中国组织工程研究与临床康复
中國組織工程研究與臨床康複
중국조직공정연구여림상강복
JOURNAL OF CLINICAL REHABILITATIVE TISSUE ENGINEERING RESEARCH
2009年
46期
9066-9070
,共5页
米坤龙%段王平%李兵%李鹏翠%焦强%卫小春
米坤龍%段王平%李兵%李鵬翠%焦彊%衛小春
미곤룡%단왕평%리병%리붕취%초강%위소춘
自体骨膜%骨髓间充质干细胞%诱导分化%移植%关节软骨%缺损
自體骨膜%骨髓間充質榦細胞%誘導分化%移植%關節軟骨%缺損
자체골막%골수간충질간세포%유도분화%이식%관절연골%결손
背景:自体骨髓间充质干细胞及骨膜移植等方法可促进关节软骨缺损的修复,但其各自的成软骨能力有限,治疗效果欠佳.目的:观察自体骨膜复合向软骨细胞诱导的骨髓间充质干细胞移植修复软骨缺损的疗效.设计、时间及地点:随机分组对照动物实验,于2005-08/2007-03在山西医科大学第二医院骨科实验室完成.材料:6～8月龄新西兰白兔18只,按随机数字表法分为3组,即骨膜复合骨髓间充质干细胞组、单纯骨膜组及空白对照组,每组6只12个膝关节标本.方法:骨膜复合骨髓间充质干细胞组采用胰酶消化法采集骨髓间充质干细胞进行贴壁培养,加入转化生长因子β1向软骨细胞进行诱导分化,同时进行PKH-26细胞膜免疫荧光标记.在全部兔双侧股骨内侧髁造成直径3 mm,深度3 mm的全层软骨缺损,骨膜复合骨髓间充质干细胞组、单纯骨膜组取同侧胫骨上段内侧骨膜,骨膜生发层朝向骨髓腔覆盖软骨缺损,骨膜复合骨髓间充质干细胞组先期缝合3针,向缺损区注入1×109L-1骨髓间充质干细胞细胞悬液20μL,植入细胞后缝合最后1针.单纯骨膜组只进行单纯骨膜覆盖缺损处理;空白对照组只钻孔不作任何处理.主要观察指标:术后6,12周时,对缺损部位进行大体观察、组织学观察、Wakitani评分以及Ⅱ型胶原免疫组化及原位杂交检测.结果:所有缝合骨膜未发现脱落,骨膜复合骨髓间充质干细胞组6周时,软骨缺损已由透明软骨样修复组织填充,12周时修复组织进一步改建,且修复组织中细胞主要为带有PKH-26荧光标记的植入细胞.单纯骨膜组缺损区修复组织为乳白色,表面光滑稍微凹陷,与周围正常软骨组织之间界限清晰;空白对照组缺损区多数比较凹陷,或缺损部位形状不规则,周围软骨组织断裂.术后6,12周时,骨膜复合骨髓间充质干细胞组Wakitani组织学评分均优于单纯骨膜组和空白对照组(P＜0.05),单纯骨膜组的评分优于空白对照组(p＜0.05).同时,骨膜复合骨髓间充质干细胞组修复组织内细胞周围基质Ⅱ型胶原免疫组化及原位杂交染色阳性,证实修复组织内的细胞为植入细胞.结论:自体骨膜复合向软骨细胞诱导的骨髓间充质干细胞移植可形成透明软骨样修复组织修复关节软骨缺损.
揹景:自體骨髓間充質榦細胞及骨膜移植等方法可促進關節軟骨缺損的脩複,但其各自的成軟骨能力有限,治療效果欠佳.目的:觀察自體骨膜複閤嚮軟骨細胞誘導的骨髓間充質榦細胞移植脩複軟骨缺損的療效.設計、時間及地點:隨機分組對照動物實驗,于2005-08/2007-03在山西醫科大學第二醫院骨科實驗室完成.材料:6～8月齡新西蘭白兔18隻,按隨機數字錶法分為3組,即骨膜複閤骨髓間充質榦細胞組、單純骨膜組及空白對照組,每組6隻12箇膝關節標本.方法:骨膜複閤骨髓間充質榦細胞組採用胰酶消化法採集骨髓間充質榦細胞進行貼壁培養,加入轉化生長因子β1嚮軟骨細胞進行誘導分化,同時進行PKH-26細胞膜免疫熒光標記.在全部兔雙側股骨內側髁造成直徑3 mm,深度3 mm的全層軟骨缺損,骨膜複閤骨髓間充質榦細胞組、單純骨膜組取同側脛骨上段內側骨膜,骨膜生髮層朝嚮骨髓腔覆蓋軟骨缺損,骨膜複閤骨髓間充質榦細胞組先期縫閤3針,嚮缺損區註入1×109L-1骨髓間充質榦細胞細胞懸液20μL,植入細胞後縫閤最後1針.單純骨膜組隻進行單純骨膜覆蓋缺損處理;空白對照組隻鑽孔不作任何處理.主要觀察指標:術後6,12週時,對缺損部位進行大體觀察、組織學觀察、Wakitani評分以及Ⅱ型膠原免疫組化及原位雜交檢測.結果:所有縫閤骨膜未髮現脫落,骨膜複閤骨髓間充質榦細胞組6週時,軟骨缺損已由透明軟骨樣脩複組織填充,12週時脩複組織進一步改建,且脩複組織中細胞主要為帶有PKH-26熒光標記的植入細胞.單純骨膜組缺損區脩複組織為乳白色,錶麵光滑稍微凹陷,與週圍正常軟骨組織之間界限清晰;空白對照組缺損區多數比較凹陷,或缺損部位形狀不規則,週圍軟骨組織斷裂.術後6,12週時,骨膜複閤骨髓間充質榦細胞組Wakitani組織學評分均優于單純骨膜組和空白對照組(P＜0.05),單純骨膜組的評分優于空白對照組(p＜0.05).同時,骨膜複閤骨髓間充質榦細胞組脩複組織內細胞週圍基質Ⅱ型膠原免疫組化及原位雜交染色暘性,證實脩複組織內的細胞為植入細胞.結論:自體骨膜複閤嚮軟骨細胞誘導的骨髓間充質榦細胞移植可形成透明軟骨樣脩複組織脩複關節軟骨缺損.
배경:자체골수간충질간세포급골막이식등방법가촉진관절연골결손적수복,단기각자적성연골능력유한,치료효과흠가.목적:관찰자체골막복합향연골세포유도적골수간충질간세포이식수복연골결손적료효.설계、시간급지점:수궤분조대조동물실험,우2005-08/2007-03재산서의과대학제이의원골과실험실완성.재료:6～8월령신서란백토18지,안수궤수자표법분위3조,즉골막복합골수간충질간세포조、단순골막조급공백대조조,매조6지12개슬관절표본.방법:골막복합골수간충질간세포조채용이매소화법채집골수간충질간세포진행첩벽배양,가입전화생장인자β1향연골세포진행유도분화,동시진행PKH-26세포막면역형광표기.재전부토쌍측고골내측과조성직경3 mm,심도3 mm적전층연골결손,골막복합골수간충질간세포조、단순골막조취동측경골상단내측골막,골막생발층조향골수강복개연골결손,골막복합골수간충질간세포조선기봉합3침,향결손구주입1×109L-1골수간충질간세포세포현액20μL,식입세포후봉합최후1침.단순골막조지진행단순골막복개결손처리;공백대조조지찬공불작임하처리.주요관찰지표:술후6,12주시,대결손부위진행대체관찰、조직학관찰、Wakitani평분이급Ⅱ형효원면역조화급원위잡교검측.결과:소유봉합골막미발현탈락,골막복합골수간충질간세포조6주시,연골결손이유투명연골양수복조직전충,12주시수복조직진일보개건,차수복조직중세포주요위대유PKH-26형광표기적식입세포.단순골막조결손구수복조직위유백색,표면광활초미요함,여주위정상연골조직지간계한청석;공백대조조결손구다수비교요함,혹결손부위형상불규칙,주위연골조직단렬.술후6,12주시,골막복합골수간충질간세포조Wakitani조직학평분균우우단순골막조화공백대조조(P＜0.05),단순골막조적평분우우공백대조조(p＜0.05).동시,골막복합골수간충질간세포조수복조직내세포주위기질Ⅱ형효원면역조화급원위잡교염색양성,증실수복조직내적세포위식입세포.결론:자체골막복합향연골세포유도적골수간충질간세포이식가형성투명연골양수복조직수복관절연골결손.
BACKGROUND:Such methods as transplanting autologous periosteum or autologous bone marrow mesenchymal stem cells (BMSCs) can promote the repair of articular cartilage defects for sure. But they all have their own limits in chondrogenic abilities,which results in an unsatisfactory curative effect.OBJECTIVE:To study the effect of transplanting BMSCs (which were induced into chondrocytes) combined with autogenous periosteum on repairing articular cartilage defects in rabbits.MATERIALS:A total of 18 New Zealand rabbits,aged 6-8 months,were divided with random digits table method into 3 groups,namely,periosteum+BMSCs group,periosteum group and blank control group,with 6 ones (12 knee joint samples) in each group. METHODS:In periosteum+BMSCs group,BMSCs were harvested and adherently cultured with trypsin digestion method. Then they were induced by transforming growth factor 81 into chondrocytes. At the same time,immunofluorescence labeling was performed to BMSCs membranes with PKH-26. Full-thickness articular cartilage defects (diameter:3mm,depth:3mm) were made to bilateral condylus medialis femoris of all rabbits. In periosteum+BMSCs group and periosteum group,defects were covered by homolateral autogenous proximal tibia periosteums,with germinal layer facing to cavitas medullaris. After that,the periosteum+BMSCs group received 3 sutures,followed by injection of 20 μL BMSCs suspension (1×109/L) into the defects,after which the last suture was taken. The periosteum group underwent coverage with periosteum on defects only. The blank control group underwent perforate only.MAIN OUTCOME MEASURES:General observation,histological observation,Wakitani's score,immunohistochemical and in situ hybridization detection of collagen type Ⅱ were performed to defects at 6 and 12 weeks following operation.RESULTS:No sutured periosteums were found desquamate. In periosteum+BMSCs group,defects were filled with hyaline cartilage-like repairing tissues at week 6 following operation;Week 12 following operation saw remodeled tissues whose cells were mainly the implanted cells labeled with PKH-26. In periosteum group,repairing tissues in defect areas were ivory white,smooth with light introcession and distinctively different from the surrounding normal cartilage tissue. In the blank control group,clearer introcession or irregular appearance,even broken surrounding cartilage tissues could be seen in the defect area. Both Wakitani's score and histological score were highest in periosteum+BMSCs group at week 6 and 12 following operation (P＜0.05),with higher ones in periosteum group than in the control group (P＜0.05). What'more,matrix around cells in the repairing tissues showed positive results to both immunohistochemical and in situ hybridization staining of collagen typeⅡ,which proved that cells in repairing tissues were the implanted ones.CONCLUSION:Transplanted BMSCs (which were induced into chondrocytes) combined with autogenous periosteum can form hyaline cartilage-like repairing tissues through which articular cartilage defects are repaired.
