Dapeng Liao

Co-culture with Schwann cells is an effective way for adipose-derived stem cells neural transdifferentiation.

Introduction Adipose-derived stem cells (ADSCs) could accomplish neural transdifferentiation with the presence of certain growth factors in vitro. It has been proved that bone marrow stromal cells (BMSCs) can realize neural transdifferentiation only by being co-cultured with Schwann cells (SCs), and in our former studies we have confirmed that ADSCs could do so too. This paper aims to investigate whether the neural induction efficiency of co-culture is as high as that of other strategies using chemicals or chemicals combined with some growth factors. Material and methods We isolated and multiplied ADSCs from adult Sprague-Dawley rats, and SCs from sciatic nerves of 1-to-2-day-old Sprague-Dawley rat pups, then induced ADSCs neural transdifferentiation through 2% dimethyl sulphoxide (DMSO) and DMSO combined with growth factors. Meanwhile we co-cultured ADSCs and SCs in Transwell culture dishes without intercellular contacts. Immunostaining and RT-PCR were adopted to investigate the neural transdifferentiation of ADSCs. Then we compared the expression differences for genes S100, nestin and GFAP of the above three protocols by real-time PCR. Results Both immunostaining and RT-PCR proved that ADSCs could accomplish neural transdifferentiation through each of the above three protocols. And real-time PCR further shows that the gene expression relative quantities for the above three genes are not statistically different between co-culture and induction through DMSO combined with growth factors (p > 0.05), but both of them are statistically different from induction only by DMSO (p < 0.05). Conclusions Co-culturing ADSCs and SCs may be a simple, effective and practical way for ADSCs neural transdifferentiation.

Research on promoting periodontal regeneration with human basic fibroblast growth factor-modified bone marrow mesenchymal stromal cell gene therapy

BACKGROUND AIMS Recently, it has been found that effective periodontal regeneration can be induced by bone marrow mesenchymal stromal cell (BMSC) transplantation or local application of basic fibroblast growth factor (bFGF). The aim of the present study was to assess, in dogs, the efficacy of periodontal regeneration via the delivery of BMSC transfected with bFGF to repair destruction of periodontal tissue. METHODS BMSC from dogs were isolated, cultured and purified via density-gradient centrifugation. Polymerase chain reaction (PCR) was employed to clone bFGF cDNA from human periodontal cells, and the product was then ligated into the eukaryotic expression vector pDC316-IREs-EGFP. BMSC transfected with pDC316bFGF-IREs-EGFP were transplanted into root furcation defects of beagle dogs. After 6 weeks, regeneration in defects was assessed via clinical examination, X-ray, histologic observation and micro-CT analysis. RESULTS DNA sequence analysis showed that the bFGF sequence of recombinant plasmid pDC316bFGF-IREs-EGFP was consistent with that reported by GeneBank. bFGF expression was detected with Western blotting, and active bFGF in supernatant was also observed. Our animal experiment proved that the regenerating speed of periodontal bone tissue in groups transplanted with BMSC containing the modified bFGF gene was higher than in those transplanted with BMSC alone. CONCLUSIONS A successfully constructed eukaryotic expression vector containing human bFGF in pDC316bFGF-IREs-EGFP could produce bioactive bFGF in vitro. bFGF overexpression mediated by the recombinant plasmid pDC316bFGF- IREs-EGFP accelerated periodontal regeneration.

Auto-transplanted mesenchymal stromal cell fate in periodontal tissue of beagle dogs

BACKGROUND AIMS Mesenchymal stromal cells (MSC) possess multilineage differentiation potential and characteristics of self-renewal. It has been reported that MSC can acquire characteristics of cells in the periodontal ligament (PDL) in vitro. Moreover, the transplantation of MSC has been shown to be a promising strategy for treating periodontal defects. However, little is known about the fate of MSC in periodontal tissue in vivo. The aim of this study was to trace the paths of MSC after transplantation into periodontal tissues in vivo. METHODS MSC labeled with bromodeoxyuridine (BrdU) were transplanted into periodontal defects of beagle dogs. Six weeks after surgery, the animals were killed and decalcified specimens were prepared. Migration and differentiation of MSC were detected by single/double immunohistochemistry and a combination of immunohistochemistry and in situ hybridization. RESULTS BrdU-labeled MSC were observed distributing into periodontal tissue that included alveolar bone, PDL, cementum and blood vessels and expressing surface markers typical of osteoblasts and fibroblasts. CONCLUSIONS Cumulatively, our data suggest that MSC migrate throughout periodontal tissue and differentiate into osteoblasts and fibroblasts after transplantation into periodontal defects at 6 weeks in vivo, and have the potential to regenerate periodontal tissue.

In vitro neural/glial differentiation potential of periodontal ligament stem cells.

Introduction It is known that periodontal ligament stem cells (PDLSCs) can differentiate into cementoblast-like cells, adipocytes and collagen-forming cells. However, whether PDLSCs are able to differentiate into Schwann cells and which method is best for their neural induction remain unknown. We attempted to determine whether PDLSCs possessed the potential for neural differentiation in vitro. Materials and methods We isolated and multiplied PDLSCs from periodontal ligaments obtained from the teeth (n = 24) of 8-month-old beagle dogs. Four protocols with different chemicals and growth factors were adopted to induce the PDLSCs to differentiate into Schwann cells. Immunochemistry, RT-PCR and qRT-PCR were performed to investigate the in vitro neural differentiation potential of PDLSCs. Results We compared the 4 different protocols and showed that all 4 protocols could successfully induce PDLSCs to express nestin, GFAP and S100, markers for Schwann cells. Further, qRT-PCR revealed relative differences in the expression levels of these 3 genes in differentiated PDLSCs obtained by different protocols. Conclusions We conclude that PDLSCs have neural/glial differentiation potential in vitro and that neural/glial differentiation can be induced in PDLSCs if suitable protocols are followed. We also found that supplementing the growth medium with suitable growth factors is more effective than applying chemicals alone. While nerve growth factor is more effective than platelet-derived growth factor for inducing neural/glial differentiation in PDLSCs, pre-induction of PDLSCs with dimethyl sulphoxide yields better results than those obtained with all-trans-retinoic acid.

Effect of implant surface microtopography on proliferation, neurotrophin secretion, and gene expression of Schwann cells.

The purpose of this study was to evaluate the effect of different implant surface properties on the morphology, proliferation, neurotrophin secretion, and gene expression of Schwann cells. Four types of implant surfaces, including ground (smooth surface), sandblasted and acid-etched (SLA), hydroxyapatite-coated (HA), and titanium plasma spray (TPS) surfaces were fabricated and photographed by a scanning electron microscopy (SEM). Schwann cells derived from neonatal rats were cultured on the implant surfaces and assessed via SEM observation and methylthiazol tetrazolium (MTT) colorimetric assay. The secretions and mRNA levels of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) were measured by enzyme-linked immunosorbent assay (ELISA) and quantitative real time RT-PCR, respectively, on days 3 and 7. Tissue culture plastic was used as a control. The results demonstrated that Schwann cells exhibited typical bipolar spindle morphology on various surfaces, and proliferated faster than the control. Neurotrophin secretion and gene expression of both BDNF and NGF were also increased by implant surfaces. This study suggests that the function of Schwann cells can be enhanced by implant implants.