摘要
目的探讨软骨微环境对MSCs的影响，研究多孔聚乙烯醇(PVA)及其复合物的性能与生物相容性，初步观察新型骨软骨一体化多孔材料、聚酰胺66／纳米羟基磷灰石(PA66／n-HA)、聚乙烯醇／明胶(PVA／Gel)修复兔膝关节骨软骨缺损的效果。
方法①以兔关节软骨细胞和软骨细胞回收液模拟软骨微环境，并与MSCs进行单层共培养，采用镜下观察、组织学染色、Ⅱ型胶原原位杂交的方法观察其相互影响。②采用乳化发泡-冷冻固化-去除表面活性剂法获得多孔PVA、PVA╱n-HA、PVA／Gel和聚乙烯醇／壳聚糖(PVA／Cs)，并采用图像分析、力学测试、MTT法、流式细胞仪、ELISA法、与MSCs复合培养法、肌肉植入实验检测材料性能和生物相容性。③以多孔PVA或PVA／n-HA为上层材料，多孔PA66／n-HA为下层材料，制得一体化多孔材料PVA／PA66／n-HA和PVA／n-HA／PA66／n-HA，采用撕裂试验观察上下层间的结合强度，并植入兔膝关节骨软骨缺损处观察其修复效果。④以PA66／n-HA复合MSCs略低于周围软骨平面植入骨软骨缺损处，观察其修复效果。⑤以Tween 20为致孔剂制得多孔PVA／Gel，并植入兔膝关节缺损处，2周后，行第2次手术注射MSCs到材料上，观察其修复效果。
结果①MSCs与软骨细胞共培养可以发生相互影响，当MSCs与软骨细胞比例为2：1时，对维持软骨细胞外基质的分泌和促进软骨细胞Ⅱ型胶原mRNA的表达是较有利的；软骨细胞的培养回收液可以引起MSCs形态上的轻度类软骨样改变和诱导MSCsⅡ型胶原mRNA的强表达，但细胞有肥大现象；MSCs回收液则对软骨细胞Ⅱ型胶原表型的维持有一定作用。
②制得的多孔PVA及其复合物，亲水性好，有100～3001ma的大孔和10μm以下的微孔两级孔结构，孔与孔之间相互相互贯通，孔隙率在80％左右，有一定韧性和弹性。MTT法、肌肉植入、ELISA法、流式细胞仪检测表明，材料清洗3天，会有一定的表面活性剂(OP)残留，对材料生物相容性有一定负面影响；PVA复合物中以PVA／Gel、PVA／n-HA、PVA的生物相容性较好，其中，PVA／Gel与MSCs黏附较好，PVA／Cs也能与MSCs黏附，但肌肉植入该材料早期炎性反应较重，且在体外对软骨细胞增殖不利。
③一体化多孔PVA／PA66／n-HA上下层之间的结合强度约4．00±2．21N。植入膝关节4月后，PVA／n-HA／PA66／n-HA的软骨修复效果优于PVA／PA66／n-HA，两组的下层材料均有胶原和骨组织长入。
④以PA66／n-HA复合MSCs植入骨软骨缺损处，并略低于周围软骨平面，4个月后软骨修复效果较为理想，修复的软骨主要表达Ⅱ型胶原，少量表达Ⅰ型胶原。
⑤制得的多孔PVA／Gel也有100μm以上的大孔和10μm以下的微孔两级孔结构，孔隙率约76％，孔与孔间相互贯通；采用2次手术的方法注射MSCs到关节缺损处的材料上，可以有效的促进软骨修复，修复的软骨已十分接近透明软骨(术后4月)，但材料稳定性还不够。
结论软骨细胞与软骨细胞回收液所模拟的软骨微环境对MSCs向软骨方向分化有一定的诱导作用，这为采用MSCs作为种子细胞修复软骨损伤提供了证据，也为诱导MSCs向软骨细胞方向分化提供了一个新思路；PA66／n-HA在骨软骨损伤的修复中起了关键作用，能够维持即刻稳定和远期稳定；PVA／Gel是一种较好的软骨修复材料，未来需进一步寻找增加PVA黏附性的新方法，如进行表面修饰、添加其他材料等或制成可降解PVA；并与PA66／nHA结合，研制新的一体化多孔材料。
Objective The purposes of this study were to investigate the mesenchymal stem cells(MSCs) affected by microenvironment of cartilage and to research the features and biocompatibility of porous Poly(vinyl alcohol) (PVA)and its composite. In addition,we wanted to preliminary observed the repairing results of knee osteochondral defects in the rabbit by using new porous integrative interlocked biomaterial, Polyamide 66/nano-Hydroxyapatite(PA66/n-HA) and Poly(vinylalcohol)/Gelatin(PVA/Gel) composite.
Methods①Chondrocyte of rabbit and its disused culture medium were used to simulate the cartilaginous microenvironment. We observed the effect cocultue chondrocyte or its disused cultue medium with MSCs on cartilaginous phenotype in vitro by microscope observation, toluidine blue staining and in situ hybridization for type II collagen mRNA.②Porous PVA , PVA/n-HA,PVA/Gel,Poly(vinyl alcohol)/Chitosan(PVA/Cs) materials were prepared by emusifier-foaming, freeze-drying and surfactant-cleaning method. The features and biocompatibility of above materials were further studyed by image analysis,mechanics test,MTT,ELISA assay,cocultue with MSCs and muscle implanting and so on.③prepared new porous integrative interlocked PVA/PA66/n-HA or PVA/n-HA/PA66/n-HA biomaterials,which upper layer material is PVA or PVA/n-HA and underlayer is PA66/n-HA. We evaluated their mechanics feature and the effects of repairing knee joint osteochondral defect of rabbits.④PA66/n-HA scaffold,which was seeded with MSCs in vitro,was implanted to knee joint osteochondral defect of rabbits.At the same time, the scaffold was lower than normal cartilage a little. After 1 and 4 months,we investigate the repairing effects.⑤We have also explored repairing effects of knee joint osteochondral defect of rabbits by implanting porous PVA/Gel,which was prepare by Tween-20 foaming agent.Differented to above method, MSCs were injected to PVA/Gel after materials implanting to defect 2 weeks.
Results①Cocultue chondrocyte with MSCs would affect each other.Perhaps the 2:1 ratio of MSCs and chondrocytes was beneficial to maintenance cartilaginous matrix and increasing collagen II mRNA expression. The disused culture medium of chondrocytes could also induce MSCs to chondrocyte-like change and express collagen II mRNA,but the cells appeared hypertrophic.②The materials obtained by this method had high hydrophilicity,porosity,pore interconnectivity and proper elasticity. Scanning electron microscopy (SEM) showed that the material had a lot of micropores (less than 10 urn) on the walls of macropores (100～300μm). Porous PVA,PVA/n-HA and PVA/Gel had relative better biocompatibility,compared with control materials. PVA/Gel and PVA/Cs had better cellular compatibility with MSCs,but the latter had bigger inflammatory reaction when muscle implanting.③PVA/n-HA/PA66/n-HA had better repairing effects of knee joint osteochondral defect of rabbits.Underlayer material was stable.④PA66/n-HA scaffold,which was seeded with MSCs , had better repairing effects of knee joint osteochondral defect of rabbits.After 4 months,repaired cartilage main expressed collagen II ,also expressed collagen I .⑤The PVA/Gel obtained by this method also had good porosity(76%),pore interconnectivity. Scanning electron microscopy (SEM) showed that the material had micropores and macropores. Afer 4 months, porous PVA/Gel,which was injected MSCs,had a satisfactory repairing results.But the stability of material was not enough.
Conclusion Our study suggests that microenvironment of cartilage,to some extent,can indue or maintain cartilaginous phenotype in vitro. So,our data provide clues to induce MSCs differentiation to cartilaginous direction.PA66/n-HA plays a key role in keeping instantly long-term stability of materials.Porous PVA/Gel is a better material for cartilage repairing.In the future, seeking for new methods such as surface modification ,adding other materials or preparing degradation PVA are required.
引文
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