生物水凝胶体系治疗兔股骨缺损的效果

发布时间：2018-08-13 20:31

【摘要】：目的:建立兔骨髓间充质干细胞(BMSCs)采用全骨髓法体外分离,检测其生物学特性和多向诱导分化特征,并观察其对细胞形态和增殖性能的影响。制备透明质酸水凝胶,检测其生物相容性和抗降解能力。通过构建兔股骨中下段缺损模型,以浓缩骨髓血液、HA冷冻解冻水凝胶和异体骨条作为内植物修复兔骨缺损,探讨生物水凝胶体系修复兔股骨中下段缺损的效果。方法:(1)采用全骨髓贴壁细胞分离法提取纯化兔骨髓间充质干细胞,用含10%胎牛血清培养基培养BMSCs;(2)采用流式细胞仪检测细胞表面特异性受体表达,进行鉴定,并且在不同诱导培养基的作用下向成骨细胞、成软骨细胞和脂肪细胞分化,采用特异性染色并观察其结果;(3)采用冷冻解冻成胶的方法制备透明质酸(HA)水凝胶,将BMSCs种植于HA水凝胶后通过观察其中的细胞形态分布来检测HA水凝胶的生物相容性,采用体外降解和动物体内包埋的方式检测其抗降解能力;(4)实验动物40只,其中4只分为2组作为造模位置选择的研究,分别在右侧桡骨和股骨中下段造成相同面积的缺损,其余36只随机分为4组,建立股骨缺损创伤模型;浓缩骨髓血液和制备HA水凝胶备用;A组植入生物水凝胶体系,B组植入异体骨-HA水凝胶复合物,C组单纯植入异体骨,D组植入自体髂骨。术后通过X线和大体标本骨检测修复效果,并且将修复区制作切片和HE染色观察微观成骨效果。结果:(1)体外培养的原代BMSCs接种1d开始贴壁,形态呈长梭形或类圆形;8d-9d后可首次传代;(2)获得的细胞高表达间充质干细胞的表面抗原CD29、CD44、CD90,不表达CD45、CD34,证明为间充质干细胞;(3)BMSCs在三种诱导液的诱导下,经过21d的增殖分化,分别可被茜素红、番红O和油红O染色,证明获得三种成体细胞;(4)造模动物一月后X线显示兔股骨中下段自身的修复能力更弱,适合造模;(5)内置材料的动物术后X线结果显示:4weeks A组缺损面积减小,24weeks A组和D组完整修复骨缺损,B、C两组骨修复区与正常骨骨质界限清晰,修复面积未达到完整结果;大体标本观察结果与X线结果一致;切片HE染色显示4weeks时A、B、D三组观察到缺损区单侧骨膜形成,8weeks只有A组具备完整性内外骨膜连续性的动物,A、D组优于B、C组,24weeks A、D组修复区与正常骨质无差别,未见与正常骨质界限,而B、C组则修复较差。结论:应用全骨髓贴壁法可以获得纯度较高、增殖能力强、表型稳定均一、具备多向分化的能力的BMSCs;冷冻解冻成胶法制备的透明质酸水凝胶可作为一种无细胞毒性的三维细胞支架材料,具备一定的抗降解能力;股骨中下段可作为骨缺损造模的选择,浓缩骨髓-HA水凝胶-异体骨合成的生物水凝胶体系对于骨缺损的修复有积极作用,可促进早期组织再生和血管再通,是一项临床应用潜力的新技术。
[Abstract]:Aim: to establish rabbit bone marrow mesenchymal stem cells (BMSCs) isolated by whole bone marrow method in vitro, to detect the biological characteristics and multidirectional differentiation characteristics, and to observe its effects on the morphology and proliferation of rabbit bone marrow mesenchymal stem cells (BMSCs). Hyaluronic acid hydrogel was prepared and its biocompatibility and anti-degradation ability were tested. A rabbit model of middle and lower femur defect was established. The bone defects were repaired with concentrated bone marrow hemoglobin freezing and thawing hydrogel and allogeneic bone strips as implants, and the effect of biological hydrogel system on repairing rabbit femur middle and lower segment defect was discussed. Methods: (1) Rabbit bone marrow mesenchymal stem cells (BMSCs) were isolated and purified by whole bone marrow adherent cells. BMSCs were cultured in 10% fetal bovine serum culture medium. (2) the expression of specific receptors on cell surface was detected by flow cytometry. In addition, osteoblasts, chondroblasts and adipocytes were differentiated into osteoblasts, chondroblasts and adipocytes in different culture medium. (3) Hyaluronic acid (HA) hydrogel was prepared by freezing and thawing gelation. The biocompatibility of HA hydrogels was detected by observing the morphological distribution of HA hydrogels, and the anti-degradation ability of HA hydrogels was tested by in vitro degradation and in vivo embedding of BMSCs. (4) 40 experimental animals were used to study the biocompatibility of HA hydrogels. Four of them were divided into 2 groups to select the location of the model. The same area of defect was created in the right radius and the middle and lower femur, and the other 36 were randomly divided into 4 groups to establish the model of femoral defect trauma. Bone marrow blood was concentrated and HA hydrogel was prepared. Group A was implanted with biological hydrogel system. Group B was implanted with allogeneic bone and HA hydrogel complex. Group C was implanted with allogeneic bone only. Group D was implanted with autogenous iliac bone. The effect of bone repair was detected by X-ray and gross bone after operation, and the microscopic osteogenic effect was observed by making sections of repair area and HE staining. Results: (1) the primary BMSCs inoculated in vitro began to adhere to the wall 1 day after inoculation. (2) the surface antigen CD29, CD44, CD90, not CD45, CD34, was proved to be mesenchymal stem cells. (3) BMSCs was induced by three kinds of inducers and differentiated after 21 days. It was proved that three kinds of adult cells were obtained by alizarin red, phannin O and oil red O, respectively. (4) X-ray showed that the repair ability of rabbit femur was weaker after 1 month. The results of X-ray showed that the defect area of group A and group D were reduced and the boundary between bone repair area and normal bone in group A and group D were clear, and the area of repair was not up to the complete result. The results were as follows: (1) the results of X-ray showed that the defect area of group A was less than that of group A, and that of group B (group D) was significantly lower than that of group B (P < 0.05). The results of gross specimen observation were consistent with those of X-ray. Section HE staining showed that only A group A with complete internal and external periosteum continuity was found to have no difference between the repair area and the normal bone in group A and B C group during 4weeks, and there was no significant difference between the repair area and the normal bone in the group A and B C, and there was no difference between group A and B C, and no difference was found between the repair area and the normal bone in the group A and B C, and there was no significant difference between the repair area and the normal bone in group A. In group C, the repair was poor. Conclusion: the whole bone marrow adherent method can obtain high purity, strong proliferative ability, stable and uniform phenotype. BMSCs with multi-differentiation ability; hyaluronic acid hydrogel prepared by freezing and thawing gelation method can be used as a non-cytotoxic three-dimensional cell scaffold material with certain anti-degradation ability; the middle and lower femur can be used as a choice for modeling bone defect. The biomedical hydrogel system which concentrates bone marrow hydrogel and allograft bone synthesis has a positive effect on the repair of bone defect and can promote early tissue regeneration and vascular recanalization. It is a potential new technique for clinical application.
【学位授予单位】：宁波大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R68

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