组织工程技术治疗股骨头骨坏死的探索性研究

发布时间：2018-03-05 09:45

  本文选题：骨软骨损伤　切入点：组织工程　出处：《中国人民解放军医学院》2015年硕士论文　论文类型：学位论文

【摘要】：研究背景：股骨头骨坏死是由多种原因引起、病理机制不清的骨科常见难治性疾病，对于股骨头骨坏死的治疗仍存在很多的争议,目前除晚期骨坏死行全髋关节置换外,对早中期骨坏死尚无统一、有效的治愈方法。所以根据早期及中期股骨头骨坏死的病理特征,怎样阻断早期股骨头坏死进一步发生塌陷,以及塌陷后骨软骨损伤或骨软骨分离后,怎样重新构建功能良好的骨软骨单元重建关节面就显得十分重要。研究目的：本课题应用聚乳酸-羟基乙酸共聚物(poly lactic-co-glycolic acid,PLGA)作为药物载体局部释放特定浓度的唑来膦酸(zoledronate acid, ZOL)抑制骨吸收,促进骨形成,探索其防止早期股骨头坏死塌陷的效果。通过将猪关节骨软骨单元用激光制孔并脱细胞而制备出含有脱细胞软骨基质、骨基质和完整骨软骨界面的双相支架,了解其附和软骨细胞后能否有效的促进其增值,并通过兔骨软骨缺损模型验证其在体内对缺损的修复情况,初步探索其修复骨软骨损伤的效果,为组织工程技术治疗中晚期骨坏死骨软骨损伤及骨软骨分离提供良好的支架选择。方法：通过在体外应用低浓度的唑来膦酸与成破骨细胞共培养,应用抗酒石酸酸性磷酸酶染色(TRAP)、图像分析计算骨吸收陷窝面积检测破骨细胞形态及骨吸收情况。碱性磷酸酶染色(ALP)、四甲基偶氮唑盐比色法(MTT)了解成骨细胞的形态及增值情况。选用PLGA作为局部应用ZOL的载体,制备含有30 u g的ZOL的PLGA棒。进行Micro-CT扫描，，分别计算比较各组的骨矿密度(bone mineral density,BMD)、骨体积分数(bone volume fraction,BVF)、骨小梁厚度(trabecular plate thickness,Tb.Th)和骨小梁间隙(trabecular spacing,Tb.Sp)扫描后进行HE和Masson三色染色了解各组间组织学改变情况。将天然猪骨软骨单元进行激光打孔,化学脱细胞的方法获得三维多孔的脱细胞骨软骨基质源性支架,并通过组织学、力学等测试了解支架的理化性质;分离培养兔关节软骨细胞接种于本支架上了解其对细胞增殖的影响；将免软骨细胞接种于支架上构建细胞-支架复合体,移植于免膝关节骨软骨缺损,并于12周和24周两个时间点分次取材,对标本进行大体观察、组织学评价、显微CT、生物力学等各方面进行评估。结果：培养1周后破骨细胞具有典型的形态特征,并在骨片上形成了吸收陷窝；ZOL组与对照组相比,破骨细胞数量及生成吸收陷窝的数目和面积减少,差异有统计学意义(p0.05)。成骨细胞有典型的梭形、ALP染色阳性特征,培养至第7天ZOL组成骨细胞光吸收值(3.37±0.11)高于对照组(2.87±0.12),差异有统计学意义(p0.05)。与打孔组和PLGA组相比,PLGA-ZOL组中BMD (535.95±15.08,p0.05),BVF(0.77±0.04,p0.05)和 Tb.Th(0.64±0.05,p0.05)显著增加,而Tb.sp(0.37±0.06,p0.05)显著降低,而打孔组和PLGA组并无统计学差异。而且PLGA-ZOL组股骨头有更好的形态,骨小梁保留更完整,新生骨更多。实验所构建的新型骨软骨支保留了完整的骨、软骨及骨软骨界面,可在软骨层见平行纵向排列的孔隙结构,孔间距300μm,孔径形状呈上宽下窄的锥形结构。压缩模量是根据应力应变曲线所得。结果显示：所制备的骨软骨支架的压缩模量1.56±0.288MPa与正常的关节软骨的压缩模量1.983±0.354MPa两者之间无统计学的差异(p0.05,n=7)。所制备支架的孔隙率为所制备的多孔骨软骨支架的孔隙率为：59.75±3.73%。通过CCK-8检测观察软骨细胞在支架上的增殖结果,发现在1,3,5,7天,细胞在支架上随着培养时间的增加而增加,同一种细胞在种植后各时间点之间增殖变化均有统计学差F=577.7265,P=0.0000。体内缺损修复实验,组织学结果发现软骨细胞附和骨软骨支架的修复效果显著优于单纯支架组和旷置组,差异有统计学意义(p0.05),术后24周,缺损区修复的软骨组织厚度基本正常,特异性染色阳性,软骨下骨及骨软骨界面再生完好,与周围正常组织整合较好：Micro-CT及骨计量学分析显示软骨下骨形成较好;压痕试验显示修复区域新生的软骨组织有较好的力学特性,与正常软骨无统计学差异(p0.05)。结论：低浓度(10-6mol/L)的ZOL能够抑制破骨细胞的增殖和活性,促进成骨细胞的增殖,选择恰当给药方式和剂量能够在抑制破骨的同时促进成骨。局部使用ZOL能够抑制局部骨吸收活动,促进了局部骨生成,预防早期股骨头塌陷,保留更好的髋关节功能。通过激光打孔和脱细胞技术,以天然关节骨软骨单元为材料而制备的骨软骨支架,既保留了天然的骨、软骨基质,也保留了完整的骨软骨界面,良好的仿生了正常骨软骨组织的结构。脱细胞多孔的骨软骨支架有良好的细胞相容性,能促进细胞的增殖,为种子细胞提供了良好的生长环境;可作为体内进行组织工程修复骨软骨损伤的良好支架材料。多孔脱细胞骨软骨支架成功修复了骨软骨缺损,可作为组织工程修复骨软骨缺损的良好支架材料。是中期股骨头骨坏死引起骨软骨损伤的一个新的选择。
[Abstract]:Background: femoral head necrosis is caused by a variety of reasons, the common pathological mechanism is unclear in the Department of orthopedics refractory disease, there are still a lot of controversy for the treatment of osteonecrosis of the femoral head, in addition to the current advanced osteonecrosis of total hip replacement, on the early stage of osteonecrosis is no uniform, so according to the effective cure. The pathological characteristics of early and mid-term osteonecrosis of the femoral head, how to block the early avascular necrosis of the femoral head further collapse, collapse of bone or cartilage damage and bone cartilage after separation, it is very important how to construct osteochondral articular surface reconstruction unit good function. Objective: in this study, application of PLGA (poly lactic-co-glycolic acid, PLGA) as a drug carrier local release of specific concentrations of zoledronic acid (zoledronate, acid, ZOL) inhibits bone resorption and promote bone formation, prevent early exploration The effect of femoral head necrosis. The porcine articular cartilage cells by laser drilling and acellular and prepared with acellular cartilage matrix, biphasic scaffold of bone matrix and bone cartilage integrity of the interface, understand the echo of cartilage cells can effectively promote the added value, and through the model of rabbit cartilage defect verification in the case of defect repair in vivo, explore the repair of osteochondral injury effect, advanced technology for the treatment of tissue engineering bone necrosis of bone cartilage and bone cartilage separation provides a good selection of stent. Methods: the co cultured in low concentration in vitro application of zoledronic acid and into osteoclasts, the application of anti tartrate resistant acid phosphatase (TRAP) staining, analysis of bone resorption area and bone cell morphology to detect broken absorption image. Alkaline phosphatase (ALP), four methyl thiazolyl tetrazolium colorimetric assay (MTT). The solution of osteoblast morphology and proliferation. PLGA was chosen as the carrier for topical application of ZOL, preparation containing 30 u g ZOL PLGA bar. Micro-CT scan, calculated the bone mineral density were compared (bone mineral, density, BMD), bone volume fraction (bone volume, fraction, BVF), trabecular bone Liang Houdu (trabecular plate, thickness, Tb.Th) and small bone (trabecular spacing, Liang Jianxi Tb.Sp) scan was performed after HE and Masson staining to understand the histological changes between groups. The natural bone cartilage unit for laser drilling, the chemical method of acellular cartilage obtained acellular bone matrix derived scaffold three-dimensional porous, and through the organization study, the physicochemical properties of mechanical test about stent; isolation and culture of rabbit articular chondrocytes seeded on the scaffold to understand its effect on cell proliferation; free chondrocytes seeded on the bracket construction of cell scaffold 澶嶅悎浣

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