Bong-Wook Park

In vitro and in vivo osteogenesis of human mesenchymal stem cells derived from skin, bone marrow and dental follicle tissues

The present study evaluated the human mesenchymal stem cells (hMSCs) isolated from skin (hSMSC), bone marrow (hBMSC) and dental follicle (hDFMSC) tissues on their in vitro and in vivo osteogenic potential using demineralized bone matrix (DBM) and fibrin glue scaffold. Cells originated from three distinct tissues showed positive expressions of CD44, CD73, CD90, CD105 and vimentin, and differentiation ability into osteocytes, adipocytes and chondrocytes. hMSCs from all tissues co-cultured with a mixed DBM and fibrin glue scaffold in non-osteogenic induction media were positively stained by von Kossa and expressed osteoblast-related genes, such as osteocalcin (OC), osteonectin (ON), runt-related transcription factor 2 (Runx2) and osterix. For in vivo osteogenic evaluation, PKH26 labeled hMSCs were implanted into the subcutaneous spaces of athymic mice with a mixed scaffold. At 4 weeks of implantation, PKH26 labeled cells were detected in all hMSC-implanted groups. Bone formation with OC expression and radio-opacity intensity were observed around DBM scaffold in all hMSC-implanted groups. Interestingly, hDFMSCs-implanted group showed the highest OC expression and calcium content. These findings demonstrated that hDFMSCs could be a potential alternative autologous cell source for bone tissue engineering.

Multilineage potential and proteomic profiling of human dental stem cells derived from a single donor.

Dental tissues provide an alternative autologous source of mesenchymal stem cells (MSCs) for regenerative medicine. In this study, we isolated human dental MSCs of follicle, pulp and papilla tissue from a single donor tooth after impacted third molar extraction by excluding the individual differences. We then compared the morphology, proliferation rate, expression of MSC-specific and pluripotency markers, and in vitro differentiation ability into osteoblasts, adipocytes, chondrocytes and functional hepatocyte-like cells (HLCs). Finally, we analyzed the protein expression profiles of undifferentiated dental MSCs using 2DE coupled with MALDI-TOF-MS. Three types of dental MSCs largely shared similar morphology, proliferation potential, expression of surface markers and pluripotent transcription factors, and differentiation ability into osteoblasts, adipocytes, and chondrocytes. Upon hepatogenic induction, all MSCs were transdifferentiated into functional HLCs, and acquired hepatocyte functions by showing their ability for glycogen storage and urea production. Based on the proteome profiling results, we identified nineteen proteins either found commonly or differentially expressed among the three types of dental MSCs. In conclusion, three kinds of dental MSCs from a single donor tooth possessed largely similar cellular properties and multilineage potential. Further, these dental MSCs had similar proteomic profiles, suggesting their interchangeable applications for basic research and call therapy.

Peripheral nerve regeneration using autologous porcine skin-derived mesenchymal stem cells.

Porcine skin‐derived mesenchymal stem cells (pSMSCs) were evaluated on their biological MSC characterizations and differentiation into mesenchymal lineages, along with in vitro and in vivo neural inductions. Isolated pSMSCs showed plate‐adherent growth, expression of various MSC‐marker proteins and transcriptional factors, and differentiation potential into mesenchymal lineages. Neuron‐like cell morphology and various neural markers were highly detected at 6 h and 24 h after in vitro neural induction of pSMSCs, but their neuron‐like characteristics disappeared as induction time extended to 48 and 72 h. To evaluate the in vivo peripheral nerve regeneration potential of pSMSCs, a total of 5 × 106 autologous pSMSCs labelled with tracking dye, supplemented with fibrin glue scaffold and collagen tubulization, were transplanted into the peripheral nerve defected miniature pigs. At 2 and 4 weeks after cell transplantation, well‐preserved transplanted cells and remarkable in vivo nerve regeneration, including histologically complete nerve bundles, were observed in the regenerated nerve tissues. Moreover, S‐100 protein and p75 nerve growth factor receptor were more highly detected in regenerated nerve fibres compared to non‐cell grafted control fibres. These results suggest that autologous pSMSCs transplanted with fibrin glue scaffold can induce prominent nerve regeneration in porcine peripheral nerve defect sites. Copyright

Cryopreservation of human dental follicle tissue for use as a resource of autologous mesenchymal stem cells.

The main purpose of this study was to develop a cryopreservation method for human dental follicle tissue to maintain autologous stem cells as a resource. A modified cryoprotectant, consisting of 0.05 m glucose, 0.05 m sucrose and 1.5 m ethylene glycol in phosphate‐buffered saline (PBS) was employed, with a slow‐ramp freezing rate. We observed > 70% of cell survival rate after 3 months of tissue storage. Isolated and cultured human dental stem cells (hDSCs) from cryopreserved dental follicles expressed mesenchymal stem cell markers at a level similar to that of hDSCs from fresh tissue. They also successfully differentiated in vitro into the mesenchymal lineage, osteocytes, adipocytes and chondrocytes under specific inductions. Using immunohistochemistry, the early transcription factors OCT4, NANOG and SOX2 were moderately or weakly detected in the nucleus of both fresh and cryopreserved dental follicles. In addition, p63, CCND1, BCL2 and BAX protein expression levels were the same in both fresh and cryopreserved tissues. However, the positive‐cell ratio and intensity of p53 protein was higher in cryopreserved tissues than in fresh tissues, indicating direct damage of the freeze–thawing process. Real‐time PCR analysis of hDSCs at passage 2 from both fresh and cryopreserved dental follicles showed similar levels of mRNA for apoptosis‐ and transcription‐related genes. Based on these results, a newly developed cryoprotectant, along with a slow ramp rate freezing procedure allows for long‐term dental tissue preservation for later use as an autologous stem cell resource in regenerative cell therapy. Copyright

Comparative Analysis of Telomere Length, Telomerase and Reverse Transcriptase Activity in Human Dental Stem Cells:

Stem cells from dental tissues have been isolated and established for tooth regenerative applications. However, basic characterization on their biological properties still needs to be investigated before employing them for effective clinical trials. In this study, we compared the telomere length, relative telomerase activity (RTA), and relative reverse transcriptase activity (RRA) as well as the surface antigen profile and mesenchymal differentiation ability in human dental papilla stem cells (DPaSCs), dental pulp stem cells (DPuSCs), and dental follicle stem cells (DFSCs) with mesenchymal stem cells (MSCs) derived from bone marrow. Dental stem cells (DSCs) were strongly positive for cell surface markers, such as CD44 and CD90. However, slightly lower expression of CD105 was observed in DPaSCs and DPuSCs compared to DFSCs and MSCs. Following specific induction, DPaSCs, DFSCs, and MSCs were successfully differentiated into adipocytes and osteocytes. However, DPuSCS, in particular, were able to differentiate into adipocytes but failed to induce into osteogenic differentiation. Further, all DSCs, MSCs, and MRC-5 fibroblasts as control were investigated for telomere length by nonradioactive chemiluminescent assay, RTA by relative-quantitative telomerase repeat amplification protocol (RQ-TRAP), and RRA by PCR-based assay. Mean telomere lengths in DPaSCs, DPuSCs, DFSCs, and MSCs was ~11 kb, and the values did not differ significantly (p < 0.05) among the cells analyzed. RTA levels in DPaSCs were significantly (p < 0.05) higher than in MSCs, DPuSCs, DFSCs, and MRC-5 fibroblasts and among DSCs, DFSCs showed a significantly (p < 0.05) lower RTA. Moreover, RRA levels were significantly (p < 0.05) higher in DPaSCs, DPuSCs, and MSCs than in DFSCs. Based on these observations, we conclude that among DSCs, DPaSCs possessed ideal characteristics on telomere length, telomerase activity and reverse transcriptase (RTase) activity, and may serve as suitable alternative candidates for regenerative medicine.

In Vitro Osteogenic Differentiation of Cultured Human Dental Papilla-Derived Cells

PURPOSE This study examined the osteogenic phenotypes and mineralization of cultured human dental papilla-derived cells. MATERIALS AND METHODS Dental papillae were harvested from mandibles during surgical extraction of lower impacted third molars from 3 patients aged 13 to 15 years. The dental papilla-derived cells were introduced into the cell culture. After passage 3, the dental papilla-derived cells were further cultured for 42 days in an osteogenic inductive culture medium containing dexamethasone, ascorbic acid, and beta-glycerophosphate. We examined the histochemical detection of alkaline phosphatase (ALP), the reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for ALP and osteocalcin, and von Kossa staining in the dental papilla-derived cells. RESULTS It was observed that ALP was strongly expressed in the earlier stage of osteoblastic differentiation, whereas osteocalcin was mainly expressed and secreted into the medium at the later stage. Von Kossa-positive mineralization nodules were first observed on day 14, which increased in number during the entire culture period. CONCLUSIONS These results suggest that dental papilla-derived cell have osteogenic potential and could be used as an additional source of cells for bone tissue engineering.

Tissue-engineered bone formation using periosteal-derived cells and polydioxanone/pluronic F127 scaffold with pre-seeded adipose tissue-derived CD146 positive endothelial-like cells.

The aim of this study was to generate tissue-engineered bone formation using periosteal-derived cells seeded into a polydioxanone/pluronic F127 (PDO/Pluronic F127) scaffold with adipose tissue-derived CD146 positive endothelial-like cells. Considering the hematopoietic and mesenchymal phenotypes of adipose tissue-derived cells cultured in EBM-2 medium, CD146 positive adipose tissue-derived cells was sorted to purify more endothelial cells in characterization. These sorted cells were referred to as adipose tissue-derived CD146 positive endothelial-like cells. Periosteum is a good source of osteogenic cells for tissue-engineered bone formation. Periosteal-derived cells were found to have good osteogenic capacity in a PDO/Pluronic F127 scaffold, which could provide a suitable environment for the osteoblastic differentiation of these cells. Through the investigation of capillary-like tube formation on matrigel and the cellular proliferation of adipose tissue-derived CD146 positive endothelial-like cells cultured in different media conditions, we examined these cells could be cultured in EBM-2 with osteogenic induction factors. We also observed that the osteogenic activity of periosteal-derived cells could be good in EBM-2 with osteogenic induction factors, in the early period of culture. The experimental results obtained in the miniature pig model suggest that tissue-engineered bone formation using periosteal-derived cells and PDO/Pluronic F127 scaffold with pre-seeded adipose tissue-derived CD146 positive endothelial-like cells can be used to restore the bony defects of the maxillofacial region when used in clinics.

Positive expression of NANOG, mutant p53, and CD44 is directly associated with clinicopathological features and poor prognosis of oral squamous cell carcinoma

BackgroundIn order to predict long-term prognosis and define individual treatment modalities for patients with oral squamous cell carcinoma (OSCC), more reliable tumor biomarkers are needed during the pretreatment workup period. The present study aimed to identify more reliable immunohistochemical tumor prognostic markers in the pretreatment biopsy specimens of patients with OSCC.MethodsWe selected 57 patients who were diagnosed with primary OSCC through histopathological analysis. Pretreatment biopsy specimens were immunohistochemically analyzed for the transcription factor NANOG, cancer stem cell marker CD44, and mutant tumor protein 53 (mutant p53). The immunostaining patterns were assessed for their association with the clinicopathological features of OSCC and overall survival rates.ResultsLate tumor stage, positive neck node metastasis, and high-grade differentiation were associated with significantly poorer survival rates. Enhanced expression of NANOG and mutant p53 positivity were significantly associated with clinically late-stage tumors, positive neck node metastasis, histologically high-grade tumors, and poor overall survival rates. OSCCs with strong co-detection of NANOG and mutant p53 were linked to significantly lower survival rates than those with both weak NANOG expression and p53 negativity. Increased expression of CD44 had a limited correlation with unfavorable clinicopathological features.ConclusionHigh expression of NANOG and positive expression of mutant p53 in the pretreatment biopsy specimens of patients with OSCC were associated with poor survival rates and unfavorable clinicopathological features. These results demonstrate that NANOG, mutant p53, and CD44 could be used as immunohistochemical markers in the pretreatment specimens of OSCC. In particular, analysis for co-expression of NANOG and mutant p53 should be made highly available as a tool for prognosis and selecting individual treatment modalities.

Vascular endothelial growth factor stimulates osteoblastic differentiation of cultured human periosteal-derived cells expressing vascular endothelial growth factor receptors

Angiogenesis plays an important role in bone development and postnatal bone fracture repair. Vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptors (VEGFRs) are primarily involved in angiogenesis. This study investigated the expression of VEGF isoforms, VEGFR-1, and VEGFR-2 during the osteoblastic differentiation of cultured human periosteal-derived cells. In addition, the effect of exogenous VEGF on the osteoblastic differentiation of cultured human periosteal-derived cells was also examined. The expression of the VEGF isoforms (VEGF121, VEGF165, VEGF189, and VEGF206), VEGFR-1, and VEGFR-2 was observed in the periosteal-derived cells. Administration of KRN633, a VEGFR-1 and VEGFR-2 inhibitor, decreased the alkaline phosphatase (ALP) activity during the osteoblastic differentiation of cultured human periosteal-derived cells. However, the administration of VEGFR2 Kinase Inhibitor IV, a VEGFR-2 inhibitor, did not affect the ALP activity. The addition of recombinant human VEGF165 elevated the ALP activity and increased the calcium content in the periosteal-derived cells. Treating the periosteal-derived cells with recombinant human VEGF165 resulted in an increase in Runx2 transactivation in the periosteal-derived cells. These results suggest that exogenous VEGF stimulates the osteoblastic differentiation of cultured human periosteal-derived cells and VEGF might act as an autocrine growth factor for the osteoblastic differentiation of cultured human periosteal-derived cells.

Ghost cell odontogenic carcinoma of the mandible: a case report demonstrating expression of tartrate-resistant acid phosphatase (TRAP) and vitronectin receptor.

INTRODUCTION Ghost cell odontogenic carcinoma is a rare neoplastic variant of calcifying odontogenic cyst, with aggressive growth characteristics. A painful swelling in the jaws with local paraesthesia is the most common symptom. Although it often causes irregular destruction of the adjacent bone, immunohistochemical expression of tartrate-resistant acid phosphatase (TRAP) and vitronectin receptor has not previously been described in this carcinoma. CASE REPORT This article describes a ghost cell odontogenic carcinoma affecting the mandible of a 55-year-old man. The patient was treated by segmental mandibulectomy and there was no evidence of recurrence or metastasis for 1.8 years. Cytological features including the immunohistochemical expression of TRAP and vitronectin receptor were studied. CONCLUSION Specimens revealed varying sized islands of anucleate cell clusters with homogenous, pale eosinophilic cytoplasm, so called ghost cells, admixed with sheets of tumour. TRAP and vitronectin receptor were detected in the ghost cells, but they were not expressed in the tumour cells. Our findings suggest that some of the cytokines produced by ghost cells may play important roles in causing extensive bone resorption in the ghost cell odontogenic carcinoma.