骨髓基质细胞修复猪膝关节非负重区软骨与骨复合缺损的实验研究

Repairing porcine knee joint osteochondral defects at non-weight bearing area by autologous BMSC

目的 探讨猪骨髓基质细胞 (bonemarrowstromalcells ,BMSC)复合聚羟基乙酸 /聚乳酸(PGA/PLA)支架修复关节软骨与骨复合缺损的可行性。方法 杂交猪 18只 ,抽取股骨骨髓 ,体外培养、扩增BMSC并分别经地塞米松诱导 (A组 )或地塞米松与转化生长因子 β1(TGFβ1)联合诱导 (B组 ) ,以免疫组织化学、逆转录 聚合酶链反应 (RT PCR)检测其软骨分化表型。其中有 2只猪部分BMSC经绿色荧光蛋白 (GFP)基因转染标记。诱导后的细胞分别接种到PGA/PLA支架 ,体外培养 1周后植入猪自体股骨下端非负重面软骨及骨复合缺损处 ,单纯支架植入 (C组 )及空白不处理 (D组 )作为对照。上述动物分别于 3( 6只 )、6 ( 10只 )个月时取材 ,进行大体观察、修复结果分级、组织学检查、葡糖氨基聚糖 (GAG)含量测定及生物力学测定。含GFP标记细胞的 2只猪 7个月时取材 ,共聚焦显微镜观察植入细胞的分布。结果 诱导后BMSC均能表达软骨特征性的Ⅱ型胶原与聚集蛋白聚糖 (aggrecan) ,两组细胞均与支架材料黏附良好。术后大体观察与组织学检查显示 :A组缺损以不完全修复为主 ,多数缺损软骨修复不良 ,而骨缺损基本修复 ,组织学主要为纤维性软骨及松质骨 ;B组缺损以完全修复为主 ,组织学表现为透明软骨及松质骨 ,少部分标本修复组织中含有纤维?

Objective To test the possibility of using bone marrows tromal cells (BMSC )and biodegradable polymers to repairarticluar osteochondral defects at non weight bearingarea of porcine knee joints. Methods Bonemarrows were harevested from 18 hybridpigs.BMSC were cultured and invitroexpanded and induced with dexame thas one (groupA) or with dexamethasone and trans for ming growth factor β1(TGF β1) (group B)respectively .Immunohis to chemistryand RT PCR were used to evaluatechondrogenic differentiation of induced cells.Part of BMSC of 2animals were retrovirally labeled withgreen fluores centprotein(GFP) .A fterinduction andlabel,cells were seeded on aconstruct of polyglycolicacid (PGA)and polylacticacid (PLA)and cocultured for1 week before implantation .Total 4osteochondraldefects ( 8 mmindiameter,5 mmindepth)ineachanimal were createdat the non weight bearingareas of knee jointson both sides .The defects were repaired with dexame thas one induced BMSC PGA/PLA constructingroup A ,with dexamethasone and TGF β1 in duced BMSC PGA/PLA constructin groupB ,with PGA/PLA constructalone (groupC)or leftuntreated (groupD) as controls.Animals were sacrificed at 3 months (n =6 )or 6 months (n =10 )post repair.Gross observation , histology ,glycosaminogly can (GAG)quant ificationandbiome chanicaltest were applied to analy zetheresults .The two animalswithGFP labeledcellsweresacrificedat 7 month spost repair too bserve with confocalmicroscope the distribution of GFP labeled cell sinrepairedtissue .Results Strongerexpression of typeⅡcollagenandaggrecan wereobservedin BMSC sinduced with both dexamethasone and TGF β1.Atbothtimepoints,Gros so bservation and histology showed thatthedefectsinmost of group A were repaired by engineeredfibrocartilageandcancellous bonewi than irregular surface ,minority de fects were repaired by engineered hyaline cartilageand can cell ous bone. However,in most of group B ,the defects were completelyrepaired by engi neered hyalinecartilageand cancellous bone.Nore pairoronlyfi broustissue were observed in groups Cand D .Besides,the compressivemoduli of repaired cartilageingroups A and Breached 30 37% and 4 3 82 % of normala mountat 3 months and 6 2 6 9% and 80 2 7% at 6month srespectively ,which was further supportedbythehighlevels of GAGcontentsin engineered cartilage of group A ( 78 0 3% of normalcontents)andgroupB (nostatistical difference from normal contents) .Moreimportantly ,confocalmicroscoperevealed the presence of GFP labeledcell sinengi neered cartilagelacuna and repairedunderlyingcancellousbone.Conclusion There sults demonstratedthatimplanted BMSC can different iateintoei the rchondrocytes or osteoblastsat different localenvironments and repaira complex articulardefect with bothengi neered cartilage and bone.TGF β1and dexamethasone in vitro induction can promote chondrogenic differentation of BMSC and thus improve the results of repairing articular defects.