[국내논문]배양연골막이 피복된 고효능 인공연골의 생체내 효과 The Effect of Cultured Perichondrial Cell Sheet Covered Highly Active Engineered Cartilage: in vivo Comparative Assessment원문보기
조직공학적 인공연골재생에 대한 관심이 증가함에 따라 많은 연구들이 활발히 수행되고 있으나 임상적인 적용의 한계를 극복하기위한 고효능을 보유한 양질의 연골조직생산의 필요성이 증가되고 있다. 인공연골은 자연연골과는 달리 '연골막(perichondrium)'을 포함하고 있지 않기 때문에 장기간 생체 내에 삽입된 후에 서서히 흡수 또는 변형으로 임상적 활용에 한계가 있다고 있다. 이에 본 연구는 양질의 연골조직생산을 목적으로, 세포판 제작기법(cell sheet engineering technique) 을 기반으로 한 인체유래의 배양 연골막(cultured perichondrium)을 이용하여 만든 인공연골막 세포판(cultured perichondrial cell sheet)의 생체 내 특성을 비교 분석하고, 배양된 연골막을 피복하여 고효능화를 유도한 인공연골복합체의 생체내 재생효능 및 조직특성을 비교 평가하고자 하였다. 본 연구에서는 Athymic nude mouse의 피하이식모델(study 1, n = 12)을 이용하여 담체로 hydrogel을 이용한 배양연골막 복합체의 생체내 효능을 분석하였고, 중대형동물의 대량연골 결손시의 재생효능을 평가하기 위하여 개의 무릎연골에 $1{\times}2cm$의 대량연골 결손모델(study 2, n = 12)을 통하여 인공배양세포판을 이식하였다. 이식12주 후 이식편을 회수하여 생화학, 분자생물학 및 면역조직학 분석을 시행한 결과, 배양연골막 복합체의 생체내 효능이 단독이식군에 비해 변형이나 과증식 없이 우수한 결과를 나타내었다. 본 연구의 결과로 토대로 배양연골막을 피복한 인공연골막의 관절내 효과를 규명하여 실제 임상적용을 조기화하는 기반을 제공하고 인공연골의 문제점이었던 변형과 흡수를 줄인 고효능 인공연골 제작기법을 제공하는데 유용할 것으로 기대된다.
조직공학적 인공연골재생에 대한 관심이 증가함에 따라 많은 연구들이 활발히 수행되고 있으나 임상적인 적용의 한계를 극복하기위한 고효능을 보유한 양질의 연골조직생산의 필요성이 증가되고 있다. 인공연골은 자연연골과는 달리 '연골막(perichondrium)'을 포함하고 있지 않기 때문에 장기간 생체 내에 삽입된 후에 서서히 흡수 또는 변형으로 임상적 활용에 한계가 있다고 있다. 이에 본 연구는 양질의 연골조직생산을 목적으로, 세포판 제작기법(cell sheet engineering technique) 을 기반으로 한 인체유래의 배양 연골막(cultured perichondrium)을 이용하여 만든 인공연골막 세포판(cultured perichondrial cell sheet)의 생체 내 특성을 비교 분석하고, 배양된 연골막을 피복하여 고효능화를 유도한 인공연골복합체의 생체내 재생효능 및 조직특성을 비교 평가하고자 하였다. 본 연구에서는 Athymic nude mouse의 피하이식모델(study 1, n = 12)을 이용하여 담체로 hydrogel을 이용한 배양연골막 복합체의 생체내 효능을 분석하였고, 중대형동물의 대량연골 결손시의 재생효능을 평가하기 위하여 개의 무릎연골에 $1{\times}2cm$의 대량연골 결손모델(study 2, n = 12)을 통하여 인공배양세포판을 이식하였다. 이식12주 후 이식편을 회수하여 생화학, 분자생물학 및 면역조직학 분석을 시행한 결과, 배양연골막 복합체의 생체내 효능이 단독이식군에 비해 변형이나 과증식 없이 우수한 결과를 나타내었다. 본 연구의 결과로 토대로 배양연골막을 피복한 인공연골막의 관절내 효과를 규명하여 실제 임상적용을 조기화하는 기반을 제공하고 인공연골의 문제점이었던 변형과 흡수를 줄인 고효능 인공연골 제작기법을 제공하는데 유용할 것으로 기대된다.
A special mesenchymal tissue layer called perichondrium has a chondrogenic capacity and is a candidate tissue for engineering of cartilage. To overcome limited potential for chondrocyte proliferation and re-absorption, we studied a method of cartilage tissue engineering comprising chondrocyte-hydrog...
A special mesenchymal tissue layer called perichondrium has a chondrogenic capacity and is a candidate tissue for engineering of cartilage. To overcome limited potential for chondrocyte proliferation and re-absorption, we studied a method of cartilage tissue engineering comprising chondrocyte-hydrogel pluronic complex (CPC) and cultured perichondrial cell sheet (cPCs) which entirely cover CPC. For effective cartilage regeneration, cell-sheet engineering technique of high-density culture was used for fabrication of cPCs. Hydrogel pluronic as a biomimetic cell carrier used for stable and maintains the chondrocytes. The human cPCs was cultured as a single layer and entirely covered CPC. The tissue engineered constructs were implanted into the dorsal subcutaneous tissue pocket on nude mice (n = 6). CPC without cPCs were used as a controls (N = 6). Engineered cartilage specimens were harvested at 12 weeks after implantation and evaluated with gross morphology and histological examination. Biological analysis was also performed for glycosaminoglycan (GAG) and type II collagen. Indeed, we performed additional in vivo studies of cartilage regeneration using canine large fullthickness chondrial defect model. The dogs were allocated to the experimental groups as treated chondrocyte sheets with perichondrial cell sheet group (n = 4), and chondrocyte sheets only group (n = 4). The histological and biochemical studies performed 12 weeks later as same manners as nude mouse but additional immunofluorescence study. Grossly, the size of cartilage specimen of cPCs covered group was larger than that of the control. On histological examination, the specimen of cPCs covered group showed typical characteristics of cartilage tissue. The contents of GAG and type II collagen were higher in cPCs covered group than that of the control. These studies demonstrated the potential of such CPC/cPCs constructs to support chondrogenesis in vivo. In conclusion, the method of cartilage tissue engineering using cPCs supposed to be an effective method with higher cartilage tissue gain. We suggest a new method of cartilage tissue engineering using cultured perichondrial cell sheet as a promising strategy for cartilage tissue reconstruction.
A special mesenchymal tissue layer called perichondrium has a chondrogenic capacity and is a candidate tissue for engineering of cartilage. To overcome limited potential for chondrocyte proliferation and re-absorption, we studied a method of cartilage tissue engineering comprising chondrocyte-hydrogel pluronic complex (CPC) and cultured perichondrial cell sheet (cPCs) which entirely cover CPC. For effective cartilage regeneration, cell-sheet engineering technique of high-density culture was used for fabrication of cPCs. Hydrogel pluronic as a biomimetic cell carrier used for stable and maintains the chondrocytes. The human cPCs was cultured as a single layer and entirely covered CPC. The tissue engineered constructs were implanted into the dorsal subcutaneous tissue pocket on nude mice (n = 6). CPC without cPCs were used as a controls (N = 6). Engineered cartilage specimens were harvested at 12 weeks after implantation and evaluated with gross morphology and histological examination. Biological analysis was also performed for glycosaminoglycan (GAG) and type II collagen. Indeed, we performed additional in vivo studies of cartilage regeneration using canine large fullthickness chondrial defect model. The dogs were allocated to the experimental groups as treated chondrocyte sheets with perichondrial cell sheet group (n = 4), and chondrocyte sheets only group (n = 4). The histological and biochemical studies performed 12 weeks later as same manners as nude mouse but additional immunofluorescence study. Grossly, the size of cartilage specimen of cPCs covered group was larger than that of the control. On histological examination, the specimen of cPCs covered group showed typical characteristics of cartilage tissue. The contents of GAG and type II collagen were higher in cPCs covered group than that of the control. These studies demonstrated the potential of such CPC/cPCs constructs to support chondrogenesis in vivo. In conclusion, the method of cartilage tissue engineering using cPCs supposed to be an effective method with higher cartilage tissue gain. We suggest a new method of cartilage tissue engineering using cultured perichondrial cell sheet as a promising strategy for cartilage tissue reconstruction.
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제안 방법
After 8 and 12 weeks, engineered cartilage tissue was harvested. The entire specimens were subjected to histological analysis, quantitative analysis for glycosaminoglycan (GAG), and assay for type I collagen and proteoglycan using RT-PCR.
After 12 weeks, engineered cartilage tissue was harvested. The entire specimens were subjected to histological analysis, quantitative analysis for glycosaminoglycan (GAG), and assay for type II collagen and proteoglycan using RT-PCR (Fig 3).
In our previous study, we investigated effectiveness of cultured perichondrial cell sheet in vivo for another possibility in large amount of cartilage regeneration. In this study, we performed two track of implantation study, murine subqutaneous and canine chondral defect model. The result of murine in vivo study demonstrated that the cultured cell sheet technique has more effective to maintain chondral condition over long period.
대상 데이터
In vivo study I, twelve female six weeks old athymic nude mouse (Harumo breeding branch, Japan) were (n = 12) used for experiment. Six completed tissue engineered constructs were implanted into the subcutaneous tissue pocket of nude mice (n = 6) on the back.
The frozen section was constructed by slicing the samples into 50 mm using a cryostat, and then embedding on cover slips. The slides were examined by a fluorescence microscope and light microscope (Olympus BX60, IX70 Olympus Optical Co., Tokyo, Japan). The population of DiL and DiO-positive cells was evaluated by fluorescence microscopy.
이론/모형
In this study, chondrocytes were used to evaluate cartilage repair and chodrial regeneration supported by perichondrial cell sheet methods.
성능/효과
Levels of total GAG were significantly (p < 0.05) higher in cPCs/cCs groups than cPCs and cCs group (Fig 4a).
In conclusion, the addition of cultured perichondrium sheet coverage over the engineering cartilage tissue seems to be an effective method providing higher cartilage tissue gain and reliable success rate for cartilage tissue engineering. This method provides enough chondrocytes and prevents vascular ingrowth from the environment which is beneficial to chondrogenesis.
후속연구
This study has important limitations, however, mostly stemming from its small sample size. And it is thought to be carried out additional large randomized control study to realize clinical applications.
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