Bone Regeneration and Repair Materials.

Bone tissue has a remarkable ability to regenerate following injury and trauma [...].

Mesenchymal stem cell-based therapy for osteoporotic bones: Effects of the interaction between cells from healthy and osteoporotic rats on osteoblast differentiation and bone repair.

AIMS: Cell therapy utilizing mesenchymal stem cells (MSCs) from healthy donors (HE-MSCs) is a promising strategy for treating osteoporotic bone defects. This study investigated the effects of interaction between HE-MSCs and MSCs from osteoporotic donors (ORX-MSCs) on osteoblast differentiation of MSCs and of HE-MSCs on bone formation in calvarial defects of osteoporotic rats. MATERIALS AND METHODS: Osteoporosis was induced by orchiectomy (ORX) and its effects on the bone were evaluated by femur microtomography (µCT) and osteoblast differentiation of bone marrow MSCs. HE- and ORX-MSCs were cocultured, and osteoblast differentiation was evaluated using genotypic and phenotypic parameters. HE-MSCs were injected into the calvarial defects of osteoporotic rats, and bone formation was evaluated by µCT, histology, and gene expression of osteoblast markers. KEY FINDINGS: ORX-induced osteoporosis was revealed by reduced bone morphometric parameters and osteoblast differentiation in ORX-MSCs. HE-MSCs partially recovered the osteogenic potential of ORX-MSCs, whereas HE-MSCs were mildly affected by ORX-MSCs. Additionally, the bone morphogenetic protein and wingless-related integration site signaling pathway components were similarly modulated in cocultures involving ORX-MSCs. HE-MSCs induced meaningful bone formation, highlighting the effectiveness of cell therapy even in osteoporotic bones. SIGNIFICANCE: These results provide new perspectives on the development of cell-based therapies to regenerate bone defects in patients with disorders that affect bone tissue.

Effect of the secretome of mesenchymal stem cells overexpressing BMP-9 on osteoblast differentiation and bone repair.

The secretome present in the conditioned medium (CM) of mesenchymal stem cells (MSCs) is a promising tool to be used in therapies to promote bone regeneration. Considering the high osteogenic potential of the bone morphogenetic protein 9 (BMP-9), we hypothesized that the secretome of MSCs overexpressing BMP-9 (MSCsBMP-9 ) enhances the osteoblast differentiation of MSCs and the bone formation in calvarial defects. CM of either MSCsBMP-9 (CM-MSCsBMP-9 ) or MSCs without BMP-9 overexpression (CM-MSCsVPR ) were obtained at different periods. As the CM-MSCsBMP-9 generated after 1 h presented the highest BMP-9 concentration, CM-MSCsBMP-9 and CM-MSCsVPR were collected at this time point and used to culture MSCs and to be injected into mouse calvarial defects. The CM-MSCsBMP-9 enhanced the osteoblast differentiation of MSC by upregulating RUNX2, alkaline phosphatase (ALP) and osteopontin protein expression, and ALP activity, compared with CM-MSCsVPR . The CM-MSCsBMP-9 also enhanced the bone repair of mouse calvarial defects, increasing bone volume, bone volume/total volume, bone surface, and trabecular number compared with untreated defects and defects treated with CM-MSCsVPR or even with MSCsBMP-9 themselves. In conclusion, the potential of the MSCBMP-9 -secretome to induce osteoblast differentiation and bone formation shed lights on novel cell-free-based therapies to promote bone regeneration of challenging defects.

Effect of Mesenchymal Stem Cells Overexpressing BMP-9 Primed with Hypoxia on BMP Targets, Osteoblast Differentiation and Bone Repair.

Bone formation is driven by many signaling molecules including bone morphogenetic protein 9 (BMP-9) and hypoxia-inducible factor 1-alpha (HIF-1α). We demonstrated that cell therapy using mesenchymal stem cells (MSCs) overexpressing BMP-9 (MSCs+BMP-9) enhances bone formation in calvarial defects. Here, the effect of hypoxia on BMP components and targets of MSCs+BMP-9 and of these hypoxia-primed cells on osteoblast differentiation and bone repair was evaluated. Hypoxia was induced with cobalt chloride (CoCl2) in MSCs+BMP-9, and the expression of BMP components and targets was evaluated. The paracrine effects of hypoxia-primed MSCs+BMP-9 on cell viability and migration and osteoblast differentiation were evaluated using conditioned medium. The bone formation induced by hypoxia-primed MSCs+BMP-9 directly injected into rat calvarial defects was also evaluated. The results demonstrated that hypoxia regulated BMP components and targets without affecting BMP-9 amount and that the conditioned medium generated under hypoxia favored cell migration and osteoblast differentiation. Hypoxia-primed MSCs+BMP-9 did not increase bone repair compared with control MSCs+BMP-9. Thus, despite the lack of effect of hypoxia on bone formation, the enhancement of cell migration and osteoblast differentiation opens windows for further investigations on approaches to modulate the BMP-9-HIF-1α circuit in the context of cell-based therapies to induce bone regeneration.

Effect of osteoblasts on osteoclast differentiation and activity induced by titanium with nanotopography.

Titanium with nanotopography (Ti Nano) favors osteoblast differentiation and attenuates the osteoclast inhibitory effects on osteoblasts. Because the interactions between nanotopography and osteoclasts are underexplored, the aims of this study were to evaluate the effects of Ti Nano on osteoclast differentiation and activity, and the influence of osteoblasts on osteoclast-Ti Nano interaction. The discs were conditioned with a mixture of 10 N H2SO4 and 30% aqueous H2O2 to create Ti Nano and non-conditioned Ti discs were used as control (Ti Control). Osteoclasts were cultured on Ti Control and Ti Nano in the presence of osteoblasts in an indirect co-culture system. Also, osteoclasts were cultured on polystyrene and calcium phosphate plates in conditioned media by osteoblasts grown on Ti Control and Ti Nano. While Ti Control exhibited an irregular and smooth surface, Ti Nano presented nanopores distributed throughout the whole surface. Additionally, anisotropy was higher on Ti Nano than Ti Control. Nanotopography favored the gene expression of osteoclast markers but inhibited osteoclast differentiation and activity, and the presence of osteoblasts enhanced the effects of Ti Nano on osteoclasts. Such findings were mimicked by conditioned medium of osteoblasts cultured on Ti Nano, which reduced the osteoclast differentiation and activity. In conclusion, our results indicated that nanotopography regulates osteoblast-osteoclast crosstalk and further investigations should focus the impact of these bone cell interactions on Ti osseointegration.

Mesenchymal Stem Cells Combined with a P(VDF-TrFE)/BaTiO3 Scaffold and Photobiomodulation Therapy Enhance Bone Repair in Rat Calvarial Defects.

BACKGROUND: Tissue engineering and cell therapy have been the focus of investigations on how to treat challenging bone defects. This study aimed to produce and characterize a P(VDF-TrFE)/BaTiO3 scaffold and evaluate the effect of mesenchymal stem cells (MSCs) combined with this scaffold and photobiomodulation (PBM) on bone repair. METHODS AND RESULTS: P(VDF-TrFE)/BaTiO3 was synthesized using an electrospinning technique and presented physical and chemical properties suitable for bone tissue engineering. This scaffold was implanted in rat calvarial defects (unilateral, 5 mm in diameter) and, 2 weeks post-implantation, MSCs were locally injected into these defects (n = 12/group). Photobiomodulation was then applied immediately, and again 48 and 96 h post-injection. The µCT and histological analyses showed an increment in bone formation, which exhibited a positive correlation with the treatments combined with the scaffold, with MSCs and PBM inducing more bone repair, followed by the scaffold combined with PBM, the scaffold combined with MSCs, and finally the scaffold alone (ANOVA, p ≤ 0.05). CONCLUSIONS: The P(VDF-TrFE)/BaTiO3 scaffold acted synergistically with MSCs and PBM to induce bone repair in rat calvarial defects. These findings emphasize the need to combine a range of techniques to regenerate large bone defects and provide avenues for further investigations on innovative tissue engineering approaches.

Association of mesenchymal stem cells derived from bone marrow and adipose tissue enhances bone repair in rat calvarial defects.

Aim: We evaluated the bone repair induced by MSCs from adipose tissue (AT-MSCs) and bone marrow (BM-MSCs) injected into rat calvarial defects at two time points. Methods & results: Both cell populations expressed MSC surface markers and differentiated into adipocytes and osteoblasts. µCT showed that the combination of cells from distinct sources exhibited synergistic effects to increase bone repair with an advantage when BM-MSCs were injected prior to AT-MSCs. The higher osteogenic potential of these MSC combinations was demonstrated using an in vitro coculture system where BM-MSCs and AT-MSCs association induced higher ALP activity in MC3T3-E1 cells. Conclusion: Our findings may drive new approaches to treat bone defects and shed light on the complexity of the mechanisms involved in bone regeneration.

We evaluated the bone repair induced by cells that can develop into different types of cells (stem cells) derived from fat and spongy tissue inside the large bones and injected into defects created in rat skulls. Cells derived from both tissues developed into fat cells and bone-forming cells. The combination of cells from fat and spongy tissue exhibited cooperative effects to increase bone repair with an advantage when cells from spongy tissue were injected prior to cells from fat. Our findings may contribute to stablish new therapies based on the use of cells to treat large bone defects.

Effects of Modulation of the Hedgehog and Notch Signaling Pathways on Osteoblast Differentiation Induced by Titanium with Nanotopography.

BACKGROUND: The events of bone formation and osteoblast/titanium (Ti) interactions may be affected by Hedgehog and Notch signalling pathways. Herein, we investigated the effects of modulation of these signalling pathways on osteoblast differentiation caused by the nanostructured Ti (Ti-Nano) generated by H2SO4/H2O2. METHODS: Osteoblasts from newborn rat calvariae were cultured on Ti-Control and Ti-Nano in the presence of the Hedgehog agonist purmorphamine or antagonist cyclopamine and of the Notch antagonist N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) or agonist bexarotene. Osteoblast differentiation was evaluated by alkaline phosphatase activity and mineralization, and the expression of Hedgehog and Notch receptors was also evaluated. RESULTS: In general, purmorphamine and DAPT increased while cyclopamine and bexarotene decreased osteoblast differentiation and regulated the receptor expression on both Ti surfaces, with more prominent effects on Ti-Nano. The purmorphamine and DAPT combination exhibited synergistic effects on osteoblast differentiation that was more intense on Ti-Nano. CONCLUSION: Our results indicated that the Hedgehog and Notch signalling pathways drive osteoblast/Ti interactions more intensely on nanotopography. We also demonstrated that combining Hedgehog activation with Notch inhibition exhibits synergistic effects on osteoblast differentiation, especially on Ti-Nano. The uncovering of these cellular mechanisms contributes to create strategies to control the process of osseointegration based on the development of nanostructured surfaces.

Bioactive glass-ceramic for bone tissue engineering: an in vitro and in vivo study focusing on osteoclasts.

Despite the crucial role of osteoclasts in the physiological process of bone repair, most bone tissue engineering strategies have focused on osteoblast-biomaterial interactions. Although Biosilicate® with two crystalline phases (BioS-2P) exhibits osteogenic properties and significant bone formation, its effects on osteoclasts are unknown. This study aimed to investigate the in vitro and in vivo effects of BioS-2P on osteoclast differentiation and activity. RAW 264.7 cells were cultured in osteoclastogenic medium (OCM) or OCM conditioned with BioS-2P (OCM-BioS-2P), and the cell morphology, viability, and osteoclast differentiation were evaluated. BioS-2P scaffolds were implanted into rat calvarial defects, and the bone tissue was evaluated using tartrate-resistant acid phosphatase (TRAP) staining and RT-polymerase chain reaction (PCR) after 2 and 4 weeks to determine the gene expressions of osteoclast markers and compare them with those of the bone grown in empty defects (Control). OCM-BioS-2P favored osteoclast viability and activity, as evidenced by an increase in the TRAP-positive cells and matrix resorption. The bone tissue grown on BioS-2P scaffolds exhibited higher expression of the osteoclast marker genes (Ctsk, Mmp 9, Rank) after 2 and 4 weeks and the RankL/Opg ratio after 2 weeks. Trap gene expression was lower at 2 weeks, and a higher number of TRAP-stained areas were observed in the newly formed bone on BioS-2P scaffolds at both 2 and 4 weeks compared to the Controls. These results enhanced our understanding of the role of bioactive glass-ceramics in bone repair, and highlighted their role in the modulation of osteoclastic activities and promotion of interactions between bone tissues and biomaterials.

Human periodontal ligament stem cells with distinct osteogenic potential induce bone formation in rat calvaria defects.

Aim: This study aimed to evaluate the ability of human periodontal ligament stem cells (PDLSCs) with high (HP-PDLSCs) and low (LP-PDLSCs) osteogenic potential, in addition to mixed cells, to repair bone tissue. Methods: Cell phenotype, proliferation and differentiation were evaluated. Undifferentiated PDLSCs were injected into rat calvarial defects and the new bone was evaluated by µCT, histology and real-time PCR. Results: PDLSCs exhibited a typical mesenchymal stem cell phenotype and HP-PDLSCs showed lower proliferative and higher osteogenic potential than LP-PDLSCs. PDLSCs induced similar bone formation and histological analysis suggests a remodeling process, confirmed by osteogenic and osteoclastogenic markers, especially in tissues derived from defects treated with HP-PDLSCs. Conclusion: PDLSCs induced similar bone formation irrespective of their in vitro osteogenic potential.

Bone is one of the most transplanted tissues worldwide and cell-based therapies has been investigated as an alternative for the treatment of bone defects. Dental tissues have been investigated as sources of stem cells and the periodontal ligament has been shown to be a viable source of these cells. Stem cells from periodontal ligament induce significant bone formation in rat calvaria defects and are safe for cell-based therapies, as the cells remain at the bone defect site for up to 4 weeks and do not migrate to vital organs, such as brain, heart, lungs, spleen, kidneys, and liver in the same period. In addition, immune responses were not detected. Considering that, stem cells from periodontal ligament can be useful in cell therapy strategies to induce bone regeneration.

Texturized P(VDF-TrFE)/BT membrane enhances bone neoformation in calvaria defects regardless of the association with photobiomodulation therapy in ovariectomized rats.

OBJECTIVES: The purpose of this investigation was to evaluate in vivo the response of bone tissue to photobiomodulation when associated with texturized P(VDF-TrFE)/BT in calvaria defects of ovariectomized rats. MATERIALS AND METHODS: Wistar Hannover rats were submitted to ovariectomy/control surgery. Calvaria bone defects of 5-mm diameter were performed after 90 days of ovariectomy. The animals were divided into OVX (without laser (L) and membrane), OVX + P(VDF-TrFE)/BT, OVX + P(VDF-TrFE)/BT + L, and OVX + PTFE + L. It was utilized a low-intensity gallium-aluminum-arsenide laser (GaAlAs) with 780-nm wavelength and 30-J/cm2 energy density in 12 sessions (120 s). Thirty days after the bone defect the animals were euthanized for histological, microtomographic, and molecular evaluation. Quantitative analysis was analyzed by statistical software for p < 0.05. RESULTS: Histological parameters showed bone tissue formation at the borders of all group defects. The association of photobiomodulation and texturized P(VDF-TrFE)/BT was not synergistic and did not show significant changes in morphometric analysis and biomarkers gene expression. Nevertheless, texturized P(VDF-TrFE)/BT membrane enhanced bone repair regardless of the association with photobiomodulation therapy, with an increase of connectivity density when compared to the OVX + PTFE + L group. The association of photobiomodulation therapy and PTFE was synergistic, increasing the expression of Runx2, Alp, Bsp, Bglap, Sp7, and Rankl, even though not enough to reflect significance in the morphometric parameters. CONCLUSIONS: The utilization of texturized P (VDF-TrFE)/BT, regardless of the association with photobiomodulation therapy, enhanced bone repair in an experimental model of osteoporosis.

Bioactive glass-ceramic for bone tissue engineering: an in vitro and in vivo study focusing on osteoclasts

Abstract: Despite the crucial role of osteoclasts in the physiological process of bone repair, most bone tissue engineering strategies have focused on osteoblast-biomaterial interactions. Although Biosilicate® with two crystalline phases (BioS-2P) exhibits osteogenic properties and significant bone formation, its effects on osteoclasts are unknown. This study aimed to investigate the in vitro and in vivo effects of BioS-2P on osteoclast differentiation and activity. RAW 264.7 cells were cultured in osteoclastogenic medium (OCM) or OCM conditioned with BioS-2P (OCM-BioS-2P), and the cell morphology, viability, and osteoclast differentiation were evaluated. BioS-2P scaffolds were implanted into rat calvarial defects, and the bone tissue was evaluated using tartrate-resistant acid phosphatase (TRAP) staining and RT-polymerase chain reaction (PCR) after 2 and 4 weeks to determine the gene expressions of osteoclast markers and compare them with those of the bone grown in empty defects (Control). OCM-BioS-2P favored osteoclast viability and activity, as evidenced by an increase in the TRAP-positive cells and matrix resorption. The bone tissue grown on BioS-2P scaffolds exhibited higher expression of the osteoclast marker genes (Ctsk, Mmp 9, Rank) after 2 and 4 weeks and the RankL/Opg ratio after 2 weeks. Trap gene expression was lower at 2 weeks, and a higher number of TRAP-stained areas were observed in the newly formed bone on BioS-2P scaffolds at both 2 and 4 weeks compared to the Controls. These results enhanced our understanding of the role of bioactive glass-ceramics in bone repair, and highlighted their role in the modulation of osteoclastic activities and promotion of interactions between bone tissues and biomaterials.

Photobiomodulation therapy does not depend on the differentiation of dental pulp cells to enhance functional activity associated with angiogenesis and mineralization.

The purpose of this study is to analyze the influence of InGaAlP diode laser (660 nm) with or without an odontogenic medium (OM) in the functional activity of OD-21 cells. Undifferentiated OD-21 pulp cells were cultivated with or without OM and divided into four groups (n = 5): nonirradiated control (C -), nonirradiated + OM (C +), irradiated (L -), and irradiated + OM (L +). Laser application was performed in two sessions of a 24-h interval with an irradiance of 11.3 mW/cm2, energy density of 1 J/cm2, and total cumulative energy/well of 4.6 J. Cell proliferation, VEGF-164 expression, mineralization, and expression of Alp, Runx2, and Dmp1 genes, as well as immunolocalization of RUNX2 and MEPE proteins, were evaluated. Data were analyzed by statistical tests (α = 0.05). All studied groups showed a similar increase in cell proliferation with or without OM. After 7 and 10 days, a significatively higher concentration of VEGF-164 in L - group when compared to C - group was observed. A significant increase in mineralized nodules in the L + was noted when compared to C + in the same conditions. Photobiomodulation upregulated significantly Runx2 and Dmp1 expression after 10 days in L - and after 7 days in L + , with downregulation of Dmp1 after 10 days in L + group. Immunolocalization of RUNX2 and MEPE was expressive after 7 days of culture in the cytoplasm adjacent to the nucleus with a decrease after 10 days, regardless of the presence of OM. Photobiomodulation enhances metabolism associated with angiogenesis, gene expression, and mineralization regardless of the odontogenic medium in OD-21 cells.

The extracellular matrix protein Agrin is expressed by osteoblasts and contributes to their differentiation.

The extracellular matrix protein Agrin has been detected in chondrocytes and endosteal osteoblasts but its function in osteoblast differentiation has not been investigated yet. Thus, it is possible that Agrin contributes to osteoblast differentiation and, due to Agrin and wingless-related integration site (Wnt) sharing the same receptor, transmembrane low-density lipoprotein receptor-related protein 4 (Lrp4), and the crosstalk between Wnt and bone morphogenetic protein (BMP) signalling, both pathways could be involved in this Agrin-mediated osteoblast differentiation. Confirming this, Agrin and its receptors Lrp4 and α-dystroglycan (Dag1) were expressed during differentiation of osteoblasts from three different sources. Moreover, the disruption of Agrin impaired the expression of its receptors and osteoblast differentiation, and the treatment with recombinant Agrin slightly increase this process. In addition, whilst Agrin knockdown downregulated the expression of genes related to Wnt and BMP signalling pathways, the addition of Agrin had no effect on these genes. Altogether, these data uncover the contribution of Agrin to osteoblast differentiation and suggest that, at least in part, an Agrin-Wnt-BMP circuit is involved in this process. This makes Agrin a candidate as target for developing new therapeutic strategies to treat bone-related diseases and injuries.

In Vitro Effect of Low-Level Laser Therapy on Undifferentiated Mouse Pulp Cells / Efeito in Vitro da Laserterapia de Baixa Intensidade em Células Pulpares Indiferenciadas de Camundongo

Low-level laser therapy has been investigated as a possible stimulus for enhancement of proliferation and differentiation of various cell types, but few reports relate undifferentiated mouse pulp cells (OD-21) response to irradiation in in vitro models. The aim of this study was to analyze the influence of low-level laser therapy (λ=660 nm), with three different irradiation times, on the behavior of OD-21 cell line. The cells were cultivated and divided into three groups: non-irradiated/control (group I); irradiated with 88 s (group II); irradiated with 177 s (group III) and irradiated with 265 s (group IV). Cell growth and viability were assessed after 7 and 10 days. Data were analyzed by Kruskal-Wallis and MannWhitney tests (α=.05). At day 7, there was a higher cell growth in groups I and II, as compared to group IV (p<.01). At the 10th day, group I showed a higher cell growth as compared to group II (p<.05). Cell viability in group IV was significantly lower at the 7th day, as compared to groups I (p<.001), II (p<.01) and III (p<.001). Cell viability in all the groups was over 80%, except in group IV at day 7. Irradiation time of group I influenced positively the proliferation and viability of OD-21 cells in late cell culture period. (AU)

A terapia a laser de baixa intensidade tem sido investigada como possível estímulo para aumento da proliferação e diferenciação de vários tipos de células, mas poucos relatos relacionam a resposta de células indiferenciadas da polpa dentária de camundongos (OD-21) à irradiação em modelos in vitro. O objetivo deste estudo foi analisar a influência do laser de baixa intensidade (λ=660 nm), com três períodos de irradiação diferentes, no comportamento das células da linhagem OD-21. As células foram cultivadas e distribuídas em três grupos: não irradiado / controle (grupo I); irradiado com 88 s (grupo II); irradiado com 177 s (grupo III) e irradiado com 265 s (grupo IV). O crescimento e a viabilidade celular foram avaliados após 7 e 10 dias. Os dados foram analisados pelos testes de Kruskal-Wallis e Mann-Whitney (α = 0,05). No dia 7, houve crescimento celular maior nos grupos I e II, em comparação ao grupo IV (p <0,01). No décimo dia, o grupo I apresentou crescimento celular superior ao grupo II (p <0,05). A viabilidade celular no grupo IV foi significativamente menor no sétimo dia, em comparação aos grupos I (p <0,001), II (p <0,01) e III (p <0,001). A viabilidade celular em todos os grupos foi superior a 80%, exceto no grupo IV no dia 7. O tempo de irradiação do grupo I influenciou positivamente a proliferação e a viabilidade das células OD-21 no período mais tardio da cultura celular.A terapia a laser de baixa intensidade tem sido investigada como possível estímulo para aumento da proliferação e diferenciação de vários tipos de células, mas poucos relatos relacionam a resposta de células indiferenciadas da polpa dentária de camundongos (OD-21) à irradiação em modelos in vitro. O objetivo deste estudo foi analisar a influência do laser de baixa intensidade (λ=660 nm), com três períodos de irradiação diferentes, no comportamento das células da linhagem OD-21. As células foram cultivadas e distribuídas em três grupos: não irradiado / controle (grupo I); irradiado com 88 s (grupo II); irradiado com 177 s (grupo III) e irradiado com 265 s (grupo IV). O crescimento e a viabilidade celular foram avaliados após 7 e 10 dias. Os dados foram analisados pelos testes de Kruskal-Wallis e Mann-Whitney (α = 0,05). No dia 7, houve crescimento celular maior nos grupos I e II, em comparação ao grupo IV (p <0,01). No décimo dia, o grupo I apresentou crescimento celular superior ao grupo II (p <0,05). A viabilidade celular no grupo IV foi significativamente menor no sétimo dia, em comparação aos grupos I (p <0,001), II (p <0,01) e III (p <0,001). A viabilidade celular em todos os grupos foi superior a 80%, exceto no grupo IV no dia 7. O tempo de irradiação do grupo I influenciou positivamente a proliferação e a viabilidade das células OD-21 no período mais tardio da cultura celular. (AU)

Miniplates coated by plasma electrolytic oxidation improve bone healing of simulated femoral fractures on low bone mineral density rats.

The treatment of polytrauma patients represents a great challenge in the maxillofacial and orthopedic surgery fields. Therefore, this study tested the hypothesis that the use of a bioactive coating (by plasma electrolytic oxidation, PEO) on titanium microplates could improve the fracture healing of low bone mineral density (BMD) rats. Thirty female rats underwent bilateral ovariectomy surgery (OVX), and 35 rats underwent fake surgery (SHAM). Three months later, animals were subjected to femoral fracture simulation and were fixed with either non-coated (CONV) or coated (PEO) titanium miniplates. Eight weeks postoperatively, microplate/bone complexes were analyzed through computed microtomography, histometric, confocal microscopy, molecular, and biomechanical analysis. Bioactive elements (Ca and P) were incorporated on the PEO microplate and the surface was modified in a volcano-like structure. In the microCT analysis the OVX/PEO group had greater values for Tb.Th (bone trabecular thickness), Tb.Sp (separation of bone trabeculae) and Tb.N (number of trabeculae) parameters compared to the OVX/CONV group. According to histometric analysis, the OVX/PEO group showed significantly higher new bone formation than the OVX/CONV group (P < 0.05). For the fluorochrome area, the OVX groups (PEO and CONV) showed greater values for calcein precipitation (old bone) than alizarin red (new bone). Molecular results showed greater values for proteins related to the final phase of bone formation (P < 0.05) in the OVX/PEO group. The OVX/PEO group showed higher bone/miniplate system resilience compared to the others (P < 0.05). It was concluded that PEO coating optimizes bone healing on simulated femoral fractures in low bone mineral density rats. This sheds new light in the treatment of osteoporotic patients with bone fractures.

Jabuticaba peel extract modulates adipocyte and osteoblast differentiation of MSCs from healthy and osteoporotic rats.

INTRODUCTION: The jabuticaba peel extract (JPE) contains bioactive compounds that regulate fat metabolism. Because the negative correlation between fat accumulation and bone formation in bone marrow, we hypothesized that JPE inhibits adipocyte as well as favors osteoblast differentiation of mesenchymal stromal cells (MSCs) under healthy and osteoporotic conditions, a disease that display an imbalance between adipocyte and osteoblast differentiation resulting in reduced bone mass. MATERIAL AND METHODS: To test these hypotheses, bone marrow MSCs were harvested from healthy and osteoporotic rats and cultured in adipogenic and osteogenic media with three concentrations of JPE, 0.25, 5 and 10 µg/ml, and vehicle (control). After selecting the most efficient concentrations of JPE, we used them to evaluate adipocyte and osteoblast differentiation of MSCs from both sources. RESULTS: We observed that, in general, JPE inhibited adipocyte differentiation of MSCs with more pronounced effects in cells from healthy than osteoporotic rats. In addition, JPE increased osteoblast differentiation, exhibiting a slightly higher osteogenic potential on MSCs from osteoporotic compared to healthy condition. CONCLUSION: Our results demonstrated that JPE drives MSCs to inhibit adipocyte differentiation and toward osteoblast differentiation under healthy and osteoporotic conditions. These findings pave the way for further translational studies to investigate the therapeutic possibilities of JPE in both prevention and treatment of osteoporosis.

Green tea extract rich in epigallocatechin gallate impairs alveolar bone loss in ovariectomized rats with experimental periodontal disease.

Periodontal disease and osteoporosis are characterized by bone resorption, and researchers have shown an association between these two diseases through increasing loss of systemic bone mass and triggering alveolar bone loss. Green tea is a common and easily accessible beverage, and evidences show that flavonoid epigallocatechin gallate (EGCG) could decrease bone loss in pathologies such as osteoporosis and periodontal disease. In order to verify its possible effects and apply them in the treatment and prevention of these diseases, this investigation aimed to evaluate the influence of green tea extract (GTE) on bone metabolism of ovariectomized rats after experimental periodontal disease (EPD) by histological, morphological and microtomographic parameters. Wistar female rats were divided into Sham, Sham + EPD, Sham + EPD + GTE, OVX, OVX + EPD and OVX + EPD + GTE groups. Immediately after surgery, gavage administration of 50 mg/kg of green tea extract (GTE) was performed for 60 days, with subsequent induction of periodontal disease by ligature 15 days before euthanasia. Mandible and femur samples were collected for histological, morphometric and microtomographic analysis. The results were analysed by means of statistical software with significance set at 5%. Histological and morphometric analysis showed a significant decrease in alveolar and femoral trabecular bone loss in groups that received GTE. Microtomographic results showed that trabecular thickness and bone surface density values in alveolar bone interradicular septum of the OVX + EPD + GTE groups were similar to the Sham group. The results obtained suggest that green tea extract may improve bone metabolism in osteoporotic rats with periodontal disease.

Frizzled 6 disruption suppresses osteoblast differentiation induced by nanotopography through the canonical Wnt signaling pathway.

This study aimed to investigate if wingless-related integration site (Wnt) signaling participates in the high osteogenic potential of titanium with nanotopography (Ti-Nano). We showed that among the several components of the Wnt signaling pathway, Frizzled 6 (Fzd6) was the transcript most intensely modulated by nanotopography compared with the untreated Ti surface (Ti-Machined). Then, we investigated whether and how Fzd6 participates in the regulation of osteoblast differentiation caused by nanotopography. The Fzd6 silencing with CRISPR-Cas9 transfection in MC3T3-E1 cells induced a more pronounced inhibition of osteoblast differentiation of cells cultured on nanotopography than those cultured on Ti-Machined. The analysis of the expression of calcium-calmodulin-dependent protein kinase II and ß-catenin demonstrated that Fzd6 disruption inhibited the osteoblast differentiation induced by Ti-Nano by preventing the activation of Wnt/ß-catenin but not that of Wnt/Ca2+ signaling, which is usually triggered by the receptor Fzd6. These findings elucidate the biological function of Fzd6 as a receptor that triggers Wnt/ß-catenin signaling and the cellular mechanisms modulated by nanotopography during osteoblast differentiation.

Effect of focal adhesion kinase inhibition on osteoblastic cells grown on titanium with different topographies.

OBJECTIVE: The present study aimed to investigate the participation of focal adhesion kinases (FAK) in interactions between osteoblastic cells and titanium (Ti) surfaces with three different topographies, namely, untreated (US), microstructured (MS), and nanostructured (NS). METHODOLOGY: Osteoblasts harvested from the calvarial bones of 3-day-old rats were cultured on US, MS and NS discs in the presence of PF-573228 (FAK inhibitor) to evaluate osteoblastic differentiation. After 24 h, we evaluated osteoblast morphology and vinculin expression, and on day 10, the following parameters: gene expression of osteoblastic markers and integrin signaling components, FAK protein expression and alkaline phosphatase (ALP) activity. A smooth surface, porosities at the microscale level, and nanocavities were observed in US, MS, and NS, respectively. RESULTS: FAK inhibition decreased the number of filopodia in cells grown on US and MS compared with that in NS. FAK inhibition decreased the gene expression of Alp, bone sialoprotein, osteocalcin, and ALP activity in cells grown on all evaluated surfaces. FAK inhibition did not affect the gene expression of Fak, integrin alpha 1 ( Itga1 ) and integrin beta 1 ( Itgb1 ) in cells grown on MS, increased the gene expression of Fak in cells grown on NS, and increased the gene expression of Itga1 and Itgb1 in cells grown on US and NS. Moreover, FAK protein expression decreased in cells cultured on US but increased in cells cultured on MS and NS after FAK inhibition; no difference in the expression of vinculin was observed among cells grown on all surfaces. CONCLUSIONS: Our data demonstrate the relevance of FAK in the interactions between osteoblastic cells and Ti surfaces regardless of surface topography. Nanotopography positively regulated FAK expression and integrin signaling pathway components during osteoblast differentiation. In this context, the development of Ti surfaces with the ability to upregulate FAK activity could positively impact the process of implant osseointegration.