纳米羟基磷灰石—壳聚糖支架复合外周血间充质干细胞修复大鼠胫骨缺损的实验研究

发布时间：2018-01-12 04:33

本文关键词：纳米羟基磷灰石—壳聚糖支架复合外周血间充质干细胞修复大鼠胫骨缺损的实验研究　出处：《南方医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景:先天性畸形、复杂性创伤、肿瘤切除以及骨病等都可能造成临界性骨缺损,骨组织无法完成自我修复而往往需要大量的骨质进行重建。传统治疗临界骨缺损有自体骨移植、异体骨移植以及人工合成材料等方法。但是供体来源缺乏、免疫排斥、容易传播传染性疾病以及耐用程度有限等均不同程度地限制各自的临床应用。骨组织工程有望克服上述缺陷并成功修复骨缺损而成为研究的热点,其往往将间充质干细胞移植至有三维结构的支架材料当中,通过成骨诱导而促进新骨形成。间充质干细胞(MSCs)在体外能够自我增殖,有较强多向分化潜能并且容易向成骨细胞分化,无免疫排斥而成为骨组织工程较理想的种子细胞。骨髓间充质干细胞(BMMSCs)有较强的增殖及成骨分化能力,成为公认的修复骨缺损的细胞来源。但是其存在取材过程创伤较大、数量、增殖和分化能力随着患者年龄增大而出现明显下降等缺陷。相对而言,外周血间充质干细胞(PBMSCs)是通过极其微创和相对简单的方式来获得的,经济成本较低,因而有可能成为替代BMMSCs的较为理想的种子细胞。目前普遍的观点认为对于可以应用至骨组织工程修复的材料而言,单一材料无法满足其需求,复合材料才是更好的选择。纳米羟基磷灰石-壳聚糖(nHA-CS)为三维多孔结构的复合支架材料,具有良好的生物相容性、可降解性及骨传导性,能够满足构建组织工程化骨的要求。本实验拟将搭载PBMSCs的nHA-CS复合支架材料移植至大鼠胫骨5mm离断性骨缺损,观察其修复效果。研究目的观察纳米羟基磷灰石-壳聚糖(nHA-CS)支架材料复合外周血间充质干细胞(PBMSCs)修复大鼠临界性骨缺损的效果。研究方法1.采用密度梯度离心及贴壁生长法分离培养大鼠PBMSCs并行体外扩增,检测其表面标志物以及成骨分化潜能。2.共沉淀法制备nHA-CS支架材料,扫描电镜(SEM)观察其立体结构,测定其孔隙率及体外降解率。3.选择SD大鼠胫骨5mm离断性骨缺损作为实验模型,42只大鼠随机分为3组:A组旷置不处理;B组仅填充nHA-CS;C组移植PBMSCs-nHA-CS。在术后8周及12周分别处死动物,通过X线、Micro-CT以及组织学检测结果判断骨缺损修复情况。4.采用SPSS 20.0统计软件对所有的数据进行分析。采用均值±标准差(X±SD)表示数据结果。采用单因素方差分析(One-wayANOVA)进行多组资料比较,采用最小显著差异法(Least significant difference,LSD)进行组间比较,若方差不齐则采用Dunnett'S T3法;P值0.05认为差异有统计学意义。研究结果1.分离培养的PBMSCs呈长梭形形态,能在体外扩增,流式细胞术检测PBMSCs表面标志物符合MSCs表达。PBMSCs在体外经成骨诱导可向成骨细胞分化,ALP表达阳性,钙结节形成。2.制备的nHA-CS支架具有三维多孔网络状结构,孔径介于100-200μm,孔隙率高且彼此相通。3.X线、Micro-CT以及组织学检测结果显示PBMSCs-nHA-CS组修复临界骨缺损的效果明显优于nHA-CS组或空白组,且Lane-Sandhu X评分、BMD、BV以及成骨面积等定量检测均提示PBMSCs-nHA-CS组高于nHA-CS组或空白组,结果具有显著性差异(P0.05)。结论本实验证明PBMSCs可以成为骨组织工程良好的细胞来源,有良好的应用前景。PBMSCs-nHA-CS能够成功修复大鼠胫骨5mm离断性缺损,其修复效果优于nHA-CS或空白组。
[Abstract]:Background: congenital malformations, complexity of trauma, tumor excision and bone disease are likely critical bone defect caused by bone tissue, unable to complete the self repair and often require a large amount of bone reconstruction. The traditional treatment of critical bone defect with autogenous bone graft, allograft and artificial synthetic materials and other methods. But the lack of donor organs, immune rejection, easy to spread of infectious diseases and durability limited to varying degrees of restrictions respectively. Clinical application of bone tissue engineering is expected to overcome the defect and repair bone defect successfully has become a research hotspot, which tend to mesenchymal stem cells transplanted to the scaffold material with three-dimensional structure, and promote new bone induction by bone formation. Mesenchymal stem cells (MSCs) in vitro can have strong self proliferation, multilineage differentiation potential and easy osteoblastic differentiation, without immune rejection And become the ideal seed cells for bone tissue engineering. Bone marrow mesenchymal stem cells (BMMSCs) and osteogenic differentiation capacity strong proliferation and repair of bone defects become recognized source of cells. But there were large number of trauma, proliferation and differentiation ability of patients with increased age and decreased significantly. Defect. In contrast, peripheral blood mesenchymal stem cells (PBMSCs) are obtained through extremely minimally invasive and relatively simple way, low cost, and may become the ideal seed cells to replace BMMSCs. The general idea that can be used for bone tissue engineering to repair materials, a single material can not meet the requirements, the composite is a better choice. Nano hydroxyapatite chitosan (nHA-CS) composite scaffolds for three-dimensional porous structure, has good biological compatibility, can be Degradation and bone conduction, can meet the requirements of the construction of tissue engineered bone. The experiment will be equipped with PBMSCs of 5mm nHA-CS composite scaffold transplanted to rat severed bone defect, to observe the repair effect. Objective to study nano hydroxyapatite chitosan (nHA-CS) composite scaffold of peripheral blood mesenchymal stem cells (PBMSCs) critical bone defect repair in rats. Methods 1. by density gradient centrifugation and adherent cells were isolated and cultured rat PBMSCs parallel in vitro, the detection of surface markers and osteogenic differentiation potential of.2. co precipitation method to prepare nHA-CS scaffolds, scanning electron microscope (SEM) to observe the stereo the structure, determination of the porosity and degradation rate in vitro of.3. SD 5mm transection of rat tibial bone defect as experimental model, 42 rats were randomly divided into 3 groups: group A no exclusion treatment; group B filled nHA-CS group C PBMSCs transplantation; -nHA-CS. after 8 weeks and 12 weeks were sacrificed by animal, X-ray, Micro-CT and histological examination results to determine the repair of bone defect by.4. on all of the data were analyzed by SPSS 20 statistical software. The mean standard deviation (X + SD). The results indicates that the data with single factor analysis of variance (One-wayANOVA) of group data comparison, using the least significant difference method (Least significant difference, LSD) were compared between the two groups, if homogeneity of variance using Dunnett'S T3 method; P value of 0.05 is considered statistically significant. Results: 1. PBMSCs isolated from the long fusiform shape, can be amplified in vitro and flow cytometry PBMSCs the surface markers with MSCs expression of.PBMSCs in vitro osteogenic induction of osteogenic differentiation, the expression of ALP, the formation of calcium nodules nHA-CS scaffolds prepared by.2. with three-dimensional porous network structure, the aperture is 100-2 00 m, high porosity and shared.3. X-ray, Micro-CT and histological examination showed that the PBMSCs-nHA-CS group to repair critical bone defect is better than that of nHA-CS group and blank group, and the Lane-Sandhu X score, BMD, BV and bone area quantitative detection showed PBMSCs-nHA-CS group than in nHA-CS group or control group, the results were significant the difference (P0.05). Conclusion PBMSCs can be a good source of cells for bone tissue engineering, has good application prospects of.PBMSCs-nHA-CS can successfully repair tibia of 5mm rats isolated defects, the repair effect is better than nHA-CS or the control group.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R687

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