Microtomographic, histomorphometric, and molecular features show a normal alveolar bone healing process in iNOS-deficient mice along a compensatory upregulation of eNOS and nNOS isoforms.

METHODOLOGY: Inducible nitric oxide synthase (iNOS) is one of the enzymes responsible for the synthesis of nitric oxide (NO), which is an important signaling molecule with effects on blood vessels, leukocytes, and bone cells. However, the role of iNOS in alveolar bone healing remains unclear. This study investigated the role of iNOS in alveolar bone healing after tooth extraction in mice. C57Bl/6 wild type (WT) and iNOS genetically deficient (iNOS-KO) mice were subjected to upper incision tooth extraction, and alveolar bone healing was evaluated by micro-computed tomography (µCT) and histological/histomorphometric, birefringence, and molecular methods. RESULTS: The expression of iNOS had very low control conditions, whereas a significant increase is observed in healing sites of WT mice, where iNOS mRNA levels peak at 7d time point, followed by a relative decrease at 14d and 21d. Regarding bone healing, both WT and iNOS-KO groups showed the usual phases characterized by the presence of clots, granulation tissue development along the inflammatory cell infiltration, angiogenesis, proliferation of fibroblasts and extracellular matrix synthesis, bone neoformation, and remodeling. The overall micro-computed tomography and histomorphometric and birefringence analyses showed similar bone healing readouts when WT and iNOS-KO strains are compared. Likewise, Real-Time PCR array analysis shows an overall similar gene expression pattern (including bone formation, bone resorption, and inflammatory and immunological markers) in healing sites of WT and iNOS-KO mice. Moreover, molecular analysis shows that nNOS and eNOS were significantly upregulated in the iNOS-KO group, suggesting that other NOS isoforms could compensate the absence of iNOS. CONCLUSION: The absence of iNOS does not result in a significant modulation of bone healing readouts in iNOS-KO mice. The upregulation of nNOS and eNOS may compensate iNOS absence, explaining the similar bone healing outcome in WT and iNOS-KO strains.

Microtomographic, histomorphometric, and molecular features show a normal alveolar bone healing process in iNOS-deficient mice along a compensatory upregulation of eNOS and nNOS isoforms

Abstract Inducible nitric oxide synthase (iNOS) is one of the enzymes responsible for the synthesis of nitric oxide (NO), which is an important signaling molecule with effects on blood vessels, leukocytes, and bone cells. However, the role of iNOS in alveolar bone healing remains unclear. This study investigated the role of iNOS in alveolar bone healing after tooth extraction in mice. Methodology C57Bl/6 wild type (WT) and iNOS genetically deficient (iNOS-KO) mice were subjected to upper incision tooth extraction, and alveolar bone healing was evaluated by micro-computed tomography (μCT) and histological/histomorphometric, birefringence, and molecular methods. Results The expression of iNOS had very low control conditions, whereas a significant increase is observed in healing sites of WT mice, where iNOS mRNA levels peak at 7d time point, followed by a relative decrease at 14d and 21d. Regarding bone healing, both WT and iNOS-KO groups showed the usual phases characterized by the presence of clots, granulation tissue development along the inflammatory cell infiltration, angiogenesis, proliferation of fibroblasts and extracellular matrix synthesis, bone neoformation, and remodeling. The overall micro-computed tomography and histomorphometric and birefringence analyses showed similar bone healing readouts when WT and iNOS-KO strains are compared. Likewise, Real-Time PCR array analysis shows an overall similar gene expression pattern (including bone formation, bone resorption, and inflammatory and immunological markers) in healing sites of WT and iNOS-KO mice. Moreover, molecular analysis shows that nNOS and eNOS were significantly upregulated in the iNOS-KO group, suggesting that other NOS isoforms could compensate the absence of iNOS. Conclusion The absence of iNOS does not result in a significant modulation of bone healing readouts in iNOS-KO mice. The upregulation of nNOS and eNOS may compensate iNOS absence, explaining the similar bone healing outcome in WT and iNOS-KO strains.

FTY720 administration results in a M2 associated immunoregulatory effect that positively influences the outcome of alveolar bone repair outcome in mice.

The alveolar bone repair process may be influenced by multiple local and systemic factors, which include immune system cells and mediators. Macrophages allegedly play important roles in the repair process, and the transition of an initial inflammatory M1 profile into a pro-reparative M2 profile theoretically contributes to a favorable repair outcome. In this context, considering immunoregulatory molecules as potential targets for improving bone repair, this study evaluated the role of the immunoregulatory molecule FTY720, previously described to favor the development of the M2 phenotype, in the process of alveolar bone healing in C57Bl/6 (WT) mice. Experimental groups submitted to tooth extraction and maintained under control conditions or treated with FTY720 were evaluated by microtomographic (µCT), histomorphometric, immunohistochemical and molecular analysis to characterize healing and host response features at 0, 1, 3, 7 and 14 days. Our results demonstrated that the FTY720 group presented higher bone tissue density, higher bone tissue volume, greater tissue volume fraction, greater number and thickness of trabeculae and a higher number of osteoblasts and osteoclasts than the control group. Accordingly, the bone markers BMP2, BMP7, ALPL, SOST and RANK mRNA expressions increased in the FTY720 treated group. Furthermore, the levels of FIZZ, ARG2 and IL-10 mRNA increased in the FTY720 group together with the presence of CD206+ cells, suggesting that the boost of bone formation mediated by FTY720 involves an increased polarization and activity of M2 macrophages in healing sites. Thus, our results demonstrate that FTY720 favored the process of alveolar bone repair, probably trough a strengthened M2 response, associated with an increased expression of markers osteogenic differentiation and activity markers. Immunoregulatory strategies based in the modulation of macrophage polarization profile can comprise effective tools to improve the bone repair process.

Macrophage Polarization and Alveolar Bone Healing Outcome: Despite a Significant M2 Polarizing Effect, VIP and PACAP Treatments Present a Minor Impact in Alveolar Bone Healing in Homeostatic Conditions.

Host inflammatory immune response comprises an essential element of the bone healing process, where M2 polarization allegedly contributes to a favorable healing outcome. In this context, immunoregulatory molecules that modulate host response, including macrophage polarization, are considered potential targets for improving bone healing. This study aims to evaluate the role of the immunoregulatory molecules VIP (Vasoactive intestinal peptide) and PACAP (Pituitary adenylate cyclase activating polypeptide), which was previously described to favor the development of the M2 phenotype, in the process of alveolar bone healing in C57Bl/6 (WT) mice. Experimental groups were submitted to tooth extraction and maintained under control conditions or treated with VIP or PACAP were evaluated by microtomographic (µCT), histomorphometric, immunohistochemical, and molecular analysis at 0, 3, 7, and 14 days to quantify tissue healing and host response indicators at the healing site. Gene expression analysis demonstrates the effectiveness of VIP or PACAP in modulating host response, evidenced by the early dominance of an M2-type response, which was paralleled by a significant increase in M2 (CD206+) in treated groups. However, despite the marked effect of M1/M2 balance in the healing sites, the histomorphometric analysis does not reveal an equivalent/corresponding modulation of the healing process. µCT reveals a slight increase in bone matrix volume and the trabecular thickness number in the PACAP group, while histomorphometric analyzes reveal a slight increase in the VIP group, both at a 14-d time-point; despite the increased expression of osteogenic factors, osteoblastic differentiation, activity, and maturation markers in both VIP and PACAP groups. Interestingly, a lower number of VIP and PACAP immunolabeled cells were observed in the treated groups, suggesting a reduction in endogenous production. In conclusion, while both VIP and PACAP treatments presented a significant immunomodulatory effect with potential for increased healing, no major changes were observed in bone healing outcome, suggesting that the signals required for bone healing under homeostatic conditions are already optimal, and additional signals do not improve an already optimal process. Further studies are required to elucidate the role of macrophage polarization in the bone healing process.

Maxillary suture expansion: A mouse model to explore the molecular effects of mechanically-induced bone remodeling.

The aim of this study was to analyze the effect of rapid maxillary expansion (RME) on hard tissues. Opening loops bonded to the first and second maxillary molars on both sides were used to apply distracting forces of 0.28 N, 0.42 N and 0.56 N at the midpalatal suture for 7 and 14 days. Microcomputed tomography (MicroCT), histomorphometry and quantitative polymerase chain reaction (qPCR) analysis were performed to evaluate RME effectiveness, midpalatal suture remodeling, cell counting of osteoblasts, osteoclasts and chondrocytes and the expression of bone remodeling markers, respectively. All forces at the two different time points resulted in similar RME and enhanced of bone remodeling. Accordingly, increased number of osteoblasts and reduced chondrocytes counting and no difference in osteoclasts were seen after all RME protocols. RME yielded increased expression of bone remodeling markers as osteocalcin (Ocn), dentin matrix acidic phosphoprotein-1 (Dmp1), runt-related transcription factor 2 (Runx2), collagen type I Alpha 1 (Col1a1), alkaline phosphatase (ALP), receptor activator of nuclear factor kappa B (RANK), receptor activator of nuclear factor kappa B ligand (Rankl), osteoprotegerin (Opg), cathepsin K (Ctsk), matrix metalloproteinases 9 and 13 (Mmp9 and 13), transforming growth fator beta 1, 2 and 3 (Tgfb 1, Tgfb 2 and Tgfb3), bone morphogenetic protein 2 (Bmp-2), sclerostin (Sost), beta-catenin-like protein 1 (Ctnnbl) and Wnt signaling pathways 3, 3a and 5a (Wnt 3, Wnt 3a and Wnt 5a). These findings characterize the cellular changes and potential molecular pathways involved in RME, proving the reliability of this protocol as a model for mechanical-induced bone remodeling.

Vasoactive Intestinal Peptide Immunoregulatory Role at the Periapex: Associative and Mechanistic Evidences from Human and Experimental Periapical Lesions.

INTRODUCTION: The balance between the host proinflammatory immune response and the counteracting anti-inflammatory and reparative responses supposedly determine the outcome of periapical lesions. In this scenario, the vasoactive intestinal peptide (VIP) may exert a protective role because of its prominent immunoregulatory capacity. In this study, we investigated (in a cause-and-effect manner) the potential involvement of VIP in the development of human and experimental periapical lesions. METHODS: Periapical granulomas (n = 124) and control samples (n = 48) were comparatively assessed for VIP and multiple immunologic/activity marker expression through real-time polymerase chain reaction. Experimental periapical lesions (C57Bl/6 wild-type mice) were evaluated regarding endogenous VIP expression correlation with lesion development and the effect of recombinant VIP therapy in lesion outcome. CCR4KO and IL4KO strains and anti-glucocorticoid-induced TNFR-related protein inhibition were used to test the involvement of Treg and Th2 cells in VIP-mediated effects. RESULTS: VIP expression was more prevalent in periapical granulomas than in controls, presenting a positive association with immunoregulatory factors and an inverse association/correlation with proinflammatory mediators and the receptor activator of nuclear factor kappa B ligand/osteoprotegerin ratio. Endogenous VIP expression up-regulation was temporally associated with lesion immunoregulation and a decline of bone loss. VIP therapy in mice prompted the arrest of lesion development, being associated with an anti-inflammatory and proreparative response that limits the proinflammatory, Th1, Th17, and osteoclastogenic response in the periapex. The VIP protective effect was dependent of Treg migration and activity and independent of interleukin 4. CONCLUSIONS: Our results show that VIP overexpression in human and experimental periapical lesions is associated with lesion inactivity and that VIP therapy results in the attenuation of experimental lesion progression associated with the immunosuppressive response involving Treg cells.

HGMB1 and RAGE as Essential Components of Ti Osseointegration Process in Mice.

The release of the prototypic DAMP High Mobility Group Box 1 (HMGB1) into extracellular environment and its binding to the Receptor for Advanced Glycation End Products (RAGE) has been described to trigger sterile inflammation and regulate healing outcome. However, their role on host response to Ti-based biomaterials and in the subsequent osseointegration remains unexplored. In this study, HMGB1 and RAGE inhibition in the Ti-mediated osseointegration were investigated in C57Bl/6 mice. C57Bl/6 mice received a Ti-device implantation (Ti-screw in the edentulous alveolar crest and a Ti-disc in the subcutaneous tissue) and were evaluated by microscopic (microCT [bone] and histology [bone and subcutaneous]) and molecular methods (ELISA, PCR array) during 3, 7, 14, and 21 days. Mice were divided into 4 groups: Control (no treatment); GZA (IP injection of Glycyrrhizic Acid for HMGB1 inhibition, 4 mg/Kg/day); RAP (IP injection of RAGE Antagonistic Peptide, 4 mg/Kg/day), and vehicle controls (1.5% DMSO solution for GZA and 0.9% saline solution for RAP); treatments were given at all experimental time points, starting 1 day before surgeries. HMGB1 was detected in the Ti-implantation sites, adsorbed to the screws/discs. In Control and vehicle groups, osseointegration was characterized by a slight inflammatory response at early time points, followed by a gradual bone apposition and matrix maturation at late time points. The inhibition of HMGB1 or RAGE impaired the osseointegration, affecting the dynamics of mineralized and organic bone matrix, and resulting in a foreign body reaction, with persistence of macrophages, necrotic bone, and foreign body giant cells until later time points. While Control samples were characterized by a balance between M1 and M2-type response in bone and subcutaneous sites of implantation, and also MSC markers, the inhibition of HMGB1 or RAGE caused a higher expression M1 markers and pro-inflammatory cytokines, as well chemokines and receptors for macrophage migration until later time points. In conclusion, HMGB1 and RAGE have a marked role in the osseointegration, evidenced by their influence on host inflammatory immune response, which includes macrophages migration and M1/M2 response, MSC markers expression, which collectively modulate bone matrix deposition and osseointegration outcome.

Oral implant osseointegration model in C57Bl/6 mice: microtomographic, histological, histomorphometric and molecular characterization.

INTRODUCTION: Despite the successful clinical application of titanium (Ti) as a biomaterial, the exact cellular and molecular mechanisms responsible for Ti osseointegration remains unclear, especially because of the limited methodological tools available in this field. OBJECTIVE: In this study, we present a microscopic and molecular characterization of an oral implant osseointegration model using C57Bl/6 mice. MATERIAL AND METHODS: Forty-eight male wild-type mice received a Ti implant on the edentulous alveolar crest and the peri-implant sites were evaluated through microscopic (µCT, histological and birefringence) and molecular (RealTimePCRarray) analysis in different points in time after surgery (3, 7, 14 and 21 days). RESULTS: The early stages of osseointegration were marked by an increased expression of growth factors and MSC markers. Subsequently, a provisional granulation tissue was formed, with high expression of VEGFb and earlier osteogenic markers (BMPs, ALP and Runx2). The immune/inflammatory phase was evidenced by an increased density of inflammatory cells, and high expression of cytokines (TNF, IL6, IL1) chemokines (CXCL3, CCL2, CCL5 and CXC3CL1) and chemokine receptors (CCR2 and CCR5). Also, iNOS expression remained low, while ARG1 was upregulated, indicating predominance of a M2-type response. At later points in time, the bone matrix density and volume were increased, in agreement with a high expression of Col1a1 and Col21a2. The remodelling process was marked by peaks of MMPs, RANKL and OPG expression at 14 days, and an increased density of osteoclasts. At 21 days, intimate Ti/bone contact was observed, with expression of final osteoblast differentiation markers (PHEX, SOST), as well as red spectrum collagen fibers. CONCLUSIONS: This study demonstrated a unique molecular view of oral osseointegration kinetics in C57Bl/6 mice, evidencing potential elements responsible for orchestrating cell migration, proliferation, ECM deposition and maturation, angiogenesis, bone formation and remodeling at the bone-implant interface in parallel with a novel microscopic analysis.

Oral implant osseointegration model in C57Bl/6 mice: microtomographic, histological, histomorphometric and molecular characterization

Abstract Despite the successful clinical application of titanium (Ti) as a biomaterial, the exact cellular and molecular mechanisms responsible for Ti osseointegration remains unclear, especially because of the limited methodological tools available in this field. Objective: In this study, we present a microscopic and molecular characterization of an oral implant osseointegration model using C57Bl/6 mice. Material and Methods: Forty-eight male wild-type mice received a Ti implant on the edentulous alveolar crest and the peri-implant sites were evaluated through microscopic (μCT, histological and birefringence) and molecular (RealTimePCRarray) analysis in different points in time after surgery (3, 7, 14 and 21 days). Results: The early stages of osseointegration were marked by an increased expression of growth factors and MSC markers. Subsequently, a provisional granulation tissue was formed, with high expression of VEGFb and earlier osteogenic markers (BMPs, ALP and Runx2). The immune/inflammatory phase was evidenced by an increased density of inflammatory cells, and high expression of cytokines (TNF, IL6, IL1) chemokines (CXCL3, CCL2, CCL5 and CXC3CL1) and chemokine receptors (CCR2 and CCR5). Also, iNOS expression remained low, while ARG1 was upregulated, indicating predominance of a M2-type response. At later points in time, the bone matrix density and volume were increased, in agreement with a high expression of Col1a1 and Col21a2. The remodelling process was marked by peaks of MMPs, RANKL and OPG expression at 14 days, and an increased density of osteoclasts. At 21 days, intimate Ti/bone contact was observed, with expression of final osteoblast differentiation markers (PHEX, SOST), as well as red spectrum collagen fibers. Conclusions: This study demonstrated a unique molecular view of oral osseointegration kinetics in C57Bl/6 mice, evidencing potential elements responsible for orchestrating cell migration, proliferation, ECM deposition and maturation, angiogenesis, bone formation and remodeling at the bone-implant interface in parallel with a novel microscopic analysis.