Preparation And Properties Of Bioactive Scaffolds For Cartilage Tissue Engineering

Naturally originated macromolecules were chosen as scaffolding materials to mimic the compositions of natural cartilage matrix.To enhance chondrogenesis, growth factors,proteins and specific groups were introduced into the scaffolds or synthetic polymer microspheres for promoting cell proliferation,differentiation and ECM secretion.A collagen gradient was fabricated on poly(L-lactide)(PLLA)surface to control cell adhesion spatially.Gelatin,chitosan and hyaluronan ternary complex in a weight ratio of 82.6%, 16.5%and 0.1%were firstly used as scaffolding materials to mimic the compositions of natural cartilage matrix for cartilage tissue engineering by a a freeze-drying method. To enhance chondrogenesis,heparin was covalently immobilized onto the scaffold by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide(EDAC),through which basic fibroblast growth factor(bFGF)was further incorporated by bioaffinity force.The heparinized scaffold showed stronger ability to resist the weight loss in phosphate buffered saline(PBS,pH 7.4)at 37℃,implying that a higher crosslinking degree was achieved by incorporation of the heparin.Rabbit auricular chondrocytes were seeded onto the ternary complex scaffold containing bFGF to assess cell response. Chondrocytes seeded in the Scaffold-heparin-bFGF indeed showed significant higher viability than that on the control scaffold.These results reveal that the ternary complex scaffolds,in particular the one containing bFGF,have the good ability to support cell proliferation and ECM secretion.Lactose and heparin modified chitosan films were prepared and their physical and biological properties were studied.The molecular structure of chitosan-g-lactose was confirmed by FTIR and ¹HNMR analysis.About 66%of the available amino groups of chitosan have reacted with lactose.The chitosan-g-lactose films have the highest swelling and weight loss ratios,which could be reduced by blending with heparin. The chitosan-g-lactose film showed stronger ability to induce chondrocyte attachment, proliferation,viability and GAG secretion than that of the chitosan film.After incorporated with heparin,its biofanctionality was further improved.By introducing chitosan-g-lactose into ternary complex scaffold,we found that this modified scaffold could better support chondrocyte proliferation and GAG secretion.For enhancing mechanical property of the ternary complex scaffold, poly(lactide-co-glycotide)(PLGA)was chosen as the material to fabricate synthetic/natural polymer composite scaffold.PLGA microspheres integrated ternary complex scaffolds were fabricated by a freeze drying technique and crosslinking with EDAC.Effects of the microspheres on porosity,density,compressive modulus, phosphate buffered saline uptake ratio and weight loss of the scaffolds were evaluated. Generally,a scaffold with higher PLGA content have lower porosity,weight loss and medium uptake ratios,but larger apparent density and compressive modulus and mechanical strength.The compressive moduli of the scaffold with 50%PLGA and 70%PLGA reached to 0.53MPa and 0.68MPa,respectively.In vitro chondrocyte culture in the 50%PLGA microspheres integrated scaffold found that the cells could normally proliferate and secrete extracellular matrix as that in the control ternary complex scaffold.Therefore,the composite scaffolds have better physical performance and preserved biocompatibility to support chondrocyte growth,thus have greater potential to be used for chondrogenesis.In the next step,PLGA based cell carriers with enhanced bioactivity for chondrocyte delivery were developed,which may be used as injectable scaffold for cartilage repair.PLGA/gelatin composite microspheres were prepared by an emulsion solvent evaporation technique.RGDS peptides were further immobilized under the catalyzation of EDAC.Characterization by confocal laser scanning microscopy and transmission electron microscopy revealed that the gelatin was entrapped in the PLGA/gelatin microspheres,with a manner of separated domains.Contents of the entrapped gelatin and immobilized RGDS peptide were quantified as 0.9mg/20mg and ².1μg/20mg microspheres by hydroproline analyses and bicinchoninic acid protein assay,respectively.Difference in morphology of PLGA,PLGA/gelatin and RGDS modified PLGA/gelatin(PLGA/gelatin-RGDS)microspheres was observed by scanning electron microscopy.The PLGA/gelatin and PLGA/gelatin-RGDS microspheres lost their weight rapidly in PBS,but slowly in DMEM/fetal bovine serum.Isolated chondrocytes were seeded onto the microspheres in vitro to assess their biological performance and applicability as cell carriers.Results show that PLGA/gelatin-RGDS microspheres have a better performance on supporting chondrocyte attachment,proliferation,viability and GAGs secretion than PLGA microspheres.Therefore,the PLGA/gelatin-RGDS microspheres are a kind of promising candidates applicable as injectable scaffolds for chondrogenesis.A collagen gradient was constructed to interrogate cell adhesion on a PLLA membrane surface.Utilizing microinfusion pump,a gradient of amino groups was generated on the PLLA surface by aminolysis and then immobilization of collagen by glutaraldehyde(GA)coupling.The -NH₂ and immobilized collagen density profiles on the PLLA membrane surfaces were quantitatively determined by ninhydrin and hydroproline(Hyp)analysis,respectively.The "S" shaped increase of-NH₂ density can be described by a logistic sigmoid function,which can predict the density and total content of-NH₂ as well.The atomic force microscopy and water contact angle studies revealed that morphology and wettability of the modified membranes changed relevantly as a function of position along the gradient surface.FITC labeled collagen (FITC-Col)were immobilized on the PLLA membrane surface to confirm the gradient profile by using fluorescence microscopy.To test cell response to the collagen gradient surface,chondrocytes were cultured on the collagen gradient membrane.Results depicted that this surface strongly control the biological response of chondrocytes.This ability to spatially control cellular adhesion in a continuous manner on a biocompatible substrate is an important prospect in designing tissue inductive biomaterials.