Osteocytes are terminally differentiated osteoblasts that secrete molecules that regulate bone-tissue homeostasis. Considering that the extracellular matrix protein agrin (AGRN) is secreted by osteoblasts and modulates their differentiation, we hypothesized that AGRN is also expressed by osteocytes and plays a role in their function and therefore in bone remodeling. To test this hypothesis, we deleted agrin specifically in osteocytes using dentin matrix acidic phosphoprotein 1 (DMP1)-Cre mice (C57/BL6 background) and silenced agrin in vitro using clustered regularly interspaced short palindromic repeats/associated nuclease Cas-9 in the Ocy454 osteocyte cell line. We found that osteocytes express agrin and its receptors, low-density lipoprotein receptor-related protein 4, and α-dystroglycan, and that mice with agrin-deficient osteocytes exhibited lower bone mass and impaired mechanical and chemical properties of bone tissue. Agrin knockdown in Ocy454 cells disrupted osteocyte differentiation and function, which reduced osteoblast and increased osteoclast differentiation in a cell co-culture model. Our results showed that agrin is expressed by osteocytes, which are key regulators of bone mass and its mechanical and chemical properties. These findings indicate that agrin may be a therapeutic target because it is important to maintain the balance of the osteocyte-osteoblast-osteoclast circuit, and consequently, bone tissue homeostasis.

Titanium (Ti) implant osseointegration is regulated by the crosstalk among bone cells that are affected by epigenetic machinery, including the regulation of long non-coding RNAs (lncRNAs). Nanotopography Ti (Ti Nano) induces the differentiation of osteoblasts that are inhibited by osteoclasts through epigenetic mechanisms. Thus, we hypothesize that osteoclasts affect lncRNA expression in Ti Nano-cultivated osteoblasts. Osteoblasts were grown on Ti Nano and Ti Control that were then co-cultured with osteoclasts for 48 h. Using RNAseq, we identified 252 modulated lncRNAs in osteoblasts regulated by both surfaces of Ti, but mainly in Ti Nanocultivated osteoblasts. A negative correlation was observed between Kcnq1ot1 and the mRNAs of Alpl, Bglap, Bmp8a, Col1a1, and Vim in Ti Nano-cultivated osteoblasts with osteoclasts. The pull-down indicated that Bglap mRNA is a direct target of Kcnq1ot1, with enhanced physical interaction in Ti Nano-cultivated osteoblasts, and greater osteoclast inhibition than the Ti Control. The bone marker expression at the levels of mRNA and protein were downregulated by the Kcnq1ot1 silencing, indicating its pivotal role in osteoblast differentiation. These results showed that nanostructured Ti surface modulates the osteoblast-osteoclast crosstalk, at least in part, through the regulation of lncRNA expression in osteoblasts. We demonstrate that the lncRNA Kcnq1ot1 directly interacts with Bglap mRNA, and this interaction is enhanced by nanotopography and reduced by osteoclasts with greater intensity in Ti Nano-cultivated osteoblasts. These findings confirm the molecular mechanisms associated with the high osteogenic potential of nanotopography and can potentially support osteointegration of dental and skeletal prostheses.

Background Diabetes mellitus (DM) negatively impacts bone tissue, leading to bone loss and increased fracture risk. Many individuals need additional treatments, and therapy based on mesenchymal stromal cells (MSCs) represents a promising treatment for bone defects in patients with diabetes. Aims The present study explored the effects of interactions between MSCs from normoglycemic (NG-MSCs) and diabetic (DM-MSCs) donors on osteoblast differentiation and the effects of cell therapy using NG-MSCs on bone regeneration in defects created in diabetic rats. Methods After inducing DM with streptozotocin, we evaluated the morphometric parameters of rat femurs and the osteoblast differentiation of MSCs, as well as the effects of the interaction between NG-MSCs and DM-MSCs on their osteoblast differentiation. The efficacy of cell therapy was measured by evaluating the bone repair in calvarial defects of diabetic rats treated with local injections of either NG-MSCs or a vehicle. Results DM induced bone loss and impaired the osteoblast differentiation of MSCs, which was partially restored by NG-MSCs, while the bone formation observed in defects treated with NG-MSCs and the vehicle was similar. Conclusion These results indicate that the beneficial effect of NG-MSCs on DM-MSCs did not translate into enhanced bone repair, mainly due to a hostile environment created by hyperglycemia, which compromised the ability of MSCs to induce bone formation.

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.

OBJECTIVE:Hypertension disrupts the bone integrity and its repair ability. This study explores the efficiency of a therapy based on the application of mesenchymal stem cells (MSCs) to repair bone defects of spontaneously hypertensive rats (SHR). METHODS:First, we evaluated SHR in terms of bone morphometry and differentiation of MSCs into osteoblasts. Then, the effects of the interactions between MSCs from normotensive rats (NTR-MSCs) cocultured with SHR (SHR-MSCs) on the osteoblast differentiation of both cell populations were evaluated. Also, bone formation into calvarial defects of SHR treated with NTR-MSCs was analyzed. RESULTS:Hypertension induced bone loss evidenced by reduced bone morphometric parameters of femurs of SHR compared with NTR as well as decreased osteoblast differentiation of SHR-MSCs compared with NTR-MSCs. NTR-MSCs partially restored the capacity of SHR-MSCs to differentiate into osteoblasts, while SHR-MSCs exhibited a slight negative effect on NTR-MSCs. An enhanced bone repair was observed in defects treated with NTR-MSCs compared with control, stressing this cell therapy efficacy even in bones damaged by hypertension. CONCLUSION:The use of MSCs derived from a heathy environment can be in the near future a smart approach to treat bone loss in the context of regenerative dentistry for oral rehabilitation of hypertensive patients.

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.

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.

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.

Type 1 (T1DM) and type 2 (T2DM) diabetes mellitus are characterized by changes in glucose metabolism and cause bone damage via a variety of mechanisms, including effects on osteoblasts. We aimed to evaluate the osteoblast differentiation of mesenchymal stem cells (MSCs) from rats with T1DM or T2DM and the effects of removing the hyperglycemic stimulus on the osteogenic potential of these cells. MSCs from healthy rats were cultured in normoglycemic medium, whereas MSCs from rats with T1DM or T2DM were cultured in hyperglycemic or normoglycemic medium. T1DM and T2DM reduced osteoblast differentiation of MSCs grown in hyperglycemic media, with T1DM having a more pronounced effect, as evidenced by alkaline phosphatase activity, RUNX2 protein expression, and extracellular matrix mineralization, and modulated the gene expression of several components of the bone morphogenetic protein signaling pathway. The restoration of the normoglycemic environment partially recovers the osteogenic potential of MSCs from rats with T1DM but not with T2DM. Our findings highlight the need for specific therapies to treat T1DM- or T2DM-induced bone loss, as both disrupt osteoblast differentiation at distinct levels and likely through different mechanisms.

Diabetes mellitus (DM) is a chronic metabolic disease that affects bone metabolism, which can be related to a reduced osteogenic potential of bone marrow mesenchymal stem cells (BM-MSCs). MSCs from diabetic rats (dBM-MSC) have shown a tendency to differentiate towards adipocytes (AD) instead of osteoblasts (OB). Since photobiomodulation (PBM) therapy is a non-invasive treatment capable of recovering the osteogenic potential of dBM-MSCs, we aimed to evaluate whether PBM can modulate MSC's differentiation under hyperglycemic conditions. BM-MSCs of healthy and diabetic rats were isolated and differentiated into osteoblasts (OB and dOB) and adipocytes (AD and dAD). dOB and dAD were treated with PBM every 3 days (660 nm; 5 J/cm2; 0.14 J; 20 mW; 0.714 W/cm2) for 17 days. Cell morphology and viability were evaluated, and cell differentiation was confirmed by gene expression (RT-PCR) of bone (Runx2, Alp, and Opn) and adipocyte markers (Pparγ, C/Ebpα, and C/Ebpβ), production of extracellular mineralized matrix (Alizarin Red), and lipid accumulation (Oil Red). Despite no differences on cell morphology, the effect of DM on cells was confirmed by a decreased gene expression of bone markers and matrix production of dOB, and an increased expression of adipocyte and lipid accumulation of dAD, compared to heatlhy cells. On the other hand, PBM reversed the effects of dOB and dAD. The negative effect of DM on cells was confirmed, and PBM improved OB differentiation while decreasing AD differentiation, driving the fate of dBM-MSCs. These results may contribute to optimizing bone regeneration in diabetic patients.

This study evaluates the effects of the availability of exogenous BMP-7 on osteoblastic cells' differentiation on a nanotextured Ti surface obtained by chemical etching (Nano-Ti). The MC3T3-E1 and UMR-106 osteoblastic cell lines were cultured for 5 and 7 days, respectively, on a Nano-Ti surface and on a control surface (Control-Ti) in an osteogenic medium supplemented with either 40 or 200 ng/mL recombinant mouse (rm) BMP-7. The results showed that MC3T3-E1 cells exhibited distinct responsiveness when exposed to each of the two rmBMP-7 concentrations, irrespective of the surface. Even with 40 ng/mL rmBMP-7, important osteogenic effects were noticed for Control-Ti in terms of cell proliferation potential; Runx2, Osx, Alp, Bsp, Opn, and Smad1 mRNA expression; and in situ ALP activity. For Nano-Ti, the effects were limited to higher Alp, Bsp, and Opn mRNA expression and in situ ALP activity. On both surfaces, the osteogenic potential of UMR-106 cultures remained unaltered with 40 ng/mL rmBMP-7, but it was significantly reduced when the cultures were exposed to the 200 ng/mL concentration. The availability of rmBMP-7 to pre-osteoblastic cells at the concentrations used alters the expression profile of osteoblast markers, indicative of the acquisition of a more advanced stage of osteoblastic differentiation. This occurs less pronouncedly on the nanotextured Ti and without reflecting in higher mineralized matrix production by differentiated osteoblasts on both surfaces.

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/cm(2) 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.

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.

Since their discovery, mesenchymal stromal cells (MSCs) have received a lot of attention, mainly due to their self-renewal potential and multilineage differentiation capacity. For these reasons, MSCs are a useful tool in cell biology and regenerative medicine. In this article, we describe protocols to isolate MSCs from bone marrow (BM-MSCs) and adipose tissues (AT-MSCs), and methods to culture, characterize, and differentiate MSCs into osteoblasts, adipocytes, and chondrocytes. After the harvesting of cells from bone marrow by flushing the femoral diaphysis and enzymatic digestion of abdominal and inguinal adipose tissues, MSCs are selected by their adherence to the plastic tissue culture dish. Within 7 days, MSCs reach 70% confluence and are ready to be used in subsequent experiments. The protocols described here are easy to perform, cost-efficient, require minimal time, and yield a cell population rich in MSCs.

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 β‐catenin demonstrated that Fzd6 disruption inhibited the osteoblast differentiation induced by Ti‐Nano by preventing the activation of Wnt/β‐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/β‐catenin signaling and the cellular mechanisms modulated by nanotopography during osteoblast differentiation.

The aim of this study was to evaluate the in vitro osteogenic potential of osteoblasts from neural crest-derived frontal bone (OB-NC) and mesoderm-derived parietal bone (OB-MS) and the bone formation induced by them when injected into calvarial defects. Calvarial bones were collected from newborn Wistar rats (3-day old) and characterized as frontal and parietal prior to OB-NC and OB-MS harvesting. The cells were cultured, and several parameters of osteoblast differentiation were evaluated. These cells, or PBS without cells (control), were locally injected into 5-mm rat calvarial defects (5 × 106 cells/defect) and after 4 weeks bone formation was evaluated by morphometric and histological analyses. The characterization of frontal and parietal bones assured the different embryonic origin of both cell populations, OB-NC and OB-MS. The OB-NC presented higher proliferation while the OB-MS presented higher alkaline phosphatase (ALP) activity, extracellular matrix mineralization and gene expression of runt-related transcription factor 2, Alp, bone sialoprotein and osteocalcin revealing their high osteogenic potential. µCT analysis indicated that there was higher amount of bone formation in defects injected with both OB-NC and OB-MS compared to the control. Moreover, the bone tissue formed by both cells displayed the same histological characteristics. Despite the distinct in vitro osteogenic potential, OB-NC and OB-MS induced similar bone repair in a rat calvarial defect model. Thus, osteoblasts, irrespective of their in vitro osteogenic potential linked to embryonic origins, seem to be suitable for cell-based therapies aiming to repair bone defects.

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.