BMP9-initiated osteogenic/odontogenic differentiation of mouse tooth germ mesenchymal cells (TGMCS) requires Wnt/ß-catenin signalling activity.

Teeth arise from the tooth germ through sequential and reciprocal interactions between immature epithelium and mesenchyme during development. However, the detailed mechanism underlying tooth development from tooth germ mesenchymal cells (TGMCs) remains to be fully understood. Here, we investigate the role of Wnt/ß-catenin signalling in BMP9-induced osteogenic/odontogenic differentiation of TGMCs. We first established the reversibly immortalized TGMCs (iTGMCs) derived from young mouse mandibular molar tooth germs using a retroviral vector expressing SV40 T antigen flanked with the FRT sites. We demonstrated that BMP9 effectively induced expression of osteogenic markers alkaline phosphatase, collagen A1 and osteocalcin in iTGMCs, as well as in vitro matrix mineralization, which could be remarkably blunted by knocking down ß-catenin expression. In vivo implantation assay revealed that while BMP9-stimulated iTGMCs induced robust formation of ectopic bone, knocking down ß-catenin expression in iTGMCs remarkably diminished BMP9-initiated osteogenic/odontogenic differentiation potential of these cells. Taken together, these discoveries strongly demonstrate that reversibly immortalized iTGMCs retained osteogenic/odontogenic ability upon BMP9 stimulation, but this process required the participation of canonical Wnt signalling both in vitro and in vivo. Therefore, BMP9 has a potential to be applied as an efficacious bio-factor in osteo/odontogenic regeneration and tooth engineering. Furthermore, the iTGMCs may serve as an important resource for translational studies in tooth tissue engineering.

A thermoresponsive polydiolcitrate-gelatin scaffold and delivery system mediates effective bone formation from BMP9-transduced mesenchymal stem cells.

Successful bone tissue engineering requires at the minimum sufficient osteoblast progenitors, efficient osteoinductive factors, and biocompatible scaffolding materials. We previously demonstrated that bone morphogenetic protein 9 (BMP9) is one of the most potent factors in inducing osteogenic differentiation of mesenchymal stem cells (MSCs). Here, we investigated the potential use of a biodegradable citrate-based thermosensitive macromolecule, poly(polyethyleneglycol citrate-co-N-isopropylacrylamide) (PPCN) mixed with gelatin (PPCNG) as a scaffold for the delivery of BMP9-stimulated MSCs to promote localized bone formation. The addition of gelatin to PPCN effectively enhanced the cell adhesion and survival properties of MSCs entrapped within the gel in 3D culture. Using the BMP9-transduced MSC line immortalized mouse embryonic fibroblasts (iMEFs), we found that PPCNG facilitated BMP9-induced osteogenic differentiation of iMEFs in vivo and promoted the formation of well-ossified and vascularized trabecular bone-like structures in a mouse model of ectopic bone formation. Histologic evaluation revealed that vascularization of the bony masses retrieved from the iMEFs + PPCNG group was significantly more pronounced than that of the direct cell injection group. Accordingly, vascular endothelial growth factor (VEGF) expression was shown to be significantly higher in the bony masses recovered from the iMEFs + PPCNG group. Taken together, our results suggest that PPCNG may serve as a novel biodegradable and injectable scaffold and carrier for gene and cell-based bone tissue engineering.

Canonical Wnt signaling acts synergistically on BMP9-induced osteo/odontoblastic differentiation of stem cells of dental apical papilla (SCAPs).

Dental pulp/dentin regeneration using dental stem cells combined with odontogenic factors may offer great promise to treat and/or prevent premature tooth loss. Here, we investigate if BMP9 and Wnt/ß-catenin act synergistically on odontogenic differentiation. Using the immortalized SCAPs (iSCAPs) isolated from mouse apical papilla tissue, we demonstrate that Wnt3A effectively induces early osteogenic marker alkaline phosphatase (ALP) in iSCAPs, which is reduced by ß-catenin knockdown. While Wnt3A and BMP9 enhance each other's ability to induce ALP activity in iSCAPs, silencing ß-catenin significantly diminishes BMP9-induced osteo/odontogenic differentiation. Furthermore, silencing ß-catenin reduces BMP9-induced expression of osteocalcin and osteopontin and in vitro matrix mineralization of iSCAPs. In vivo stem cell implantation assay reveals that while BMP9-transduced iSCAPs induce robust ectopic bone formation, iSCAPs stimulated with both BMP9 and Wnt3A exhibit more mature and highly mineralized trabecular bone formation. However, knockdown of ß-catenin in iSCAPs significantly diminishes BMP9 or BMP9/Wnt3A-induced ectopic bone formation in vivo. Thus, our results strongly suggest that ß-catenin may play an important role in BMP9-induced osteo/ondontogenic signaling and that BMP9 and Wnt3A may act synergistically to induce osteo/odontoblastic differentiation of iSCAPs. It's conceivable that BMP9 and/or Wnt3A may be explored as efficacious biofactors for odontogenic regeneration and tooth engineering.

Bone morphogenetic protein-9 effectively induces osteo/odontoblastic differentiation of the reversibly immortalized stem cells of dental apical papilla.

Dental pulp/dentin regeneration using dental stem cells combined with odontogenic factors may offer great promise to treat and/or prevent premature tooth loss. We previously demonstrated that bone morphogenetic protein 9 (BMP9) is one of the most potent factors in inducing bone formation. Here, we investigate whether BMP9 can effectively induce odontogenic differentiation of the stem cells from mouse apical papilla (SCAPs). Using a reversible immortalization system expressing SV40 T flanked with Cre/loxP sites, we demonstrate that the SCAPs can be immortalized, resulting in immortalized SCAPs (iSCAPs) that express mesenchymal stem cell markers. BMP9 upregulates Runx2, Sox9, and PPARγ2 and odontoblastic markers, and induces alkaline phosphatase activity and matrix mineralization in the iSCAPs. Cre-mediated removal of SV40 T antigen decreases iSCAP proliferation. The in vivo stem cell implantation studies indicate that iSCAPs can differentiate into bone, cartilage, and, to lesser extent, adipocytes upon BMP9 stimulation. Our results demonstrate that the conditionally iSCAPs not only maintain long-term cell proliferation but also retain the ability to differentiate into multiple lineages, including osteo/odontoblastic differentiation. Thus, the reversibly iSCAPs may serve as an important tool to study SCAP biology and SCAP translational use in tooth engineering. Further, BMP9 may be explored as a novel and efficacious factor for odontogenic regeneration.

Adenovirus-mediated gene transfer in mesenchymal stem cells can be significantly enhanced by the cationic polymer polybrene.

Mesenchymal stem cells (MSCs) are multipotent progenitors, which can undergo self-renewal and give rise to multi-lineages. A great deal of attentions have been paid to their potential use in regenerative medicine as potential therapeutic genes can be introduced into MSCs. Genetic manipulations in MSCs requires effective gene deliveries. Recombinant adenoviruses are widely used gene transfer vectors. We have found that although MSCs can be infected in vitro by adenoviruses, high virus titers are needed to achieve high efficiency. Here, we investigate if the commonly-used cationic polymer Polybrene can potentiate adenovirus-mediated transgene delivery into MSCs, such as C2C12 cells and iMEFs. Using the AdRFP adenovirus, we find that AdRFP transduction efficiency is significantly increased by Polybrene in a dose-dependent fashion peaking at 8 µg/ml in C2C12 and iMEFs cells. Quantitative luciferase assay reveals that Polybrene significantly enhances AdFLuc-mediated luciferase activity in C2C12 and iMEFs at as low as 4 µg/ml and 2 µg/ml, respectively. FACS analysis indicates that Polybrene (at 4 µg/ml) increases the percentage of RFP-positive cells by approximately 430 folds in AdRFP-transduced iMEFs, suggesting Polybrene may increase adenovirus infection efficiency. Furthermore, Polybrene can enhance AdBMP9-induced osteogenic differentiation of MSCs as early osteogenic marker alkaline phosphatase activity can be increased more than 73 folds by Polybrene (4 µg/ml) in AdBMP9-transduced iMEFs. No cytotoxicity was observed in C2C12 and iMEFs at Polybrene up to 40 µg/ml, which is about 10-fold higher than the effective concentration required to enhance adenovirus transduction in MSCs. Taken together, our results demonstrate that Polybrene should be routinely used as a safe, effective and inexpensive augmenting agent for adenovirus-mediated gene transfer in MSCs, as well as other types of mammalian cells.

Characterization of chondrocyte scaffold carriers for cell-based gene therapy in articular cartilage repair.

Articular cartilage lesions in the knee are common injuries. Chondrocyte transplant represents a promising therapeutic modality for articular cartilage injuries. Here, we characterize the viability and transgene expression of articular chondrocytes cultured in three-dimensional scaffolds provided by four types of carriers. Articular chondrocytes are isolated from rabbit knees and cultured in four types of scaffolds: type I collagen sponge, fibrin glue, hyaluronan, and open-cell polylactic acid (OPLA). The cultured cells are transduced with adenovirus expressing green fluorescence protein (AdGFP) and luciferase (AdGL3-Luc). The viability and gene expression in the chondrocytes are determined with fluorescence microscopy and luciferase assay. Cartilage matrix production is assessed by Alcian blue staining. Rabbit articular chondrocytes are effectively infected by AdGFP and exhibited sustained GFP expression. All tested scaffolds support the survival and gene expression of the infected chondrocytes. However, the highest transgene expression is observed in the OPLA carrier. At 4 weeks, Alcian blue-positive matrix materials are readily detected in OPLA cultures. Thus, our results indicate that, while all tested carriers can support the survival of chondrocytes, OPLA supports the highest transgene expression and is the most conductive scaffold for matrix production, suggesting that OPLA may be a suitable scaffold for cell-based gene therapy of articular cartilage repairs.