MSCs附和PLGA电纺支架修复兔跟腱损伤的实验研究
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摘要
目的：研究兔骨髓间充质干细胞附和PLGA电纺支架修复跟腱损伤的作用。
方法：利用静电纺丝技术制备PLGA电纺丝膜状支架,对PLGA支架结构进行观察和测量,并使用高温高压、低温等离子、钻6。照射、紫外线照射、酒精浸泡等五种不同方式消毒,进行细菌培养检测消毒后效果并观察支架结构的破坏情况；采用骨髓穿刺法获取兔骨髓,并用密度梯度离心法分离并体外培养骨髓间充质干细胞(MSCs);将第3代骨髓间充质干细胞结合PLGA支架复合培养；将45只成年新西兰大白兔,随机分为3组,每组15只,将左后肢跟腱中央束切断,改良Kessler法缝合,MSCs组使用MSCs附和PLGA电纺丝膜进行包裹,PLGA支架组使用单纯PLGA电纺丝膜进行包裹,对照组只做单纯缝合。分别在2、4、6周时间点各组处死5只兔取材,组织学和生物力学分析评估跟腱的愈合情况。
结果：PLGA电纺丝膜状支架纤维直径均匀,结构规整,孔隙率82.6±3.45%；五种消毒方式均可达到灭菌目的；其中钻6。照射和紫外线照射对支架结构的破坏最小最适合PLGA电纺丝膜状支架的消毒；使用梯度离心法分离获得的MSCs纯度高,生物学特性明显,体外增殖能力较强,可以与PLGA电纺丝膜状支架较好结合;MSCs组和PLGA电纺支架组组织学显示肌腱愈合好,与周围组织粘连较轻,生物力学优于对照组。
结论：静电纺丝技术是获得纳米级纤维的一种简便方法,使用梯度离心法分离获得的MSCs纯度较高,生物学特性明显,体外增殖能力较强,可以与PLGA电纺丝膜状支架结合;MSCs附和PLGA电纺支架能够促进兔跟腱损伤的愈合,减少外源性疤痕组织长入,减少肌腱与周围组织的粘连。
Objective: To study the rabbit bone marrow mesenchymal stem cells (MSCs) combindwith PLGA electrospinning frame for repairing the Achilles tendon injuries. Methods:Made PLGA electrospinning membrane frame by using electrostatic spinning technology.To study PLGA electrospinning membrane frame with morphology observation andmeasurement. Disinfected the PLGA frame by five ways including high temperature andhigh pressure method, low temperature plasma, Co60illuminate, ultraviolet ray and ethanol.Used bacteria cultures to detect sterilizing effects and observed structure damage of PLGAframe; To harvested mesenchymal stem cells(MSCs) from rabbit bone marrow by usingdensity gradient centrifugation in vitro; Cultured P3generation of MSCs with PLGAelectrospinning frame;45adult Newzealand white rabbits were randomly divided into3groups: MSCs+PLGA frame group, PLGA frame group and control group. Each group had15r abbits. The central bundle of achilles of left hind legs of rabbit was cut off and suturedwith improved Kessler method. MSCs group used MSCs with PLGA electrospinningframe. PLGA frame group simply used PLGA electrospinning frame. The control groupsimply sutured only.5rabbits of each group were respectively put to death to obtainmaterial in2nd,4th,6th week. Achilles tendon healing were evaluated with histology andbiomechnaics analysis. Results: Fiber diameter of PLGA elctrospinning membrane framewas uniform. The structure was neat. The porosity was82.6±3.45%. Five kinds ofdisinfection way may achieve sterilization purposes. Co60illuminate and ultravioletradiation suitable for PLGA electrospinning membrane frame because of minimumdamaged to it. MSCs harvested by using gradient centrifugation separation got high purity,obvious biological characteristics and showed strong ability in vitro proliferation combinedwith PLGA electrospinning membrane frame. The histological and biomechanical evaluation tendon healing of MSCs group and PLGA frame group were better than that ofthe control group, with the surrounding tissue adhesion was lighter. Conclusion:Electrostatic spinning technology is a simple method for nanoscale fibers. MSCs harvestedby using gradient centrifugation separation got high purity, obvious biologicalcharacteristics and showed strong ability in vitro proliferation combined with PLGAelectrospinning membrane frame; MSCs combind with PLGA electricity spinning framecan promote the healing of endogenous rabbit achilles tendon injuries, reduce theexogenous healing intervention, reduce adhesion of tendons with surrounding tissue.
方法：利用静电纺丝技术制备PLGA电纺丝膜状支架,对PLGA支架结构进行观察和测量,并使用高温高压、低温等离子、钻6。照射、紫外线照射、酒精浸泡等五种不同方式消毒,进行细菌培养检测消毒后效果并观察支架结构的破坏情况；采用骨髓穿刺法获取兔骨髓,并用密度梯度离心法分离并体外培养骨髓间充质干细胞(MSCs);将第3代骨髓间充质干细胞结合PLGA支架复合培养；将45只成年新西兰大白兔,随机分为3组,每组15只,将左后肢跟腱中央束切断,改良Kessler法缝合,MSCs组使用MSCs附和PLGA电纺丝膜进行包裹,PLGA支架组使用单纯PLGA电纺丝膜进行包裹,对照组只做单纯缝合。分别在2、4、6周时间点各组处死5只兔取材,组织学和生物力学分析评估跟腱的愈合情况。
结果：PLGA电纺丝膜状支架纤维直径均匀,结构规整,孔隙率82.6±3.45%；五种消毒方式均可达到灭菌目的；其中钻6。照射和紫外线照射对支架结构的破坏最小最适合PLGA电纺丝膜状支架的消毒；使用梯度离心法分离获得的MSCs纯度高,生物学特性明显,体外增殖能力较强,可以与PLGA电纺丝膜状支架较好结合;MSCs组和PLGA电纺支架组组织学显示肌腱愈合好,与周围组织粘连较轻,生物力学优于对照组。
结论：静电纺丝技术是获得纳米级纤维的一种简便方法,使用梯度离心法分离获得的MSCs纯度较高,生物学特性明显,体外增殖能力较强,可以与PLGA电纺丝膜状支架结合;MSCs附和PLGA电纺支架能够促进兔跟腱损伤的愈合,减少外源性疤痕组织长入,减少肌腱与周围组织的粘连。
Objective: To study the rabbit bone marrow mesenchymal stem cells (MSCs) combindwith PLGA electrospinning frame for repairing the Achilles tendon injuries. Methods:Made PLGA electrospinning membrane frame by using electrostatic spinning technology.To study PLGA electrospinning membrane frame with morphology observation andmeasurement. Disinfected the PLGA frame by five ways including high temperature andhigh pressure method, low temperature plasma, Co60illuminate, ultraviolet ray and ethanol.Used bacteria cultures to detect sterilizing effects and observed structure damage of PLGAframe; To harvested mesenchymal stem cells(MSCs) from rabbit bone marrow by usingdensity gradient centrifugation in vitro; Cultured P3generation of MSCs with PLGAelectrospinning frame;45adult Newzealand white rabbits were randomly divided into3groups: MSCs+PLGA frame group, PLGA frame group and control group. Each group had15r abbits. The central bundle of achilles of left hind legs of rabbit was cut off and suturedwith improved Kessler method. MSCs group used MSCs with PLGA electrospinningframe. PLGA frame group simply used PLGA electrospinning frame. The control groupsimply sutured only.5rabbits of each group were respectively put to death to obtainmaterial in2nd,4th,6th week. Achilles tendon healing were evaluated with histology andbiomechnaics analysis. Results: Fiber diameter of PLGA elctrospinning membrane framewas uniform. The structure was neat. The porosity was82.6±3.45%. Five kinds ofdisinfection way may achieve sterilization purposes. Co60illuminate and ultravioletradiation suitable for PLGA electrospinning membrane frame because of minimumdamaged to it. MSCs harvested by using gradient centrifugation separation got high purity,obvious biological characteristics and showed strong ability in vitro proliferation combinedwith PLGA electrospinning membrane frame. The histological and biomechanical evaluation tendon healing of MSCs group and PLGA frame group were better than that ofthe control group, with the surrounding tissue adhesion was lighter. Conclusion:Electrostatic spinning technology is a simple method for nanoscale fibers. MSCs harvestedby using gradient centrifugation separation got high purity, obvious biologicalcharacteristics and showed strong ability in vitro proliferation combined with PLGAelectrospinning membrane frame; MSCs combind with PLGA electricity spinning framecan promote the healing of endogenous rabbit achilles tendon injuries, reduce theexogenous healing intervention, reduce adhesion of tendons with surrounding tissue.
引文
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[1]曲彦隆,杨志明,解慧琪.组织工程化肌腱对T淋巴细胞亚群及其受体的影响[J].中国修复重建外科杂志,2001,15：113-117.
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[5]Dietmar W H. Scaffolds in tissue engineering bone and cartilage[J]. Biomaterials,2000,21(5):2529-2543.
[6]胡蕴玉.把握契机加快我国组织工程学的应用研究.中华骨科杂志2000,20(9)：517-519.
[7]王涵,张华.组织工程用多孔支架技术的开发现状.天津工业大学学报.2006,(25)6：20-24.
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[1]曲彦隆,杨志明,解慧琪.组织工程化肌腱对T淋巴细胞亚群及其受体的影响[J].中国修复重建外科杂志,2001,15：113-117.
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