【摘要】：股骨頭缺血性壞死是由于各種病因破壞了股骨頭的血液供應,造成的以股骨頭內(nèi)骨小梁和骨髓壞死為特征的臨床常見病,治療不及時可導致終生殘疾,是骨科醫(yī)生面臨的一大難題。骨壞死區(qū)的根本問題是成骨祖細胞和血管缺乏,壞死區(qū)域骨血管的形成是骨組織修復的前提,各國學者將研究點集中于如何促進骨壞死區(qū)骨小梁再生、新生血管形成。富血小板血漿(Platelet-rich plasma,PRP)是新鮮全血經(jīng)離心后獲取的自體血小板的濃縮體,含有多種高濃度的生長因子,包括：血小板源性生長因子(platelet derived growth factor, PDGF)、轉(zhuǎn)移生長因子(transforming growth factor, TGF-β)、血管內(nèi)皮生長因子(vascular endothelial growth factor, VEGF)、類胰島素生長因子(insulin-like growthfactor,IGF)、表皮生長因子(epidermal growth factor,EGF)等。本項目擬將骨髓基質(zhì)細胞(bone marrow stromal cells, BMSCs)與p-磷酸三鈣(p-TCP)在灌注式生物反應器(bioreatctor, BO)內(nèi)復合三維培養(yǎng),利用PRP的促血管生成、成骨作用,構(gòu)建高質(zhì)量的血管化復合體,在MRI實時導引下利用氬氦冷凍消融建立兔股骨頭壞死動物模型,同時在MRI導引下通過氬氦刀探針釘?shù)缹?gòu)建的血管化復合體精確置入到股骨頭骨壞死區(qū),通過大體觀察、X線檢測、組織學評價觀察其促進骨壞死修復的作用,期望為臨床治療早期股骨頭缺血性壞死提供新的治療方法。 1MRI實時導引下氬氦冷凍消融建立新西蘭兔股骨頭缺血性壞死模型的實驗研究 目的探討MRI實時導引下氬氦冷凍消融建立新西蘭兔股骨頭缺血性壞死模型的可行性和技術(shù)方法 方法取2-3月齡的健康的新西蘭大白兔48只,靜脈麻醉成功后置于開放式0.23TMRI系統(tǒng)中,MRI導引下在大轉(zhuǎn)子下方1cm向股骨頭負重區(qū)打入2mm導引針,位置滿意后置入氬氦刀探針,左側(cè)實驗組股骨頭氬氦冷凍消融2個循環(huán)20分鐘,右側(cè)股骨頭對照組氬氦冷凍消融1個循環(huán)10分鐘。分別于術(shù)后4周、8周、12周三個時間點行X線檢查、大體觀察和組織學觀察,評估股骨頭組織的壞死和修復情況。 結(jié)果術(shù)后4、8、12周股骨頭組織學評估,實驗組1組股骨頭陷窩細胞分別是49.75士3.17,62.06±4.12,48.25±2.76,高于對照組2組39.13±4.48,50.69±3.84,37.50±3.86。術(shù)后8周實驗組陷窩細胞率為62.06%。術(shù)后12周實驗組股骨頭塌陷率43.7%,對照組股骨頭塌陷率12.5%。 結(jié)論MRI實時導引下利用氬氦冷凍消融成功的建立了兔股骨頭缺血性壞死模型,術(shù)后4周、8周、12周實驗組陷窩細胞率均明顯高于對照組。 2灌注式生物反應器應用富血小板血漿構(gòu)建細胞-支架復合體體內(nèi)血管化評估目的灌注式生物反應器內(nèi)應用富血小板血漿構(gòu)建BMSCs-β-TCP復合體并評估其體內(nèi)血管化情況。 方法從兔耳緣靜脈抽取新鮮血液,采用二次離心法制備富血小板血漿,與低糖DMEM培養(yǎng)液制備全培養(yǎng)基。體外將兔骨髓間充質(zhì)細胞與β-磷酸三鈣陶瓷復合培養(yǎng)后植入到灌注式生物反應器內(nèi),定期更換全培養(yǎng)基,3周后取出復合體,進行掃描電鏡檢測。將各組不同方式培養(yǎng)的復合體植入到兔皮下組織內(nèi),術(shù)后12周,取出復合體,免疫組織化學法檢測血管標志物血小板-內(nèi)皮細胞粘附分子(Cluster of Diffrentiation31,CD31).第Ⅷ因子關(guān)連抗原(von willebrand factor,vWF)的表達. 結(jié)果實驗組電鏡掃描顯示β—磷酸三鈣(p-TCP)上可見有細胞粘附,細胞伸出偽足和p一磷酸三鈣(β-TCP)相連接,骨髓間充質(zhì)干細胞細胞在β-TCP多孔陶瓷支架上的黏附、伸展和增殖良好。實驗組免疫組化結(jié)果顯示血管標志物CD31.第Ⅷ因子關(guān)連抗原(VWF)呈強陽性表現(xiàn),其他組CD31.vWF的表達較少。結(jié)論富血小板血漿富含多種細胞因子,灌注式生物反應器內(nèi)應用富血小板血漿構(gòu)建細胞-支架復合體可以明顯促進復合體血管化。 3灌注式生物反應器內(nèi)構(gòu)建血管化BMSCs-β-TCP復合體修復骨壞死的實驗研究 目的探討灌注式生物反應內(nèi)構(gòu)建血管化BMSCs-β-TCP復合體促進骨壞死修復的作用。 方法分別用實驗1、2描述的方法建立新西蘭兔股骨頭缺血性壞死模型和構(gòu)建血管化復合體。30只兔隨機分為六組,A組釘?shù)乐胁恢踩巳魏尾牧?B組植入β-TCP支架,C組MSCs-β-TCP, D組MSCs-β-TCP+bioreatctor, E組MSCs-β-TCP+PRP,F組MSCs-β-TCP+bioreactor+PRP。將不同方式構(gòu)建的BMSCs-β-TCP復合體沿股骨頭建模制作的釘?shù)乐踩氲焦晒穷^壞死區(qū),術(shù)后12周利用大體觀察、X線檢查和組織學觀察評估其促進骨壞死修復的作用。 結(jié)果術(shù)后12周,各組X線檢查示：A組、B組均有部分股骨頭骨皮質(zhì)不完整,局部骨密度高；C、D、E組未見有股骨頭明顯塌陷；F組未見有股骨頭塌陷,壞死區(qū)骨密度與宿主區(qū)骨密度相近。組織學結(jié)果顯示：A組、B組見較多纖維組織,骨形成少；C、D、E組少量骨形成,骨小梁排列不整齊；F組骨形成量較多,骨小梁排列良好,與正常骨小梁結(jié)構(gòu)相近。 結(jié)論生物反應器內(nèi)構(gòu)建血管化組織工程骨可以有效修復骨壞死,從而為臨床治療股骨頭缺血性壞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 鹵 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，

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