脂肪基质干细胞复合SIS治疗家兔生长板缺损的实验研究
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摘要
目的:
体外分离培养家兔脂肪血管基质部分细胞(Stromal Vascular Fractioncells,SVF cells),并检测其间充质干细胞表面抗原及多向分化潜能,证实其为脂肪基质干细胞(Adipose Derived Mesenchymal Stem Cells,ADSCs);制备猪小肠粘膜下层(Small Intestinal Submucosa,SIS),验证其组织相容性和细胞相容性;观察ADSCs-SIS复合物在体外经成软骨诱导培养基诱导分化的效果;观察经体外成软骨诱导的ADSCs-SIS复合物用于治疗家兔胫骨近端内侧生长板缺损的疗效。
方法:
第一部分切取家兔腹股沟区脂肪,Ⅰ型胶原酶消化,获取脂肪组织中血管基质部分细胞,体外培养扩增,取第3代SVF细胞,流式细胞术检测表面标记物CD29、CD44、CD45,进行成脂、成骨、成软骨诱导分化,油红O染色鉴定成脂诱导分化,碱性磷酸酶染色、茜素红染色、Von Kossa染色鉴定成骨诱导分化,Ⅱ型胶原mRNA RT-PCR检测、Ⅱ型胶原免疫组织化学染色、甲苯胺蓝染色、番红O染色鉴定成软骨诱导分化,MTT法绘制细胞生长曲线;
第二部分使用酶消化-高渗盐水脱细胞法制备猪小肠粘膜下层脱细胞基质材料,-52℃深低温冷冻干燥,伽玛射线灭菌分装,取1cm~2大小SIS植入家兔肌袋中,于术后2周和4周取材进行HE切片检测。SIS复水后,接种第3代脂肪基质干细胞于SIS的单面或双面,空白SIS支架材料和细胞支架材料复合物进行电镜和石蜡切片检测;
第三部分将第3代脂肪基质干细胞接种于复水后的SIS双面,加入成软骨诱导培养基,于体外诱导培养第7天和14天后,使用实时荧光定量RT-PCR检测Ⅱ型胶原mRNA,GAPDH作为内参基因,使用2~(-ΔΔCT)法计算CT值的标化值,比较未经诱导的ADSCs-SIS复合物、诱导7天和诱导14天的ADSCs-SIS复合物的Ⅱ型胶原mRNA量的变化,免疫组织化学染色检测Ⅱ型胶原蛋白,甲苯胺蓝染色及番红O染色观察细胞外基质,扫描电镜观察体外成软骨诱导14天后细胞在支架材料上生长状况;
第四部分将第3代同种异体脂肪基质干细胞接种于SIS的双面,在体外使用成软骨诱导培养基诱导分化14天后,准备植入家兔生长板缺损。取36只6-8周新西兰大耳白兔,随机分为A、B、C三组,每组12只,三组均造成右侧胫骨近端内侧生长板50%的缺损,A组为空白对照组,缺损不植入填充物,B组缺损填充空白SIS支架材料,C组填充在体外进行成软骨诱导14天的ADSCs-SIS复合物,左侧为正常对照组,不进行手术。于术后4、8、16周,每组各处死4只家兔,取双侧胫、腓骨,10%福尔马林固定,X线片摄片,测量双侧胫骨长度及胫骨上关节面角度,计算每只兔左、右侧胫骨上关节面角度差值及胫骨长度差值,组内比较家兔双侧胫骨长度及胫骨上关节面角度差异,组间比较胫骨长度差值及胫骨上关节面角度差值的差异。石蜡切片观察生长板软骨修复状况。
应用SPSS 13.0软件进行数据录入和统计学处理,采用配对T检验和单因素方差分析进行分析。
结果:
第一部分家兔原代脂肪基质血管部分细胞为短梭形及多角形,5-7天达90%融合,传代后,转变为长梭型,传至第6代,仍保持长梭形,且继续增殖。脂肪基质血管部分细胞成骨诱导后14天,碱性磷酸酶染色阳性,诱导28天后,茜素红染色、Von Kossa染色阳性,成脂诱导后7天,油红O染色阳性。成软骨诱导分化后,Ⅱ型胶原mRNA RT-PCR检测提示SVF细胞在178bp处出现产物条带,SVF细胞诱导3天、7天和14天后,Ⅱ型胶原mRNA RT-PCR产物在178bp处也出现条带,其中诱导7天和14后产物条带信号较强,诱导14天后Ⅱ型胶原免疫组织化学染色阳性,细胞聚集处甲苯胺蓝异染阳性,番红O染色见胞浆红染。生长曲线提示第三代细胞于接种后第3天达到指数期,第5天达到平台期,成软骨诱导时,脂肪基质干细胞生长变缓。
第二部分猪小肠粘膜下层为白色半透明膜状物,冻干后仍为白色膜状物,γ射线辐照消毒后,颜色不发生改变,电镜检测提示SIS粘膜面为光滑面,浆膜面为粗糙面,石蜡切片检测显示SIS纤维表面无细胞残留,经完全培养基复水后,SIS为柔软膜状物,复合ADSCs后,相差显微镜下可见材料表面有细胞附着生长,扫面电镜检测提示单面复合ADSCs后,上表面可见大量ADSCs附着,下表面只有少量ADSCs附着,双面复合ADSCs后,上、下表面均可见大量ADSCs附着,石蜡切片显示ADSCs附着SIS纤维生长,SIS植入家兔肌袋后,无感染及免疫排斥反应发生,石蜡切片显示2周时SIS被完全包裹,4周时,SIS被完全吸收。
第三部分Ⅱ型胶原mRNA荧光定量RT-PCR检测提示,体外成软骨诱导后7天、14天的ADSCs—SIS复合物Ⅱ型胶原mRNA的标化值与未诱导的ADSCs—SIS复合物有显著性差异(P＜0.05),诱导14天与诱导7天的ADSCs—SIS复合物Ⅱ型胶原mRNA的标化值有显著性差异(P＜0.05),ADSCs—SIS复合物成软骨诱导后14天,Ⅱ型胶原免疫组化染色为阳性,甲苯胺蓝染色可见基质异染,番红O染色可见细胞外基质致密红染,未诱导的ADSCs—SIS复合物Ⅱ型胶原免疫组化染色为阴性。扫描电镜检测显示诱导14天时,细胞长满支架材料的双面。
第四部分家兔术后饮食、活动正常,伤口无感染,无免疫排斥反应。标本大体观察提示移植后4周,A组右侧胫骨近端关节面明显成角畸形,B组右侧胫骨近端关节面稍有成角畸形,C组右侧胫骨近端关节面无明显成角畸形,三个组均无明显的胫骨短缩畸形;术后8周,A组右侧胫骨近端关节面有明显成角畸形,胫骨有短缩畸形,B组右侧胫骨近端关节面有成角畸形,胫骨有短缩畸形,C组右侧胫骨近端关节面无成角畸形,胫骨无短缩畸形;术后16周,A组右侧胫骨近端关节面有明显成角畸形,胫骨有明显短缩畸形,B组右侧胫骨近端关节面有明显成角畸形,胫骨有明显短缩畸形,C组右侧胫骨近端关节面无成角畸形,胫骨无短缩畸形。X线片测量的组内比较提示,A组在4、8、16周时,左、右侧胫骨上关节面角度和胫骨长度具有显著性差异(P＜0.05);B组在4周时,左、右侧胫骨上关节面角度无显著性差异(P＞0.05),胫骨长度有显著性差异(P＜0.05),8周时,左、右侧胫骨上关节面角度无显著性差异(P＞0.05),胫骨长度无显著性差异(P＞0.05),16周时,左、右侧胫骨上关节面角度有显著性差异(P＜0.05),胫骨长度有显著性差异(P＜0.05);C组在4、8、16周时,左、右侧胫骨上关节面角度均无显著性差异(P＞0.05),胫骨长度均无显著性差异(P＞0.05)。X线片测量的组间比较提示,术后4周时,A组胫骨近端关节面成角差值大于B组(P＜0.05)和C组(P＜0.05),而B组与C组无显著性差异(P＞0.05),三个组胫骨长度差值无显著性差异(P＞0.05);术后8周,A组胫骨近端关节面成角差值大于B组(P＜0.05)和C组(P＜0.05),而B组与C组无显著性差异(P＞0.05),A组胫骨长度差值大于B组(P＜0.05)和C组(P＜0.05),B组与C组无显著性差异(P＞0.05);16周时,A组胫骨近端关节面成角差值大于B组(P＜0.05)和C组(P＜0.05),而B组大于C组(P＜0.05),A组胫骨长度差值大于B组(P＜0.05)和C组(P＜0.05),而B组大于C组(P＜0.05)。HE染色切片示,在术后4周,A组生长板有骨桥形成,未见修复软骨,B组生长板内可见骨桥形成,有少量的修复软骨出现,C组生长板内无骨桥形成,修复软骨排列呈无序状;术后8周,A组生长板内骨桥未被吸收,生长板软骨中断,B组生长板内骨桥未被吸收,生长板软骨中断,C组生长板软骨排列成柱状;术后16周,A组生长板骨桥未被吸收,外侧生长板未闭合,B组生长板缺损内骨桥致密,生长板软骨中断,外侧生长板未闭合;C组生长板为软骨性修复,软骨呈柱状排列,生长板未闭合。
结论:
第一部分脂肪血管基质部分细胞的间充质干细胞表面标记物为阳性,具有多向诱导分化潜能,证实所获取的SVF细胞为脂肪基质干细胞。
第二部分酶消化—高渗盐水脱细胞法制备的小肠粘膜下层具有良好的组织相容性和细胞相容性,适合作为脂肪基质干细胞的支架材料。
第三部分脂肪基质干细胞复合至小肠粘膜下层后,在体外经成软骨诱导培养基成软骨诱导后,能够向成软骨细胞分化。
第四部分体外成软骨诱导分化后的ADSCs-SIS复合物,用于治疗家兔胫骨近端生长板缺损,有效避免了肢体的成角和短缩畸形,并且能够修复生长板软骨,为ADSCs-SIS复合物体外成软骨诱导分化后用于临床治疗生长板损伤提供一定参考。
Purpose:
To isolate and culture stromal vascular fraction cells(SVF cells) from adipose tissue of rabbits, and to testify SVF cells are adipose derived mesenchymal stem cells(ADSCs) by examining mesenchymal stem cell surface antigens and potential of multidirectional differentiation. To prepare porcine small intestinal submucosa, and examine its histocompatibility and cell compatibility. To observe chondrogenic effect of chondrogenic medium on ADSCs-SIS composite in vitro. To observe reparative effect of ADSCs-SIS composites induced by chondrogenic medium in vitro on treatment of growth plate defects in rabbits.
Method:
Part 1 Dissected the subcutaneous fat from groin of rabbit, digested with type I Collagenase to obtain stromal vascular fraction cells (SVF cells), the surface antigens including CD29, CD44, CD45 of 3~(rd) passsge SVF cells were analyzed by flow cytometry, multi-directional differentiation potential of SVF cells were examined by adipogenic,osteogenic,and chondrogenic differentiation, alkaline Phosphatase staining, alizarin red staining, Von Kossa staining were used to identify osteogenic differentiation, oil red staining were used to identify adipogenic differentiation, type II collagen mRNA RT-PCR, type II collagen immunohistological staining, toluidine blue staining and safranin O staining were used to identify chondrogenic differentiation.
Part 2 Small intestinal submucosa were processed by enzyme digestion-hypertonic saline decellularization, lyophilized at -52℃in high vaccum, and sterilized by gamma radiation, paraffin slices were used to observe the effect of decellularization, the surface structure of SIS were observed by scan electron microscope(SEM).SIS were implant into sacrospinous muscle pocket of rabbits, the specimens were examined by paraffin slice to observe degradation and histocompatibility of SIS, ADSCs of rabbits were isolated and cultured in vitro, 3~(rd) passage of ADSCs were seeded onto one side or both sides of SIS, after one week of cocultivation, ADSCs-SIS composite were observed by paraffin slice and SEM.
Part 3 3~(rd) passage of ADSCs were seeded onto both sides of SIS, and induced by chondrogenic medium in vitro, 7 days and 14 days after being induced, real-time fluorescent quantitative RT-PCR was used to analyze mRNA level of typeⅡcollagen, GAPDH was used as internal control gene, 2~(-△△CT) method was used to calculate. ADSCs-SIS composites which were not induced were used as control, typeⅡcollagen protein was examined by histoimmunologic staining, toluidine blue staining and safranin O staining were used to observe extracellular matrix. SEM was used to observe cellular morphology after 14 days of chondrogenic differentiation.
Part 4 3~(rd) passage of ADSCs were seeded onto both sides of SIS, and induced by chondrogenic medium in vitro for 14 days before being implanted. 36 New Zealand rabbits of 6-8 weeks old were divided into 3 groups randomly, 50% of medial growth plate in proximal right tibia was excised, group A was blank control group, the defects of growth plates were not filled, the defects of growth plates in group B were filled with SIS without cells, the defects of growth plates in group C were filled with ADSCs-SIS composites induced by chondrogenic medium in vitro for 14 days. 4 rabbits of each group were sacrificed at 4, 8 and 16 weeks, leg bones of both sides were taken off, fixed with 10% formalin solution, X ray photographs were taken to measure tibial length and proximal tibial articular surface angle. Calculated the differences of proximal articular surface angle and length of left and right tibias in each rabbit. Compared proximal tibial articular surface angle and length of left and right tibia in each group of different time interval, and compared difference of proximal articular surface angle and length of left and right tibias among 3 groups of different time interval. Paraffin slices stained with HE were used to observe reparative effect of ADSCs-SIS composites on growth plate cartilage.
Spss 13.0 statitical package was used to deal with all data. Calculate a p-Value for paired T test and one-factor analysis of variance.
Result:
Part 1 Primary SVF cells were multi-angular or short spindle shaped, 3~(rd) passage SVF cells were long spindle shaped. Cell surface antigen of 3~(rd) passage SVF cells were CD44+, CD29+, CD45-. Oil red staining was positive in adipogenic differentiation group, ALP staining, alizarin red staining and Von Kossa staining were positive in osteogenic differentiation group. RT-PCR of typeⅡcollagen mRNA showed that SVFs cells also expressed typeⅡcollagen mRNA, but the product bands were brighter in cells after 7 and 14 days of chondrogenic differentiation. After 14 days of chondrogenic differentiation, histoimmunologic staining of typeⅡcollagen was positive, toluidine blue staining showed that metachromatic staining was observed, and safranin O staining showed that cytoplasms were stained red.
Part 2 SIS was white and semi opaque membrane, paraffin slices showed that no cell was observed in SIS, loose weave structure of serosal surface and compact structure of mucosal surface were observed by SEM, after one week of cocultivation, plenty of cells could be observed on the upside and few cells were observed on the downside of SIS when ADSCs were seeded only onto upside of SIS, plenty of cells could be observed on both sides of SIS when ADSCs were seeded onto both sides of SIS. Cells adhering to the fibers of SIS could be observed in paraffin slice after ADSCs were implanted onto SIS. After SIS were implanted into the muscle pocket of rabbits, infection and immunologic reaction were not observed, paraffin slice showed that only few of SIS fibers were not absorbed at 2 weeks after implantation, and completely abosorbed at 4 weeks after implantation.
Part 3 Standardized quantity of typeⅡcollagen mRNA in ADSCs-SIS composites which were induced for 7 days and 14 days were larger than that in uninduced ADSCs-SIS composites(P < 0.05), standardized quantity of typeⅡcollagen mRNA in ADSCs-SIS composites which were induced for 14 days was larger than that in ADSCs-SIS composites which were induced for 7 days(P<0.05) . Histoimmunologic staining of typeⅡcollagen protein was positive in ADSCs-SIS composites which were induced for 14 days by chondrogenic medium, but was negative in uninduced ADSCs-SIS composites. Extracellular matrix was metachromatic stained by toluidine blue, and red stained by safranin O in ADSCs-SIS composites which were induced for 14 days by chondrogenic medium. Plenty of ADSCs adhered to both sides of SIS after 14 days of chondrogenic differentiation were observed by SEM.
Part 4 After operation, incisions were not infected, and no immunologic rejection was observed, feeding and activity of animals were normal. The direct-viewing of specimens showed that 4 weeks after operation, obvious angular deformity of tibias was observed in group A, and slight angular deformity of tibias was observed in group B, no angular deformity of tibias was observed in group C, and no length discrepancy was observed in all 3 groups; 8 weeks after operation, obvious angular deformity and length discrepancy of tibias were observed in group A, angular deformity and length discrepancy of tibias were observed in group B, no length discrepancy or angular deformity was observed in group C; 16 weeks after operation, obvious angular deformity and obvious length discrepancy of tibias were observed in group A and group B, no angular deformity or length discrepancy of tibias was observed in group C. Comparison of X ray photograph measurement in each group showed that, in group A, 4, 8, 16 weeks after operation, there were significant differences between right and left tibias in the proximal articular surface angle(P< 0.05) and length(P<0.05); in group B, 4 weeks after operation, there was significant difference between right and left tibias in length(P<0.05) , there was no significant difference between right and left tibias in the proximal articular surface angle(P> 0.05), 8 weeks after operation, there was no significant difference between right and left tibias in the proximal articular surface angle or length(P>0.05), 16 weeks after operation, there were significant differences between right and left tibias in the proximal articular surface angle and length(P<0.05) ; in group C, 4, 8, 16 weeks after operation, there was no significant difference between right and left tibias in the proximal articular surface angle(P>0.05) or length(P>0.05). Comparison of X ray photograph measurement among 3 groups showed that 4 weeks after operation, the proximal tibial articular surface angle difference of group A was larger than that of group B(P<0.05) and that of group C(P<0.05) ,there was no significant difference between group B and group C in proximal tibial articular surface angle difference(P> 0.05), and there was no significant difference among all 3 groups in tibial length difference(P>0.05); 8 weeks after operation, proximal tibial articular surface angle difference of group A was larger than that of group B(P<0.05) and that of group C(P <0.05) , tibial length difference of group A was larger than that of group B(P<0.05) and that of group C(P<0.05) , there was no significant difference between group B and group C in tibial length difference(P>0.05) or proximal tibial articular surface angle difference(P>0.05); 16 weeks after operation, proximal tibial articular surface angle difference of group A was larger than that of group B(P<0.05) and that of group C(P<0.05) , proximal tibial articular surface angle difference of group B was larger than that of group C(P<0.05) , tibial length difference of group A was larger than that of group B(P<0.05) and that of group C(P<0.05), tibial length difference of group B was larger than that of group C(P<0.05) . Paraffin slices stained with HE showed that 4 weeks after operation, bone bridges could be seen in growth plate cartilages and no reparative cartilage could be seen in growth plates of group A, bone bridges could be seen in growth plates and few reparative cartilages could be seen in growth plates of group B, and no bone bridges could be seen and reparative cartilages could be seen in growth plates of group C; 8 weeks after operation, bone bridges could still be seen in group A, bone bridges were not absorbed and growth plates were still discontinuous in group B, the reparative cartilages in growth plates rearranged in group C; 16 weeks after operation, bone bridges could be seen in growth plates of group A and group B, and cartilages rearranged like column in the medial half of growth plates in group C.
Conclusion:
Part 1 Stromal vascular fraction cells have identical surface antigens of mesenchymal stem cells and potential of multidirectional differentiation. So SVF cells were identified as adipose derived mesenchymal stem cells
Part 2 Small intestinal submucosa has good histocompatibility and good cell compatibility. It can be used as scaffold for ADSCs.
Part 3 ADSCs could differentiated into chondrogenic cells after being induced by chondrogenic medium in SIS scaffold.
Part 4 ADSCs-SIS composites induced by chondrogenic medium in vitro could prevent angular deformity and length discrepancy of tibias effectively after being implanted into medial growth plate defect of proximal tibia, and could repair cartilage of growth plates. This experimental study can give some suggestion to clinical treatment of growth plate injury by using the ADSCs-SIS composites which are induced by chondrogenic medium in vitro.
体外分离培养家兔脂肪血管基质部分细胞(Stromal Vascular Fractioncells,SVF cells),并检测其间充质干细胞表面抗原及多向分化潜能,证实其为脂肪基质干细胞(Adipose Derived Mesenchymal Stem Cells,ADSCs);制备猪小肠粘膜下层(Small Intestinal Submucosa,SIS),验证其组织相容性和细胞相容性;观察ADSCs-SIS复合物在体外经成软骨诱导培养基诱导分化的效果;观察经体外成软骨诱导的ADSCs-SIS复合物用于治疗家兔胫骨近端内侧生长板缺损的疗效。
方法:
第一部分切取家兔腹股沟区脂肪,Ⅰ型胶原酶消化,获取脂肪组织中血管基质部分细胞,体外培养扩增,取第3代SVF细胞,流式细胞术检测表面标记物CD29、CD44、CD45,进行成脂、成骨、成软骨诱导分化,油红O染色鉴定成脂诱导分化,碱性磷酸酶染色、茜素红染色、Von Kossa染色鉴定成骨诱导分化,Ⅱ型胶原mRNA RT-PCR检测、Ⅱ型胶原免疫组织化学染色、甲苯胺蓝染色、番红O染色鉴定成软骨诱导分化,MTT法绘制细胞生长曲线;
第二部分使用酶消化-高渗盐水脱细胞法制备猪小肠粘膜下层脱细胞基质材料,-52℃深低温冷冻干燥,伽玛射线灭菌分装,取1cm~2大小SIS植入家兔肌袋中,于术后2周和4周取材进行HE切片检测。SIS复水后,接种第3代脂肪基质干细胞于SIS的单面或双面,空白SIS支架材料和细胞支架材料复合物进行电镜和石蜡切片检测;
第三部分将第3代脂肪基质干细胞接种于复水后的SIS双面,加入成软骨诱导培养基,于体外诱导培养第7天和14天后,使用实时荧光定量RT-PCR检测Ⅱ型胶原mRNA,GAPDH作为内参基因,使用2~(-ΔΔCT)法计算CT值的标化值,比较未经诱导的ADSCs-SIS复合物、诱导7天和诱导14天的ADSCs-SIS复合物的Ⅱ型胶原mRNA量的变化,免疫组织化学染色检测Ⅱ型胶原蛋白,甲苯胺蓝染色及番红O染色观察细胞外基质,扫描电镜观察体外成软骨诱导14天后细胞在支架材料上生长状况;
第四部分将第3代同种异体脂肪基质干细胞接种于SIS的双面,在体外使用成软骨诱导培养基诱导分化14天后,准备植入家兔生长板缺损。取36只6-8周新西兰大耳白兔,随机分为A、B、C三组,每组12只,三组均造成右侧胫骨近端内侧生长板50%的缺损,A组为空白对照组,缺损不植入填充物,B组缺损填充空白SIS支架材料,C组填充在体外进行成软骨诱导14天的ADSCs-SIS复合物,左侧为正常对照组,不进行手术。于术后4、8、16周,每组各处死4只家兔,取双侧胫、腓骨,10%福尔马林固定,X线片摄片,测量双侧胫骨长度及胫骨上关节面角度,计算每只兔左、右侧胫骨上关节面角度差值及胫骨长度差值,组内比较家兔双侧胫骨长度及胫骨上关节面角度差异,组间比较胫骨长度差值及胫骨上关节面角度差值的差异。石蜡切片观察生长板软骨修复状况。
应用SPSS 13.0软件进行数据录入和统计学处理,采用配对T检验和单因素方差分析进行分析。
结果:
第一部分家兔原代脂肪基质血管部分细胞为短梭形及多角形,5-7天达90%融合,传代后,转变为长梭型,传至第6代,仍保持长梭形,且继续增殖。脂肪基质血管部分细胞成骨诱导后14天,碱性磷酸酶染色阳性,诱导28天后,茜素红染色、Von Kossa染色阳性,成脂诱导后7天,油红O染色阳性。成软骨诱导分化后,Ⅱ型胶原mRNA RT-PCR检测提示SVF细胞在178bp处出现产物条带,SVF细胞诱导3天、7天和14天后,Ⅱ型胶原mRNA RT-PCR产物在178bp处也出现条带,其中诱导7天和14后产物条带信号较强,诱导14天后Ⅱ型胶原免疫组织化学染色阳性,细胞聚集处甲苯胺蓝异染阳性,番红O染色见胞浆红染。生长曲线提示第三代细胞于接种后第3天达到指数期,第5天达到平台期,成软骨诱导时,脂肪基质干细胞生长变缓。
第二部分猪小肠粘膜下层为白色半透明膜状物,冻干后仍为白色膜状物,γ射线辐照消毒后,颜色不发生改变,电镜检测提示SIS粘膜面为光滑面,浆膜面为粗糙面,石蜡切片检测显示SIS纤维表面无细胞残留,经完全培养基复水后,SIS为柔软膜状物,复合ADSCs后,相差显微镜下可见材料表面有细胞附着生长,扫面电镜检测提示单面复合ADSCs后,上表面可见大量ADSCs附着,下表面只有少量ADSCs附着,双面复合ADSCs后,上、下表面均可见大量ADSCs附着,石蜡切片显示ADSCs附着SIS纤维生长,SIS植入家兔肌袋后,无感染及免疫排斥反应发生,石蜡切片显示2周时SIS被完全包裹,4周时,SIS被完全吸收。
第三部分Ⅱ型胶原mRNA荧光定量RT-PCR检测提示,体外成软骨诱导后7天、14天的ADSCs—SIS复合物Ⅱ型胶原mRNA的标化值与未诱导的ADSCs—SIS复合物有显著性差异(P＜0.05),诱导14天与诱导7天的ADSCs—SIS复合物Ⅱ型胶原mRNA的标化值有显著性差异(P＜0.05),ADSCs—SIS复合物成软骨诱导后14天,Ⅱ型胶原免疫组化染色为阳性,甲苯胺蓝染色可见基质异染,番红O染色可见细胞外基质致密红染,未诱导的ADSCs—SIS复合物Ⅱ型胶原免疫组化染色为阴性。扫描电镜检测显示诱导14天时,细胞长满支架材料的双面。
第四部分家兔术后饮食、活动正常,伤口无感染,无免疫排斥反应。标本大体观察提示移植后4周,A组右侧胫骨近端关节面明显成角畸形,B组右侧胫骨近端关节面稍有成角畸形,C组右侧胫骨近端关节面无明显成角畸形,三个组均无明显的胫骨短缩畸形;术后8周,A组右侧胫骨近端关节面有明显成角畸形,胫骨有短缩畸形,B组右侧胫骨近端关节面有成角畸形,胫骨有短缩畸形,C组右侧胫骨近端关节面无成角畸形,胫骨无短缩畸形;术后16周,A组右侧胫骨近端关节面有明显成角畸形,胫骨有明显短缩畸形,B组右侧胫骨近端关节面有明显成角畸形,胫骨有明显短缩畸形,C组右侧胫骨近端关节面无成角畸形,胫骨无短缩畸形。X线片测量的组内比较提示,A组在4、8、16周时,左、右侧胫骨上关节面角度和胫骨长度具有显著性差异(P＜0.05);B组在4周时,左、右侧胫骨上关节面角度无显著性差异(P＞0.05),胫骨长度有显著性差异(P＜0.05),8周时,左、右侧胫骨上关节面角度无显著性差异(P＞0.05),胫骨长度无显著性差异(P＞0.05),16周时,左、右侧胫骨上关节面角度有显著性差异(P＜0.05),胫骨长度有显著性差异(P＜0.05);C组在4、8、16周时,左、右侧胫骨上关节面角度均无显著性差异(P＞0.05),胫骨长度均无显著性差异(P＞0.05)。X线片测量的组间比较提示,术后4周时,A组胫骨近端关节面成角差值大于B组(P＜0.05)和C组(P＜0.05),而B组与C组无显著性差异(P＞0.05),三个组胫骨长度差值无显著性差异(P＞0.05);术后8周,A组胫骨近端关节面成角差值大于B组(P＜0.05)和C组(P＜0.05),而B组与C组无显著性差异(P＞0.05),A组胫骨长度差值大于B组(P＜0.05)和C组(P＜0.05),B组与C组无显著性差异(P＞0.05);16周时,A组胫骨近端关节面成角差值大于B组(P＜0.05)和C组(P＜0.05),而B组大于C组(P＜0.05),A组胫骨长度差值大于B组(P＜0.05)和C组(P＜0.05),而B组大于C组(P＜0.05)。HE染色切片示,在术后4周,A组生长板有骨桥形成,未见修复软骨,B组生长板内可见骨桥形成,有少量的修复软骨出现,C组生长板内无骨桥形成,修复软骨排列呈无序状;术后8周,A组生长板内骨桥未被吸收,生长板软骨中断,B组生长板内骨桥未被吸收,生长板软骨中断,C组生长板软骨排列成柱状;术后16周,A组生长板骨桥未被吸收,外侧生长板未闭合,B组生长板缺损内骨桥致密,生长板软骨中断,外侧生长板未闭合;C组生长板为软骨性修复,软骨呈柱状排列,生长板未闭合。
结论:
第一部分脂肪血管基质部分细胞的间充质干细胞表面标记物为阳性,具有多向诱导分化潜能,证实所获取的SVF细胞为脂肪基质干细胞。
第二部分酶消化—高渗盐水脱细胞法制备的小肠粘膜下层具有良好的组织相容性和细胞相容性,适合作为脂肪基质干细胞的支架材料。
第三部分脂肪基质干细胞复合至小肠粘膜下层后,在体外经成软骨诱导培养基成软骨诱导后,能够向成软骨细胞分化。
第四部分体外成软骨诱导分化后的ADSCs-SIS复合物,用于治疗家兔胫骨近端生长板缺损,有效避免了肢体的成角和短缩畸形,并且能够修复生长板软骨,为ADSCs-SIS复合物体外成软骨诱导分化后用于临床治疗生长板损伤提供一定参考。
Purpose:
To isolate and culture stromal vascular fraction cells(SVF cells) from adipose tissue of rabbits, and to testify SVF cells are adipose derived mesenchymal stem cells(ADSCs) by examining mesenchymal stem cell surface antigens and potential of multidirectional differentiation. To prepare porcine small intestinal submucosa, and examine its histocompatibility and cell compatibility. To observe chondrogenic effect of chondrogenic medium on ADSCs-SIS composite in vitro. To observe reparative effect of ADSCs-SIS composites induced by chondrogenic medium in vitro on treatment of growth plate defects in rabbits.
Method:
Part 1 Dissected the subcutaneous fat from groin of rabbit, digested with type I Collagenase to obtain stromal vascular fraction cells (SVF cells), the surface antigens including CD29, CD44, CD45 of 3~(rd) passsge SVF cells were analyzed by flow cytometry, multi-directional differentiation potential of SVF cells were examined by adipogenic,osteogenic,and chondrogenic differentiation, alkaline Phosphatase staining, alizarin red staining, Von Kossa staining were used to identify osteogenic differentiation, oil red staining were used to identify adipogenic differentiation, type II collagen mRNA RT-PCR, type II collagen immunohistological staining, toluidine blue staining and safranin O staining were used to identify chondrogenic differentiation.
Part 2 Small intestinal submucosa were processed by enzyme digestion-hypertonic saline decellularization, lyophilized at -52℃in high vaccum, and sterilized by gamma radiation, paraffin slices were used to observe the effect of decellularization, the surface structure of SIS were observed by scan electron microscope(SEM).SIS were implant into sacrospinous muscle pocket of rabbits, the specimens were examined by paraffin slice to observe degradation and histocompatibility of SIS, ADSCs of rabbits were isolated and cultured in vitro, 3~(rd) passage of ADSCs were seeded onto one side or both sides of SIS, after one week of cocultivation, ADSCs-SIS composite were observed by paraffin slice and SEM.
Part 3 3~(rd) passage of ADSCs were seeded onto both sides of SIS, and induced by chondrogenic medium in vitro, 7 days and 14 days after being induced, real-time fluorescent quantitative RT-PCR was used to analyze mRNA level of typeⅡcollagen, GAPDH was used as internal control gene, 2~(-△△CT) method was used to calculate. ADSCs-SIS composites which were not induced were used as control, typeⅡcollagen protein was examined by histoimmunologic staining, toluidine blue staining and safranin O staining were used to observe extracellular matrix. SEM was used to observe cellular morphology after 14 days of chondrogenic differentiation.
Part 4 3~(rd) passage of ADSCs were seeded onto both sides of SIS, and induced by chondrogenic medium in vitro for 14 days before being implanted. 36 New Zealand rabbits of 6-8 weeks old were divided into 3 groups randomly, 50% of medial growth plate in proximal right tibia was excised, group A was blank control group, the defects of growth plates were not filled, the defects of growth plates in group B were filled with SIS without cells, the defects of growth plates in group C were filled with ADSCs-SIS composites induced by chondrogenic medium in vitro for 14 days. 4 rabbits of each group were sacrificed at 4, 8 and 16 weeks, leg bones of both sides were taken off, fixed with 10% formalin solution, X ray photographs were taken to measure tibial length and proximal tibial articular surface angle. Calculated the differences of proximal articular surface angle and length of left and right tibias in each rabbit. Compared proximal tibial articular surface angle and length of left and right tibia in each group of different time interval, and compared difference of proximal articular surface angle and length of left and right tibias among 3 groups of different time interval. Paraffin slices stained with HE were used to observe reparative effect of ADSCs-SIS composites on growth plate cartilage.
Spss 13.0 statitical package was used to deal with all data. Calculate a p-Value for paired T test and one-factor analysis of variance.
Result:
Part 1 Primary SVF cells were multi-angular or short spindle shaped, 3~(rd) passage SVF cells were long spindle shaped. Cell surface antigen of 3~(rd) passage SVF cells were CD44+, CD29+, CD45-. Oil red staining was positive in adipogenic differentiation group, ALP staining, alizarin red staining and Von Kossa staining were positive in osteogenic differentiation group. RT-PCR of typeⅡcollagen mRNA showed that SVFs cells also expressed typeⅡcollagen mRNA, but the product bands were brighter in cells after 7 and 14 days of chondrogenic differentiation. After 14 days of chondrogenic differentiation, histoimmunologic staining of typeⅡcollagen was positive, toluidine blue staining showed that metachromatic staining was observed, and safranin O staining showed that cytoplasms were stained red.
Part 2 SIS was white and semi opaque membrane, paraffin slices showed that no cell was observed in SIS, loose weave structure of serosal surface and compact structure of mucosal surface were observed by SEM, after one week of cocultivation, plenty of cells could be observed on the upside and few cells were observed on the downside of SIS when ADSCs were seeded only onto upside of SIS, plenty of cells could be observed on both sides of SIS when ADSCs were seeded onto both sides of SIS. Cells adhering to the fibers of SIS could be observed in paraffin slice after ADSCs were implanted onto SIS. After SIS were implanted into the muscle pocket of rabbits, infection and immunologic reaction were not observed, paraffin slice showed that only few of SIS fibers were not absorbed at 2 weeks after implantation, and completely abosorbed at 4 weeks after implantation.
Part 3 Standardized quantity of typeⅡcollagen mRNA in ADSCs-SIS composites which were induced for 7 days and 14 days were larger than that in uninduced ADSCs-SIS composites(P < 0.05), standardized quantity of typeⅡcollagen mRNA in ADSCs-SIS composites which were induced for 14 days was larger than that in ADSCs-SIS composites which were induced for 7 days(P<0.05) . Histoimmunologic staining of typeⅡcollagen protein was positive in ADSCs-SIS composites which were induced for 14 days by chondrogenic medium, but was negative in uninduced ADSCs-SIS composites. Extracellular matrix was metachromatic stained by toluidine blue, and red stained by safranin O in ADSCs-SIS composites which were induced for 14 days by chondrogenic medium. Plenty of ADSCs adhered to both sides of SIS after 14 days of chondrogenic differentiation were observed by SEM.
Part 4 After operation, incisions were not infected, and no immunologic rejection was observed, feeding and activity of animals were normal. The direct-viewing of specimens showed that 4 weeks after operation, obvious angular deformity of tibias was observed in group A, and slight angular deformity of tibias was observed in group B, no angular deformity of tibias was observed in group C, and no length discrepancy was observed in all 3 groups; 8 weeks after operation, obvious angular deformity and length discrepancy of tibias were observed in group A, angular deformity and length discrepancy of tibias were observed in group B, no length discrepancy or angular deformity was observed in group C; 16 weeks after operation, obvious angular deformity and obvious length discrepancy of tibias were observed in group A and group B, no angular deformity or length discrepancy of tibias was observed in group C. Comparison of X ray photograph measurement in each group showed that, in group A, 4, 8, 16 weeks after operation, there were significant differences between right and left tibias in the proximal articular surface angle(P< 0.05) and length(P<0.05); in group B, 4 weeks after operation, there was significant difference between right and left tibias in length(P<0.05) , there was no significant difference between right and left tibias in the proximal articular surface angle(P> 0.05), 8 weeks after operation, there was no significant difference between right and left tibias in the proximal articular surface angle or length(P>0.05), 16 weeks after operation, there were significant differences between right and left tibias in the proximal articular surface angle and length(P<0.05) ; in group C, 4, 8, 16 weeks after operation, there was no significant difference between right and left tibias in the proximal articular surface angle(P>0.05) or length(P>0.05). Comparison of X ray photograph measurement among 3 groups showed that 4 weeks after operation, the proximal tibial articular surface angle difference of group A was larger than that of group B(P<0.05) and that of group C(P<0.05) ,there was no significant difference between group B and group C in proximal tibial articular surface angle difference(P> 0.05), and there was no significant difference among all 3 groups in tibial length difference(P>0.05); 8 weeks after operation, proximal tibial articular surface angle difference of group A was larger than that of group B(P<0.05) and that of group C(P <0.05) , tibial length difference of group A was larger than that of group B(P<0.05) and that of group C(P<0.05) , there was no significant difference between group B and group C in tibial length difference(P>0.05) or proximal tibial articular surface angle difference(P>0.05); 16 weeks after operation, proximal tibial articular surface angle difference of group A was larger than that of group B(P<0.05) and that of group C(P<0.05) , proximal tibial articular surface angle difference of group B was larger than that of group C(P<0.05) , tibial length difference of group A was larger than that of group B(P<0.05) and that of group C(P<0.05), tibial length difference of group B was larger than that of group C(P<0.05) . Paraffin slices stained with HE showed that 4 weeks after operation, bone bridges could be seen in growth plate cartilages and no reparative cartilage could be seen in growth plates of group A, bone bridges could be seen in growth plates and few reparative cartilages could be seen in growth plates of group B, and no bone bridges could be seen and reparative cartilages could be seen in growth plates of group C; 8 weeks after operation, bone bridges could still be seen in group A, bone bridges were not absorbed and growth plates were still discontinuous in group B, the reparative cartilages in growth plates rearranged in group C; 16 weeks after operation, bone bridges could be seen in growth plates of group A and group B, and cartilages rearranged like column in the medial half of growth plates in group C.
Conclusion:
Part 1 Stromal vascular fraction cells have identical surface antigens of mesenchymal stem cells and potential of multidirectional differentiation. So SVF cells were identified as adipose derived mesenchymal stem cells
Part 2 Small intestinal submucosa has good histocompatibility and good cell compatibility. It can be used as scaffold for ADSCs.
Part 3 ADSCs could differentiated into chondrogenic cells after being induced by chondrogenic medium in SIS scaffold.
Part 4 ADSCs-SIS composites induced by chondrogenic medium in vitro could prevent angular deformity and length discrepancy of tibias effectively after being implanted into medial growth plate defect of proximal tibia, and could repair cartilage of growth plates. This experimental study can give some suggestion to clinical treatment of growth plate injury by using the ADSCs-SIS composites which are induced by chondrogenic medium in vitro.
引文
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