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Cartilage Matrix Mimic Scaffolds Based on Poly(L-glutamic Acid) for Cartilage Tissue Engineering

Author: ZhangKunZuo
Tutor: YinJingBo
School: Shanghai University
Course: Materials Science
Keywords: poly(L-glutamic acid) chitosan matrix mimic adiposederived stem cells cell spheroid cell adhesion
CLC: R318.08
Type: PhD thesis
Year: 2013
Downloads: 32
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Abstract

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.

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