Efficacy of mesenchymal stem cell-based therapy on the bone repair of hypertensive rats.

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.

Normoglycemia partially recovers the disrupted osteoblast differentiation of mesenchymal stem cells induced by type 1 but not type 2 diabetes mellitus.

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.

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.

Role of embryonic origin on osteogenic potential and bone repair capacity of rat calvarial osteoblasts.

INTRODUCTION: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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 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.

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.

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.