Objective： In our previous work, we prepared a type of chitosan hydrogel with excellent biocompatibility. In this study, tissue-engineered cartilage constructed with this chitosan hydrogel and costal chondrocytes was used to repair the articular cartilage defects. Methods： Chitosan hydrogels were prepared with a crosslinker formed by combining 1,6-diisocyanatohexane and polyethylene glycol. Chitosan hydrogel scaffold was seeded with rabbit chondrocytes that had been cultured for one week in vitro to form the preliminary tissue-engineered cartilage. This preliminary tissue-engineered cartilage was then transplanted into the defective rabbit articular cartilage. There were three treatment groups： the experimental group received preliminary tissue-engineered cartilage; the blank group received pure chitosan hydrogels; and, the control group had received no implantation. The knee joints were harvested at predetermined time. The repaired cartilage was analyzed through gross morphology, histologically and immunohistochemically. The repairs were scored according to the international cartilage repair society （ICRS） standard. Results： The gross morphology results suggested that the defects were repaired completely in the experimental group after twelve weeks. The regenerated tissue connected closely with subchondral bone and the boundary with normal tissue was fuzzy. The cartilage lacuna in the regenerated tissue was similar to normal cartilage lacuna. The results of ICRS gross and histological grading showed that there were significant differences among the three groups （P〈0.05）. Conclusions： Chondrocytes implanted in the scaffold can adhere, proliferate, and secrete extracellular matrix. The novel tissue-engineered cartilage constructed in our research can completely repair the structure of damaged articular cartilage.
Ming ZHAOZhu CHENKang LIUYu-qing WANXu-dong LIXu-wei LUOYi-guang BAIZe-long YANGGang FENG
Objective： To construct a recombinant adenovirus vector-carrying human growth and differentiation factor-5 （GDF-5） gene, investigate the biological effects of adenovirus-mediated GDF-5 （Ad-GDF-5） on extracellular matrix （ECM） expression in human degenerative disc nucleus pulposus （NP） cells, and explore a candidate gene therapy method for intervertebral disc degeneration （IDD）. Methods： Human NP cells of a degenerative disc were isolated, cultured, and infected with Ad-GDF-5 using the AdEasy-1 adenovirus vector system. On Days 3, 7, 14, and 21, the contents of the sulfated glycosaminoglycan （sGAG）, deoxyribonucleic acid （DNA） and hydroxyproline （Hyp）, synthesis of proteoglycan and collagen II, gene expression of collagen II and aggrecan, and NP cell proliferation were assessed. Results： The adenovirus was an effective vehicle for gene delivery with prolonged expression of GDF-5. Biochemical analysis revealed increased sGAG and Hyp contents in human NP cells infected by Ad-GDF-5 whereas there was no conspicuous change in basal medium （BM） or Ad-green fluorescent protein （GFP） groups. Only cells in the Ad-GDF-5 group promoted the production of ECM, as demonstrated by the secretion of proteoglycan and up-regulation of collagen II and aggrecan at both protein and mRNA levels. The NP cell proliferation was significantly promoted. Conclusions： The data suggest that Ad-GDF-5 gene therapy is a potential treatment for IDD, which restores the functions of degenerative intervertebral disc through enhancing the ECM production of human NP ceils.
Objective:To construct a novel non-viral vector loaded with growth and differentiation factor-5(GDF-5) plasmid using chitosan,hyaluronic acid,and chondroitin sulfate for osteoarthritis (OA)gene therapy.Methods: Nano-microspheres (NMPs)were prepared by mixing chitosan,hyaluronic acid,and chondreitin sulfate.GDF-5 plasmid was encapsulated in the NMPs through electrostatic adsorption.The basic characteristics of the NMPs were observed,and then they were co-cultured with chondrocytes to observe their effects on extracellular matrix (ECM) protein expression.Finally,NMPs loaded with GDF-5were inje.cted into the articular cavities of rabbits to observe their therapeutic effects on OA in vivo.Results:NMPs exhibited good physicochemical properties and low cytotoxicity.Their average diameter was (0.61±0.20)μm,and encapsulation efficiency was (38.19±0.36)%.According to Cell Counting Kit-8(CCK-8)assay,relative cell viability was 75%-99%when the total weight of NMPs was less than 560μg. Transfection efficiency was (62.0±2.1)% in a liposome group,and (60.0±1.8)% in the NMP group.There was no sig- nificant difference between the two groups (P>0.05).Immunohistochemical staining results suggested that NMPs can successfully transfect chondrocytes and stimulate ECM protein expression in vitro.Compared with the control groups, the NMP group significantly promoted the expression of chondrocyte ECM in vivo (P<0.05),as shown by analysis of the biochemical composition of chondrocyte ECM.When NMPs were injected into OA model rabbits,the expression of ECM proteins in chondrocytes was significantly promoted and the progression of OA was slowed down.Conclusions: Based on these data,we think that these NMPs with excellent physicochemical and biological properties could be promising non-viral vectors for OA gene therapy.
