• Polymer(Korea)
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• v.29 no.5
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• pp.501-507
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• 2005

Small intestinal submucosa (SIS) had been widely used as a biomaterial without immune rejection responses. SIS sponges prepared by crosslinking with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). SIS powders dissolved in $3\%(v/v)$ acetic acid aqueous solution for 48hrs and freeze-dried. EDC solution ($H_2O$ : ethanol = 5 : 95) as a crosslink agent was used in concentration of 100mM. In vitro, rat-BMSCs seeded in SIS sponges and induced the osteogenesis for 28 days. We have characterized the osteogenic potential of rat-BMSCs in SIS sponges by alkaline phosphatase activity(ALP), n assay, SEM and RT-PCR for osteogenic phenotype. In SEM, all morphology of SIS sponges was regular and showed interconnected pore structure. By RT-PCR analysis, we observed type I collagen expression. These results demonstrate osteogenic differentiation of rat-BMSCs. In conclusion, we confirmed that the morphology of surface, cross-section, and side of SIS sponges were highly porous with good interconnections between each pores, which can support the surface of cell growth, proliferation, and differentiation. This result indicates that SIS sponge is useful for osteogenesis of BMSCs.

• Archives of Plastic Surgery
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• v.37 no.3
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• pp.207-212
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• 2010

Purpose: The object of this study is to develop a novel BMP-2 delivery system for continuous osteogenic differentiation and to induce osteogenesis of stem cells using a bi-phase alginate carrier in vitro. Methods: Alginate nanoparticle loaded BMP-2 was prepared by the reverse emulsification-diffusion technique. Physical properties and release profiles of alginate carriers were measured by Instron and ELISA kit, respectively. Cell viability and alkaline phosphate activity of hBMSCs differentiation was also evaluated by MTS and Metra BAP assays, respectively. Results: Optimal concentration for bi-phase alginate carrier was determined as 2 wt% by evaluating mechanical and biological properties, and differentiation of BMSCs for bone regeneration. The 2% bi-phase alginate carrier had the lowest initial and final release ratio. In addition, the 2% bi-phase alginate carrier had a little higher ALP activity than the homogeneous carrier. An improved controlled release profile was obtained by combining alginate hydrogel with lyophilized particles. Conclusion: Bi-phase alginate carrier has many advantages such as biocompatibility and controlled release capability. It is expected to be effective as a scaffold and carrier in bone tissue engineering.

• Molecules and Cells
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• v.24 no.2
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• pp.255-260
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• 2007

Bone marrow (BM) derived mesenchymal stem cells (MSC) are pluripotent cells which can differentiate into osteogenic, adipogenic and other lineages. In spite of the broad interest, the information about the changes in BM cell composition, in particularly about the variation of MSC number and their properties in relation to the age of the donor is still controversial. The aim of this study was to investigate the age associated changes in variations of BM cell composition, phenotype and differentiation capacities of MSC using a rat model. Cell populations were characterized by flow cytometry using light scattering parameters, DNA content and a set of monoclonal antibodies. Single cell analysis was performed by conventional fluorescent microscopy. In vitro culture of MSC was established and their phenotype and capability for in vitro differentiation into osteogenic and adipogenic cells was shown. Age related changes in tibiae and femurs, amount of BM tissue, BM cell composition, proportions of separated MSC and yield of MSC in 2 weeks of in vitro culture were found. At the same time, neither change in phenotype no in differentiation capacities of MSC was registered. Age-related changes of the number of MSC should be taken into account whenever MSC are intended to be used for investigations.

• Journal of Periodontal and Implant Science
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• v.38 no.1
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• pp.41-50
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• 2008

Purpose: Bone morphogenetic protein-2(BMP-2) has been shown to possess significant osteoinducitve potential. There have been attempts to overcome a limitation of mass production, and economical efficiency of BMP. The aim of this study was to produce recombinant human BMP-2(rhBMP-2) from E. coli in a large scale and evaluate its biological activity. Materials and Methods: The E.coli strain BL21(DE3) was used as a host for rhBMP-2 production. Dimerized rhBMP-2 was purified by affinity chromatography using Heparin column. To determine the physicochemical properties of the rhBMP-2 expressed in E. coli, we examined the HPLC profile and performed Western blot analysis. The effect of the purified rhBMP-2 dimer on osteoblast differentiation was examined by alkaline phosphatase (ALP) activity and representing morphological change using C2C12 cell. Results: E. coli was genetically engineered to produce rhBMP-2 in a non-active aggregated form. We have established a method which involves refolding and purifying a folded rhBMP-2 dimer from non-active aggregates. The purified rhBMP-2 homodimer was characterized by SDS-PAGE as molecular weight of about 28kDa and eluted at 34% acetonitrile, 13.27 min(retention time) in the HPLC profile and detected at Western blot. The purified rhBMP-2 dimer stimulated ALP activity and induced the transformation from myogenic differentiation to osteogenic differentiation. Conclusion: rhBMP-2 was produced in E. coli using genetic engineering. The purified rhBMP-2 dimer stimulated ALP activity and induced the osteogenic differentiation of C2C12 cells.

• Journal of Microbiology and Biotechnology
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• v.18 no.5
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• pp.975-982
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• 2008

We investigated whether transplantation of osteogenically differentiated bone marrow-derived mesenchymal stem cells (BMMSCs) and the use of an hydroxyapatite (HAp) scaffold can enhance the in vivo bone formation efficacy of human BMMSCs. Three months after implantation to the subcutaneous dorsum of athymic mice, transplantation of osteogenically differentiated human BMMSCs increased the bone formation area and calcium deposition to 7.1- and 6.2-folds, respectively, of those of transplantation of undifferentiated BMMSCs. The use of the HAp scaffold increased the bone formation area and calcium deposition to 3.7- and 3.5-folds, respectively, of those of a polymer scaffold. Moreover, a combination of transplantation of osteogenically differentiated BMMSCs and HAp scaffold further increased the bone formation area and calcium deposition to 10.6- and 9.3-folds, respectively, of those of transplantation of undifferentiated BMMSCs seeded onto polymer scaffolds. The factorial experimental analysis showed that osteogenic differentiation of BMMSCs prior to transplantation has a stronger positive effect than the HAp scaffold on in vivo bone formation.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.36 no.1
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• pp.7-15
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• 2010

Complex human tissues harbor stem cells and precursor cells, which are responsible for tissue development or repair. Recently, dental tissues such as dental pulp, periodontal ligament (PDL), dental follicle have been identified as easily accessible sources of undifferentiated cells. These tissues contain mesenchymal stem cells that can be differentiate into bone, cartilage, fat or muscle by exposing them to specific growth conditions. In this study, the authors procured the stem cell from pulp, PDL, and dental follicle and differentiate them into osteoblast and examine the bone induction capacity. Dental pulp stem cell (DPSC), periodontal ligament stem cell (PDLSC), and dental follicle precursor cell (DFPC) were obtained from human 3rd molar and cultured. Each cell was analyzed for presence of stem cell by fluorescence activated cell sorter (FACs) against CD44, CD105 and CD34, CD45. Each stem cell was cultured, expanded and grown in an osteogenic culture medium to allow formation of a layer of extracellular bone matrix. Osteogenic pathway was checked by alizarin red staining, alkaline phosphatase (ALP) activity test and RT-PCR for ALP and osteocalcin (OCN) gene expression. According to results from FACs, mesenchymal stem cell existed in pulp, PDL, and dental follicle. As culturing with bone differentiation medium, stem cells were differentiated to osteoblast like cell. Compare with stem cell from pulp, PDL and dental follicle-originated stem cell has more osteogenic effect and it was assumed that the character of donor cell was able to affect on differential potency of stem cell. From this article, we are able to verify the pulp, PDL, and dental follicle from extracted tooth, and these can be a source of osteoblast and stem cell for tissue engineering.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.36 no.3
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• pp.172-176
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• 2010

Postirradiation extraosseous osteogenic sarcomas are uncommon in the head and neck, despite the extensive use of high-dose radiation. It has been described as de novo radiation-induced neoplasm. We present a 73-year-old male who had been treated by radiotherapy for gingival cancer 7 years earlier and later developed extraosseous osteogenic sarcomas (EOSs) of the neck. Microscopically, the neck mass was composed with mesenchymal malignant cells with cartilaginous and osteogenic differentiation. Immunohistochemical stain demonstrated strong positivity of tumor cells for Snail, the one of major epithelial-mesenchymal transition (EMT) inducer. The E-cadherin expression was scarce, showing inverse relationship to Snail expression. Compared with previous squamous cell carcinoma (SCC) of the gingiva, the present EOS sample revealed the remained epithelial cells on cytokeratin immunohistochemistry, suggesting the tumor arise from the cells of epithelial origin. We have also reviewed the previous 6 cases of head and neck EOSs carefully. The clinicopathologic features of the unusual lesion suggest that it is an incomplete EMT of precedent epithelial malignancy rather than de novo pathology.

• Molecules and Cells
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• v.39 no.3
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• pp.186-194
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• 2016

Epidemiological evidence suggests that bone is especially sensitive to oxidative stress, causing bone loss in the elderly. Previous studies indicated that human amnion-derived mesenchymal stem cells (HAMSCs), obtained from human amniotic membranes, exerted osteoprotective effects in vivo. However, the potential of HAMSCs as seed cells against oxidative stress-mediated dysfunction is unknown. In this study, we systemically investigated their antioxidative and osteogenic effects in vitro. Here, we demonstrated that HAMSCs significantly promoted the proliferation and osteoblastic differentiation of $H_2O_2$-induced human bone marrow mesenchymal stem cells (HBMSCs), and down-regulated the reactive oxygen species (ROS) level. Further, our results suggest that activation of the ERK1/2 MAPK signal transduction pathway is essential for both HAMSCs-mediated osteogenic and protective effects against oxidative stress-induced dysfunction in HBMSCs. U0126, a highly selective inhibitor of extracellular ERK1/2 MAPK signaling, significantly suppressed the antioxidative and osteogenic effects in HAMSCs. In conclusion, by modulating HBMSCs, HAMSCs show a strong potential in treating oxidative stress- mediated bone deficiency.

• International Journal of Oral Biology
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• v.42 no.2
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• pp.71-78
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• 2017

BMP-2 is a well-known TGF-beta related growth factor, having a significant role in bone and cartilage formation. It has been employed to promote bone formation in some clinical trials, and to differentiate mesenchymal stem cells into osteoblasts. However, it is difficult to obtain this protein in its soluble and active form. hBMP-2 is expressed as an inclusion body in the bacterial system. To continuously supply hBMP-2 for research, we optimized the refolding of recombinant hBMP-2 expressed in E. coli, and established an efficient method by using detergent and alkali. Using a heparin column, the recombinant hBMP-2 was purified with the correct refolding. Although combinatorial refolding remarkably enhanced the solubility of the inclusion body, a higher yield of active dimer form of hBMP-2 was obtained from one-step refolding with detergent. The refolded recombinant hBMP-2 induced alkaline phosphatase activity in mouse myoblasts, at $ED_{50}$ of 300-480ng/ml. Furthermore, the expressions of osteogenic markers were upregulated in hPDLSCs and hDPSCs. Therefore, using the process described in this study, the refolded hBMP-2 might be cost-effectively useful for various differentiation experiments in a laboratory.

• Journal of Periodontal and Implant Science
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• v.49 no.2
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• pp.114-126
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• 2019

Purpose: The aim of this study was to evaluate the enhancement of osteogenic potential of biphasic calcium phosphate (BCP) bone substitute coated with Escherichia coli-derived recombinant human bone morphogenetic protein-2 (ErhBMP-2) and epigallocatechin-3-gallate (EGCG). Methods: The cell viability, differentiation, and mineralization of osteoblasts was tested with ErhBMP-2-/EGCG solution. Coated BCP surfaces were also investigated. Standardized, 6-mm diameter defects were created bilaterally on the maxillary sinus of 10 male New Zealand white rabbits. After removal of the bony windows and elevation of sinus membranes, ErhBMP-2-/EGCG-coated BCP was applied on one defect in the test group. BCP was applied on the other defect to form the control group. The animals were sacrificed at 4 or 8 weeks after surgery. Histologic and histometric analyses of the augmented graft and surrounding tissue were performed. Results: The 4-week and 8-week test groups showed more new bone (%) than the corresponding control groups (P<0.05). The 8-week test group showed more new bone (%) than the 4-week test group (P<0.05). Conclusions: ErhBMP-2-/EGCG-coated BCP was effective as a bone graft material, showing enhanced osteogenic potential and minimal side effects in a rabbit sinus augmentation model.