不連通雙層PLGA支架負(fù)載自體骨髓間充質(zhì)干細(xì)胞與富血小板血漿的復(fù)合體修復(fù)骨軟骨缺損的實(shí)驗(yàn)研究
發(fā)布時(shí)間：2018-08-24 08:30
【摘要】：一直以來(lái),骨科學(xué)界內(nèi)普遍認(rèn)為軟骨的巨大缺損難以自愈,需要進(jìn)行積極治療以恢復(fù)其結(jié)構(gòu)和功能。目前,臨床現(xiàn)有的治療方法包括關(guān)節(jié)腔內(nèi)藥物注射、基于骨髓刺激技術(shù)的微骨折術(shù)和鉆孔術(shù)、自體軟骨細(xì)胞移植、骨軟骨移植術(shù)以及關(guān)節(jié)置換術(shù)等手段。這些治療方法均有各自的優(yōu)缺點(diǎn)和適應(yīng)癥,重建和修復(fù)后的關(guān)節(jié)軟骨與正常關(guān)節(jié)軟骨在結(jié)構(gòu)和功能上存在一定的差異。探索一種徹底治愈此類疾患的技術(shù)方法是當(dāng)前骨科基礎(chǔ)和臨床研究的熱點(diǎn)和難點(diǎn)之一。組織工程學(xué)和再生醫(yī)學(xué)的研究不斷深入為徹底治愈關(guān)節(jié)軟骨缺損和改善缺損重加后的關(guān)節(jié)軟骨結(jié)構(gòu)和功能提供了一個(gè)嶄新的思路。 應(yīng)用胞外基質(zhì)材料、仿生材料和人工合成材料構(gòu)建支架作為種子細(xì)胞和細(xì)胞因子的載體進(jìn)行體內(nèi)外構(gòu)建組織工程化的組織是當(dāng)前組織工程研究的重要內(nèi)容之一。由于自體或異體基質(zhì)來(lái)源的支架的取材來(lái)源相對(duì)短缺和可能帶來(lái)的疾病傳播風(fēng)險(xiǎn)問(wèn)題以及生物力學(xué)性能欠佳等不足一定程度上限制了其廣泛應(yīng)用。人工合成的生物可降解材料具有容易獲取并對(duì)其塑性使其與缺損的形狀相匹配的優(yōu)點(diǎn)逐漸受到研究者的親睞。已有人工合成的PLGA支架用于肌腱、韌帶、軟骨以及骨組織等缺損修復(fù)和重建的研究報(bào)道。對(duì)于關(guān)節(jié)骨軟骨缺損的重建,研究者認(rèn)為應(yīng)用雙層的支架進(jìn)行構(gòu)建關(guān)節(jié)軟骨和軟骨下骨是必要的。因此,構(gòu)建雙層的的PLGA支架可能是構(gòu)建骨軟骨的一個(gè)良好選擇。 考慮到正常組織的再生和修復(fù)需要眾多細(xì)胞因子的參與和協(xié)同作用,將不同的細(xì)胞因子負(fù)載于支架可能是必要的。盡管有學(xué)者通過(guò)負(fù)載不同的重組細(xì)胞因子進(jìn)行重建組織工程骨軟骨獲得了較理想的結(jié)果,但是,依然存在一些不足之處。比如單一細(xì)胞因子的參與難以真正模擬組織的再生修復(fù)過(guò)程,而且在體內(nèi)降解迅速。同時(shí),細(xì)胞因子的價(jià)格相對(duì)昂貴以及可供臨床選擇的種類依然較少等等。近年來(lái),富血小板血漿(platelet-rich plasma, PRP)因激活后可釋放血小板源性生長(zhǎng)因子(platelet-derived growth factor PDGF),血管內(nèi)皮生長(zhǎng)因子(vascular endothelial growth factor VEGF),轉(zhuǎn)化生長(zhǎng)因子(transforming growth factor-p TGF-p),胰島素生長(zhǎng)因子(insulin growth factor (IGF)以及成纖維生長(zhǎng)因子(basic fibroblast growth factor b-FGF)等而廣受關(guān)注。PRP被作為自體的生長(zhǎng)因子的重要來(lái)源,具有避免疾病傳播風(fēng)險(xiǎn)、價(jià)格低廉以及含有生長(zhǎng)因子種類較多等有優(yōu)點(diǎn)。研究結(jié)果表明PRP可以提高間充質(zhì)干細(xì)胞(mesenchymal stem cells MSC)等增殖并向成軟骨方向分化；PRP還可以促進(jìn)軟骨下骨內(nèi)的祖細(xì)胞的遷出和成軟骨分化。體內(nèi)研究結(jié)果表明PRP聯(lián)合MSC可以促進(jìn)骨組織的形成及其在骨軟骨修復(fù)中的積極作用。骨髓來(lái)源的MSC可以方便地獲取和體外擴(kuò)增,且具有多向分化潛能。因此,BMSCs是構(gòu)建組織工程骨軟骨復(fù)合體的較為理想的種子細(xì)胞之一。另外,通過(guò)采集新西蘭兔的自體外周血制備PRP可以獲取自體來(lái)源的多種生長(zhǎng)因子用于誘導(dǎo)BMSCs的增殖與分化并形成成熟的骨與軟骨組織。 在關(guān)節(jié)負(fù)重部位的骨軟骨缺損修復(fù)中,PRP的力學(xué)支撐性能相對(duì)不足。為此,我們?cè)O(shè)計(jì)了不連通的雙層PLGA支架作為自體BMSCs和PRP的載體,將其復(fù)合體植入體內(nèi)用于骨軟骨缺損的重建與修復(fù)。本實(shí)驗(yàn)中,主要探討不連通的雙層PLGA支架作為自體BMSCs和PRP的載體支架修復(fù)兔骨軟骨缺損的可行性,并觀察負(fù)載BMSCs和PRP在骨軟骨缺損重建和修復(fù)中的作用。該實(shí)驗(yàn)主要的實(shí)驗(yàn)材料、方法、內(nèi)容和結(jié)論主要包括以下幾個(gè)部分： 第1部分不連通雙層PLGA支架負(fù)載自體富血小板血漿修復(fù)骨軟骨缺損的實(shí)驗(yàn)研究 目的： 初步明確不連通雙層PLGA支架構(gòu)建骨軟骨復(fù)合體的可行性,觀察負(fù)載自體PRP在兔膝關(guān)節(jié)骨軟骨缺損修復(fù)中的作用 方法： 1. PLGA支架的準(zhǔn)備與消毒 雙層不連通PLGA支架采用常溫模壓粒子浸出制備技術(shù)制備。材料分為三段,上段模擬軟骨段孔徑為50-100μm,孔隙率為92%,厚度為0.3mm；中間段模擬骨軟骨交界處,是厚度為0.3mm PLGA膜；下段模擬骨段孔徑為300-450μm,孔隙率為92%,厚度為3.4mm。支架的各段都是分別制備,最后通過(guò)二氯甲烷粘合起來(lái),然后裁剪成直徑和高度均為4mm的圓柱形支架。將致孔劑用去離子水浸出以后,就得到研究所需的雙層支架。本研究所用支架均由復(fù)旦大學(xué)高分子材料系生物醫(yī)用材料課題組暨聚合物分子工程國(guó)家重點(diǎn)實(shí)驗(yàn)室提供。 在獲得研究所需的多孔的不連通雙層PLGA支架后,75%乙醇浸泡30min, PBS反復(fù)漂洗3次,每次5min。 2.自體PRP的制備 經(jīng)新西蘭兔耳中央動(dòng)脈穿刺術(shù)采集新鮮外周血10ml(含1m13.8%的枸櫞酸鈉溶液作為抗凝劑),在室溫條件下,采用二次離心法進(jìn)行制備。首次給予400g的離心力離心15min分離紅細(xì)胞和血漿,再給予800g的離心力10min分離血漿中的血小板,棄去大部分的貧血小板血漿,剩余約0.8m1液體并輕微混懸即為PRP。每次制備均在手術(shù)前完成。制備前后手工計(jì)數(shù)全血和外周血來(lái)源的PRP中的血小板濃度。實(shí)施骨髓穿刺術(shù)抽取骨髓5m1進(jìn)行制備骨髓來(lái)源的RPP,制備方法與過(guò)程同上。比較外周血來(lái)源的PRP與骨髓來(lái)源PRP的濃度差異。 3. PLGA/PRP復(fù)合體的構(gòu)建 將無(wú)菌的不連通雙層PLGA支架置入6孔板(2例／孔),每個(gè)孔內(nèi)加入0.8ml的外周血來(lái)源的自體PRP和40μ1的10%CaCl2。 4.新西蘭兔股骨內(nèi)髁骨軟骨缺損模型的制備與PLGA/PRP復(fù)合體的植入 健康新西蘭兔18只,分為三組。直徑4mm的環(huán)鉆在股骨內(nèi)髁鉆取深度4mm的新鮮缺損,將體外構(gòu)建的PLGA/PRP復(fù)合體植入缺損,對(duì)照組僅植入PLGA支架,空白組缺損處不植入材料。術(shù)后4周和12周進(jìn)行大體觀察、組織學(xué)、免疫組化、q-PCR檢測(cè)相關(guān)基因表達(dá)和Micro-CT掃描觀察等。 結(jié)果： 獲取了具有不同孔徑的不連通雙層PLGA支架,制備的PRP中的血小板濃度為全血中的4.9倍,骨髓來(lái)源PRP的濃度為外周血來(lái)源PRP濃度的1.41倍。在術(shù)后4周和12周,PLGA/PRP組的大體評(píng)分和組織學(xué)評(píng)分均高于PLGA組和空白缺損組(P0.05)。12周時(shí),PLGA/PRP組內(nèi)的Ⅱ型膠原和aggrecan的相對(duì)表達(dá)水平明顯高于PLGA組和空白缺損組(P0.05); Micro-CT掃描可見(jiàn)PLGA/PRP組的軟骨下骨缺損區(qū)域有礦化組織存在較多。 結(jié)論： 在新西蘭兔骨軟骨缺損修復(fù)模型中,不連通雙層PLGA支架可以用于骨軟骨缺損的修復(fù),負(fù)載PRP促進(jìn)了軟骨缺損的修復(fù)以及軟骨下骨缺損內(nèi)礦化組織的形成。 第2部分不連通雙層PLGA支架負(fù)載自體BMSCs與PRP修復(fù)骨軟骨缺損的實(shí)驗(yàn)研究 目的： 初步觀察不連通雙層PLGA支架負(fù)載自體BMSCs和PRP在兔骨軟骨缺損修復(fù)中的作用 方法： 1. BMSCs的獲取、培養(yǎng) 于新西蘭兔髂后上棘行骨髓穿刺術(shù)抽取3-4ml骨髓,采用全骨髓貼壁培養(yǎng)法進(jìn)行體外擴(kuò)增培養(yǎng),選用第三代(P3)的自體BMSCs進(jìn)行體內(nèi)植入研究。 2.細(xì)胞接種 將培養(yǎng)至第三代的BMSCs制備成濃度為4.0×106/ml和4×105/ml的細(xì)胞懸液各1ml,使用自制的細(xì)胞接種離心管,采用分次離心的方法將BMSCs接種至雙層的PLGA支架的軟骨層和骨層。接種前后使用細(xì)胞計(jì)數(shù)板鏡下手工計(jì)數(shù)懸液中接種前后的細(xì)胞數(shù)量計(jì)算接種的效率。將支架-細(xì)胞復(fù)合體轉(zhuǎn)移至6孔板繼續(xù)培養(yǎng),電鏡掃描觀察細(xì)胞在PLGA支架上的粘附。 3.自體PRP的負(fù)載舞 按照上述方法制備新西蘭兔的自體PRP,將PLGA/BMSCs復(fù)合體轉(zhuǎn)移至6孔板內(nèi)(2例/孔),加入0.8ml自體PRP與40μl的10%CaC12。 4.新西蘭兔股骨內(nèi)髁骨軟骨缺損模型制備與PLGA/BMSCs/PRP復(fù)合體植入 健康成年新西蘭兔16只,分為4組。按照上述的骨軟骨缺損制備直徑和深度4mm的缺損,實(shí)驗(yàn)組植入PLGA/BMSCs/PRP,對(duì)照組分別植入PLGA/PRP和PLGA,空白組缺損處不植入任何材料。于術(shù)后6個(gè)月進(jìn)行大體外觀評(píng)估、組織學(xué)評(píng)估、免疫組化染色觀察、q-PCR檢測(cè)相關(guān)基因表達(dá)水平以及micro-CT掃描和定量評(píng)估軟骨下骨缺損區(qū)域的BV/TV值。 結(jié)果： 骨髓穿刺獲取骨髓貼壁培養(yǎng)可以獲取自體的BMSCs,電鏡觀察表明分次離心接種法可以將不同濃度的BMSCs接種于PLGA支架的軟骨層和骨層并粘附與支架。骨層和軟骨層的接種效率分別為(81.474±2.53)%和(85.27±1.79)。術(shù)后6個(gè)月,PLGA/BMSCs/PRP組的大體評(píng)分和組織學(xué)評(píng)分均高于PLGA組和空白缺損組(P0.05), PLGA/BMSCs/PRP組與PLGA/PRP組的大體評(píng)分間無(wú)明顯統(tǒng)計(jì)學(xué)差異(P0.05)；Ⅱ型膠原和aggrecan在PLGA/BMSCs/PRP組的相對(duì)表達(dá)水平均高于PLGA組和空白缺損組(P0.05),在PLGA/BMSCs/PRP組和PLGA/PRP組的表達(dá)水平無(wú)明顯差異(P0.05)；新生骨組織在缺損處所用組織內(nèi)的百分比即(BV/TV),PLGA/BMSCs/PRP組的BV/TV為(57±14)%,明顯高于PLGA/PRP組((42.3±2.6)%)、PLGA組((28.8±5.9)%)和空白缺損組((34.8±5.7)%)(P0.05)。 結(jié)論： 在新西蘭兔骨軟骨缺損修復(fù)模型中,不連通雙層PLGA支架負(fù)載自體BMSCs和PRP的復(fù)合體的植入促進(jìn)了軟骨組織和軟骨下骨的再生,是一種便捷有效的骨軟骨缺損修復(fù)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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R318.08
本文編號(hào)：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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R318.08
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