Notch pathway activation promotes the differentiation of beagle dog periodontal ligament stem cells to Schwann cells.

BACKGROUND: Periodontal ligament stem cells (PDLSCs) have demonstrated the potential for differentiation into many cell types, though the molecular mechanism of their neural differentiation in particular remains largely unknown. OBJECTIVES: The Notch signaling pathway plays a key role in regulating cell differentiation and development. In this article, we explore its potential role in the differentiation of PDLSCs to Schwann cells (SCs). MATERIAL AND METHODS: The PDLSCs were either transfected with viral vectors carrying genetic material for Notch Delta ligands, thereby induced their overexpression, or treated with DAPT (a Notch-pathway-specific inhibitor) to inhibit γ-secretase. The potential effects of Notch signaling on myelination and SCs differentiation were then investigated using western blotting, immunostaining and reverse transcriptase polymerase chain reaction (RT-PCR) to detect the expression of SC-specific marker genes. RESULTS: Specifically inhibiting Notch signaling with DAPT decreased the expression of SC-specific marker genes GFAP, S100 and P75, as well as of SC-myelin-related genes PMP22, MBP, connexin, and P0 in cells undergoing induced differentiation from PDLSCs. Conversely, activating Notch signaling through overexpression of Delta ligands enhanced the expression of SC-specific marker genes as well as myelin-related genes in cells undergoing induced differentiation from PDLSCs. This promotion was reversed by DAPT. CONCLUSIONS: The Notch signaling pathway positively regulated the process of PDLSC differentiation into SCs, and the activation of this signaling was important in maintaining the differentiation of PDLSCs to SCs, and then SC myelination. These results may improve the method of obtaining pure SCs from PDLSCs for transplantation application.

Odontogenesis and neuronal differentiation characteristics of periodontal ligament stem cells from beagle dog.

Periodontal ligament stem cells (PDLSCs) from beagle dogs had the characteristics of multi-directional differentiation and had great application potential in tissue engineering and cell regenerative medicine. In this study, we analysed the odontogenesis and neuronal differentiation characteristics of PDLSCs in vitro. Results showed that the calcined tooth powder (CTP) and silver nanoparticles (AgNPs) additives could induce the PDLSCs into odontogenesis differentiation; besides, the immunofluorescence staining identified that the high dosage calcined tooth powder (400 µg/mL) significantly facilitated the odontogenesis associated with BMP4 expression. While the nutritional factor (L-glutamine, NGF (nerve growth factor), bFGF (basic fibroblast growth factor), IGF-1 (insulin-like growth factor-1) and EGF (epidermal growth factor)) additives were prior to induce the PDLSCs into neuronal differentiation. Simultaneously, PDLSCs had high proliferation ability with the different supplemented additives. Importantly, the Western blot results also proved the BMP4 and SMAD1 proteins were highly expressed in the induced odontoblast, while the SOX1, NCAM1, GFAP and VEGFA proteins were all obviously expressed in the induced neurons. Hence, PDLSCs had characteristics of both odontogenesis and neuronal differentiation.

The Role of Wnt/ß-Catenin Signaling Pathway in the Transdifferentiation from Periodontal Ligament Stem Cells to Schwann Cells.

This study was aimed to investigate the role of Wnt/ß-catenin signaling pathway in the differentiation from periodontal ligament stem cells (PDLSCs) to Schwann cells (SCs) and the possible mechanisms. FzB was applied to inhibit the Wnt/ß-catenin signaling pathway of differentiated PDLSCs (dPDLSCs), and then immunofluorescence, polymerase chain reaction (PCR), and Western blotting analysis were performed to detect SC marker genes and proteins such as S100, glial fibrillary acidic protein (GFAP), and P75NTR. Results showed that when the Wnt/ß-catenin signaling pathway was inhibited, the expression of S100, GFAP, and P75NTR protein significantly decreased in dPDLSCs, p < 0.05, whereas PCR results showed that expression of SC myelinogenesis-related genes krox-20, Oct-6, P0, and PMP-22 was significantly downregulated at the same time, p < 0.05. These results showed that Wnt/ß-catenin signaling pathway participated in the differentiation from PDLSCs to SCs, and inhibiting it could inhibit the differentiation process.

Biological behavior of neurally differentiated periodontal ligament stem cells on different titanium implant surfaces.

We investigated the biological behavior of periodontal ligament stem cells (PDLSCs) induced to differentiate into Schwann cells (SCs) on the surfaces of titanium discs. Two types of titanium surfaces, sand blasted and acid etched (SA) and smooth polished, were prepared on titanium discs, and the behavior of SC-like cells on these discs was investigated. Cell morphology was examined by scanning electron microscopy, and cell proliferation was assessed using assays for methylthiazol tetrazolium metabolism and total protein content. Real-time polymerase chain reaction and Western blotting analyses were used to determine the gene and protein expression, respectively, of SC markers by PDLSCs. Differentiated PDLSCs could adhere, proliferate, differentiate, and express SC marker genes and proteins on the prepared titanium surfaces, and the highest levels of SC marker protein expression were observed in PDLSCs on SA titanium surfaces. SA titanium surfaces show good biocompatibility for the SC-like cells, which is important for the application of these cells in peri-implant nerve tissue engineering, through which they are expected to improve the osseoperception of dental implants.

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.

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.

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.

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.

[A fundamental study on bioreactions of Sr-HA].

OBJECTIVE: Sr-HA, a new type of hydroxyapatite biomaterial, was implanted into animals to study the bioreaction and character, which would be helpful for the further clinical applications in the future. METHODS: Totally 24 rabbits were divided into 3 groups. The bone defect of 6 mm x 12 mm x 4 mm was made at both mandibular angles of rabbits and Sr-HA of different proportion (10%, 5%, 0) was applied to reform the defects. One group of animals were killed randomly at 1, 3 and 6 months after operation to evaluate the material biological compatibility using anatomic, X-ray examination, histological and ECT methods. RESULTS: The histological photographs showed that Sr-HA caused little infection around implanted area and, almost was not repulsed by hosts. With the degradation of biomaterial, there was more apparent new bone growth in the area around Sr-HA than that around HA and some ossification can be found in soft tissue nearby. Also a tight osteointegrity was gradually got after the operation, according to the results of X-ray and, the border between Sr-HA and bone was hardly discovered at the 6th month after the operation. A more obvious nuclide assembling was observed at the side of Sr-HA by ECT images. With the biodegradation of Sr-HA, more new bone was intruded into the spare space of the biomaterial. CONCLUSION: Sr-HA has better biocompatibility and higher biodegradation than that of pure HA. It holds an excellent osteoinductivity and fair osteoconductivity to some degree too. So a more satisfying effect of bone defect rehabilitation was gained with the increasing new bone depositing in the free space of the material, when it degraded gradually.