• Journal of Veterinary Clinics
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• v.23 no.4
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• pp.411-415
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• 2006

Ascorbic acid has been used widely as a medium supplement to stimulate cell proliferation, but its effects on cell proliferation have not yet been elucidated, and no reports have analyzed effects on subcultured chondrocytes. Subcultured canine chondrocytes of passage one, two and four were cultured in monolayer and alginate beads with and without ascorbic acid. Cell proliferation was examined by 2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt) (XTT) colormetric assay. Ascorbic acid stimulated cell proliferation significantly in both culture methods (p<0.05). The increased cell numbers by stimulation with ascorbic acid were significantly high in passage one cells compared to that of other passages. Differences in cell proliferative capacity by subculturing were not determined. These results suggest that ascorbic acid stimulated the proliferation of subcultured canine chondrocytes and enhanced it more in low-passage cells than in the other cells tested.

• Journal of Acupuncture Research
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• v.25 no.2
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• pp.105-117
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• 2008

목적 : 만성 염증성 질환에 대한 전침효과를 알아보기 위해 complete Freund's adjuvant(CFA) 유발 관절염 모델의 거퇴 관절 내 염증관련 단백질 및 복합당질의 변화를 살펴보았다. 방법 : Sprague-Dawley계 흰쥐의 족부에 CFA를 주사한 다음 3일 간격으로 2Hz, 15Hz 및 120Hz 전침자극을 주며 부종 형성여부를 plethysmometer로 측정하여 판정하였으며 30일째 거퇴관절을 취하여 4% paraformaldehyde에 고정하고 EDTA용액에서 탈회시켜 파라핀연속 절편을 얻어 $NF-{\kappa}B$를 비롯한 5종의 염증관련 단백질의 발현 및 복합당질 변화를 살펴보았다. 결과 : 관절연골 내 면역반응 중 연골기질은 반응이 없거나 약하고 연골세포는 $NF-{\kappa}Bp65$, $I-{\kappa}B{\alpha}$, iNOS 반응이 강하며 특히 유리연골층에서 더 현저하였으나 염증 및 전침자극에 따른 변화는 없었다. 관절낭에서 면역반응을 살펴보면 염증유발시 활액세포의 면역반응세포는 $I-{\kappa}B{\alpha}$가 감소한 반면 iNOS, $IL-1{\beta}$는 증가하며 특히 iNOS 증가가 현저하였으며 전침자극에 의해 iNOS 가 감소하였다. 활액막조직에서 모든 면역반응이 증가하며 특히 $NF-{\kappa}Bp65$, $I-{\kappa}B{\alpha}$, iNOS 반응이 현저한데 전침자극에 의해 $IL-1{\beta}$를 제외한 모든 반응이 감소하였다. 복합당질 염색성은 CFA를 주사한 염증유발 흰쥐군이 정상군에 비해 감소하였다. 관절연골 중 구역간질의 중성복합당질 및 연골세포피막의 산성복합당질이 현저히 감소하였다 Lectin반응도 DBA을 제외한 모든 발현이 염증유발시 감소하였다. 그러나 전침처리에 의해 정상군과 유사한 염색성과 lectin반응을 유지하였다. 특히 구역간질의 중성복합당질과 연골세포의 sWGA와 RCA-1 반응이 현저하였다. 결론 : 만성 염증성 동물모델의 거퇴 관절 내 염증관련 단백질은 관절연골보다 관절낭에서, 복합당질의 변화는 관절연골에서 큰 변화를 보였으며 전침의 자극에 의해 이들 변화가 억제되는 것을 알 수 있다. 이상의 결과로 보아 전침처치는 염증관련 단백질 발현 및 복합당질의 변화 억제를 통해 만성 관절염 질환에 효과적임을 알 수 있다.

• Journal of Veterinary Clinics
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• v.27 no.2
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• pp.147-153
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• 2010

Bio-integration of cartilage grafts with subchondral bone is a significant clinical challenge. To date, the use of demineralized bone matrix (DBM) has been one of the most effective strategies for bone cell proliferation in vivo. Here, we investigated whether coculture of chondrocytes and DBM could serve as a single-platform system containing all the essential elements for purposive bone and cartilage induction. The aim of this study was to evaluate and compare the phenotype and proliferation of bovine chondrocytes cocultured with DBM in two different culture systems, pellet and alginate bead culture. In alginate bead culture, we observed an increase in chondrocyte number and formation of cell clusters. Typical chondrocytic phenotype was maintained for entire eight weeks. Histological analysis showed that chondrocytes maintained a typical round, plump morphology and there was a gradual increase in lacunae. Both coculture systems yielded an expanded cell population as compared to the controls (chondrocytes alone). The production of glycosaminoglycans was also increased in the coculture systems as compared to controls.

• Journal of Acupuncture Research
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• v.22 no.4
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• pp.55-63
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• 2005

목적 : 만성 염증성 질환에 대한 전침효과를 알아보기 위해 complete Freund's adjuvant (CA) 유발 관절염 모델에서 염증관련 단백질의 변화를 살펴보았다. 방법 : Sprague-Dawley계 흰쥐의 족부에 CFA를 주사한 다음 3일 간격으로 2 Hz, 15 Hz 및 120 Hz 전침 자극을 주며 부종 형성여부를 plethysmometer로 측정하여 판정하였으며 30일 째 거퇴관절을 취하여 4% paraformaldehyde에 고정하고 EDTA용액에서 탈회시켜 파라핀연속 절편을 얻어 $NF-{\kappa}B$를 비롯한 5종의 염증관련 단백질의 발현을 면역조직화학적으로 살펴보았다. 결과 : 관절연골내 면역반응 중 연골기질은 반응이 없거나 약하고 연골세포는 $NF-{\kappa}Bp65,\;I-{\kappa}B{\alpha},\;iNOS$반응이 강하며 특히 유리연골층에서 더 현저하였으나 염증 및 전침자극에 따른 변화는 없었다. 관절낭에서 면역반응을 살펴보면 염증유발시 활액세포의 면역반응세포는 $I-{\kappa}B{\alpha}$가 감소한 반면 iNOS, $IL-1{\beta}$는 증가하며 특히 iNOS 증가가 현저하였으며 전침자극에 의해 iNOS가 감소하였다. 활액막조직에서 모든 면역반응이 증가하며 특히 $NF-{\kappa}Bp65,\;I-{\kappa}B{\alpha},\;iNOS$ 반응이 현저한데 전침자극에 의해 $IL-1{\beta}$를 제외한 모든 반응이 감소하였다. 결론 : 만성 염증성 동물모델의 거퇴관절 내 염증관련 단백질은 관절연골보다 관절낭에서 큰 변화를 보이며 전침처치에 의해 이들 단백질 발현이 억제되는 것으로 보아 전침이 만성 염증성 질환에 효과적임을 알 수 있다.

• The Journal of the Korean bone and joint tumor society
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• v.13 no.2
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• pp.105-112
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• 2007

Purpose: Recent studies have shown increased levels of cyclooxygenase-2 (COX-2) in various human malignancies to include various bone and soft tissue tumors. However, little is known with regard to COX-2 expression patterns in chondroid tumors. Materials and Methods: Immunohistochemistry assays were performed for COX-2 in enchondromas (n=10), chondroblastomas (n=11), chondromyxoid fibromas (n=5), conventional chondrosarcomas (n=17), clear cell chondrosarcomas (n=7), and mesenchymal chondrosarcomas (n=6). Results: Among the benign chondroid tumors, chondroblastomas revealed characteristic strong positivity in 6 of 11 cases(54.5%). All enchondromas and chondromyxoid fibromas were negative except in one case. In conventional chondrosarcomas, three cases(17.6%) were strongly reactive with COX-2 and all positive cases represented grade III chondrosarcomas. Clear cell chondrosarcomas were found to be focally positive in two cases(28.5%), while all mesenchymal chondrosarcomas were negative. Conclusions: These findings suggest that COX-2 overexpression in conventional chondrosarcoma may represent an advanced histologic grade. Interestingly, expression of COX-2 in chondroblastomas could be an important factor for inducing peritumoral inflammatory changes in these specific tumors.

• The Journal of the Korean bone and joint tumor society
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• v.15 no.2
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• pp.155-159
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• 2009

Clear cell chondrosarcoma is a rare, low-grade variant of chondrosarcoma that comprises approximately 2% of all chondrosarcomas. This tumor usually involves the epiphysis and epimetaphysis of long bones, especially the proximal part of the femur or humerus, whereas involvement of the scapula is rare. It occurs at any age, but the peak is third to fifth decade, and is rarely seen in the first and second decades of life. Histologically, tumor cells with abundant clear cytoplasm and benign giant cells are usually found. We report on a case of clear cell chondrosarcoma of the scapula in an 8-year-old girl.

• Development and Reproduction
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• v.15 no.2
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• pp.99-111
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• 2011

The present experiment was performed to evaluate the chondrogenic differentiation potential of human adipose tissue-derived mesenchymal stem cells (ATMSCs) in the chondrogenic induction medium (CIM) with transforming growth factor-${\beta}1$ (TGF-${\beta}1$) and to evaluate the chondrogenic differentiation of ATMSCs seeded in gelatin-chondroitinglucosamine scaffold (GCG-scaffold). ATMSCs and mouse chondrocytes were cultured in the basic medium and CIM without TGF-${\beta}1$ (CIM1) or with TGF-${\beta}1$ (CIM2) for chondrogenic differentiation potential. The chondrogenic differentiation of ATMSCs was evaluated by glycosaminoglycan (GAG) synthesis and histochemical staining. In pellet culture, GAG synthesis of ATMSCs and chondrocyte was increased in culture on 14 days, but higher in CIM1 than basic medium, especially highest in CIM2. Cartilage matrix was observed in ATMSCs cultured in CIM2 on 14 days by Safranin O and trichrome staining. In well plate culture, proliferation of ATMSCs was continuously increased in culture on 10 days and higher in CIM than basic medium. The cell adhesion rate of ATMSCs seeded in flask or scaffolds was continuously increased during culture period, but higher in scaffold than flask. GAG synthesis of ATMSCs seeded in scaffolds showed no change in control group. In the CIM groups, GAG synthesis of ATMSCs was continuously increased than control group during culture period, especially very high in CIM2 and in the GCG-scaffold was slightly higher than the gelatin scaffold (G-scaffold). The present results demonstrated that ATMSCs showed an low chondrogenic differentiation potential, compared to mouse chondrocytes for 14 days of culture. TGF-${\beta}1$ is important factor in chondrogenic differentiation of ATMSCs. Gelatin scaffold was considered to increasing the effective chondrogenic differentiation environment. ATMSCs seeded in GCG-scaffold was more effective in chondrogenesis than in G-scaffold. Conclusively, the present results demonstrated that the treatment of chondroitin and glucosamine in the scaffold was more effective to promote the cartilage matrix formation.

• Journal of Periodontal and Implant Science
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• v.34 no.1
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• pp.93-100
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• 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 Life Science
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• v.21 no.4
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• pp.534-541
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• 2011

Retinoic acid (RA), a metabolite of vitamin A, is known to regulate dedifferentiation of rabbit articular chondrocytes. The regulatory mechanism of dedifferentiation by RA is not yet understood. Thus, the effect of RA on the regulation of nitric oxide (NO)-induced dedifferentiation was investigated in rabbit articular chondrocytes. RA caused loss of the differentiated chondrocyte phenotype as demonstrated by inhibition of type II collagen expression and proteoglycan synthesis. RA also accelerated NO-induced dedifferentiation in rabbit articular chondrocytes as detected by expression of type II collagen and Sox-9 using Western blot analysis and production of sulfated proteoglycan using Alcain blue staining. Further, RA potentiated NO-induced activation of ERK. Inhibition of ERK with PD98059 (PD) recovered the expression of type II collagen and Sox-9 and production of sulfate proteoglycan in NO-induced dedifferentiated chondrocytes by RA treatment. Our findings suggest that RA accelerates NO-induced dedifferentiation of rabbit articular chondrocytes via the ERK pathway.

• Polymer(Korea)
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• v.35 no.6
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• pp.499-504
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• 2011

Articular cartilage has an intrinsic difficulty in recovering damages, which requires its tissue engineering treatment. Demineralized bone particle (DBP) contains various bioactive molecules. It is widely used biomaterials in the field of tissue engineering. We developed the synthetic/natural hybrid scaffolds with poly(lactide-co-glycolide) (PLGA) and solution of DBP. The chondrocytes were seeded on the PLGA-DBP scaffolds and MTT assay, morphological observation, biological assay for collagen, sGAG, and RT-PCR were performed to analyze the effect of the DBP on cell viability and extracellular matrix secretion. In SEM observation, we observed that PLGA-DBP scaffolds had uniform porosity. As MTT assay showed scaffolds containing DB solution had higher cell viability then only PLGA scaffolds. The PLGA-DBP scaffolds had better ECM production than PLGA scaffold. It was proven by the higher specific mRNA expression in the PLGA-DBP scaffold than that in PLGA scaffold. These results indicated that PLGA-DBP scaffolds might serve as potential cell delivery vehicles and structural bases for in vitro tissue engineered articular cartilage.