CAJ | 학술논문

目的：软骨组织多处于人体骨骼的重要部位，其缺损修复一直为临床急待解决的难题，用组织工程方法修复关节软骨缺损是近年来正在研究的新途径。其中绝大多数研究几乎均着重体外培养条件的研究[1,2]，而忽略了对于改善局部微环境的探讨，为此，本实验试图在载体复合物植入软骨缺损微环境内时，增加能促进MSCs分裂、增殖、分化及血管新生的bFGF及参与和活跃成软骨细胞合成软骨基质及纤维的维生素C等，从而达到提高软骨缺损修复疗效的目的。方法从24只3月龄新西兰大耳白兔髂骨处抽取骨髓，以密度梯度离心法分离出骨髓间充质干细胞（MSCs）作为种子细胞进行扩增培养至第三代，制成细胞悬液，而后在自制模具中与藻酸钙制备成与兔膝关节软骨全层缺损（直径4mm、深度4mm）相一致的载体复合物同时加入bFGF和维生素C，将该载体复合物植入兔左膝关节软骨全层缺损处作为A组，而右膝关节的关节软骨全层缺损处则植入MSCs与藻酸钙的载体复合物作为B组。另取兔6只，左膝按上述方法作一缺损植入单纯藻酸钙载体作为C组；右膝缺损则作空白对照D组。待术后不同时间点（30 d，60 d及90 d）取材，常规石蜡包埋后制成切片，分别用于HE、Masson、番红O、透射电镜、免疫组化等指标以观察软骨缺损修复效果。结果 A，B，C三组软骨缺损均被透明软骨和纤维组织充填，只是A组的透明软骨组织较B组多，C组最少。随植入时间后移，A组几乎均被透明软骨样组织填平并且充填组织与周边正常关节软骨的交界逐渐分辨不出，并与深层软骨下骨相连接，软骨细胞间质的胶原纤维从表层向深层呈放射状排列于软骨细胞基质中。而B组的缺损处表面尚有较多的纤维组织且充填组织与周边正常关节软骨的分界较A组清晰，C组缺损处表面可见有较多纤维组织和未降解的藻酸钙，与周边正常关节软骨分界较A，B组清晰。A组填充的软骨细胞具有明显陷窝的，互相距离较远的3~5个细胞排列呈柱状的成熟软骨细胞明显较B组多，C组则很难观察到成行排列的软骨细胞。功能旺盛的软骨细胞（Ⅱ型胶原阳性细胞，胞质粗面内质网扩张等）则属A组最多，C组最少。D组术后90 d时缺损处为大量纤维组织填充且与周边正常关节软骨和软骨下骨整合较差。结论用自体MSC藻酸钙载体复合物植入膝关节软骨缺损同时向该缺损微环境内加bFGF和维生素C有利于软骨修复，能提高软骨缺损修复效果。目的：軟骨組織多處于人體骨骼的重要部位，其缺損脩複一直為臨床急待解決的難題，用組織工程方法脩複關節軟骨缺損是近年來正在研究的新途徑。其中絕大多數研究幾乎均著重體外培養條件的研究[1,2]，而忽略瞭對于改善跼部微環境的探討，為此，本實驗試圖在載體複閤物植入軟骨缺損微環境內時，增加能促進MSCs分裂、增殖、分化及血管新生的bFGF及參與和活躍成軟骨細胞閤成軟骨基質及纖維的維生素C等，從而達到提高軟骨缺損脩複療效的目的。方法從24隻3月齡新西蘭大耳白兔髂骨處抽取骨髓，以密度梯度離心法分離齣骨髓間充質榦細胞（MSCs）作為種子細胞進行擴增培養至第三代，製成細胞懸液，而後在自製模具中與藻痠鈣製備成與兔膝關節軟骨全層缺損（直徑4mm、深度4mm）相一緻的載體複閤物同時加入bFGF和維生素C，將該載體複閤物植入兔左膝關節軟骨全層缺損處作為A組，而右膝關節的關節軟骨全層缺損處則植入MSCs與藻痠鈣的載體複閤物作為B組。另取兔6隻，左膝按上述方法作一缺損植入單純藻痠鈣載體作為C組；右膝缺損則作空白對照D組。待術後不同時間點（30 d，60 d及90 d）取材，常規石蠟包埋後製成切片，分彆用于HE、Masson、番紅O、透射電鏡、免疫組化等指標以觀察軟骨缺損脩複效果。結果 A，B，C三組軟骨缺損均被透明軟骨和纖維組織充填，隻是A組的透明軟骨組織較B組多，C組最少。隨植入時間後移，A組幾乎均被透明軟骨樣組織填平併且充填組織與週邊正常關節軟骨的交界逐漸分辨不齣，併與深層軟骨下骨相連接，軟骨細胞間質的膠原纖維從錶層嚮深層呈放射狀排列于軟骨細胞基質中。而B組的缺損處錶麵尚有較多的纖維組織且充填組織與週邊正常關節軟骨的分界較A組清晰，C組缺損處錶麵可見有較多纖維組織和未降解的藻痠鈣，與週邊正常關節軟骨分界較A，B組清晰。A組填充的軟骨細胞具有明顯陷窩的，互相距離較遠的3~5箇細胞排列呈柱狀的成熟軟骨細胞明顯較B組多，C組則很難觀察到成行排列的軟骨細胞。功能旺盛的軟骨細胞（Ⅱ型膠原暘性細胞，胞質粗麵內質網擴張等）則屬A組最多，C組最少。D組術後90 d時缺損處為大量纖維組織填充且與週邊正常關節軟骨和軟骨下骨整閤較差。結論用自體MSC藻痠鈣載體複閤物植入膝關節軟骨缺損同時嚮該缺損微環境內加bFGF和維生素C有利于軟骨脩複，能提高軟骨缺損脩複效果。
목적：연골조직다처우인체골격적중요부위，기결손수복일직위림상급대해결적난제，용조직공정방법수복관절연골결손시근년래정재연구적신도경。기중절대다수연구궤호균착중체외배양조건적연구[1,2]，이홀략료대우개선국부미배경적탐토，위차，본실험시도재재체복합물식입연골결손미배경내시，증가능촉진MSCs분렬、증식、분화급혈관신생적bFGF급삼여화활약성연골세포합성연골기질급섬유적유생소C등，종이체도제고연골결손수복료효적목적。방법종24지3월령신서란대이백토가골처추취골수，이밀도제도리심법분리출골수간충질간세포（MSCs）작위충자세포진행확증배양지제삼대，제성세포현액，이후재자제모구중여조산개제비성여토슬관절연골전층결손（직경4mm、심도4mm）상일치적재체복합물동시가입bFGF화유생소C，장해재체복합물식입토좌슬관절연골전층결손처작위A조，이우슬관절적관절연골전층결손처칙식입MSCs여조산개적재체복합물작위B조。령취토6지，좌슬안상술방법작일결손식입단순조산개재체작위C조；우슬결손칙작공백대조D조。대술후불동시간점（30 d，60 d급90 d）취재，상규석사포매후제성절편，분별용우HE、Masson、번홍O、투사전경、면역조화등지표이관찰연골결손수복효과。결과 A，B，C삼조연골결손균피투명연골화섬유조직충전，지시A조적투명연골조직교B조다，C조최소。수식입시간후이，A조궤호균피투명연골양조직전평병차충전조직여주변정상관절연골적교계축점분변불출，병여심층연골하골상련접，연골세포간질적효원섬유종표층향심층정방사상배렬우연골세포기질중。이B조적결손처표면상유교다적섬유조직차충전조직여주변정상관절연골적분계교A조청석，C조결손처표면가견유교다섬유조직화미강해적조산개，여주변정상관절연골분계교A，B조청석。A조전충적연골세포구유명현함와적，호상거리교원적3~5개세포배렬정주상적성숙연골세포명현교B조다，C조칙흔난관찰도성행배렬적연골세포。공능왕성적연골세포（Ⅱ형효원양성세포，포질조면내질망확장등）칙속A조최다，C조최소。D조술후90 d시결손처위대량섬유조직전충차여주변정상관절연골화연골하골정합교차。결론용자체MSC조산개재체복합물식입슬관절연골결손동시향해결손미배경내가bFGF화유생소C유리우연골수복，능제고연골결손수복효과。
Objective Cartilage tissue is mostly located at the important site of bones in human body, and the repair of its defect remains in suspense in clinical. With the development of the tissue engineering, it brings new approach for the repair of the defect of the articular cartilage. The researchs emphases on the ex vivo condition. Accordingly, scholars ignore the effect of improving the local micro-circumstances. Consequ-ently we add the bFGF and VitC in the micro-circumstances when embedding the carrier compound in order to improve the effect of the repair. Methods Bone marrow is obtained from the iliac bone of 20 rabbits respectively. As the seed cells, Mesenchymal Stem Cells are purified with the density gradient centrifugation and ampilifid. The cell suspension is prepared with the 3rd generation and bFGF/VitC, then finishing the carrier compound which is coincident with the articular cartilage full-thickness defect (diameter 4mm, depth 4mm) and embedding. The left articular genu defects are embedded with carrier compound as the experimental group (A), and the right embedded without bFGF/VitC as the group (B). The left articular genu defects of another 6 rabbits are given the implantation of calcium alginate exclusively as group (C), and the right defects remain blank as control group (D). Sacrificing the responding rabbits in the different time after operation(30d,60d,90d), and paraffin imbedding with routine methods and preparing the microtome section. Then observing the recovery result with the index of HE, Masson, Saffrine O, EM, Immunity-histchemistry. Results The chondro-defects in group A, B and C are filling with hyaline cartilage-similar tissue and fibre tissue. Hyaline cartilage-similar tissue in group A is much more than group B, and it is the lest in group C. With the time extending, the defects in group A are mostly fell with hyaline cartilage-similar. The boundary between normal circum-articular cartilage and filling tissue becomes illegible and the connecting with subcartilage-bone is visible. There are many collagen fibre presented emission-like in the extracellular matrix. On the surface of defects in group B, however, there are still plenty of fibre tissue and the boundary remains distinctive. 3-5 chondrocytes possessing obvious lacunes stand in line in the filling tissue of group A, and much more than that in group B. The phenomenon above can be scarcely observed in group C. The number of vigorous chondrocytes-collagen typeⅡ (+), rough endoplasmic reticulum in cytoplasm dialating etc. In group A is more than that of group B. It is the lest in group C. Considerable fibra tissue can be seen in the defect of group D after 90 days. The integration between filling tissue and subcartilage-bone or normal circum-articular cartilage is un-ideal. Conclusion Adding bFGF and VitC has positive function in improving the repairing effect of the defect location when embedding the compound composed of auto-MSCs and calcium alginate to the defect of knee joint.