• Maxillofacial Plastic and Reconstructive Surgery
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• v.39
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• pp.8.1-8.8
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• 2017

Background: For an effective bone graft for reconstruction of the maxillofacial region, an adequate vascular network will be required to supply blood, osteoprogenitor cells, and growth factors. We previously reported that the secretomes of bone marrow-derived mesenchymal stem cells (MSC-CM) contain numerous growth factors such as insulin-like growth factor (IGF)-1, transforming growth factor $(TGF)-{\beta}1$, and vascular endothelial growth factor (VEGF), which can affect the cellular characteristics and behavior of regenerating bone cells. We hypothesized that angiogenesis is an important step for bone regeneration, and VEGF is one of the crucial factors in MSC-CM that would enhance its osteogenic potential. In the present study, we focused on VEGF in MSC-CM and evaluated the angiogenic and osteogenic potentials of MSC-CM for bone regeneration. Methods: Cytokines in MSC-CM were measured by enzyme-linked immunosorbent assay (ELISA). Human umbilical vein endothelial cells (HUVECs) were cultured with MSC-CM or MSC-CM with anti-VEGF antibody (MSC-CM + anti-VEGF) for neutralization, and tube formation was evaluated. For the evaluation of bone and blood vessel formation with micro-computed tomography (micro-CT) and for the histological and immunohistochemical analyses, a rat calvarial bone defect model was used. Results: The concentrations of IGF-1, VEGF, and $TGF-{\beta}1$ in MSC-CM were $1515.6{\pm}211.8pg/mL$, $465.8{\pm}108.8pg/mL$, and $339.8{\pm}14.4pg/mL$, respectively. Tube formation of HUVECs, bone formation, and blood vessel formation were increased in the MSC-CM group but decreased in the MSC-CM + anti-VEGF group. Histological findings suggested that new bone formation in the entire defect was observed in the MSC-CM group although it was decreased in the MSC-CM + anti-VEGF group. Immunohistochemistry indicated that angiogenesis and migration of endogenous stem cells were much more abundant in the MSC-CM group than in the MSC-CM + anti-VEGF group. Conclusions: VEGF is considered a crucial factor in MSC-CM, and MSC-CM is proposed to be an adequate therapeutic agent for bone regeneration with angiogenesis.

• Journal of Embryo Transfer
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• v.33 no.2
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• pp.85-97
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• 2018

The trans-differentiation potential of mesenchymal stem cells (MSCs) is employed, but there is little understanding of the cell source-dependent trans-differentiation potential of MSCs into corneal epithelial cells. In the present study, we induced trans-differentiation of MSCs derived from umbilical cord matrix (UCM-MSCs) and from dental tissue (D-MSCs), and we comparatively evaluated the in vitro trans-differentiation properties of both MSCs into corneal epithelial-like cells. Specific cell surface markers of MSC (CD44, CD73, CD90, and CD105) were detected in both UCM-MSCs and D-MSCs, but MHCII and CD119 were significantly lower (P < 0.05) in UCM-MSCs than in D-MSCs. In UCM-MSCs, not only expression levels of Oct3/4 and Nanog but also proliferation ability were significantly higher (P < 0.05) than in D-MSCs. In vitro differentiation abilities into adipocytes and osteocytes were confirmed for both MSCs. UCM-MSCs and D-MSCs were successfully trans-differentiated into corneal epithelial cells, and expression of lineage-specific markers (Cytokeratin-3, -8, and -12) were confirmed in both MSCs using immunofluorescence staining and qRT-PCR analysis. In particular, the differentiation capacity of UCM-MSCs into corneal epithelial cells was significantly higher (P < 0.05) than that of D-MSCs. In conclusion, UCM-MSCs have higher differentiation potential into corneal epithelial-like cells and have lower expression of CD119 and MHC class II than D-MSCs, which makes them a better source for the treatment of corneal opacity.

• The Korean Journal of Physiology and Pharmacology
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• v.21 no.2
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• pp.153-160
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• 2017

In this study, we aim to determine the in vivo effect of human umbilical cord blood-derived multipotent stem cells (hUCB-MSCs) on neuropathic pain, using three, principal peripheral neuropathic pain models. Four weeks after hUCB-MSC transplantation, we observed significant antinociceptive effect in hUCB-MSC-transplanted rats compared to that in the vehicle-treated control. Spinal cord cells positive for c-fos, CGRP, p-ERK, p-p 38, MMP-9 and MMP 2 were significantly decreased in only CCI model of hUCB-MSCs-grafted rats, while spinal cord cells positive for CGRP, p-ERK and MMP-2 significantly decreased in SNL model of hUCB-MSCs-grafted rats and spinal cord cells positive for CGRP and MMP-2 significantly decreased in SNI model of hUCB-MSCs-grafted rats, compared to the control 4 weeks or 8weeks after transplantation (p<0.05). However, cells positive for TIMP-2, an endogenous tissue inhibitor of MMP-2, were significantly increased in SNL and SNI models of hUCB-MSCs-grafted rats. Taken together, subcutaneous injection of hUCB-MSCs may have an antinociceptive effect via modulation of pain signaling during pain signal processing within the nervous system, especially for CCI model. Thus, subcutaneous administration of hUCB-MSCs might be beneficial for improving those patients suffering from neuropathic pain by decreasing neuropathic pain activation factors, while increasing neuropathic pain inhibition factor.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.36 no.4
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• pp.243-249
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• 2010

Tissue engineered bone (TEB) can replace an autogenous bone graft requiring an secondary operation site as well as avoid complications like inflammation or infection from xenogenic or synthetic bone graft. Adult mesenchymal stem cells (MSC) for TEB are considered to have various ranges of differentiation capacity or multipotency by the donor site and age. This study examined the effect of age on proliferation capacity, differentiation capacity and bone morphogenetic protein-2 (BMP-2) responsiveness of human bone marrow stromal cells (hBMSC) according to the age. In addition, to evaluate the effect on enhancement for osteoblast differentiation, the hBMSC were treated with Trichostatin A (TSA) and 5-Azacitidine (5-AZC) which was HDAC inhibitors and methyltransferase inhibitors respectively affecting chromatin remodeling temporarily and reversibly. The young and old group of hBMSC obtained from the iliac crest from total 9 healthy patients, showed similar proliferation capacity. Cell surface markers such as CD34, CD45, CD90 and CD105 showed uniform expression regardless of age. However, the young group showed more prominent transdifferentiation capacity with adipogenic differentiation. The osteoblast differentiation capacity or BMP responsiveness was low and similar between young and old group. TSA and 5-AZC showed potential for enhancing the BMP effect on osteoblast differentiation by increasing the expression level of osteogenic master gene, such as DLX5, ALP. More study will be needed to determine the positive effect of the reversible function of HDAC inhibitors or methyltransferase inhibitors on enhancing the low osteoblast differentiation capacity of hBMSC.

• Molecules and Cells
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• v.39 no.11
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• pp.790-796
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• 2016

Dental pulp is a highly vascularized tissue requiring adequate blood supply for successful regeneration. In this study, we investigated the functional role of stem cells from human exfoliated deciduous teeth (SHEDs) as a perivascular source for in vivo formation of vessel-like structures. Primarily isolated SHEDs showed mesenchymal stem cell (MSC)-like characteristics including the expression of surface antigens and in vitro osteogenic and adipogenic differentiation potentials. Moreover, SHEDs were positive for NG2, ${\alpha}$-smooth muscle actin (SMA), platelet-derived growth factor receptor beta ($PDGFR{\beta}$), and CD146 as pericyte markers. To prove feasibility of SHEDs as perivascular source, SHEDs were transplanted into immunodeficient mouse using Matrigel with or without human umbilical vein endothelial cells (HUVECs). Transplantation of SHEDs alone or HUVECs alone resulted in no formation of vessel-like structures with enough red blood cells. However, when SHEDs and HUVECs were transplanted together, extensive vessel-like structures were formed. The presence of murine erythrocytes within lumens suggested the formation of anastomoses between newly formed vessel-like structures in Matrigel plug and the host circulatory system. To understand underlying mechanisms of in vivo angiogenesis, the expression of angiogenic cytokine and chemokine, their receptors, and MMPs was compared between SHEDs and HUVECs. SHEDs showed higher expression of1VEGF, SDF-$1{\alpha}$, and $PDGFR{\beta}$ than HUVECs. On the contrary, HUVECs showed higher expression of VEGF receptors, CXCR4, and PDGF-BB than SHEDs. This differential expression pattern suggested reciprocal interactions between SHEDs and HUVECs and their involvement during in vivo angiogenesis. In conclusion, SHEDs could be a feasible source of perivascular cells for in vivo angiogenesis.

• Molecules and Cells
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• v.40 no.6
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• pp.386-392
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• 2017

Periodontal ligament stem cells (PDLSCs) are multipotent stem cells derived from periodontium and have mesenchymal stem cell (MSC)-like characteristics. Recently, the perivascular region was recognized as the developmental origin of MSCs, which suggests the in vivo angiogenic potential of PDLSCs. In this study, we investigated whether PDLSCs could be a potential source of perivascular cells, which could contribute to in vivo angiogenesis. PDLSCs exhibited typical MSC-like characteristics such as the expression pattern of surface markers (CD29, CD44, CD73, and CD105) and differentiation potentials (osteogenic and adipogenic differentiation). Moreover, PDLSCs expressed perivascular cell markers such as NG2, ${\alpha}-smooth$ muscle actin, platelet-derived growth factor receptor ${\beta}$, and CD146. We conducted an in vivo Matrigel plug assay to confirm the in vivo angiogenic potential of PDLSCs. We could not observe significant vessel-like structures with PDLSCs alone or human umbilical vein endothelial cells (HUVECs) alone at day 7 after injection. However, when PDLSCs and HUVECs were co-injected, there were vessel-like structures containing red blood cells in the lumens, which suggested that anastomosis occurred between newly formed vessels and host circulatory system. To block the $SDF-1{\alpha}$ and CXCR4 axis between PDLSCs and HUVECs, AMD3100, a CXCR4 antagonist, was added into the Matrigel plug. After day 3 and day 7 after injection, there were no significant vessel-like structures. In conclusion, we demonstrated the perivascular characteristics of PDLSCs and their contribution to in vivo angiogenesis, which might imply potential application of PDLSCs into the neovascularization of tissue engineering and vascular diseases.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.31 no.4
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• pp.281-286
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• 2009

Introduction: Enamel matrix derivative (EMD) is a protein which is secreted by Hertwig root sheath and plays a major role in the formation of cementum and attachment of peridontium. Several studies have shown that EMD promoted the proliferation and differentiation of preosteoblasts, osteoblasts and periodontal ligament cells in vitro: however, reports showing the inhibition of osteogenic differentiation by EMD also existed. This study was designed to simultaneously evaluate the effect of EMD on the two cell lines (human mesenchymal stem cells: hMSC, human periodontal ligament derived fibroblasts: hPDLCs) by means of quantitative analysis of some bone related matrices (Alkaline phosphatase : ALP, osteopontin ; OPN, osteocalcin ; OC). Materials and Methods: hMSCs and hPDLCs were expanded and cells in the 4${\sim}$6 passages were adopted to use. hMSc and hPDLCs were cultured during 1,2,7, and 14 days with 0, 50 and 100 ${\mu}g/ml$ of EMD, respectively. ALP activity was assessed by SensoLyte ALP kit and expressed as values of the relative optical density. Among the matrix proteins of the bony tissue, OC and OPN were assessed and quantification of these proteins was evaluated by means of human OC immunoassay kit and human OPN assay kit, respectively. Results: ALP activity maintained without EMD at $1,2^{nd}$ day. The activity increased at $7^{th}$ day but decreased at $14^{th}$ day. EMD increased the activity at $14^{th}$ day in the hPDLCs culture. In the hMSCs, rapid decrease was noted in $7^{th}$ and $14^{th}$ days without regard to EMD concentrations. Regarding the OPN synthesis in hPDLCs, marked decrease of OPN was noted after EMD application. Gradual decrease tendency of OPN was shown over time. In hMSCs, marked decrease of OPN was also noted after EMD application. Overall concentration of OPN was relatively consistent over time than that in hPDLCs. Regarding the OC synthesis, in both of hPDLCs and hMSCs, inhibition of OC formation was noted after EMD application in the early stages but EMD exerted minimal effect at the later stages. Conclusion: In this experimental condition, EMD seemed to play an inhibitory role during the differentiation of hMSCs and hPDLCs in the context of OC and OPN formation. In the periodontium, there are many kinds of cells contributing to the regeneration of oral tissue. EMD enhanced ALP activity in hPDLCs rather than in hMSCs and this may imply that EMD has a positive effect on the differentiation of cementoblasts compared with the effect on hMSCs. The result of our research was consistent with recent studies in which the authors showed the inhibitory effect of EMD in terms of the differentiation of mineral colony forming cells in vitro. This in vitro study may not stand for all the charateristics of EMD; thus, further studies involving many other bone matrices and cellular attachment will be necessary.

• Journal of Life Science
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• v.21 no.5
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• pp.631-646
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• 2011

Mesenchymal stem cells (MSC) are multipotent and can be isolated from diverse human tissues including bone marrow, fat, placenta, dental pulp, synovium, tonsil, and the thymus. They function as regulators of tissue homeostasis. Because of their various advantages such as plasticity, easy isolation and manipulation, chemotaxis to cancer, and immune regulatory function, MSCs have been considered to be a potent cell source for regenerative medicine, cancer treatment and other cell based therapy such as GVHD. However, relating to its supportive feature for surrounding cell and tissue, it has been frequently reported that MSCs accelerate tumor growth by modulating cancer microenvironment through promoting angiogenesis, secreting growth factors, and suppressing anti-tumorigenic immune reaction. Thus, clinical application of MSCs has been limited. To understand the underlying mechanism which modulates MSCs to function as tumor supportive cells, we co-cultured human adipose tissue derived mesenchymal stem cells (ASC) with cancer cell lines H460 and U87MG. Then, expression data of ASCs co-cultured with cancer cells and cultured alone were obtained via microarray. Comparative expression analysis was carried out using DAVID (Database for Annotation, Visualization and Integrated Discovery) and PANTHER (Protein ANalysis THrough Evolutionary Relationships) in divers aspects including biological process, molecular function, cellular component, protein class, disease, tissue expression, and signal pathway. We found that cancer cells alter the expression profile of MSCs to cancer associated fibroblast like cells by modulating its energy metabolism, stemness, cell structure components, and paracrine effect in a variety of levels. These findings will improve the clinical efficacy and safety of MSCs based cell therapy.

• Transactions of the Korean Society of Mechanical Engineers A
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• v.34 no.6
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• pp.675-681
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• 2010

It is well known that mesenchymal stem cell(MSCs) can be differentiated into fibroblasts, chondrocytes, and osteoblasts and that they develop into fibrous tissue, cartilage, or bone, as a result of mechanical stimulation. In this study, we developed a bioreactor system, which is composed of a reactor vessel that provides the required cell culture environment, an environment controlling chamber to control the media, a gas mixer, and a reactor motion control subsystem to apply mechanical stimuli to the cells. For the MSC culture, We used a poly-urethane (PU) scaffold, with a collagen coating to ensure improved cohesion ratio. Then, we transferred the cultivated MSCs in the PU scaffold, cultured the cells in the bioreactor system, and confirmed the proliferation, differentiation, and ossification processes, resulting from mechanical stimuli.