生物反应器内应用富血小板血浆构建血管化复合体修复骨坏死的研究
发布时间：2018-10-22 11:34
【摘要】：股骨头缺血性坏死是由于各种病因破坏了股骨头的血液供应,造成的以股骨头内骨小梁和骨髓坏死为特征的临床常见病,治疗不及时可导致终生残疾,是骨科医生面临的一大难题。骨坏死区的根本问题是成骨祖细胞和血管缺乏,坏死区域骨血管的形成是骨组织修复的前提,各国学者将研究点集中于如何促进骨坏死区骨小梁再生、新生血管形成。富血小板血浆(Platelet-rich plasma,PRP)是新鲜全血经离心后获取的自体血小板的浓缩体,含有多种高浓度的生长因子,包括：血小板源性生长因子(platelet derived growth factor, PDGF)、转移生长因子(transforming growth factor, TGF-β)、血管内皮生长因子(vascular endothelial growth factor, VEGF)、类胰岛素生长因子(insulin-like growthfactor,IGF)、表皮生长因子(epidermal growth factor,EGF)等。本项目拟将骨髓基质细胞(bone marrow stromal cells, BMSCs)与p-磷酸三钙(p-TCP)在灌注式生物反应器(bioreatctor, BO)内复合三维培养,利用PRP的促血管生成、成骨作用,构建高质量的血管化复合体,在MRI实时导引下利用氩氦冷冻消融建立兔股骨头坏死动物模型,同时在MRI导引下通过氩氦刀探针钉道将构建的血管化复合体精确置入到股骨头骨坏死区,通过大体观察、X线检测、组织学评价观察其促进骨坏死修复的作用,期望为临床治疗早期股骨头缺血性坏死提供新的治疗方法。 1MRI实时导引下氩氦冷冻消融建立新西兰兔股骨头缺血性坏死模型的实验研究 目的探讨MRI实时导引下氩氦冷冻消融建立新西兰兔股骨头缺血性坏死模型的可行性和技术方法 方法取2-3月龄的健康的新西兰大白兔48只,静脉麻醉成功后置于开放式0.23TMRI系统中,MRI导引下在大转子下方1cm向股骨头负重区打入2mm导引针,位置满意后置入氩氦刀探针,左侧实验组股骨头氩氦冷冻消融2个循环20分钟,右侧股骨头对照组氩氦冷冻消融1个循环10分钟。分别于术后4周、8周、12周三个时间点行X线检查、大体观察和组织学观察,评估股骨头组织的坏死和修复情况。 结果术后4、8、12周股骨头组织学评估,实验组1组股骨头陷窝细胞分别是49.75士3.17,62.06±4.12,48.25±2.76,高于对照组2组39.13±4.48,50.69±3.84,37.50±3.86。术后8周实验组陷窝细胞率为62.06%。术后12周实验组股骨头塌陷率43.7%,对照组股骨头塌陷率12.5%。 结论MRI实时导引下利用氩氦冷冻消融成功的建立了兔股骨头缺血性坏死模型,术后4周、8周、12周实验组陷窝细胞率均明显高于对照组。 2灌注式生物反应器应用富血小板血浆构建细胞-支架复合体体内血管化评估目的灌注式生物反应器内应用富血小板血浆构建BMSCs-β-TCP复合体并评估其体内血管化情况。 方法从兔耳缘静脉抽取新鲜血液,采用二次离心法制备富血小板血浆,与低糖DMEM培养液制备全培养基。体外将兔骨髓间充质细胞与β-磷酸三钙陶瓷复合培养后植入到灌注式生物反应器内,定期更换全培养基,3周后取出复合体,进行扫描电镜检测。将各组不同方式培养的复合体植入到兔皮下组织内,术后12周,取出复合体,免疫组织化学法检测血管标志物血小板-内皮细胞粘附分子(Cluster of Diffrentiation31,CD31).第Ⅷ因子关连抗原(von willebrand factor,vWF)的表达. 结果实验组电镜扫描显示β—磷酸三钙(p-TCP)上可见有细胞粘附,细胞伸出伪足和p一磷酸三钙(β-TCP)相连接,骨髓间充质干细胞细胞在β-TCP多孔陶瓷支架上的黏附、伸展和增殖良好。实验组免疫组化结果显示血管标志物CD31.第Ⅷ因子关连抗原(VWF)呈强阳性表现,其他组CD31.vWF的表达较少。结论富血小板血浆富含多种细胞因子,灌注式生物反应器内应用富血小板血浆构建细胞-支架复合体可以明显促进复合体血管化。 3灌注式生物反应器内构建血管化BMSCs-β-TCP复合体修复骨坏死的实验研究 目的探讨灌注式生物反应内构建血管化BMSCs-β-TCP复合体促进骨坏死修复的作用。 方法分别用实验1、2描述的方法建立新西兰兔股骨头缺血性坏死模型和构建血管化复合体。30只兔随机分为六组,A组钉道中不植人任何材料,B组植入β-TCP支架,C组MSCs-β-TCP, D组MSCs-β-TCP+bioreatctor, E组MSCs-β-TCP+PRP,F组MSCs-β-TCP+bioreactor+PRP。将不同方式构建的BMSCs-β-TCP复合体沿股骨头建模制作的钉道植入到股骨头坏死区,术后12周利用大体观察、X线检查和组织学观察评估其促进骨坏死修复的作用。 结果术后12周,各组X线检查示：A组、B组均有部分股骨头骨皮质不完整,局部骨密度高；C、D、E组未见有股骨头明显塌陷；F组未见有股骨头塌陷,坏死区骨密度与宿主区骨密度相近。组织学结果显示：A组、B组见较多纤维组织,骨形成少；C、D、E组少量骨形成,骨小梁排列不整齐；F组骨形成量较多,骨小梁排列良好,与正常骨小梁结构相近。 结论生物反应器内构建血管化组织工程骨可以有效修复骨坏死,从而为临床治疗股骨头缺血性坏死提供了可行的方法。
[Abstract]:Ischemic necrosis of avascular necrosis of the femoral head is due to various causes that destroy the blood supply of the femoral head, resulting in the clinical common disease characterized by the small beam and bone marrow necrosis of the femoral head, which can lead to lifelong disability in a timely manner, which is a difficult problem faced by an orthopedic surgeon. The basic problem of osteonecrosis is that bone progenitor cells and blood vessels lack, and the formation of bone vessels in necrotic areas is the prerequisite for bone tissue repair, and researchers focus on how to promote the regeneration of bone trabecula and neovascularization in osteonecrosis area. Platelet-rich plasma (PRP) is an autoplatelet concentrate obtained after centrifugation of fresh whole blood, containing a variety of high concentrations of growth factors, including platelet derived growth factor (PDGF), transfer growth factor (PAF), Vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), epidermal growth factor (EGF), etc. In this project, bone marrow stromal cells (BMSCs) and p-phosphate (p-TCP) are combined in three-dimensional culture in a perfusion bioreactor (BO), and a high-quality vascularized complex is constructed by using PRP's pro-angiogenic and bone-forming effects. The rabbit femoral head necrosis animal model is established by using argon helium cryoablation under the real-time MRI guidance, and the constructed blood vessel complex is accurately placed into the osteonecrosis area of the femoral head under MRI guidance, It is expected to provide a new treatment method for the treatment of ischemic necrosis of the femoral head in the early stage of clinical treatment. Real-time MRI guided argon-helium cryoablation to establish a model of ischemic necrosis of femoral head in New Zealand rabbits Objective To explore the feasibility of establishing a new Zealand rabbit femoral head ischemic necrosis model with real-time MRI guided argon-helium cryoablation The method of sexual and technical method takes 2-3 48 healthy New Zealand white rabbits were placed in open 0. 23TMRI system after intravenous anesthesia. Under MRI guidance, a 2mm guide needle was driven into the femoral head under the guidance of MRI under the guidance of MRI under the guidance of MRI. Argon helium knife probe was placed after the position was satisfied, and argon-helium cryoablation was performed on the left experimental group. 2 cycles 20 minutes, right femoral head control argon-helium cryoablation One cycle was performed for 10 minutes. X-ray examination, gross observation and histological observation were performed at three time points, 4 weeks, 8 weeks, 12 weeks, respectively, and the femoral head tissue was evaluated. The necrosis and repair of the femoral head in the experimental group were 49. 75 卤 3.17, 62. 06, 4.12, 48. 25 and 2.76, respectively, compared with control group 2, 39. 13, 4.48, 50. 69, 3.84, respectively., 37. 50, 3.86. Experimental group 8 weeks after operation The rate of collapse of femoral head was 62. 06%. The collapse rate of femoral head in experimental group was 43. 7% in 12 weeks after operation and control group. The ischemic necrosis model of femoral head was successfully established by using argon-helium cryoablation in real-time MRI, 4 weeks, 8 weeks and 12 weeks after operation. group trapping cell rate was significantly higher than that in control group. 2 perfusion bioreactor was used to construct the blood vessel in platelet-rich plasma to construct a perfusion bioreactor to construct BMSCs-7721 cells with platelet-rich plasma. In order to extract fresh blood from rabbit ear vein, the method of secondary centrifugation was used to prepare blood-rich blood. The whole culture medium is prepared from the plasma of the plate and the low-sugar DMEM culture solution. The rabbit bone marrow mesenchymal cells and the calcium phosphate-phosphate three-calcium ceramic are combined and cultured in vitro to be implanted into the perfusion bioreactor, and the whole culture is periodically replaced. The complex was taken out after 3 weeks. The complex was implanted into the subcutaneous tissue of rabbit. After 12 weeks, the complex was taken out, and the blood vessel marker platelet-endothelial cell adhesion molecule (Cluster of Di) was detected by immunohistochemistry. fferentiation31, CD31). Factor 鈪，
本文编号：2287041
[Abstract]:Ischemic necrosis of avascular necrosis of the femoral head is due to various causes that destroy the blood supply of the femoral head, resulting in the clinical common disease characterized by the small beam and bone marrow necrosis of the femoral head, which can lead to lifelong disability in a timely manner, which is a difficult problem faced by an orthopedic surgeon. The basic problem of osteonecrosis is that bone progenitor cells and blood vessels lack, and the formation of bone vessels in necrotic areas is the prerequisite for bone tissue repair, and researchers focus on how to promote the regeneration of bone trabecula and neovascularization in osteonecrosis area. Platelet-rich plasma (PRP) is an autoplatelet concentrate obtained after centrifugation of fresh whole blood, containing a variety of high concentrations of growth factors, including platelet derived growth factor (PDGF), transfer growth factor (PAF), Vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), epidermal growth factor (EGF), etc. In this project, bone marrow stromal cells (BMSCs) and p-phosphate (p-TCP) are combined in three-dimensional culture in a perfusion bioreactor (BO), and a high-quality vascularized complex is constructed by using PRP's pro-angiogenic and bone-forming effects. The rabbit femoral head necrosis animal model is established by using argon helium cryoablation under the real-time MRI guidance, and the constructed blood vessel complex is accurately placed into the osteonecrosis area of the femoral head under MRI guidance, It is expected to provide a new treatment method for the treatment of ischemic necrosis of the femoral head in the early stage of clinical treatment. Real-time MRI guided argon-helium cryoablation to establish a model of ischemic necrosis of femoral head in New Zealand rabbits Objective To explore the feasibility of establishing a new Zealand rabbit femoral head ischemic necrosis model with real-time MRI guided argon-helium cryoablation The method of sexual and technical method takes 2-3 48 healthy New Zealand white rabbits were placed in open 0. 23TMRI system after intravenous anesthesia. Under MRI guidance, a 2mm guide needle was driven into the femoral head under the guidance of MRI under the guidance of MRI under the guidance of MRI. Argon helium knife probe was placed after the position was satisfied, and argon-helium cryoablation was performed on the left experimental group. 2 cycles 20 minutes, right femoral head control argon-helium cryoablation One cycle was performed for 10 minutes. X-ray examination, gross observation and histological observation were performed at three time points, 4 weeks, 8 weeks, 12 weeks, respectively, and the femoral head tissue was evaluated. The necrosis and repair of the femoral head in the experimental group were 49. 75 卤 3.17, 62. 06, 4.12, 48. 25 and 2.76, respectively, compared with control group 2, 39. 13, 4.48, 50. 69, 3.84, respectively., 37. 50, 3.86. Experimental group 8 weeks after operation The rate of collapse of femoral head was 62. 06%. The collapse rate of femoral head in experimental group was 43. 7% in 12 weeks after operation and control group. The ischemic necrosis model of femoral head was successfully established by using argon-helium cryoablation in real-time MRI, 4 weeks, 8 weeks and 12 weeks after operation. group trapping cell rate was significantly higher than that in control group. 2 perfusion bioreactor was used to construct the blood vessel in platelet-rich plasma to construct a perfusion bioreactor to construct BMSCs-7721 cells with platelet-rich plasma. In order to extract fresh blood from rabbit ear vein, the method of secondary centrifugation was used to prepare blood-rich blood. The whole culture medium is prepared from the plasma of the plate and the low-sugar DMEM culture solution. The rabbit bone marrow mesenchymal cells and the calcium phosphate-phosphate three-calcium ceramic are combined and cultured in vitro to be implanted into the perfusion bioreactor, and the whole culture is periodically replaced. The complex was taken out after 3 weeks. The complex was implanted into the subcutaneous tissue of rabbit. After 12 weeks, the complex was taken out, and the blood vessel marker platelet-endothelial cell adhesion molecule (Cluster of Di) was detected by immunohistochemistry. fferentiation31, CD31). Factor 鈪，
本文编号：2287041
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