Gelatin-chondroitin-glucosamine Scaffold에 접종한 인간지방조직-유래 중간엽 줄기세포의 연골형성
Chondrogenesis of Human Adipose Tissue Derived Mesenchymal Stem Cells (ATMSCs) Seeded in Gelatin-chondroitin-glucosamine Scaffold
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본 실험은 transforming growth factor-β1(TGF-β1)이 첨가된 chondrogenic induction medium(CIM)을 이용하여 인간지방조직에서 유래된 중간엽 줄기세포(human adipose tissue-derived mesenchymal stem cells, ATMSCs)의 연골형성능과 gelatin-chondroitin-glucosamine scaffold(GCG-scaffold)에 접종시킨 ATMSCs의 연골형성능을 알아보고자 수행하였다. ATMSCs와 생쥐 chondrocyte를 기본배양액과 TGF-β1이 첨가되지 않은 CIM1 및 TGF-β1이 첨가된 CIM2에서 배양하여 연골형성능을 비교하였다. ATMSCs의 연골형성은 gelatin scaffold(G-scaffold)와 GCG-scaffold를 사용하여 glycosaminoglycan (GAG) 합성과 조직화학적 염색으로 연골기질형성 여부를 확인하였다. 펠렛 배양에서 ATMSCs와 chondrocyte의 GAG 합성은 대조군에서 14일의 배양기간 동안 약간 증가하였으나, CIM1과 CIM2배양군은 배양 14일에 대조군에 비해 크게 증가하였으며, CIM2 배양군에서 가장 높았다. 그러나 연골기질은 배양 14일에 CIM2 배양군에서만 Safranin O와 trichrome에 의해 염색되었다. Well plate에서 배양된 ATMSCs의 증식은 모든 배양군에서 배양 10일까지 계속적인 세포 증식이 일어났으며, 대조군에 비해 CIM 배양군이 높았다. ATMSCs 접종 후 플라스크나 scaffold의 세포부착률은 배양시간이 길어짐에 따라 모든 배양군에서 증가하였고, 플라스크에 비해 scaffold에서 더욱 높게 나타났다. Scaffold에 ATMSCs 접종후 GAG 합성은 28일의 배양기간 동안 대조군에서는 별 변화가 없었으나, CIM1, CIM2 배양군은 대조군보다 GAG 합성이 증가되었다. CIM2 배양군에서 GAG 합성이 매우 높게 나타났다. 또한 G-scaffold보다 GCG-scaffold에서 GAG 합성이 약간 높게 나타났다. 조직화학적으로 관찰한 ATMSCs의 연골기질형성도 CIM2 배양군의 GCG-scaffold에서 가장 높게 나타났다. 본 실험의 결과들을 종합해 볼 때, 펠렛 배양에서 ATMSCs는 생쥐 chondrocyte보다 낮은 연골형성능을 보였고, CIM2 배양군에서만 연골기질이 형성된 것으로 보아 TGF-β1이 연골분화에 중요한 요소로 작용한 것이라 사료된다. G-scaffold는 효과적인 연골분화 환경을 제공해 줌으로써 ATMSCs의 세포부착률과 GAG 합성을 증가시키는 것으로 보여지며, G-scaffold보다 GCG-scaffold에서 많은 연골기질이 형성된 것은 GCG-scaffold에 첨가된 chondroitin과 glucosamine이 연골기질 형성을 촉진시키는 물질로 작용한 것으로 사료된다.
Abstract
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-β1 (TGF-β1) and to evaluate the chondrogenic differentiation of ATMSCs seeded in gelatin-chondroitin- glucosamine scaffold (GCG-scaffold). ATMSCs and mouse chondrocytes were cultured in the basic medium and CIM without TGF-β1 (CIM1) or with TGF-β1 (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-β1 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.
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