瓦楞状组织工程骨支架对种子细胞接种和成骨的影响

Effects of corrugated tissue engineered bone scaffold on cell seeding and osteogenesis

背景:不同形状工程骨支架负载种子细胞修复骨缺损的研究效果评价不一,而负载细胞数量的多少是影响疗效的重要因素之一,目前该方面研究证据不多。目的:自制瓦楞状组织工程骨支架和其他3种形状的支架,比较4种不同形状支架负载种子细胞的数量,以及瓦楞状组织工程骨支架体内成骨时凹槽的优势及特点。方法:①体外实验:将体积和样本数相同的4组支架分为单纯瓦楞状支架组、无瓦楞支架组、圆柱状支架组和带中空管柱状支架组,分别以相同密度、相同容积的成骨诱导兔骨髓间充质干细胞悬液接种于支架表面,孵育、培养、消化、收集,进行细胞计数、吸光度值检测以及碱性磷酸酶和茜素红染色。②体内实验:将兔随机分为重组人骨形态发生蛋白2/瓦楞状自固化磷酸钙人工骨组,瓦楞状自固化磷酸钙人工骨组和松质骨组,将体积相同的3组支架植入兔L_(5-6)两侧横突间,植入后4,8,12周行大体、组织学观察。结果与结论:体外实验显示,瓦楞状工程骨支架上滴注的细胞液能充分停留在表面,由于表面瓦楞状凹槽和液体的表面张力以及支架本身的无孔隙性使得细胞液不向培养皿流失,每个样本平均消化下来的正常形态的细胞数量高于其他3组(P<0.05),吸光度值差异无显著性意义(P>0.05)。体内实验显示,各时间点重组人骨形态发生蛋白2/瓦楞状自固化磷酸钙人工骨组的成骨量比瓦楞状自固化磷酸钙人工骨组明显多(P<0.05),与松质骨组之间比较差异无显著性意义(P>0.05)。结果证实,实验自制的瓦楞状工程骨支架的形状特点有利于种子细胞负载,从数量上保证了接种种子细胞的有效性,可促进支架大量成骨和段状骨缺损的愈合。

BACKGROUND：The effects of engineered bone scaffold containing seeding cels with different shapes to repair bone defect are varied, while the loaded cellquantity is the important factor influencing the curative effect, but which is rarely reported.OBJECTIVE：By preparing self-made corrugated tissue-engineered bone scaffold and other three forms of bone tissue engineering scaffolds, to study the quantity of loaded cels on different scaffolds and osteogenesis of corrugated tissue-engineered bone scaffold so as to discuss the advantages and features of self-made corrugated tissue-engineered bone scaffold. METHODS： （1） Experimentin vitro： There were four kinds of scaffolds with the same volume and samples： calcium phosphate cement （CPC） corrugated surface scaffold group, smooth surface scaffold group, cylindrical scaffold group and porous cylindrical scaffold with holow tubes group, in which the latter three groups are control ones. A certain volume with same density of rabbit bone marrow mesenchymal stem cels （BMSCs） suspension after osteogenesis induction was seeded onto the scaffolds. After incubation, culture, digestion and colection, cellquantity was counted, absorbance value was finaly detected and cellactivity was proofed by alkaline phosphatase and alizarin red staining. （2） Experimentin vivo： New Zealand rabbits were randomly and equaly divided into recombinant human bone morphogenetic protein-2 （rhBMP-2）/CPC/BMSCs corrugated scaffold group, pure CPC corrugated scaffold group and cancelous bone implant group. Three kinds of scaffold implants with the same volume were inserted into the area between rabbit’s L5, 6 transverse processes bilateraly. At 4, 8, 12 weeks postoperatively, gross and histological observation was performed. RESULTS AND CONCLUSION：（1）Experimentin vitro： The drip of cellsuspension steadily stayed on the surface of corrugated scaffold because of corrugated shape groove and the surface tension of the liquid. The amount of cels per sample digested down from the CPC corrugated surface scaffold was significantly more than that from the other three groups （P 〈 0.05）, while the absorbance values did not differ （P 〉 0.05）. （2） Experimentin vivo： At each time point the osteogenesis quantity of rhBMP-2/CPC/BMSCs corrugated scaffold group was more than that of the pure CPC corrugated scaffold group （P 〈 0.05）, while there was no difference from the cancelous bone implant group （P 〉 0.05）. These findings indicate that the characteristics of the self-made corrugated engineered bone scaffold are beneficial to seed cellloading, which supports a large number of osteogenesis and provides feasibility to promote the healing of segmental bone defects.