聚L-谷氨酸基软骨基质模拟支架的构建及其软骨组织工程应用
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摘要
创伤、关节炎等导致的软骨缺损是临床亟待解决的问题，软骨组织工程为自愈能力极为有限的软骨损伤提供了有效的修复策略。软骨组织工程中作为临时基质的支架，应协调诱导因子，促进干细胞的软骨向分化，维持软骨细胞表型，或通过对细胞所处环境的模拟，刺激软骨组织的再生。本论文从模拟软骨基质组分和生理环境出发，构建组织工程支架，旨在促进干细胞软骨向分化以及软骨组织再生。
通过聚L-谷氨酸(PLGA)和壳聚糖(CS)间静电作用，结合相分离技术构建细胞粘附型静电复合多孔支架，PLGA和CS分别模拟软骨基质中的蛋白质和多糖。研究冷冻温度和固含量对支架孔结构影响，发现冷冻温度越低支架孔径越小；固含量越高，孔径越小，且内部贯通性变差。考察固含量对支架溶胀性能，力学性能和降解性能影响，发现固含量增加，支架溶胀度降低，压缩模量和储能模量升高，降解速率变慢。控制冷冻温度为-20oC和固含量为2%时，可获得孔径为150-200μm，内部贯通，孔隙率90%以上，综合性能优异的类海绵支架，支架溶胀率达760±45%(mass%)，含溶菌酶的PBS中12w可降解50%，压缩测试和动态力学测试显示支架具有良好弹性。
体外扩增兔脂肪干细胞(ASCs)并接种到PLGA/CS静电复合支架，通过扫描电镜观察发现，ASCs可粘附至支架孔壁并伸展成长梭形，同时分泌大量细胞外基质。激光共聚焦和DNA定量检测显示，细胞在支架中具有良好的增殖性能。以TGF-β1和IGF-1对细胞-支架复合物进行体外软骨向诱导，通过免疫酶联吸附测定法在蛋白水平定量检测软骨特异性基质糖胺多糖(GAG)和二型胶原(COLⅡ)含量，并进一步以实时定量PCR技术在基因水平定量分析软骨特异性基因Sox9，aggrecan和COL Ⅱ的表达，结果表明ASCs在支架内可以合成软骨特异性基质并表达相应基因，证实PLGA/CS静电复合支架支持ASCs软骨向分化。
体外诱导2w后，将细胞-支架复合物作为实验组，植入自体兔膝关节非负重区全层软骨缺损处(缺损直径4mm，深至软骨下骨)，进行体内软骨组织重建。对照组植入空白支架。对体内样品进行大体观察和组织学检测发现，6w后，实验组缺损处填充新生组织，其色泽、质地与正常软骨相近，但厚度不均一，且存在明显连接界限。12w后，新生组织与周围正常软骨组织和软骨下骨整合性良好，组织结构更加成熟。对新生组织基质中的GAG和COL Ⅱ进行定量检测显示，随体内修复时间延长，软骨特异性基质沉积增多，12w后样品中GAG和COL Ⅱ含量均接近正常软骨，分别为正常软骨的84%和70%；通过宏观压缩测试和纳米压痕测试检测新生组织的力学性能，发现12w后新生组织压缩模量为正常软骨的78%，弹性模量为正常软骨的83%。对照组软骨缺损未修复。实验组和对照组缺损处均未发现材料残留和炎症反应。
为进一步模拟软骨细胞生理环境，以EDC和NHS活化PLGA羧基，与CS氨基共价交联形成酰胺键，构建可模拟蛋白聚糖精细结构的水凝胶，通过冷冻相分离技术得到多孔支架。研究冷冻温度对支架孔径的影响，发现冷冻温度越低，支架孔径越小。对比研究固含量为2%和3%支架的孔结构、溶胀度、力学性能和降解性能，与2%的支架相比，固含量3%的支架压缩模量和储能模量较高，但内部贯通性差，溶胀度和降解速率较低。控制冷冻温度为-80oC，固含量为2%，得到孔径为200-300μm，内部贯通，孔隙率90%以上，平衡溶胀度高达1652±70%(mass%)的非细胞粘附型化学交联支架，高溶胀度支架为细胞提供近似天然的生理环境。同时，该支架具有良好的弹性和可降解性，含溶菌酶的PBS中12w可降解67%。
通过扫描电镜观测发现，ASCs接种于化学交联支架4h后未在孔壁表面粘附，而随培养时间延长，细胞迁移聚集成直径约50-100μm细胞微团，并分泌大量细胞外基质，包裹微团细胞。以Dio标记ASCs，通过激光共聚焦显微镜监测细胞在支架孔内成团过程，结果显示接种24h后细胞聚集成微团。Hoechst332558DNA定量和活-死细胞染色分析显示，微团中细胞具有良好活性。细胞在支架孔内聚集成大量细胞微团，构建出细胞微团-支架复合物，以TGF-β1、IGF-1对复合物进行软骨向诱导，通过免疫酶联吸附测定法在蛋白水平检测GAG和COL Ⅱ含量，并以实时定量PCR技术在基因水平监测ASCs微团的软骨向分化，结果证实，细胞微团-支架复合物在成软骨诱导因子作用下具有更强的软骨向分化能力。
体外诱导2w后，将细胞微团-支架复合物作为实验组，进行体内的软骨组织重建，对照组植入空白支架。组织学检测显示6w后新生组织内细胞已具有明显的软骨陷窝结构，形态上更接近于透明软骨。12w和18w后，新生组织结构更加完整，组织内细胞数量、排列均与正常软骨组织相似。对体内样品进行生化成分检测，发现随修复时间延长，软骨特异性基质沉积增多，18w后GAG和COL Ⅱ含量更加接近正常软骨，分别达到正常软骨的94%和91%。宏观压缩测试结果显示，12w后新生软骨组织的压缩模量达到正常软骨的87%，18w后达到正常软骨的92%。对照组缺损处6w时填充纤维状软骨组织，但12w和18w后塌陷，缺损未修复。实验组和对照组缺损处均未发现材料残留和炎症反应。体内12w结果证实，与静电复合支架实验组相比，PLGA/CS化学交联支架修复的软骨组织中GAG和COL Ⅱ含量分别高7%和18%；新生组织的压缩模量高9%。因此PLGA/CS化学交联支架较静电复合支架更利于ASCs的软骨向分化和软骨组织再生。
为解释PLGA/CS化学交联支架内ASCs成团现象，本文考察了PLGA/CS化学交联二维膜表面电荷与亲水性对ASCs粘附行为影响。通过设定羧基与氨基比例为3:1、1:1、1:3和1:5，改变膜的表面电荷；通过冷冻干燥和热干燥方法改变膜的亲水性。分别检测膜表面的zeta电势、接触角和细胞粘附性。结果显示，冷冻干燥膜和热干燥膜的表面电荷量相近，但亲水性差别显著。冷冻干燥膜的亲水性明显高于热干燥膜，且均不支持ASCs粘附(1:5膜除外)。动态接触角测试显示不支持ASCs粘附的冷冻干燥膜与水相接触后极易被润湿，接触角迅速降低，推测超亲水表面与水分子结合后迅速形成水化壳层，阻止细胞粘附，使细胞相互吸引聚集成微团。1:5冷冻干燥膜因其适宜的亲水性支持ASCs粘附。热干燥均支持ASCs粘附，且随PLGA组分增多，膜亲水性增加，导致粘附细胞数目增多。因此亲水性主导PLGA/CS化学交联体系细胞粘附性，表面电荷未对细胞粘附产生规律性影响。
Cartilage tissue engineering was designed to achieve the regeneration ofcartilage tissue with limited self-repairing capability. Chondrocytes and stem cellswere employed in cartilage tissue engineering. Chondrogenic differentiation of thesecells should be explored. Scaffolds should be able to coordinate the inductive factorsto promote the chondrogenic differentiation of stem cells, and maintain thephenotype of chondrocytes. Therefore, in this paper, simulation components ofscaffold were selected to construct matrix mimic scaffold in order to promote celldifferentiation and cartilage tissue formation.
As a synthetic polypeptide, water soluble poly(L-glutamic acid)(PLGA)/wasdesigned to fabricate scaffold for cartilage tissue engineering. Chitosan (CS) wasemployed as the physical cross-linking composition to realize the construction ofscaffold. The pore structure, swelling ratio, degradation and mechanical performanceof PLGA/CS electrostatic complex were affected by freezing temperature and solidcontent. The PLGA/CS scaffold with2%solid content, freezed at-20oC was sign asan excellent water imbibition material, swelling ratio of which reached760±45%(mass%), showing certain biomechanics and proper biodegradation.
Autologous ASCs were expanded and seeded on the PLGA/CS scaffold. SEMimages showed the adherence of ASCs on scaffold with deposition of ECM. CLSMimages and Hoechst333258DNA quantitative analysis showed proliferation of ASCsin scaffold. ASCs/scaffold constructs were then subjected to chondrogenic inductionin vitro for2weeks. The results of GAG and COL Ⅱ quantitative analysis showed thatPLGA/CS scaffold could support chondrogenic differentiation of ASCs.
After that, the ASCs/scaffold constructs were transplanted to repair full thicknessarticular cartilage defects (4mm in diameter, deep to subchondral bone) created inrabbit femur trochlea. It was found that articular defect was covered with newly formed cartilage at6weeks post-implantation. After12weeks, the regeneratedcartilage integrated well with surrounding native cartilage and subchondral bone.Similar accumulation of GAG and type Ⅱ collagen was achieved in engineeredcartilage at12weeks post-implantation as in the native one through the toluidine blueand immunohistochemical staining, respectively, and was further confirmed by thequantitative analysis of ECM deposition. Biomechanical test showed analogousbiomechanical behavior of engineered cartilage as the native one. Results thusdemonstrated the potentiality of using this polyelectrolyte PLGA/CS as scaffold forcartilage tissue engineering.
While in cartilage, the GAGs exist mostly as hydrodynamically largeaggregating proteoglycans, which show the combination of GAG and peptide chainsthrough covalent bond. Thus, PLGA linked with chitosan through amide bonds wasdesigned to mimic the component of proteoglycan. After lyophilization, a3-Dporous scaffold with pore size of200-300μm was obtained. The pore structure,swelling ratio, degradation and mechanical performance of PLGA/CS electrostaticcomplex were controlled by freezing temperature and solid content. The swellingratio of scaffold with2%solid content and-80oC freezing temperature was1652±70%(mass%), which also showed elasticity.
High swelling ratio leads to non-adhesion of ASCs at4h post-seeding. Whilethe cell-cell interaction inducing the formation of cell spheroids with diameter of50-100μm after24h post-seeding. Live-dead assay showed favorable survival abilityin spheroid. Compared to the traditional two dimensional monolayer culture, SEMimages showed that cells in three-dimensional spheroids could depose moreextracellular matrix. Detection of cartilage expression on protein level and gene levelshowed that ASCs in spheroids displayed better differentiation capacities uponinduction, and could be immediately differentiate into chondroblasts.
The ASCs spheroids/scaffold constructs were transplanted to repair full thickness articular cartilage defects (4mm in diameter, deep to subchondral bone)created in rabbit femur trochlea.6w,12w and18w samples showed that ASCs inspheroids had sufficient chondrogenic differentiation in vivo, leading to regeneratethe hyaline-like cartilage tissue with lacuna structure. The in vitro and in vivo resultsdemonstrated that PLGA/CS chemical cross-linked scaffold which mimics theproteoglycan structure can induce the formation of ASCs spheroids, leading to betterchondrogenic differentiation and cartilage regeneration. The results in vitro and invivo confirmed that PLGA/CS chemical crosslinked scaffold was more efficient thanPLGA/CS polyelectrolyte complex scaffold in chondrogenic differentiation of ASCs.The expression of GAG and COL Ⅱ in vivo at12w post-implantation were7%and18%higher than those in polyelectrolyte complex scaffold group, respectively.Compression modulus of new born was9%higher than that in polyelectrolytecomplex scaffold group.
The reason of cell adhesion and non-adhesion in different PLGA/CS chemicalcross-linked scaffold was explored by investigating the influence of hydrophilicityand surface charge on ASCs adhesion. At first, the surface charge was adjusted bychanging the content of carboxyl group and amino group. The hydrophilicity wasadjusted by freeze-drying method and thermal-drying method. The result showedfilms made by freeze-drying owned higher hydrophilicity than films made bythermal-drying method (except1:5films) could not support ASCs adhesion. Thesefilms could be wetted quickly after contact with water, with a rapidly decreasedcontact angle. The water interacted with film to form a hydration shell on the film,preventing cell adhesion. Besides, with the increase of contact angle, the films withthe contact angle greater than80o, the number of adhered cells was decreased.Surface charge is not the regular effect on cell adhesion.
通过聚L-谷氨酸(PLGA)和壳聚糖(CS)间静电作用，结合相分离技术构建细胞粘附型静电复合多孔支架，PLGA和CS分别模拟软骨基质中的蛋白质和多糖。研究冷冻温度和固含量对支架孔结构影响，发现冷冻温度越低支架孔径越小；固含量越高，孔径越小，且内部贯通性变差。考察固含量对支架溶胀性能，力学性能和降解性能影响，发现固含量增加，支架溶胀度降低，压缩模量和储能模量升高，降解速率变慢。控制冷冻温度为-20oC和固含量为2%时，可获得孔径为150-200μm，内部贯通，孔隙率90%以上，综合性能优异的类海绵支架，支架溶胀率达760±45%(mass%)，含溶菌酶的PBS中12w可降解50%，压缩测试和动态力学测试显示支架具有良好弹性。
体外扩增兔脂肪干细胞(ASCs)并接种到PLGA/CS静电复合支架，通过扫描电镜观察发现，ASCs可粘附至支架孔壁并伸展成长梭形，同时分泌大量细胞外基质。激光共聚焦和DNA定量检测显示，细胞在支架中具有良好的增殖性能。以TGF-β1和IGF-1对细胞-支架复合物进行体外软骨向诱导，通过免疫酶联吸附测定法在蛋白水平定量检测软骨特异性基质糖胺多糖(GAG)和二型胶原(COLⅡ)含量，并进一步以实时定量PCR技术在基因水平定量分析软骨特异性基因Sox9，aggrecan和COL Ⅱ的表达，结果表明ASCs在支架内可以合成软骨特异性基质并表达相应基因，证实PLGA/CS静电复合支架支持ASCs软骨向分化。
体外诱导2w后，将细胞-支架复合物作为实验组，植入自体兔膝关节非负重区全层软骨缺损处(缺损直径4mm，深至软骨下骨)，进行体内软骨组织重建。对照组植入空白支架。对体内样品进行大体观察和组织学检测发现，6w后，实验组缺损处填充新生组织，其色泽、质地与正常软骨相近，但厚度不均一，且存在明显连接界限。12w后，新生组织与周围正常软骨组织和软骨下骨整合性良好，组织结构更加成熟。对新生组织基质中的GAG和COL Ⅱ进行定量检测显示，随体内修复时间延长，软骨特异性基质沉积增多，12w后样品中GAG和COL Ⅱ含量均接近正常软骨，分别为正常软骨的84%和70%；通过宏观压缩测试和纳米压痕测试检测新生组织的力学性能，发现12w后新生组织压缩模量为正常软骨的78%，弹性模量为正常软骨的83%。对照组软骨缺损未修复。实验组和对照组缺损处均未发现材料残留和炎症反应。
为进一步模拟软骨细胞生理环境，以EDC和NHS活化PLGA羧基，与CS氨基共价交联形成酰胺键，构建可模拟蛋白聚糖精细结构的水凝胶，通过冷冻相分离技术得到多孔支架。研究冷冻温度对支架孔径的影响，发现冷冻温度越低，支架孔径越小。对比研究固含量为2%和3%支架的孔结构、溶胀度、力学性能和降解性能，与2%的支架相比，固含量3%的支架压缩模量和储能模量较高，但内部贯通性差，溶胀度和降解速率较低。控制冷冻温度为-80oC，固含量为2%，得到孔径为200-300μm，内部贯通，孔隙率90%以上，平衡溶胀度高达1652±70%(mass%)的非细胞粘附型化学交联支架，高溶胀度支架为细胞提供近似天然的生理环境。同时，该支架具有良好的弹性和可降解性，含溶菌酶的PBS中12w可降解67%。
通过扫描电镜观测发现，ASCs接种于化学交联支架4h后未在孔壁表面粘附，而随培养时间延长，细胞迁移聚集成直径约50-100μm细胞微团，并分泌大量细胞外基质，包裹微团细胞。以Dio标记ASCs，通过激光共聚焦显微镜监测细胞在支架孔内成团过程，结果显示接种24h后细胞聚集成微团。Hoechst332558DNA定量和活-死细胞染色分析显示，微团中细胞具有良好活性。细胞在支架孔内聚集成大量细胞微团，构建出细胞微团-支架复合物，以TGF-β1、IGF-1对复合物进行软骨向诱导，通过免疫酶联吸附测定法在蛋白水平检测GAG和COL Ⅱ含量，并以实时定量PCR技术在基因水平监测ASCs微团的软骨向分化，结果证实，细胞微团-支架复合物在成软骨诱导因子作用下具有更强的软骨向分化能力。
体外诱导2w后，将细胞微团-支架复合物作为实验组，进行体内的软骨组织重建，对照组植入空白支架。组织学检测显示6w后新生组织内细胞已具有明显的软骨陷窝结构，形态上更接近于透明软骨。12w和18w后，新生组织结构更加完整，组织内细胞数量、排列均与正常软骨组织相似。对体内样品进行生化成分检测，发现随修复时间延长，软骨特异性基质沉积增多，18w后GAG和COL Ⅱ含量更加接近正常软骨，分别达到正常软骨的94%和91%。宏观压缩测试结果显示，12w后新生软骨组织的压缩模量达到正常软骨的87%，18w后达到正常软骨的92%。对照组缺损处6w时填充纤维状软骨组织，但12w和18w后塌陷，缺损未修复。实验组和对照组缺损处均未发现材料残留和炎症反应。体内12w结果证实，与静电复合支架实验组相比，PLGA/CS化学交联支架修复的软骨组织中GAG和COL Ⅱ含量分别高7%和18%；新生组织的压缩模量高9%。因此PLGA/CS化学交联支架较静电复合支架更利于ASCs的软骨向分化和软骨组织再生。
为解释PLGA/CS化学交联支架内ASCs成团现象，本文考察了PLGA/CS化学交联二维膜表面电荷与亲水性对ASCs粘附行为影响。通过设定羧基与氨基比例为3:1、1:1、1:3和1:5，改变膜的表面电荷；通过冷冻干燥和热干燥方法改变膜的亲水性。分别检测膜表面的zeta电势、接触角和细胞粘附性。结果显示，冷冻干燥膜和热干燥膜的表面电荷量相近，但亲水性差别显著。冷冻干燥膜的亲水性明显高于热干燥膜，且均不支持ASCs粘附(1:5膜除外)。动态接触角测试显示不支持ASCs粘附的冷冻干燥膜与水相接触后极易被润湿，接触角迅速降低，推测超亲水表面与水分子结合后迅速形成水化壳层，阻止细胞粘附，使细胞相互吸引聚集成微团。1:5冷冻干燥膜因其适宜的亲水性支持ASCs粘附。热干燥均支持ASCs粘附，且随PLGA组分增多，膜亲水性增加，导致粘附细胞数目增多。因此亲水性主导PLGA/CS化学交联体系细胞粘附性，表面电荷未对细胞粘附产生规律性影响。
Cartilage tissue engineering was designed to achieve the regeneration ofcartilage tissue with limited self-repairing capability. Chondrocytes and stem cellswere employed in cartilage tissue engineering. Chondrogenic differentiation of thesecells should be explored. Scaffolds should be able to coordinate the inductive factorsto promote the chondrogenic differentiation of stem cells, and maintain thephenotype of chondrocytes. Therefore, in this paper, simulation components ofscaffold were selected to construct matrix mimic scaffold in order to promote celldifferentiation and cartilage tissue formation.
As a synthetic polypeptide, water soluble poly(L-glutamic acid)(PLGA)/wasdesigned to fabricate scaffold for cartilage tissue engineering. Chitosan (CS) wasemployed as the physical cross-linking composition to realize the construction ofscaffold. The pore structure, swelling ratio, degradation and mechanical performanceof PLGA/CS electrostatic complex were affected by freezing temperature and solidcontent. The PLGA/CS scaffold with2%solid content, freezed at-20oC was sign asan excellent water imbibition material, swelling ratio of which reached760±45%(mass%), showing certain biomechanics and proper biodegradation.
Autologous ASCs were expanded and seeded on the PLGA/CS scaffold. SEMimages showed the adherence of ASCs on scaffold with deposition of ECM. CLSMimages and Hoechst333258DNA quantitative analysis showed proliferation of ASCsin scaffold. ASCs/scaffold constructs were then subjected to chondrogenic inductionin vitro for2weeks. The results of GAG and COL Ⅱ quantitative analysis showed thatPLGA/CS scaffold could support chondrogenic differentiation of ASCs.
After that, the ASCs/scaffold constructs were transplanted to repair full thicknessarticular cartilage defects (4mm in diameter, deep to subchondral bone) created inrabbit femur trochlea. It was found that articular defect was covered with newly formed cartilage at6weeks post-implantation. After12weeks, the regeneratedcartilage integrated well with surrounding native cartilage and subchondral bone.Similar accumulation of GAG and type Ⅱ collagen was achieved in engineeredcartilage at12weeks post-implantation as in the native one through the toluidine blueand immunohistochemical staining, respectively, and was further confirmed by thequantitative analysis of ECM deposition. Biomechanical test showed analogousbiomechanical behavior of engineered cartilage as the native one. Results thusdemonstrated the potentiality of using this polyelectrolyte PLGA/CS as scaffold forcartilage tissue engineering.
While in cartilage, the GAGs exist mostly as hydrodynamically largeaggregating proteoglycans, which show the combination of GAG and peptide chainsthrough covalent bond. Thus, PLGA linked with chitosan through amide bonds wasdesigned to mimic the component of proteoglycan. After lyophilization, a3-Dporous scaffold with pore size of200-300μm was obtained. The pore structure,swelling ratio, degradation and mechanical performance of PLGA/CS electrostaticcomplex were controlled by freezing temperature and solid content. The swellingratio of scaffold with2%solid content and-80oC freezing temperature was1652±70%(mass%), which also showed elasticity.
High swelling ratio leads to non-adhesion of ASCs at4h post-seeding. Whilethe cell-cell interaction inducing the formation of cell spheroids with diameter of50-100μm after24h post-seeding. Live-dead assay showed favorable survival abilityin spheroid. Compared to the traditional two dimensional monolayer culture, SEMimages showed that cells in three-dimensional spheroids could depose moreextracellular matrix. Detection of cartilage expression on protein level and gene levelshowed that ASCs in spheroids displayed better differentiation capacities uponinduction, and could be immediately differentiate into chondroblasts.
The ASCs spheroids/scaffold constructs were transplanted to repair full thickness articular cartilage defects (4mm in diameter, deep to subchondral bone)created in rabbit femur trochlea.6w,12w and18w samples showed that ASCs inspheroids had sufficient chondrogenic differentiation in vivo, leading to regeneratethe hyaline-like cartilage tissue with lacuna structure. The in vitro and in vivo resultsdemonstrated that PLGA/CS chemical cross-linked scaffold which mimics theproteoglycan structure can induce the formation of ASCs spheroids, leading to betterchondrogenic differentiation and cartilage regeneration. The results in vitro and invivo confirmed that PLGA/CS chemical crosslinked scaffold was more efficient thanPLGA/CS polyelectrolyte complex scaffold in chondrogenic differentiation of ASCs.The expression of GAG and COL Ⅱ in vivo at12w post-implantation were7%and18%higher than those in polyelectrolyte complex scaffold group, respectively.Compression modulus of new born was9%higher than that in polyelectrolytecomplex scaffold group.
The reason of cell adhesion and non-adhesion in different PLGA/CS chemicalcross-linked scaffold was explored by investigating the influence of hydrophilicityand surface charge on ASCs adhesion. At first, the surface charge was adjusted bychanging the content of carboxyl group and amino group. The hydrophilicity wasadjusted by freeze-drying method and thermal-drying method. The result showedfilms made by freeze-drying owned higher hydrophilicity than films made bythermal-drying method (except1:5films) could not support ASCs adhesion. Thesefilms could be wetted quickly after contact with water, with a rapidly decreasedcontact angle. The water interacted with film to form a hydration shell on the film,preventing cell adhesion. Besides, with the increase of contact angle, the films withthe contact angle greater than80o, the number of adhered cells was decreased.Surface charge is not the regular effect on cell adhesion.
引文
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