BMSCs来源细胞外基质支架对微骨折骨髓刺激术后血凝块体外软骨化分化的作用研究

EFFECT OF BONE MARROW MESENCHYMAL STEM CELLS-DERIVED EXTRACELLULAR MATRIX SCAFFOLD ON CHONDROGENIC DIFFERENTIATION OF MARROW CLOT AFTER MICROFRACTURE OF BONE MARROW STIMULATION IN VITRO

目的探讨BMSCs来源细胞外基质(extracellular matrix,ECM)支架结合微骨折骨髓刺激术后血凝块在体外软骨化分化的可行性及有效性。方法选用5～6月龄新西兰大白兔40只,随机取20只兔骨髓分离、培养BMSCs并行多向分化鉴定;利用冻干技术制备BMSCs来源的ECM支架,通过扫描电镜观察其微结构。于兔股骨滑车处作直径6 mm软骨缺损,垂直缺损面作5个直径1 mm、深3 mm的微骨折孔洞。将未穿刺的20只动物随机分为2组(n=10):A组取微骨折术后渗出的血凝块直接体外培养;B组在A组基础上用TGF-β3诱导其成软骨分化。将抽取骨髓的20只动物随机分为2组(n=10):C组用制备的ECM支架植入软骨缺损处,待ECM支架与渗出的骨髓血充分融合后取出体外直接培养;D组在C组基础上给于TGF-β3诱导其成软骨分化。体外培养1、2、4、8周时分别行大体、组织学、免疫组织化学及生化成分分析观察,检测其分化效果。结果分离、培养的细胞经多向分化鉴定,呈成骨细胞、软骨细胞、脂肪细胞的表型特征。扫描电镜观察示ECM支架呈三维多孔状结构。在培养过程中,A组组织块逐渐降解消失;C组组织块逐渐形成质地柔软的疏松样结构;B、D组组织块逐渐形成表面平滑的新生软骨样组织。体外培养4、8周,C、D组组织块面积均大于B组,差异有统计学意义(P<0.05);D组大于C组,差异亦有统计学意义(P<0.05)。A、C组HE、番红O及免疫组织化学染色示组织内仅有少量细胞分布,未见糖胺多糖(glycosaminoglycan,GAG)及Ⅱ型胶原的积累;B、D组可见组织内出现许多软骨陷窝样结构,且GAG及Ⅱ型胶原分布逐渐增多。生化成分分析示,培养过程中,B、D组GAG及总胶原含量逐渐增加,其中D组增加更明显,培养至4、8周,D组与B组比较差异有统计学意义(P<0.05)。结论 BMSCs来源的ECM支架结合微骨折骨髓刺激术后血凝块在体外给予TGF-β3诱导培养时具有良好的软骨化分化潜能。

Objective To evaluate the feasibility and validity of chondrogenic differentiation of marrow clot after microfracture of bone marrow stimulation combined with bone marrow mesenchymal stem cells （BMSCs）-derived extracellular matrix （ECM） scaffold in vitro. Methods BMSCs were obtained and isolated from 20 New Zealand white rabbits （5-6 months old）. The 3rd passage cells were cultured and induced to osteoblasts, chondrocytes, and adipocytes in vitro, respectively. ECM scaffold was manufactured using the 3rd passage cells via a freeze-dying method. Microstructure was observed by scanning electron microscope （SEM）. A full-thickness cartilage defect （6 mm in diameter） was established and 5 microholes （1 mm in diameter and 3 mm in depth） were created with a syringe needle in the trochlear groove of the femur of rabbits to get the marrow clots. Another 20 rabbits which were not punctured were randomly divided into groups A （n=10） and B （n=10）： culture of the marrow clot alone （group A） and culture of the marrow clot with transforming growth factor 03 （TGF-/33） （group B）. Twenty rabbits which were punctured were randomly divided into groups C （n= 10） and D （n= 10）： culture of the ECM scaffold and marrow clot composite （group C） and culture of the ECM scaffold and marrow clot composite with TGF-B3 （group D）. The cultured tissues were observed and evaluated by gross morphology, histology, immunohistochemistry, and biochemical composition at 1, 2, 4, and 8 weeks after culture. Results Cells were successfully induced into osteoblasts, chondrocytes,and adipocytes in vitro. Highly porous microstructure of the ECM scaffold was observed by SEM. The cultured tissue gradually reduced in size with time and disappeared at 8 weeks in group A. Soft and loose structure developed in group C during culturing. Chondroid tissue with smooth surface developed in groups B and D with time. The cultured tissue size of groups C and D were significantly larger than that of group B at 4 and 8 weeks （P 〈 0.05）; group D was significantly larger than group C in size （P 〈 0.05）. Few cells were seen, and no glycosaminoglycan （GAG） and collagen type II accumulated in groups A and C; many cartilage lacunas containing cells were observed and more GAG and collagen type II were synthesized in groups B and D. The contents of GAG and collagen increased gradually with time in groups B and D, especially in group D, and significant difference was found between groups B and D at 4 and 8 weeks （P 〈 0.05）. Conclusion The BMSCs-derived ECM scaffold combined with the marrow clot after microfracture of bone marrow stimulation is effective in TGF-B3-induced chondrogenic differentiation in vitro.