Expression of Transforming Growth Factor β1 in Mesenchymal Stem Cells: Potential Utility in Molecular Tissue Engineering for Osteochondral Repair

Summary: The feasibility of using gene therapy to treat full-thickness articular cartilage defects was investigated with respect to the transfection and expression of exogenous transforming growth factor (TGF)-β1 genes in bone marrow-derived mesenchymal stem cells (MSCs) in vitro. The full-length rat TGF-β1 cDNA was transfected to MSCs mediated by lipofectamine and then selected with G418,a synthetic neomycin analog. The transient and stable expression of TGF-β1 by MSCs was detected by using immunohistochemical staining. The lipofectamine-mediated gene therapy efficiently trans fected MSCs in vitro with the TGF-β1 gene causing a marked up-regulation in TGF-β1 expression as compared with the vector-transfected control groups, and the increased expression persisted for at least 4 weeks after selected with G418. It was suggested that bone marrow-derived MSCs were sus ceptible to in vitro lipofectamine mediated TGF-β1 gene transfer and that transgene expression persist-ed for at least 4 weeks. Having successfully combined the existing techniques of tissue engineering with the novel possibilities offered by modern gene transfer technology, an innovative concept, I.e.molecular tissue engineering, are put forward for the first time. As a new branch of tissue engineer-ing, it represents both a new area and an important trend in research. Using this technique, we have a new powerful tool with which: (1) to modify the functional biology of articular tissue repair along defined pathways of growth and differentiation and (2) to affect a better repair of full-thickness artic ular cartilage defects that occur as a result of injury and osteoarthritis.     

Study of Rat Osteoblasts Transfected by Transforming Growth Factor β1 Gene

Summary: In order to investigate the effect of TGFβ1 gene transfer on the biological characteristics,the effects of gene transfer and supernatant of transfected osteoblasts on the proliferation and ALP activity of osteoblasts were detected by 3H-TdR and MTT. Our results showed that TGFβ1 gene transfer had no effect on the biological characteristics and the activated supernatant of transfected os-teoblasts stimulated proliferation and inhibited ALP activity of osteoblasts. TGFβ1 gene transfer could promote the expression of TGFβ1 and the biological characteristics of transfected osteoblasts were sta-ble, which might be helpful for gene therapy of bone defects in vivo.     

Osteogenic Potential of Cultured Bone Marrow Stromal Cells Transfected with Transforming Growth Factor β_1 Gene in vitro

Bone tissue engineering has a broad prospect intreating bone defects. It is a crucial link to chooseand optimize seed cells.In recent years,bone mar-row stromal cells(BMSCs) have been regarded to beone kind of preferred seed cells[1,2 ] . We have trans-fected BMSCs with TGF- β1gene and then examinedtheirosteogenic potential in orderto improve theirbi-ological properties.1　 MATERIALSAND METHODS1 .1　 Experimental MaterialsThe materials used in the experiment includedpc DNA3 - T…     

Influence of Exogenous TGFβ_1 on the Expression of Smad2 and Smad3 in Rat Bone Marrow-derived Mesenchymal Stem Cells

Bone marrow derived mesenchymal stem cells(MSCs) have multipotential to differentiate intoosteogenic, myogenic, and adipogenic cell linea ges, depending on signals derived from the cellularenvironment. At present, MSCs have been regar ded to be one kind of preferred seed cells in the re generation of bone defects. The TGFβ1 protein ismultifunctional peptide and has a broad range ofcellular activities including the control of cell pro liferation and differentiation …     

Cloning and Expression of Rat Transforming Growth Factor β1 cDNA in Osteoblasts

In order to study transforming growth factor81 (TGF51) in molecular level and investigate thefeasibility of TGF51 gene therapy for bone defects,an eukaryotic expression vector containing ratTGF51 cDNA was constructed. The expression ofTGF51 in the transfected osteoblasts was observed.1 MATERIALS AND METHODS1. 1 Animals and Cell CultureSprague--Dawley rats, I adult and 5 newborn,were purchased from Animal Center of Tong nMedical University. Lymphocytes, isolated fromperipheral…     

Construction of Antisense Transforming Growth Factorβ_1 Gene and Its Effect on the Proliferation by Expression in Osteosarcoma Cells

Summary:To construct the antisense transforming growth factorβ1(TGFβ1)gene and investigatethe effect of TGFβ1 autocrine loop blockage on the proliferation of osteosarcoma cells.TGFβ1 cDNAwas cloned by RT-PCR from human osteosarcoma cells(MG-63)and inserted into pcDNA_3 to con-struct an antisense expression vector,which was dubbed pcDNA_3-TGFβ1(-).MTT was used to de-tect the proliferation of osteosarcoma cells transfected by antisense TGFβ1 gene.Our results showedthat the proliferation of the transfected osteosarcoma cells was suppressed markedly.It is concludedthat TGFβ1 autocrine loop blockage in osteosarcoma cells could inhibit cell proliferation,which mightbe helpful for gene therapy of osteosarcoma.     

Modulation of Matrix Metalloproteinase and TIMP-1 Expression by TGF-β1 in Cultured Human RPE Cells

Matrix metalloproteinases(MMPs)are a familyof zinc binding,calcium-dependent endopeptidases thatfunction by degrading extracellular matrix(ECM)components.The functional effects of these enzymesare in part controlled byinteractions withtissueinhibi-tors of metalloproteinases(TI MPs)acting as naturalMMPinhibitors.Aprecise balance between MMPandTI MPactivities may be i mportant for the integrity ofECMcomponents[1].It shows that the expression andactivity of MMPs could be regulated by va…     

COA: Bone Morphogenetic Protein 2 Promoted Transforming Growth Factor β3 Induced Chondrogenesis of Human Osteoarthritic Synovium-Derived Stem Cells

Background Synovium-derived stem cells (SDSCs) with greater chondrogenic potential are attracting more considerable attention as a cell source for cartilage regeneration. The aim of this study was to investigate the effect of bone morphogenetic protein-2 (BMP-2) on transforming growth factor-beta3 (TGF-β3)-induced chondrogenesis of SDSCs isolated from human osteoarthritic synovium in a pellet culture system. Methods Nucleated cells isolated from human osteoarthritic synovium were plated at an optimal cell density to allow the selective proliferation of SDSCs. The clonogenicity, stem cell marker expression and multi-differentiation potential were determined by CFU assay, flow cytometry assay and specific staining including alizarin red S staining, Oil red staining and alcian blue staining, respectively. SDSCs pellet was cultured in chondrogenic medium without or with TGF-β3 or/and BMP-2. At day 21, the diameter and the weight of the pellets were measured. Chondrogenic differentiation of SDSCs was evaluated by Safranin O staining, immunohistochemical staining of collagen type II, sulfated glycosaminoglycan (sGAG) synthesis and mRNA expression of collagen type II (COL2A1), aggrecan (ACAN), SOX9, link-protein (HAPLN1), collagen type X (COL10A1) and BMP receptor II (BMPR-II). Results Cells isolated under the optimized culturing density (104/60cm2) showed clonogenicity and multi-differentiation potential. These cells were positive (>99% positive) for CD44, CD90, CD105 and negative (<10% positive) for CD34 and CD71. SDSCs differentiated to a chondrocytic phenotype in chondrogenic medium containing TGF-β3 with or without BMP-2. Metachromatic staining of the extracellular matrix with Safarnin O was positive and the expression of collagen type II was detected. The combination of TGF-β3 and BMP-2 produced cell pellets with larger diameter and weight, produced more sGAGs, expression higher levels of collagen type II and chondrogenic markers, except COL10A1, than medium with TGF-β3 alone. Conclusions SDSCs could be isolated from human osteoarthritic synovium. Supplementation of BMP-2 significantly promoted the in vitro TGF-β3-induced chondrogenic differentiation of SDSCs.