• Journal of Periodontal and Implant Science
• /
• v.34 no.1
• /
• pp.93-100
• /
• 2004

많은 연구들에서 hMSC를 얻기 위해 centrifugation, fluoroscence activated cell sorter(FACS), magnetic activated cell sorter(MACS)가 이용되어져 왔다. 그러나 centrifugation만을 이용한 경우 순도가 떨어지며 FACS나 MACS의 경우에는 비용, 시간이 많이 드는 단점이 있다. 따라서 이 연구에서는 antibody cocktail을 이용하여 hMSC를 좀더 쉽게 얻어내는 방법에 대해 알아보았다. 사람의 골반에서 12G의 바늘을 이용하여 골수를 흡입한 후 heparin이 들어있는 시험관에 넣고 처리과정을 시행하기 전에 냉장고에 보관하며 가능한 한 빨리 처리 과정을 실시한다. 얻은 골수에 적당량의 RosetteSep( Stemcell Technologies)을 첨가한 후 실온에서 20분간 반응시킨다. 그 후 적당량의 Ficoll-paque위에 골수와 RosetteSep의 혼합물을 섞이지 않게 올리고 원심분리를 이용하여 원하는 세포층을 얻어낸다. 이 세포층을 따로 분리한 뒤 배양한다. 배양 시 세포가 80%이상 차기 전에 계속 passage를 시행하며 배양한다. 이는 세포가 밀도가 높아져 원치 않는 세포로 분화되는 것을 막기 위함이다. 배양된 세포가 다양한 분화능력을 가지고 있는지 알아보기 위해 세 가지로 분화를 유도하였다. 적절한 배지와 적절한 환경에서 배양함으로써 얻어진 세포를 osteoblast, chondroblast, adipocyte로 분화를 유도하였다. 분화된 세포가 원하는 형질의 세포로 분화되었는지를 확인하기 위하여 osteoblast의 경우 alizarin red staining, alkaline phosphatase activity, chondroblast의 경우 toluidine blue staining, adipocyte의 경우 Oil-Red-O staining으로 염색하여 분화를 확인하였다. 분리해낸 세포는 각각 세 가지 세포로 분화가 되었으며 이는 RosetteSep이 hMSC를 성공적으로 분리해냈다는 것을 보여준다. 그러나 모든 세포가 분화를 보이지는 않았으며 따라서 hMSC의 순도를 높이기 위한 연구가 더 필요하다. RosetteSep을 이용하면 다른 방법들 보다 쉽게 hMSC를 얻을 수 있으나 기존의 방법과 순도의 측면에서 더 비교할 필요가 있다.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
• /
• v.32 no.6
• /
• pp.524-529
• /
• 2006

For the repairing of bone defect, autogenous or allogenic bone grafting remains the standard. However, these methods have numerous disadvantages including limited amount, donor site morbidity and spread of diseases. Tissue engineering technique by culturing stem cells may allow for a smart solution for this problem. Adipose tissue contains 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 buccal fat pad and differentiate them into osteoblast and are to examine the bone induction capacity. Buccal fat-derived cells (BFDC) were obtained from human buccal fat pad and cultured. BFDC were analyzed for presence of stem cell by immunofluorescent staining against CD-34, CD-105 and STRO-1. After BFDC were differentiated in osteogenic medium for three passages, their ability to differentiate into osteogenic pathway were checked by alkaline phosphatase (ALP) staining, Alizarin red staining and RT-PCR for osteocalcin (OC) gene expression. Immunofluorescent and biochemical assays demonstrated that BFDC might be a distinguished stem cells and mineralization was accompanied by increased activity or expression of ALP and OC. And calcium phosphate deposition was also detected in their extracelluar matrix. The current study supports the presence of stem cells within the buccal fat pad and the potential implications for human bone tissue engineering for maxillofacial reconstruction.

• Restorative Dentistry and Endodontics
• /
• v.39 no.3
• /
• pp.187-194
• /
• 2014

Objectives: The effects of bone morphogenetic protein-2 (BMP-2) and enamel matrix derivative (EMD) respectively with mineral trioxide aggregate (MTA) on hard tissue regeneration have been investigated in previous studies. This study aimed to compare the osteogenic effects of MTA/BMP-2 and MTA/EMD treatment in MC3T3-E1 cells. Materials and Methods: MC3T3-E1 cells were treated with MTA (ProRoot, Dentsply), BMP-2 (R&D Systems), EMD (Emdogain, Straumann) separately and MTA/BMP-2 or MTA/EMD combination. Mineralization was evaluated by staining the calcium deposits with alkaline phosphatase (ALP, Sigma-Aldrich) and Alizarin red (Sigma-Aldrich). The effects on the osteoblast differentiation were evaluated by the expressions of osteogenic markers, including ALP, bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN) and osteonectin (OSN), as determined by reverse-transcription polymerase chain reaction analysis (RT-PCR, AccuPower PCR, Bioneer). Results: Mineralization increased in the BMP-2 and MTA/BMP-2 groups and increased to a lesser extent in the MTA/EMD group but appeared to decrease in the MTA-only group based on Alizarin red staining. ALP expression largely decreased in the EMD and MTA/EMD groups based on ALP staining. In the MTA/BMP-2 group, mRNA expression of OPN on day 3 and BSP and OCN on day 7 significantly increased. In the MTA/EMD group, OSN and OCN gene expression significantly increased on day 7, whereas ALP expression decreased on days 3 and 7 (p < 0.05). Conclusions: These results suggest the MTA/BMP-2 combination promoted more rapid differentiation in MC3T3-E1 cells than did MTA/EMD during the early mineralization period.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
• /
• v.34 no.4
• /
• pp.412-418
• /
• 2008

Bone morphogenetic proteins (BMPs) in combination with stem cells gain more significance for their use in bone tissue engineering. The mesenchymal stem cell can be differentiated into osteoblast by the treatment of BMP. The aim of this study is to characterize the osteogenic differentiation process of adult stem cells derived from buccal fat pad according to BMP-2 within culture media and decide the appropriate concentration of BMP-2 to facilitate osteogenesis. The authors procured the stem cell from buccal fat pad and analyzed for presence of stem cell by flow cytomety against CD-34, CD-105 and STRO-1. The buccal fat derived stem cells (BFDC) were treated by application of the different concentration with BMP-2 of 0, 10, 50, 100 and 200ng/ml, respectively. And their ability to differentiate into osteogenic pathway were checked by alkaline phosphatase(ALP) staining, Alizarin red staining and RT-PCR for osteocalcin(OC) gene expression at 7, 14 and 21day of culture. Flow cytometric analysis and biochemical assays demonstrated that BFDC might be a distinguished stem cells, and mineralization was accompanied in proportion to BMP-2 concentration. However, with 100ng/ml concentration of BMP-2, the BFDC demonstrated most efficient staining pattern of ALP and Alizarin red. The feasibility of the osteogenic differentiation in the group of both 50ng/ml and 200ng/ml of BMP-2 showed similar activity and relatively weaker than that of 100ng/ml. These results suggest that the BMP-2 stimulate osteogenesis by BFDC effectively and that bone induction might be controlled through negative regulatory feedback in higher concentration.

• Food Science of Animal Resources
• /
• v.41 no.6
• /
• pp.1022-1035
• /
• 2021

This study estimated the effect of exposure to propiconazole through implementation and residues in finishing pigs. We analyzed the expression of fibrosis-related genes and performed histological analysis of the blood, liver, kidney, muscle, ileum, and fat tissues. The animals were exposed for 28 d to different concentrations of propiconazole (0.09, 0.44, 0.88, 4.41, and 8.82 mg/kg bw/d). Quantitative, gene expression, and histological analyses in tissues were performed using liquid chromatography mass spectrometry, real-time PCR, and Masson's trichrome staining, respectively. Final body weight did not differ among groups. However, genes involved in fibrosis were significantly differentially regulated in response to propiconazole concentrations. Glucose, alanine aminotransferase, and total bilirubin levels were significantly increased compared with those in the control group, while alkaline phosphatase level was decreased (p<0.05) after exposure to propiconazole. The residue limits of propiconazole were increased in the finishing phase at 4.41 and 8.82 mg/kg bw/d. The liver, kidney, and ileum showed blue staining after propiconazole treatment, confirmed by Masson's trichrome staining. In conclusion, these findings suggest that propiconazole exposure disturbs the expression of fibrosis-related genes. This study on dietary propiconazole in pigs can provide a basis for determining maximum residue limits and a better understanding of metabolism in pigs and meat products.

• Korean Journal of Veterinary Research
• /
• v.29 no.3
• /
• pp.215-222
• /
• 1989

In order to investigate the morphological characteristics of epididymal duct of the squirrel, the histological and histochemical studies were carried out. The results obtained were summarized as follows: The epididymal duct can be divided into 9 segments by histological and histochemical features. Segments 1 to 5 were located in the head, segments 6 and 7 in the body, and segments 8 and 9 in the tail of the epididymis. The apical cells were numerous in the segment 1. Clear cells which has a compact, deeply staining nucleus and a characteristically clear cytoplasm were scattered in the epithelium throughout the duct. Interepithelial clear cells which had PAS-positive granules tended to increase in number caudally. Strong PAS-positive reaction was detected at the intralumen of the segments 3,8 and 9. Acid phosphatase activity was relatively high in the basal cytoplasm of the segment 7, and then in the supranuclear region of the segments 8 and 9. Alkaline phosphatase activity was weakly positive or negative except the segments 3 and 4. ATPase activity was strong in the free surface of the epithelium in the head and the entire cytoplasm in the body and tail, a,nd SDH activity was generally weak except for the body where it was more intense.

• Journal of Korean Medicine Rehabilitation
• /
• v.30 no.3
• /
• pp.1-21
• /
• 2020

Objectives This study was designed to evaluate the bone regeneration effects of Sintongchukea-tang (SC) on rib fractured rats. Methods Rats were randomly divided into 5 groups (normal, control, positive control, SC low [SC-L] and SC high [SC-H]). All groups were subject to fractured rib except normal group. Normal group received no treatment at all. Control group was orally fed with phosphate buffered saline, and positive control group was medicated with tramadol (20 mg/kg). SC group was orally medicated with SC (50 mg/kg, 100 mg/kg) once a day for 14 days. The fracture healing process was observed by x-ray, micro CT and fracture tissue slide was observed by immunohistochemical staining. We analysed levels of transforming growth factor-β1, Ki67, alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), receptor activator of nuclear factor kappa-β, tartrate resistant acid phosphatase (TRAP) and analysed levels of Osteocalcin in plasma. We measured levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), ALP, blood urea nitrogen (BUN) and creatinine in plasma, for hepatotoxicity and nephrotoxicity of SC. Results Though X-ray and micro-computed tomography, more callus formation was observed and bone union was progressing. Through Hematoxylin and Eosin, callus formation was increased compared to the control group. Runx2 level at SC-H was significantly increased and TRAP level at SC-L was significantly decreased compared with the control group. AST, ALT, ALP, BUN and creatinine were not statistically different from the control group. Conclusions As described above, SC promoted fracture healing by stimulating the bone regeneration factor. And SC shows no hepatotoxicity and nephrotoxicity. In conclusion, it seems that SC helps to promote fracture regeneration and it can be used clinically to patients with fracture.

• Biomolecules & Therapeutics
• /
• v.20 no.1
• /
• pp.89-95
• /
• 2012

Poncirus trifoliata fruit (PTF) affects the digestive and cardiovascular systems, and kidney function. The authors studied the effects of ethyl acetate (EtOAc) extract of PTF on the activities of osteoblasts and in an animal model. The main compounds of the EtOAc extract, naringin and poncirin have been confirmed by HPLC and NMR analysis. Effects of osteoblastic differentiation were measured by alkaline phosphatase (ALP) activity, osteopontin (OPN) protein expression and osteoprotegerin (OPG) mRNA expression in MC3T3-E1 cells. Also, osteoclast differentiation was measured by multinucleated cells (MNCs) formation through tartrate resistance acid phosphatase (TRAP)-positive staining. Bone mineral density (BMD) was measured before and after treatment with EtOAc extract of PTF in prednisolone-induced osteoporotic mice. Dexamethasone (DEX) decreased OPN and OPG expression level in MC3T3-E1 cells and ALP activity was decreased by DEX dose-dependently. EtOAc extract of PTF recovered the levels of ALP activity, and the expression of OPN and OPG in MC3T3-E1 cells treated with DEX. In osteoclast differentiation, multinucleated TRAP-positive cell formation was significantly suppressed by the EtOAc extract of PTF. Total body BMD was restored by EtOAc extract of PTF in prednisolone-induced osteoporotic mice. In conclusion, EtOAc extract of PTF recovered DEX-mediated deteriorations in osteoblastic and osteoclastic functions, and increased BMD in glucocorticoid-induced osteoporosis.

• Journal of Korean Medicine Rehabilitation
• /
• v.30 no.2
• /
• pp.19-35
• /
• 2020

Objectives The aim of this study is to evaluate the fracture healing effect of Jinmu-tang (JM) on femur fractured rats. Methods Rats were randomly divided into 5 groups (normal, control, positive control, JM extract with low concentration and JM extract with high concentration). All group except normal group went through both femur fracture. Normal and control group received no treatment at all. Positive control group were medicated with tramadol (20 mg/kg) once a day for 14 days. Experimental group was orally medicated with JM extract (10 mg/kg for low concentration, 50 mg/kg for high concentration) once a day for 14 days. In order to investigate fracture healing process, plasma and serum were obtained. Also, micro-computed tomography was conducted to see the frature site visually. Immunohistochemistry for transforming growth factor-β1, Ki67, alkaline phosphatase, runt-related transcription factor 2, receptor activator of nuclear factor kappa-β, tartrate resistant acid phosphatase was conducted to observe bone healing progress after 14 days since fracture occured. Aspartate aminotransferase, alanine aminotransferase, blood urea nitrogen and creatinine levels were measured in plasma, for hepatotoxicity and nephrotoxicity of JM extract. Osteocalcin was measured to observe activity of osteoblast. Results Through Micro-CT, more fracture healing was observed on both experimental group than control and positive control group. Through Hematoxylin & Eosin and safranin O staining showed bone cell proliferation and bone formation in the experimental group. RANK was significantly increased in the experimental groups. JM with high concentration showed statistically significant of TGF-β and Osteocalcin. NO, TRAP and ALP were not significantly changed. Liver toxicity was not significantly observed. Creatinine significantly increased in both experimental groups after 28 days. Conclusions As described above, JM extract showed anti-inflammatory effect, promoted fracture healing by stimulating the bone regeneration factor, and showed little hepatotoxicity and nephrotoxicity. In conclusion, JM extract can promote fracture healing and it can be used clinically to patients with fracture.

• Biomolecules & Therapeutics
• /
• v.17 no.4
• /
• pp.353-361
• /
• 2009

Recent in vitro and in vivo animal studies have reported that statin, a cholesterol-lowering drug, stimulate osteogenic differentiation. In the present study, we investigated the effect of simvastatin on osteogenic and adipogenic differentiation in primarily cultured human adipose-derived stem cells (hADSCs). The simvastatin treatment significantly increased the positive cell numbers in alkaline phosphatase and von Kossa staining, and enhanced the expression levels of bone morphogenic protein (BMP)-2, core binding factor alpha 1 (cbfa1), collgen type I and osteonectin mRNAs. Lastly, hADSCs were cultured in the adipogenic media with or without simvastatin to examine the effect of simvastatin on adipogenic differentiation. In the RT-PCR analysis, there were notable decreases in mRNA expression of aP1, C/EBP-$\alpha$ and PPAR-$\gamma$ in hADSCs cultivated in simvastatin-added medium, compared to those in simvastatin-free medium. It suggests that the adipogenic differentiation was significantly inhibited by simvastatin treatment. These observations indicate that simvastatin induces osteogenic differentiation and suppresses adipogenic differentiation in hADSCs.