不连通双层PLGA支架负载自体骨髓间充质干细胞与富血小板血浆的复合体修复骨软骨缺损的实验研究
发布时间：2018-08-24 08:30
【摘要】：一直以来,骨科学界内普遍认为软骨的巨大缺损难以自愈,需要进行积极治疗以恢复其结构和功能。目前,临床现有的治疗方法包括关节腔内药物注射、基于骨髓刺激技术的微骨折术和钻孔术、自体软骨细胞移植、骨软骨移植术以及关节置换术等手段。这些治疗方法均有各自的优缺点和适应症,重建和修复后的关节软骨与正常关节软骨在结构和功能上存在一定的差异。探索一种彻底治愈此类疾患的技术方法是当前骨科基础和临床研究的热点和难点之一。组织工程学和再生医学的研究不断深入为彻底治愈关节软骨缺损和改善缺损重加后的关节软骨结构和功能提供了一个崭新的思路。 应用胞外基质材料、仿生材料和人工合成材料构建支架作为种子细胞和细胞因子的载体进行体内外构建组织工程化的组织是当前组织工程研究的重要内容之一。由于自体或异体基质来源的支架的取材来源相对短缺和可能带来的疾病传播风险问题以及生物力学性能欠佳等不足一定程度上限制了其广泛应用。人工合成的生物可降解材料具有容易获取并对其塑性使其与缺损的形状相匹配的优点逐渐受到研究者的亲睐。已有人工合成的PLGA支架用于肌腱、韧带、软骨以及骨组织等缺损修复和重建的研究报道。对于关节骨软骨缺损的重建,研究者认为应用双层的支架进行构建关节软骨和软骨下骨是必要的。因此,构建双层的的PLGA支架可能是构建骨软骨的一个良好选择。 考虑到正常组织的再生和修复需要众多细胞因子的参与和协同作用,将不同的细胞因子负载于支架可能是必要的。尽管有学者通过负载不同的重组细胞因子进行重建组织工程骨软骨获得了较理想的结果,但是,依然存在一些不足之处。比如单一细胞因子的参与难以真正模拟组织的再生修复过程,而且在体内降解迅速。同时,细胞因子的价格相对昂贵以及可供临床选择的种类依然较少等等。近年来,富血小板血浆(platelet-rich plasma, PRP)因激活后可释放血小板源性生长因子(platelet-derived growth factor PDGF),血管内皮生长因子(vascular endothelial growth factor VEGF),转化生长因子(transforming growth factor-p TGF-p),胰岛素生长因子(insulin growth factor (IGF)以及成纤维生长因子(basic fibroblast growth factor b-FGF)等而广受关注。PRP被作为自体的生长因子的重要来源,具有避免疾病传播风险、价格低廉以及含有生长因子种类较多等有优点。研究结果表明PRP可以提高间充质干细胞(mesenchymal stem cells MSC)等增殖并向成软骨方向分化；PRP还可以促进软骨下骨内的祖细胞的迁出和成软骨分化。体内研究结果表明PRP联合MSC可以促进骨组织的形成及其在骨软骨修复中的积极作用。骨髓来源的MSC可以方便地获取和体外扩增,且具有多向分化潜能。因此,BMSCs是构建组织工程骨软骨复合体的较为理想的种子细胞之一。另外,通过采集新西兰兔的自体外周血制备PRP可以获取自体来源的多种生长因子用于诱导BMSCs的增殖与分化并形成成熟的骨与软骨组织。 在关节负重部位的骨软骨缺损修复中,PRP的力学支撑性能相对不足。为此,我们设计了不连通的双层PLGA支架作为自体BMSCs和PRP的载体,将其复合体植入体内用于骨软骨缺损的重建与修复。本实验中,主要探讨不连通的双层PLGA支架作为自体BMSCs和PRP的载体支架修复兔骨软骨缺损的可行性,并观察负载BMSCs和PRP在骨软骨缺损重建和修复中的作用。该实验主要的实验材料、方法、内容和结论主要包括以下几个部分： 第1部分不连通双层PLGA支架负载自体富血小板血浆修复骨软骨缺损的实验研究 目的： 初步明确不连通双层PLGA支架构建骨软骨复合体的可行性,观察负载自体PRP在兔膝关节骨软骨缺损修复中的作用 方法： 1. PLGA支架的准备与消毒 双层不连通PLGA支架采用常温模压粒子浸出制备技术制备。材料分为三段,上段模拟软骨段孔径为50-100μm,孔隙率为92%,厚度为0.3mm；中间段模拟骨软骨交界处,是厚度为0.3mm PLGA膜；下段模拟骨段孔径为300-450μm,孔隙率为92%,厚度为3.4mm。支架的各段都是分别制备,最后通过二氯甲烷粘合起来,然后裁剪成直径和高度均为4mm的圆柱形支架。将致孔剂用去离子水浸出以后,就得到研究所需的双层支架。本研究所用支架均由复旦大学高分子材料系生物医用材料课题组暨聚合物分子工程国家重点实验室提供。 在获得研究所需的多孔的不连通双层PLGA支架后,75%乙醇浸泡30min, PBS反复漂洗3次,每次5min。 2.自体PRP的制备 经新西兰兔耳中央动脉穿刺术采集新鲜外周血10ml(含1m13.8%的枸橼酸钠溶液作为抗凝剂),在室温条件下,采用二次离心法进行制备。首次给予400g的离心力离心15min分离红细胞和血浆,再给予800g的离心力10min分离血浆中的血小板,弃去大部分的贫血小板血浆,剩余约0.8m1液体并轻微混悬即为PRP。每次制备均在手术前完成。制备前后手工计数全血和外周血来源的PRP中的血小板浓度。实施骨髓穿刺术抽取骨髓5m1进行制备骨髓来源的RPP,制备方法与过程同上。比较外周血来源的PRP与骨髓来源PRP的浓度差异。 3. PLGA/PRP复合体的构建 将无菌的不连通双层PLGA支架置入6孔板(2例／孔),每个孔内加入0.8ml的外周血来源的自体PRP和40μ1的10%CaCl2。 4.新西兰兔股骨内髁骨软骨缺损模型的制备与PLGA/PRP复合体的植入 健康新西兰兔18只,分为三组。直径4mm的环钻在股骨内髁钻取深度4mm的新鲜缺损,将体外构建的PLGA/PRP复合体植入缺损,对照组仅植入PLGA支架,空白组缺损处不植入材料。术后4周和12周进行大体观察、组织学、免疫组化、q-PCR检测相关基因表达和Micro-CT扫描观察等。 结果： 获取了具有不同孔径的不连通双层PLGA支架,制备的PRP中的血小板浓度为全血中的4.9倍,骨髓来源PRP的浓度为外周血来源PRP浓度的1.41倍。在术后4周和12周,PLGA/PRP组的大体评分和组织学评分均高于PLGA组和空白缺损组(P0.05)。12周时,PLGA/PRP组内的Ⅱ型胶原和aggrecan的相对表达水平明显高于PLGA组和空白缺损组(P0.05); Micro-CT扫描可见PLGA/PRP组的软骨下骨缺损区域有矿化组织存在较多。 结论： 在新西兰兔骨软骨缺损修复模型中,不连通双层PLGA支架可以用于骨软骨缺损的修复,负载PRP促进了软骨缺损的修复以及软骨下骨缺损内矿化组织的形成。 第2部分不连通双层PLGA支架负载自体BMSCs与PRP修复骨软骨缺损的实验研究 目的： 初步观察不连通双层PLGA支架负载自体BMSCs和PRP在兔骨软骨缺损修复中的作用 方法： 1. BMSCs的获取、培养 于新西兰兔髂后上棘行骨髓穿刺术抽取3-4ml骨髓,采用全骨髓贴壁培养法进行体外扩增培养,选用第三代(P3)的自体BMSCs进行体内植入研究。 2.细胞接种 将培养至第三代的BMSCs制备成浓度为4.0×106/ml和4×105/ml的细胞悬液各1ml,使用自制的细胞接种离心管,采用分次离心的方法将BMSCs接种至双层的PLGA支架的软骨层和骨层。接种前后使用细胞计数板镜下手工计数悬液中接种前后的细胞数量计算接种的效率。将支架-细胞复合体转移至6孔板继续培养,电镜扫描观察细胞在PLGA支架上的粘附。 3.自体PRP的负载舞 按照上述方法制备新西兰兔的自体PRP,将PLGA/BMSCs复合体转移至6孔板内(2例/孔),加入0.8ml自体PRP与40μl的10%CaC12。 4.新西兰兔股骨内髁骨软骨缺损模型制备与PLGA/BMSCs/PRP复合体植入 健康成年新西兰兔16只,分为4组。按照上述的骨软骨缺损制备直径和深度4mm的缺损,实验组植入PLGA/BMSCs/PRP,对照组分别植入PLGA/PRP和PLGA,空白组缺损处不植入任何材料。于术后6个月进行大体外观评估、组织学评估、免疫组化染色观察、q-PCR检测相关基因表达水平以及micro-CT扫描和定量评估软骨下骨缺损区域的BV/TV值。 结果： 骨髓穿刺获取骨髓贴壁培养可以获取自体的BMSCs,电镜观察表明分次离心接种法可以将不同浓度的BMSCs接种于PLGA支架的软骨层和骨层并粘附与支架。骨层和软骨层的接种效率分别为(81.474±2.53)%和(85.27±1.79)。术后6个月,PLGA/BMSCs/PRP组的大体评分和组织学评分均高于PLGA组和空白缺损组(P0.05), PLGA/BMSCs/PRP组与PLGA/PRP组的大体评分间无明显统计学差异(P0.05)；Ⅱ型胶原和aggrecan在PLGA/BMSCs/PRP组的相对表达水平均高于PLGA组和空白缺损组(P0.05),在PLGA/BMSCs/PRP组和PLGA/PRP组的表达水平无明显差异(P0.05)；新生骨组织在缺损处所用组织内的百分比即(BV/TV),PLGA/BMSCs/PRP组的BV/TV为(57±14)%,明显高于PLGA/PRP组((42.3±2.6)%)、PLGA组((28.8±5.9)%)和空白缺损组((34.8±5.7)%)(P0.05)。 结论： 在新西兰兔骨软骨缺损修复模型中,不连通双层PLGA支架负载自体BMSCs和PRP的复合体的植入促进了软骨组织和软骨下骨的再生,是一种便捷有效的骨软骨缺损修复方法。
[Abstract]:It has long been recognized in the orthopedic community that the huge defects of cartilage are difficult to heal and require active treatment to restore its structure and function. These treatments have their own advantages, disadvantages and indications. There are some differences in structure and function between the reconstructed and repaired articular cartilage and the normal articular cartilage. To explore a technique to cure these diseases thoroughly is one of the hotspots and difficulties in basic and clinical orthopedic research. And the research of regenerative medicine provides a new way to cure articular cartilage defect thoroughly and improve the structure and function of articular cartilage.
Tissue engineering using extracellular matrix materials, biomimetic materials and synthetic materials as scaffolds for seed cells and cytokines in vitro and in vivo is one of the most important research fields in tissue engineering. Synthetic biodegradable materials are easy to obtain and their plasticity matches the shape of the defect. Synthetic PLGA scaffolds have been used in tendons, ligaments, cartilages and so on. For the reconstruction of articular cartilage defects, it is necessary to construct articular cartilage and subchondral bone with bilayer scaffolds. Therefore, the construction of bilayer PLGA scaffolds may be a good choice for the construction of osteochondral defects.
Considering that the regeneration and repair of normal tissues require the participation and synergy of many cytokines, it may be necessary to load different cytokines on scaffolds. In recent years, platelet-rich plasma (PRP) can release platelet-derived growth factors because of its activation. Plaelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor-p TGF-p, insulin growth factor (IGF) and basic fibroblast growth factor b-FGF have attracted much attention. As an important source of autologous growth factors, PRP has many advantages, such as avoiding the risk of disease transmission, low price and many kinds of growth factors. In vivo studies have shown that PRP combined with MSC can promote the formation of bone tissue and play an active role in osteochondral repair. MSCs derived from bone marrow can be easily obtained and amplified in vitro, and have the potential of multidirectional differentiation. In addition, PRP can be obtained from the peripheral blood of New Zealand rabbits to induce the proliferation and differentiation of BMSCs and to form mature bone and cartilage tissue.
In this study, we designed a disconnected double-layer PLGA scaffold as the carrier of autologous BMSCs and PRP, and implanted its complex in vivo for the reconstruction and repair of osteochondral defects. The feasibility of repairing rabbit osteochondral defects with BMSCs and PRP-loaded scaffolds in vivo and the role of BMSCs and PRP-loaded scaffolds in the reconstruction and repair of osteochondral defects were observed.
Part 1 Repair of Osteochondral Defects with Autologous Platelet-rich Plasma Loaded on Disconnected Bilayer PLGA Scaffolds
Objective:
The feasibility of constructing osteochondral complex with disconnected bilayer PLGA scaffolds was preliminarily clarified, and the role of PRP loaded autograft in repairing osteochondral defects of rabbit knee joint was observed.
Method:
Preparation and disinfection of 1. PLGA stent
Two-layer disconnected PLGA scaffolds were prepared by normal-temperature molded particle leaching technique.The material was divided into three sections.The pore size of the upper simulated cartilage segment was 50-100 micron,the porosity was 92% and the thickness was 0.3 mm.The thickness of the middle simulated bone-cartilage interface was 0.3 mm PLGA membrane.The pore size of the lower simulated bone segment was 300-450 micron,the porosity was 92% and the thickness was 3.4 mm. Each segment of the scaffold was prepared separately, and then bonded by dichloromethane. The scaffolds were cut into cylindrical scaffolds with diameter and height of 4 mm. After the porogens were leached in deionized water, the double-layer scaffolds needed for the study were obtained. The State Key Laboratory of molecular engineering.
After the porous disconnected bilayer PLGA scaffolds were obtained, 75% ethanol was soaked for 30 minutes and PBS was rinsed three times for 5 minutes each time.
2. preparation of autologous PRP
Fresh peripheral blood 10ml (containing 1 m 13.8% sodium citrate solution as anticoagulant) was collected by central artery puncture in New Zealand rabbit ear and prepared by twice centrifugation at room temperature. Each preparation was performed before and after the preparation. Platelet concentrations in whole blood and peripheral blood-derived PRPs were counted manually. Bone marrow puncture was performed to extract 5 mm of bone marrow for preparation of bone marrow-derived RPPs. The preparation method and procedure were the same as above. The concentration of PRP was different from that of bone marrow derived PRP.
Construction of 3. PLGA/PRP complex
Sterile disconnected bilayer PLGA scaffolds were implanted into 6-well plates (2 cases/hole) with 0.8 ml of autologous PRP and 40 U 1 10% CaCl2 in each hole.
Preparation of PLGA/PRP New Zealand rabbit femoral condyle cartilage defect model and implantation of PLGA/PRP complex
Eighteen healthy New Zealand rabbits were divided into three groups.Four mm diameter trephine was used to drill fresh defects in the femoral medial condyle with a depth of 4 mm.PLGA/PRP complex was implanted into the defect in vitro. Da and Micro-CT scan observation.
Result:
The platelet concentration in the prepared PRP was 4.9 times higher than that in the whole blood and the PRP concentration in the bone marrow was 1.41 times higher than that in the peripheral blood. The relative expression levels of type II collagen and aggrecan in PLGA group were significantly higher than those in PLGA group and blank defect group (P 0.05). Micro-CT scan showed that there were more mineralized tissue in subchondral bone defect area of PLGA/PRP group.
Conclusion:
In the New Zealand rabbit model of osteochondral defect repair, disconnected bilayer PLGA scaffolds can be used to repair osteochondral defect. PRP loading promotes the repair of cartilage defect and the formation of mineralized tissue in subchondral bone defect.
The second part is an experimental study of repair of osteochondral defects with autologous BMSCs and PRP with unconnected double PLGA stent.
Objective:
Preliminary observation of the role of unattached double PLGA scaffolds loaded with autologous BMSCs and PRP in repairing bone and cartilage defects in rabbits
Method:
1. acquisition and cultivation of BMSCs
3-4 ml bone marrow was extracted from the posterior superior iliac spine of New Zealand rabbits by bone marrow puncture. The whole bone marrow adherent culture was used to amplify the bone marrow in vitro. The third generation (P3) autologous BMSCs were used for in vivo implantation.
2. cell vaccination
BMSCs cultured to the third generation were prepared into cell suspensions with concentrations of 4.0 *106/ml and 4 *105/ml, respectively. BMSCs were inoculated into the cartilage and bone layers of the bilayer PLGA scaffolds by a self-made centrifugal tube. The number of cells before and after inoculation was counted by hand under a cell counting plate microscope. The efficiency of inoculation was calculated quantitatively. The scaffold-cell complex was transferred to 6-well plate for further culture. The adhesion of cells on PLGA scaffolds was observed by electron microscopy.
3. load dance with autologous PRP
Autologous PRP of New Zealand rabbits was prepared according to the above method. PLGA/BMSCs complex was transferred into 6-well plate (2 cases/hole) and 0.8 ml PRP and 40 ml CaC12 were added.
4. New Zealand rabbit femoral internal condyle cartilage defect model preparation and PLGA/BMSCs/PRP complex implantation
Sixteen healthy adult New Zealand rabbits were divided into four groups. According to the above-mentioned defects, PLGA/BMSCs/PRP was implanted in the experimental group, PLGA/PRP and PLGA were implanted in the control group, and no material was implanted in the blank group. The expression level of related genes and the BV/TV value of subchondral bone defect area were detected and quantitatively evaluated by micro-CT scan.
Result:
BMSCs could be obtained by bone marrow aspiration. Electron microscopic observation showed that BMSCs with different concentrations could be seeded into the cartilage layer and bone layer of PLGA scaffolds and adhered to the scaffolds. The inoculation efficiency of bone layer and cartilage layer were (81.474 2.53)% and (85.27 1.79). Six months after operation, the PLGA / BMSCs / PRP group was inoculated with BMSCs. The gross score and histological score of PLGA / BMSCs / PRP group and PLGA / PRP group were higher than those of PLGA group and blank defect group (P 0.05). There was no significant difference in gross score between PLGA / BMSCs / PRP group and PLGA / PRP group (P 0.05). The relative expression levels of type II collagen and aggrecan in PLGA / BMSCs / PRP group were higher than those in PLGA group and blank defect group (P 0.05). There was no significant difference in the expression level (P 0.05). The percentage of BV/TV in the defect tissues of the new bone tissue was (57
Conclusion:
In the New Zealand rabbit model of osteochondral defect repair, the implantation of a disconnected bilayer PLGA scaffold loaded with autologous BMSCs and PRP composite promotes the regeneration of cartilage tissue and subchondral bone, which is a convenient and effective method for repairing osteochondral defect.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R318.08
本文编号：2200213
[Abstract]:It has long been recognized in the orthopedic community that the huge defects of cartilage are difficult to heal and require active treatment to restore its structure and function. These treatments have their own advantages, disadvantages and indications. There are some differences in structure and function between the reconstructed and repaired articular cartilage and the normal articular cartilage. To explore a technique to cure these diseases thoroughly is one of the hotspots and difficulties in basic and clinical orthopedic research. And the research of regenerative medicine provides a new way to cure articular cartilage defect thoroughly and improve the structure and function of articular cartilage.
Tissue engineering using extracellular matrix materials, biomimetic materials and synthetic materials as scaffolds for seed cells and cytokines in vitro and in vivo is one of the most important research fields in tissue engineering. Synthetic biodegradable materials are easy to obtain and their plasticity matches the shape of the defect. Synthetic PLGA scaffolds have been used in tendons, ligaments, cartilages and so on. For the reconstruction of articular cartilage defects, it is necessary to construct articular cartilage and subchondral bone with bilayer scaffolds. Therefore, the construction of bilayer PLGA scaffolds may be a good choice for the construction of osteochondral defects.
Considering that the regeneration and repair of normal tissues require the participation and synergy of many cytokines, it may be necessary to load different cytokines on scaffolds. In recent years, platelet-rich plasma (PRP) can release platelet-derived growth factors because of its activation. Plaelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor-p TGF-p, insulin growth factor (IGF) and basic fibroblast growth factor b-FGF have attracted much attention. As an important source of autologous growth factors, PRP has many advantages, such as avoiding the risk of disease transmission, low price and many kinds of growth factors. In vivo studies have shown that PRP combined with MSC can promote the formation of bone tissue and play an active role in osteochondral repair. MSCs derived from bone marrow can be easily obtained and amplified in vitro, and have the potential of multidirectional differentiation. In addition, PRP can be obtained from the peripheral blood of New Zealand rabbits to induce the proliferation and differentiation of BMSCs and to form mature bone and cartilage tissue.
In this study, we designed a disconnected double-layer PLGA scaffold as the carrier of autologous BMSCs and PRP, and implanted its complex in vivo for the reconstruction and repair of osteochondral defects. The feasibility of repairing rabbit osteochondral defects with BMSCs and PRP-loaded scaffolds in vivo and the role of BMSCs and PRP-loaded scaffolds in the reconstruction and repair of osteochondral defects were observed.
Part 1 Repair of Osteochondral Defects with Autologous Platelet-rich Plasma Loaded on Disconnected Bilayer PLGA Scaffolds
Objective:
The feasibility of constructing osteochondral complex with disconnected bilayer PLGA scaffolds was preliminarily clarified, and the role of PRP loaded autograft in repairing osteochondral defects of rabbit knee joint was observed.
Method:
Preparation and disinfection of 1. PLGA stent
Two-layer disconnected PLGA scaffolds were prepared by normal-temperature molded particle leaching technique.The material was divided into three sections.The pore size of the upper simulated cartilage segment was 50-100 micron,the porosity was 92% and the thickness was 0.3 mm.The thickness of the middle simulated bone-cartilage interface was 0.3 mm PLGA membrane.The pore size of the lower simulated bone segment was 300-450 micron,the porosity was 92% and the thickness was 3.4 mm. Each segment of the scaffold was prepared separately, and then bonded by dichloromethane. The scaffolds were cut into cylindrical scaffolds with diameter and height of 4 mm. After the porogens were leached in deionized water, the double-layer scaffolds needed for the study were obtained. The State Key Laboratory of molecular engineering.
After the porous disconnected bilayer PLGA scaffolds were obtained, 75% ethanol was soaked for 30 minutes and PBS was rinsed three times for 5 minutes each time.
2. preparation of autologous PRP
Fresh peripheral blood 10ml (containing 1 m 13.8% sodium citrate solution as anticoagulant) was collected by central artery puncture in New Zealand rabbit ear and prepared by twice centrifugation at room temperature. Each preparation was performed before and after the preparation. Platelet concentrations in whole blood and peripheral blood-derived PRPs were counted manually. Bone marrow puncture was performed to extract 5 mm of bone marrow for preparation of bone marrow-derived RPPs. The preparation method and procedure were the same as above. The concentration of PRP was different from that of bone marrow derived PRP.
Construction of 3. PLGA/PRP complex
Sterile disconnected bilayer PLGA scaffolds were implanted into 6-well plates (2 cases/hole) with 0.8 ml of autologous PRP and 40 U 1 10% CaCl2 in each hole.
Preparation of PLGA/PRP New Zealand rabbit femoral condyle cartilage defect model and implantation of PLGA/PRP complex
Eighteen healthy New Zealand rabbits were divided into three groups.Four mm diameter trephine was used to drill fresh defects in the femoral medial condyle with a depth of 4 mm.PLGA/PRP complex was implanted into the defect in vitro. Da and Micro-CT scan observation.
Result:
The platelet concentration in the prepared PRP was 4.9 times higher than that in the whole blood and the PRP concentration in the bone marrow was 1.41 times higher than that in the peripheral blood. The relative expression levels of type II collagen and aggrecan in PLGA group were significantly higher than those in PLGA group and blank defect group (P 0.05). Micro-CT scan showed that there were more mineralized tissue in subchondral bone defect area of PLGA/PRP group.
Conclusion:
In the New Zealand rabbit model of osteochondral defect repair, disconnected bilayer PLGA scaffolds can be used to repair osteochondral defect. PRP loading promotes the repair of cartilage defect and the formation of mineralized tissue in subchondral bone defect.
The second part is an experimental study of repair of osteochondral defects with autologous BMSCs and PRP with unconnected double PLGA stent.
Objective:
Preliminary observation of the role of unattached double PLGA scaffolds loaded with autologous BMSCs and PRP in repairing bone and cartilage defects in rabbits
Method:
1. acquisition and cultivation of BMSCs
3-4 ml bone marrow was extracted from the posterior superior iliac spine of New Zealand rabbits by bone marrow puncture. The whole bone marrow adherent culture was used to amplify the bone marrow in vitro. The third generation (P3) autologous BMSCs were used for in vivo implantation.
2. cell vaccination
BMSCs cultured to the third generation were prepared into cell suspensions with concentrations of 4.0 *106/ml and 4 *105/ml, respectively. BMSCs were inoculated into the cartilage and bone layers of the bilayer PLGA scaffolds by a self-made centrifugal tube. The number of cells before and after inoculation was counted by hand under a cell counting plate microscope. The efficiency of inoculation was calculated quantitatively. The scaffold-cell complex was transferred to 6-well plate for further culture. The adhesion of cells on PLGA scaffolds was observed by electron microscopy.
3. load dance with autologous PRP
Autologous PRP of New Zealand rabbits was prepared according to the above method. PLGA/BMSCs complex was transferred into 6-well plate (2 cases/hole) and 0.8 ml PRP and 40 ml CaC12 were added.
4. New Zealand rabbit femoral internal condyle cartilage defect model preparation and PLGA/BMSCs/PRP complex implantation
Sixteen healthy adult New Zealand rabbits were divided into four groups. According to the above-mentioned defects, PLGA/BMSCs/PRP was implanted in the experimental group, PLGA/PRP and PLGA were implanted in the control group, and no material was implanted in the blank group. The expression level of related genes and the BV/TV value of subchondral bone defect area were detected and quantitatively evaluated by micro-CT scan.
Result:
BMSCs could be obtained by bone marrow aspiration. Electron microscopic observation showed that BMSCs with different concentrations could be seeded into the cartilage layer and bone layer of PLGA scaffolds and adhered to the scaffolds. The inoculation efficiency of bone layer and cartilage layer were (81.474 2.53)% and (85.27 1.79). Six months after operation, the PLGA / BMSCs / PRP group was inoculated with BMSCs. The gross score and histological score of PLGA / BMSCs / PRP group and PLGA / PRP group were higher than those of PLGA group and blank defect group (P 0.05). There was no significant difference in gross score between PLGA / BMSCs / PRP group and PLGA / PRP group (P 0.05). The relative expression levels of type II collagen and aggrecan in PLGA / BMSCs / PRP group were higher than those in PLGA group and blank defect group (P 0.05). There was no significant difference in the expression level (P 0.05). The percentage of BV/TV in the defect tissues of the new bone tissue was (57
Conclusion:
In the New Zealand rabbit model of osteochondral defect repair, the implantation of a disconnected bilayer PLGA scaffold loaded with autologous BMSCs and PRP composite promotes the regeneration of cartilage tissue and subchondral bone, which is a convenient and effective method for repairing osteochondral defect.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R318.08
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