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.

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.

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.

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.

Inhibitory effects of dabigatran etexilate, a direct thrombin inhibitor, on osteoclasts and osteoblasts.

INTRODUCTION: Anticoagulants are widely used in orthopedic surgery to decrease the risk of deep vein thrombosis. While significant bone impairment is induced by long-term heparin therapy, little is known about the effects of direct oral anticoagulants (DOACs). Herein, we investigated the effects of dabigatran etexilate (Pradaxa®), a DOAC inhibitor of thrombin, on bone cells using in vitro and ex vivo cell culture models. MATERIALS AND METHODS: Osteoblasts and osteoclasts exposed to different concentrations of dabigatran etexilate and untreated cells were assayed for cell differentiation and activity. Favorable osteogenic conditions for osteoblasts were tested using titanium with nanotopography (Ti-Nano). In addition, mice treated with a dabigatran etexilate solution had bone marrow cells analyzed for the ability to generate osteoclasts. RESULTS: Dabigatran etexilate at concentrations of 1 µg/mL and 2 µg/mL did not impact osteoclast or osteoblast viability. The drug inhibited osteoclast differentiation and activity as observed by the reduction of TRAP+ cells, resorption pits and gene and protein expression of cathepsin K. Consistently, osteoclasts from mice treated with dabigatran showed decreased area, resorptive activity, as well as gene and protein expression of cathepsin K. In osteoblast cultures, grown both on polystyrene and Ti-Nano, dabigatran etexilate reduced alkaline phosphatase (ALP) activity, matrix mineralization, gene expression of ALP and osteocalcin. CONCLUSIONS: Dabigatran etexilate inhibited osteoclast differentiation in ex vivo and in vitro models in a dose-dependent manner. Moreover, the drug reduced osteoblast activity even under optimal osteogenic conditions. This study provides new evidence regarding the negative overall impact of DOACs on bone cells.

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

Abstract 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.

Effect of stem cells combined with a polymer/ceramic membrane on osteoporotic bone repair.

Cell therapy associated with guided bone regeneration (GBR) can be used to treat bone defects under challenging conditions such as osteoporosis. This study aimed to evaluate the effect of mesenchymal stem cells (MSCs) in combination with a poly(vinylidene-trifluoroethylene)/barium titanate (PVDF-TrFE/BT) membrane on bone repair in osteoporotic rats. Osteoporosis was induced in female rats by bilateral removal of the ovaries (OVX) or sham surgery (SHAM), and the osteoporotic condition was characterized after 5 months by microtomographic and morphometric analyses. Calvarial defects were created in osteoporotic rats that immediately received the PVDF-TrFE/BT membrane. After 2 weeks, bone marrow-derived MSCs from healthy rats, characterized by the expression of surface markers using flow cytometry, or phosphate-buffered saline (PBS) (Control) were injected into the defects and bone formation was evaluated 4 weeks post-injection by microtomographic, morphometric, and histological analyses. A reduction in the amount of bone tissue in the femurs of OVX compared with SHAM rats confirmed the osteoporotic condition of the experimental model. More bone formation was observed when the defects were injected with MSCs compared to that with PBS. The modification that we are proposing in this study for the classical GBR approach where cells are locally injected after a membrane implantation may be a promising therapeutic strategy to increase bone formation under osteoporotic condition.

Effect of cell source and osteoblast differentiation on gene expression profiles of mesenchymal stem cells derived from bone marrow or adipose tissue.

Mesenchymal stem cells (MSCs) have been used in therapies for bone tissue healing. The aim of this study was to investigate the effect of cell source and osteoblast differentiation on gene expression profiles of MSCs from bone marrow (BM-MSCs) or adipose tissue (AT-MSCs) to contribute for selecting a suitable cell population to be used in cell-based strategies for bone regeneration. BM-MSCs and AT-MSCs were cultured in growth medium to keep MSCs characteristics or in osteogenic medium to induce osteoblast differentiation (BM-OBs and AT-OBs). The transcriptomic analysis was performed by microarray covering the entire rat functional genome. It was observed that cells from bone marrow presented higher expression of genes related to osteogenesis, whereas cells from adipose tissue showed a higher expression of genes related to angiogenesis and adipocyte differentiation, irrespective of cell differentiation. By comparing cells from the same source, MSCs from both sources exhibited higher expression of genes involved in angiogenesis, osteoblast differentiation, and bone morphogenesis than osteoblasts. The clustering analysis showed that AT-OBs exhibited a gene expression profile closer to MSCs from both sources than BM-OBs, suggesting that BM-OBs were in a more advanced stage of differentiation. In conclusion, our results suggest that in cell-based therapies for bone regeneration AT-MSCs could be considered for angiogenic purposes, whereas BM-MSCs and osteoblasts differentiated from either source could be better for osteogenic approaches.

Effect of stem cells combined with a polymer/ceramic membrane on osteoporotic bone repair

Abstract Cell therapy associated with guided bone regeneration (GBR) can be used to treat bone defects under challenging conditions such as osteoporosis. This study aimed to evaluate the effect of mesenchymal stem cells (MSCs) in combination with a poly(vinylidene-trifluoroethylene)/barium titanate (PVDF-TrFE/BT) membrane on bone repair in osteoporotic rats. Osteoporosis was induced in female rats by bilateral removal of the ovaries (OVX) or sham surgery (SHAM), and the osteoporotic condition was characterized after 5 months by microtomographic and morphometric analyses. Calvarial defects were created in osteoporotic rats that immediately received the PVDF-TrFE/BT membrane. After 2 weeks, bone marrow-derived MSCs from healthy rats, characterized by the expression of surface markers using flow cytometry, or phosphate-buffered saline (PBS) (Control) were injected into the defects and bone formation was evaluated 4 weeks post-injection by microtomographic, morphometric, and histological analyses. A reduction in the amount of bone tissue in the femurs of OVX compared with SHAM rats confirmed the osteoporotic condition of the experimental model. More bone formation was observed when the defects were injected with MSCs compared to that with PBS. The modification that we are proposing in this study for the classical GBR approach where cells are locally injected after a membrane implantation may be a promising therapeutic strategy to increase bone formation under osteoporotic condition.

Menopause transition promotes distinct modulation of mRNAs and miRNAs expression in calvaria and bone marrow osteoblastic cells.

Investigation on functional genome research may contribute to the knowledge of functional roles of different mRNAs and miRNAs in bone cells of osteoporotic animals. Currently, few studies indicate the changes in gene modulation that osteoporosis causes in osteoblastic cells from different sites. Thus, the purpose of this investigation was to evaluate cell viability, alkaline phosphatase activity and modulation of mRNAs/miRNAs in osteoblastic cells from calvaria and bone marrow by means of microarray technology. Wistar female rats were divided in sham operated and ovariectomized groups. After 150 days of ovariectomy, cells were isolated from both sites to perform cell culture. Results showed that calvaria cells from ovariectomized rats had a decrease in viability when compared to control groups and to bone marrow cells from osteoporotic rats after 3 days. Alkaline phosphatase activity decreased in calvaria cells from ovariectomized rats whereas it was increased in bone marrow osteoblastic cells in the same group. Microarray data analysis showed 5447 differentially expressed mRNAs and 82 differentially expressed miRNAs in calvaria cells. The same way, 4399 mRNAs and 54 miRNAs were expressed in bone marrow cells. mRNAs associated with bone metabolism such as Anxa5, Sp7, Spp1, Notch1 were distinctively modulated in both sites, as well as miRNAs such as miR-350, miR-542-3p, miR-204-5p, and miR-30e-3p. The RNA species identified in this study could be further used as targets for treatment or prevention of osteoporosis.

Poly(Vinylidene Fluoride-Trifluorethylene)/barium titanate membrane promotes de novo bone formation and may modulate gene expression in osteoporotic rat model.

Osteoporosis is a chronic disease that impairs proper bone remodeling. Guided bone regeneration is a surgical technique that improves bone defect in a particular region through new bone formation, using barrier materials (e.g. membranes) to protect the space adjacent to the bone defect. The polytetrafluorethylene membrane is widely used in guided bone regeneration, however, new membranes are being investigated. The purpose of this study was to evaluate the effect of P(VDFTrFE)/BT [poly(vinylidene fluoride-trifluoroethylene)/barium titanate] membrane on in vivo bone formation. Twenty-three Wistar rats were submitted to bilateral ovariectomy. Five animals were subjected to sham surgery. After 150 days, bone defects were created and filled with P(VDF-TrFE)/BT membrane or PTFE membrane (except for the sham and OVX groups). After 4 weeks, the animals were euthanized and calvaria samples were subjected to histomorphometric and computed microtomography analysis (microCT), besides real time polymerase chain reaction (real time PCR) to evaluate gene expression. The histomorphometric analysis showed that the animals that received the P(VDF-TrFE)/BT membrane presented morphometric parameters similar or even better compared to the animals that received the PTFE membrane. The comparison between groups showed that gene expression of RUNX2, BSP, OPN, OSX and RANKL were lower on P(VDF-TrFE)/BT membrane; the gene expression of ALP, OC, RANK and CTSK were similar and the gene expression of OPG, CALCR and MMP9 were higher when compared to PTFE. The results showed that the P(VDF-TrFE)/BT membrane favors bone formation, and therefore, may be considered a promising biomaterial to support bone repair in a situation of osteoporosis.

Aging impairs osteoblast differentiation of mesenchymal stem cells grown on titanium by favoring adipogenesis.

OBJECTIVE: We investigated the osteoblast and adipocyte differentiation of mesenchymal stem cells (MSCs) from young and aged rats cultured on Ti. MATERIAL AND METHODS: Bone marrow MSCs derived from 1-month and 21-month rats were cultured on Ti discs under osteogenic conditions for periods of up to 21 days and osteoblast and adipocyte markers were evaluated. RESULTS: Cell proliferation, alkaline phosphatase (ALP) activity, extracellular matrix mineralization and gene expression of RUNX2, osterix, ALP, bone sialoprotein, osteopontin, and osteocalcin were reduced in cultures of 21-month rats compared with 1-month rats grown on Ti. Gene expression of PPAR-γ , adipocyte protein 2, and resistin and lipid accumulation were increased in cultures of 21-month rats compared with 1-month rats grown on the same conditions. CONCLUSIONS: These results indicate that the lower osteogenic potential of MSCs derived from aged rats compared with young rats goes along with the higher adipogenic potential in cultures grown on Ti surface. This unbalance between osteoblast and adipocyte differentiation should be considered in dental implant therapy to the elderly population.

Aging impairs osteoblast differentiation of mesenchymal stem cells grown on titanium by favoring adipogenesis

ABSTRACT Aging negatively affects bone/titanium implant interactions. Our hypothesis is that the unbalance between osteogenesis and adipogenesis induced by aging may be involved in this phenomenon. Objective We investigated the osteoblast and adipocyte differentiation of mesenchymal stem cells (MSCs) from young and aged rats cultured on Ti. Material and Methods Bone marrow MSCs derived from 1-month and 21-month rats were cultured on Ti discs under osteogenic conditions for periods of up to 21 days and osteoblast and adipocyte markers were evaluated. Results Cell proliferation, alkaline phosphatase (ALP) activity, extracellular matrix mineralization and gene expression of RUNX2, osterix, ALP, bone sialoprotein, osteopontin, and osteocalcin were reduced in cultures of 21-month rats compared with 1-month rats grown on Ti. Gene expression of PPAR-γ , adipocyte protein 2, and resistin and lipid accumulation were increased in cultures of 21-month rats compared with 1-month rats grown on the same conditions. Conclusions These results indicate that the lower osteogenic potential of MSCs derived from aged rats compared with young rats goes along with the higher adipogenic potential in cultures grown on Ti surface. This unbalance between osteoblast and adipocyte differentiation should be considered in dental implant therapy to the elderly population.

Osteopontin expression in co-cultures of human squamous cell carcinoma-derived cells and osteoblastic cells and its effects on the neoplastic cell phenotype and osteoclastic activation.

This study evaluated the temporal expression of osteopontin (OPN) in co-cultures of human osteoblastic cells (SAOS-2) and oral squamous cell carcinoma (OSCC)-derived cells (SCC9) and examined the effects of osteoblast-derived OPN on the neoplastic cell phenotype. Additionally, the effects of these co-cultures on subsequent osteoclastic activity were explored. SCC9 cells were plated on Transwell® membranes that were either coated or not coated with Matrigel and were then co-cultured with SAOS-2 cells during the peak of OPN expression. SCC9 cells exposed to OPN-silenced SAOS-2 cultures and SCC9 cells cultured alone served as controls. SCC9 cells were quantitatively evaluated for cell adhesion, proliferation, migration, and invasion into Matrigel. The impact of co-culturing SAOS-2 and SCC9 cells on the resorptive capacity of U-937-derived osteoclastic cells was also investigated. Furthermore, a reciprocal induction of SAOS-2 and SCC9 cells in terms of OPN expression over the co-culture interval was identified. SAOS-2-secreted OPN altered the SCC9 cell phenotype, leading to enhanced cell adhesion and proliferation and higher Matrigel invasion. This invasion was also enhanced, albeit to a lesser degree, by co-culture with OPN-silenced SAOS-2 cells. Cell migration was not affected. Co-culture with SAOS-2 cells-mainly during the period of peak OPN expression-promoted over-expression of IL-6 and IL-8 by SCC9 cells and enhanced the resorptive capacity of osteoclastic cells. Taken together, these results suggest that osteoblast-derived OPN affects the interactions among OSCC-derived epithelial cells, osteoblasts, and osteoclasts, which could contribute to the process of bone destruction during bone invasion by OSCC.

Effect of collagen sponge and fibrin glue on bone repair

ABSTRACT The ability of hemostatic agents to promote bone repair has been investigated using in vitro and in vivo models but, up to now, the results are inconclusive. Objective In this context, the aim of this study was to compare the potential of bone repair of collagen sponge with fibrin glue in a rat calvarial defect model. Material and Methods Defects of 5 mm in diameter were created in rat calvariae and treated with either collagen sponge or fibrin glue; untreated defects were used as control. At 4 and 8 weeks, histological analysis and micro-CT-based histomorphometry were carried out and data were compared by two-way ANOVA followed by Student-Newman-Keuls test when appropriated (p≤0.05). Results Three-dimensional reconstructions showed increased bone formation in defects treated with either collagen sponge or fibrin glue compared with untreated defects, which was confirmed by the histological analysis. Morphometric parameters indicated the progression of bone formation from 4 to 8 weeks. Additionally, fibrin glue displayed slightly higher bone formation rate when compared with collagen sponge. Conclusion Our results have shown the benefits of using collagen sponge and fibrin glue to promote new bone formation in rat calvarial bone defects, the latter being discreetly more advantageous.