• Journal of Periodontal and Implant Science
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• v.42 no.3
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• pp.73-80
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• 2012

Purpose: The purpose of this study was to investigate the effects of the immunosuppressants FK506 and cyclosporin A (CsA) on the osteogenic differentiation of rat mesenchymal stem cells (MSCs). Methods: The effect of FK506 and CsA on rat MSCs was assessed in vitro. The MTT assay was used to determine the deleterious effect of immunosuppressants on stem cell proliferation at 1, 3, and 7 days. Alkaline phosphatase (ALP) activity was analyzed on days 3, 7, and 14. Alizarin red S staining was done on day 21 to check mineralization nodule formation. Real-time polymerase chain reaction (RT-PCR) was also performed to detect the expressions of bone tissue-specific genes on days 1 and 7. Results: Cell proliferation was promoted more in the FK506 groups than the control or CsA groups on days 3 and 7. The FK506 groups showed increased ALP activity compared to the other groups during the experimental period. The ALP activity of the CsA groups did not differ from the control group in any of the assessments. Mineralization nodule formation was most prominent in the FK506 groups at 21 days. RT-PCR results of the FK506 groups showed that several bone-related genes-osteopontin, osteonectin, and type I collagen (Col-I)-were expressed more than the control in the beginning, but the intensity of expression decreased over time. Runx2 and Dlx5 gene expression were up-regulated on day 7. The effects of 50 nM CsA on osteonectin and Col-I were similar to those of the FK506 groups, but in the 500 nM CsA group, most of the genes were less expressed compared to the control. Conclusions: These results suggest that FK506 enhances the osteoblastic differentiation of rat MSCs. Therefore, FK506 might have a beneficial effect on bone regeneration when immunosuppressants are needed in xenogenic or allogenic stem cell transplantation to treat bone defects.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.32 no.2
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• pp.97-106
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• 2010

Aim of the study: An alternative source of adult stem cells that could be obtained in large quantities, under local anesthesia, with minimal discomfort would be advantageous. Adipose tissue could be processed to obtain a fibroblast-like population of cells or adipose tissue-derived stromal cells (ATSCs). This study was performed to confirm the availability of ATSCs in bone tissue engineering. Materials amp; Methods: In this study, adipose tissue-derived mesenchymal stem cell was extracted from the liposuctioned abdominal fat of 24-old human and cultivated, and the stem cell surface markers of CD 105 and SCF-R were confirmed by immunofluorescent staining. The proliferation of bone marrow mesenchymal stem cell and ATSCs were compared, and evaluated the osteogenic differentiation of ATSCs in a specific osteogenic induction medium. Osteogenic differentiation was assessed by von Kossa and alkaline phosphatase staining. Expression of osteocyte specific BMP-2, ALP, Cbfa-1, Osteopontin and osteocalcin were confirmed by RT-PCR. With differentiation of ATSCs, calcium concentration was assayed, and osteocalcin was evaluated by ELISA (Enzyme-linked immunosorbant assay). The bone formation by 5-week implantation of HA/TCP block loaded with bone marrow mesenchymal stem cells and ATSCs in the subcutaneous pocket of nude mouse was evaluated by histologic analysis. Results: ATSCs incubated in the osteogenic medium were stained positively for von Kossa and alkaline phosphatase staining. Expression of osteocyte specific genes was also detected. ATSCs could be easily identified through fluorescence microscopy, and bone formation in vivo was confirmed by using ATSC-loaded HA/TCP scaffold. Conclusions: The present results show that ATSCs have an ability to differentiate into osteoblasts and formed bone in vitro and in vivo. So ATSCs may be an ideal source for further experiments on stem cell biology and bone tissue engineering.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.27 no.1
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• pp.1-8
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• 2005

This study was aimed to characterize osteogenic potential of rat bone marrow stromal cells (BMSC) isolated with standard flushing method and investigate the plasticity of transdifferentiation between osteoblastic and adipocytic lineage of cultured BMSC. Unlike aspiration method in human, rat bone marrow was extracted by means of irrigation with culture media that elevates the possibility of co-extraction of committed osteoprogenitor, or preosteoblast or other progenitor cells of several types present inside bone marrow. The cultured stromal cells showed high ALP activity which is representative marker of osteoblast without any treatment. Osteogenic inducers such as Dex and BMP-2 were examined for the evaluation of their effect on osteogenic and adipocytic differentiation of stromal cells, because they function as osteoinductive agent in stromal cells, but simultaneously induce adipogenic differentiation. Osteogenic differentiation was evaluated by measuring alkaline phosphatase activity or mRNA expression of osteoblast markers such as osteopontin, bone sialoprotein, collagen type I and CbfaI, and in vitro matrix mineralization by von Kossa staining. Oil red staining method was used to detect adipocyte and adipocytic marker, aP2 and $PPAR{\gamma}2$ expression was examined using RT-PCR. It can be supposed that irrigation procedure resulted in high portion of already differentiation-committed osteoprogenitor cell showing elevated ALP activity and strong mineralization only under the supplement of $100{\mu}M$ ascorbic 2-phosphate and 10mM ${\beta}$-glycerophosphate without any treatment of osteogenic inducers such as Dex and BMP-2. Dex and BMP-2 seemed to transdifferentiate osteoprogenitor cells having high ALP activity into adipocytes temporarily, but continuous treatment redifferentiated into osteoblast and developed in vitro matrix mineralization. This property must be considered either in tissue engineering for bone regeneration, or in research of characterization of osteogenic differentiation, with rat BMSC isolated by the standard irrigation method.

• Journal of Nutrition and Health
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• v.55 no.6
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• pp.617-629
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• 2022

Purpose: Osteoporosis is characterized by structural deterioration of the bone tissue because of the loss of osteoblastic activity or the increase in osteoclastic activity, resulting in bone fragility and an increased risk of fractures. Hederagenin (Hed) is a pentacyclic triterpenoid saponin isolated from Dipsaci Radix, the dried root of Dipsacus asper Wall. Dipsaci Radix has been used in Korean herbal medicine to treat bone fractures. In this study, we attempted to demonstrate the potential anti-osteoporotic effect of Hed by examining its effect on osteoblast differentiation in MC3T3-E1 cells. Methods: Osteoblastic MC3T3-E1 cells were cultured in 0, 1, and 10 ㎍/mL Hed for 3 and 7 days. The activity of alkaline phosphatase (ALP), bone nodule formation and level of expression of bone-related genes and proteins were measured in MC3T3-E1 cells exposed to Hed. The western blot test was used to detect the activation of the bone morphogenetic protein-2 (BMP2)/ Suppressor of Mothers against Decapentaplegic (SMAD)1 pathway. Results: Hed significantly increased the proliferation of MC3T3-E1 cells. Intracellular ALP activity was significantly increased in the 1 ㎍/mL Hed-treated group. Hed significantly increased the concentration of calcified nodules. Furthermore, Hed significantly upregulated the expression of genes and proteins associated with osteoblast proliferation and differentiation, such as Runt-related transcription factor 2 (Runx2), ALP, osteopontin (OPN), and type I procollagen (ProCOL1). Induction of osteoblast differentiation by Hed was associated with increased BMP2. In addition, Hed induced osteoblast differentiation by increasing the activity of SMAD1/5/8. These results suggest that Hed has the potential to prevent osteoporosis by promoting osteoblastogenesis in osteoblastic MC3T3-E1 cells via the modulation of the BMP2/SMAD1 pathway. Conclusion: The results presented in this study indicate that Hed isolated from Dipsaci Radix has the potential to be developed as a healthcare food and functional material possessing anti-osteoporosis effects.

• Journal of Periodontal and Implant Science
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• v.32 no.2
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• pp.389-402
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• 2002

BMP can induce ectopic bone formation when implanted into sites such as rat muscle and can greatly enhance healing of bony defects when applied exogenously. In addition, BMP stimulated osteoblastic differentiation in vitro in various types of cells. The aim of this study was to investigate the effect of recombinant human bone morphogenetic protein(rhBMP-2) on the proliferation and osteoblastic differentiation of human periodontal ligament cells and gingival fibroblasts. The cell number and alkaline phosphatase activity were measured in 3 experimental groups of human periodontal ligament cells and gingival fibroblasts (control group, rhBMP-2 50ng/ml group, and rhBMP-2 100ng/ml group) at 1 and 2 weeks after culture. At the same time, total RNA of cultured cells were extracted and reverse trascription polymerase chain reaction(RT-PCR) was performed to determine the expression of mRNA of bone matrix protein. RhBMP-2 had no effect on the cell proliferation of human periodontal ligament cells and gingival fibroblasts. Alkaline phosphatase activity was elevated significantly by rhBMP-2 in both cells. And periodontal ligament cells showed significantly higher alkaline phosphatase activity than gingival fibroblasts. ${\beta}$-actin, type I collagen, alkaline phosphatase, BMP-2 mRNA were expressed in all of the samples. Osteopontin, osteocalcin mRNA were expressed in all periodontal ligament cell groups, and rhBMP-2 50ng/ml group, rhBMP-2 100ng/ml group of 2 week culture period of gingival fibroblasts. Bone sialoprotein mRNA was only expressed in rhBMP-2 50ng/ml group and rhBMP-2 100ng/ml group of 2-week culture period. These results suggest that rhBMP-2 stimulates osteoblastic differentiation in human periodontal ligament cells and gingival fibroblasts in vitro.

• Molecules and Cells
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• v.41 no.12
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• pp.1016-1023
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• 2018

Regenerative orthopedics needs significant devices to transplant human stem cells into damaged tissue and encourage automatic growth into replacements suitable for the human skeleton. Soft biomaterials have similarities in mechanical, structural and architectural properties to natural extracellular matrix (ECM), but often lack essential ECM molecules and signals. Here we engineer mineralized polysaccharide beads to transform MSCs into osteogenic cells and osteoid tissue for transplantation. Bone morphogenic proteins (BMP-2) and indispensable ECM proteins both directed differentiation inside alginate beads. Laminin and collagen IV basement membrane matrix proteins fixed and organized MSCs onto the alginate matrix, and BMP-2 drove differentiation, osteoid tissue self-assembly, and small-scale mineralization. Augmentation of alginate is necessary, and we showed that a few rationally selected small proteins from the basement membrane (BM) compartment of the ECM were sufficient to up-regulate cell expression of Runx-2 and osteocalcin for osteoid formation, resulting in Alizarin red-positive mineral nodules. More significantly, nested BMP-2 and BM beads added to a non-union skull defect, self-generated osteoid expressing osteopontin (OPN) and osteocalcin (OCN) in a chain along the defect, at only four weeks, establishing a framework for complete regeneration expected in 6 and 12 weeks. Alginate beads are beneficial surgical devices for transplanting therapeutic cells in programmed (by the ECM components and alginate-chitosan properties) reaction environments ideal for promoting bone tissue.

• Journal of Periodontal and Implant Science
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• v.35 no.2
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• pp.321-333
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• 2005

1. 목적 in vitro 상에서 법랑기질유도체가 치주인대섬유아세포, 불멸화 조골세포와 백악질 유래세포의 증식과 유전자 발현에 미치는 영향을 알아보고자 하였다. 2. 연구방법 및 재료 <세포증식 연구> 교정을 목적으로 발거한 치아에서 분리, 배양한 치주인대섬유아세포와 백악질유래세포, 그리고 $SaOs_2$ 세포를 이용하였다. 법랑기질유도체가 세포 증식에 미치는 영향을 알아보기 위해, 35 mm Petri dish에 dish 당 $5{\times}10^3$ 개의 세포를 접종하였다. 대조군은 1% 항생제와 10% FBS를 포함한 DMEM 배지를 이용했고, 5mM 초산을 첨가한 군과 첨가하지 않은 두 개의 대조군이 이용되었다. 실험군은 100 ${\mu}g/ml$의 법랑기질유도제를 첨가한 군과 100 ${\mu}g/ml$의 법랑기질유도체와 5 mM의 초산을 첨가한 2개의 실험군이 이용되었다. 각 군은 세 개의 배양접시에 행해졌고, 1, 3, 8일에 세포의 수를 각각 측정하였다. 결과는 repeated measures ANOVA로 통계 처리하였다. <유전자 발현 연구> 각 세포의 형질 특성을 알아보기 위해 RT-PCR을 실시하여 조골세포 분화 표식자와 연관된 Human collagen type I(COL I), human osteopontin(OP), human osteocalcin(OC), human alkaline phosphatase(ALP)와 human bone sialoprotein(BSP)의 mRNA 발현을 실험 1, 3, 8일에 걸쳐, 세 군의 차이를 비교 관찰하였다. 3. 결과 <세포증식 연구> 치주인대세포와 백악질유래세포, 그리고 $SaOs_2$ 세포의 증식은 법랑기질유도체에 의해 영향을 받지 않았다. 대조군과 초산이 포함된 대조군 그리고 법랑기질유도체와 초산이 포함된 실험군에서 유의할 만한 세포 수의 차이가 실험 기간 1, 3, 8일에 걸쳐 나타나지 않았다(p<0.05). <유전자 발현 연구> ALP와 COL I은 세 군의 세포에서 모두 발현되었고, 발현 정도는 EMD에 영향을 받지 않았다. OC은 세 군에서 모두 비교적 약하게 발현되었고, 특히 $SaOs_2$ cell과 백악질유래세포에서 약하게 발현되었다. EMD는 OC의 발현정도를 약하게 하였다. OP은 백악질유래세포에서 1, 3, 8일에 걸쳐 EMD 유무에 관련 없이 발현되지 않았다. 그러나 치주인대세포와 $SaOs_2$ cell에서는 강하게 발현되었다. BSP는 치주인대세포와 $SaOs_2$ cell에서 1, 3, 8일에 걸쳐 비교적 고르게 발현되었다. EMD 배지에서 배양된 백악질유래세포는 8일에는 BSP가 발현되지 않았다. 4 결론 이번 실험 결과에 의하면 법랑기질유도체는 치주인대세포, 불멸화 조골세포와 백악질 유래세포의 증식에 있어 유의성 있는 효과를 나타내지 않았다. 그러나, 유전자 발현에 있어서는, 치주인대세포와 백악질유래세포, 그리고 $SaOs_2$ 세포 모두에서 OC mRNA의 발현을 억제하는 효과를 나타내었다. EMD는 세포의 증식에는 영향을 미치지 않지만, 유전자 발현에 있어 일부 영향을 미치는 것으로 보인다. 법랑기질유도체가 세포의 증식과 유전자 발현에 미치는 영향은 배양된 세포의 형질특성, 배양환경, 배양일수 등에 따라 달라질 수 있다. 그러므로 법랑기질유도체가 in vitro 상에서 세포에 미치는 영향은 보다 정량화된 연구가 필요하다.