulated human like-macrophages. It also exhibited antioxidant properties and decreased LPS induced lipid peroxidation in gingival epithelial cells. These findings support promising use of these products in future preventive or therapeutic strategies against periodontal diseases.