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Constructing Tissue-engineering Cartilage With TGF-β1 Gene Modified Mesenchymal Stem Cells Loaded In Chitosan

Posted on:2006-09-26Degree:DoctorType:Dissertation
Country:ChinaCandidate:J Z HuoFull Text:PDF
GTID:1104360155960444Subject:Bone surgery
Abstract/Summary:PDF Full Text Request
Articular cartilages are avascular hyaline cartilage providing absorbing shock and decreasing abrasion, being composed of a unique type of cell, i.e. the chondroctyes, embedded within a dense extracellular matrix. Articular cartilage has a limited capacity for self-regeneration after injury and degenerative arthritis. Instrinsic biological features of the tissue itself are the limited factors. While many repair techniques have been proposed over the past four decades, none have successfully regenerated long-lasting hyaline cartilage tissue to replace damaged cartilage[1]. Tissue engineering approach provides alternative route to repair cartilage defect. In 1994, Brittberg et al[1] stated that they had successfully repaired full-thickness defect of the articular cartilage with autologous articular chondrocytes .which had been isolated and expanded by a monolayer culture in vitro and then seeded into the defect. However, the findings of many laboratories have confirmed that the chondrocytes grown as a monolayer dedifferentiate with passaging and loose both the chondrogenic phenotype and their biochemical characteristics[2,3]. Then, the sources of autologous chondrocytes are limited, because there are not redundant donor sites on the surfaces of the articular cartilages. Differentiated chondrocytes can be obtained also from non-articular, hyaline cartilage tissues such as nasal or rib cartilage. However, additional work will be necessary to assess whether cells of non-articular origin can be successfully used for articular cartilage repair. Moreover, the proliferative potential and the number of cell division of the articular chondrocytes decrease in vitro with the donor aging[4,5]. Immunological rejection induced by allogenic cartilage transplantation has not been perfectly solved. These factors limit theclinical application of chondrocytes transplantation for repairing articular cartilage defects. Although artificial prosthesis is the main and effective choice, high cost and complication could not be neglected.With cell biology developing, in particular within the field of the stem/progenitor cells, tissue engineering is progressing rapidly. In most cases, differentiated cells released from adult tissues exhibit a very limited proliferation capacity. This poses serious limitations to their expansion in culture and their use for in vitro reconstruction of engineered tissues to be transplanted in patients[4]. A stem cell is a cell from the embryo, fetus, or adult that, under certain conditions, can reproduce for long periods. It can also give rise to specialized cells of body tissues and organs. Although embryonic stem cells (ES) derived from the preimplantation embryo have higher proliferative and pluripotent differentiation capacity, the use of ES cells poses ethical problems, immunological rejection and so ont6]. The adult stem cells are generally well accepted by the society. Mesenchymal stem cells (MSCs) are undifferentiated cells present in a differentiated tissue, which renew themselves, and can be stimulated to differentiate into bone, cartilage, adipose, muscle, marrow stromal, a variety of connective tissues, new stem cells^'89101 and, under certain conditions, also into cell types derived from different blastodermic cells[11|12]; The harvest of a limited bone marrow sample is an easy and relatively safe procedure, without immunological rejection and ethic problems. Large numbers of MSCs can be obtained in culture, making it possible to engineer transplantable constructs composed of these cells in appropriate scaffolds stimulated by special cell growth factors . As progenitor cell of many tissues, the properties of MSCs are deeply influenced by the micro environmental conditions. MSCs can in some circumstances contribute to cell types very different from those in their tissue of origin[13Io Stimulated by different growth factors in the microenvironment, MSCs can differentiate into different tissue cells. To obtain a large number of chondroprogenitors, the effects of several growth factors on proliferation and differentiation of MSCs have been investigated. The transforming growth factor-(31 (TGF-(31) not only greatly promote MSCs differentiation along the chondrogenic phenotype, but also increases the rate of cell proliferation and maintains the ability of cells toredifferentiate upon transfer into a three-dimensional environment1141.As discussed in the previous paragraphs, MSCs having higher proliferative and pluripotent differentiation capacity are suitable to be seed cells for tissue engineering cartilage. As the same reason, it is more interesting to modify the intrinsic biological potential of MSCs than those of differentiated cells by gene transfer before expanded in vitro or transplanted into the tissue defect. Being the one member of transforming growth factor (TGF) family, TGF-J31 is strong cartilage-inducing factor (CIF) and contributes to maintain the cells self-renewal capacity1141. Therefore, it is of great value to construct tissue engineering cartilage to repair articular cartilage defect with the new seed cells MSCs modified by TGF-(31 gene, which induced by autocrined TGF-(31 can differentiate into chondrocytes, and maintain higher proliferative and pluripotent differentiation capacity.Tissue engineering of articular cartilage involves the isolation of seed cells and the choice of a biocompatible matrix, or scaffold, and the cultivation of the cells seeded into the scaffold. Engineered cartilage tissues are avascular when implanted and transplanted cells are exposed to an initial phase of hypoxia and insufficient nutrition until blood vessels invade the engineered construct. Thus it would be wiser to implant stem/progenitor cells with scaffolds specifically designed to provide a valid space in that engineered cells can interchange nutrition and metabolic products, and a model for engineered cells to regenerate an articular cartilage in the defect site, and to promote blood vessels to invade the engineered construct. From the point of tissue engineering, cartilage tissues are constituted of chondrocytes and natural scaffold supporting and nourishing the chondrocytes. Thus the ideal scaffold should be the one that most closely mimics the naturally occurring environment in the articular cartilage matrix, and be un-immunogenic, innocuity, biocompatible, degradable and easy be processed. Chitosan is a semi-crystalline polymer constituted of glucosamine and A/-acetylglucosamine, structurally, sharing some characteristics with glycosaminoglycan (GAG) in articular-specific extracellular matrix (ECM), and have been extensively being scaffold for repairing the articular cartilage defect with tissue-engineeringcartilage[15,16,17,18]The final purpose of constructing tissue-engineering cartilages is to repair the articular cartilage defects and to restore the surface and function of the joints. It is an effective method to implant the seed cells of the tissue-engineering cartilage with scaffold into the articular cartilage defect in vivo. As we knowledge, there are no reports on implanting the MSCs transferred by TGF-31 gene with chitosan into the articular cartilage defects. The aim of the present study was to assess the possibility of new suitable culturing system of tissue-engineering cartilage. In order to establish experiment basis for future clinical therapy, we cultured MSCs modified by TGF-p1 gene with chitosan in vitro, then implanted into the cartilage defects.Part I observation of chondrogenesis among passaged mesenchymaI stem ceI IsObjective To investigate different methods of isolation and culture how to have impact on the purity of MSCs and to observe the chondrogenesis among passaged MSCs.Method mesenchymal stem cells were isolated from bone marrow harvested from 14 New Zealand white rabbits divided into two groups at random and cultured by density gradient centrifugation method combinated with differential attachment technique (group A) or whole bone marrow method (group B) , respectively. The morphological changes, growth kinetics using MTT, surface antigen CD71 expression using immunohistochemical method and flow cytometry and osteogenic differentiation were investigated in group A and B. After the passage 3, 5, 7, 9 of MSCs cultured with chondrogenic supplements for 2W, growth kinetics using MTT and articular-specific extracellular matrix using toluidine blue staining were observed; At the same time, collagen II expression was examined by RT-PCR analysis and Western blot at the mRNA and protein levels.Result the purity of MSCs in group A reached 90%, and only 50% in group B; After continuously cultured with chondrogenic supplements for 2W, the chondrogenic potential of MSCs decreased with passaging. Collagen II expressed higher in the MSCs P5.Conclusions It is effective for density gradient centrifugation combinated with differential attachment technique to increase the cell purity of MSCs, and to decrease the contamination by hematocytes and fibroblasts. As seed cells, the P4, 5, 6 of MSCs are suitable for tissue engineering cartilage.Part II TGF-P1 gene transfecting mesenchymal stemeel IsObjective To establish the experiment basis for whether MSCs transfected by TGF-|31 gene are new suitable seed cells for the cartilage tissue engineering.method Recombining DNA and gene clone technique were applied to construct recombinant plasmid pcDNA3-TGF P 1. After confirmation of recombinant, lipofectin method was used to introduce TGF-(31 gene into mesenchymal stem cells (MSCs) .RT-PCR, Western blot and flow cytometry assays were used to examine TGF-@1 mRNA levels and peptides. Steady TGF-(31 gene expression was established after G418 screening. Growth kinetics and collagen II mRNAor peptides of transfected MSCs were examined by MTT, RT-PCR or Western blot assays, respectively.Results Recombinant pcDNA3-TGF-(31 was constructed successfully. After TGF-pi gene was introduced into mesenchymal stem cells (MSCs), TGF-(31 and collagen II expression were higher than control group, so did proliferative capacity.Conclusions TGF-01 gene can be introduced into MSCs by lipofectinmethod and steady express. Collagen II within transfected MSCs stimulated by autocrined TGF-(31 and growth kinetics was higher than non-transfected MSCs. It suggested that MSCs transfected by TGF-01 gene be new suitable seed cells for cartilage tissue engineering.Part III repairing the cartilage defects in rabbit joints with TGF-3 1 transfected MSCs loaded inchitosanObjective To investigate the cultivation in vitro and repairing cartilage defects of rabbit knee joins of TGF-|31 gene transfected MSCs seeded into chitosan.Method After TGF-01 gene transfected MSCs seed into three-dimensional scaffold chitosan, the MSCs adherent using scanning electron microscopic (SEM) and growth kinetics using by MTT analysis were observed, full-thickness cylindrical cartilage defects of 5 mm diameter and 5 mm depth were created using crank brace in the patellar groove of the bilateral knees of 14 rabbits divided into group A, B and C at random. In one knee the defect treated with nothing as control, in the other the defect was covered with chitosan (A), chitosan+non-transfected MSCs (B), chitosan+ TGF-(31-MSCs (C). The rabbits were sacrificed at 6 and 12 weeks after transplantation. Each cartilage defect area was evaluated both macroscopically and histologically.Results at 4 weeks after transplantation, the defects in the group A were filled with fibrous tissue. In the group B and C, the defects were covered with regenerated cartilage similar to hyaline cartilage and abundant extracellular matrix (collagen II), as shown by toluidine blue and immunohistochemical analysis, TGF-01 expression was higher in the group C than that in the group B. At the 12 weeks after transplantation, the defects in the group C were covered by regenerated hyaline-like cartilage; The defects in the group B have not been completely covered with regenerated cartilage. The defects in the group A still contained fibrous tissue and hyaline cartilage-like regeneration tissue was observed at the border of the normal cartilage. Collagen II was...
Keywords/Search Tags:Transforming growth factor-β1, gene transfer, mesenchymal stem cell (MSCs), collagen II, tissue engineering cartilage, seed cells, chitosan
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