Du Jingyuan

Experimental study of vascular endothelial growth factor gene therapy for avascular necrosis of the femoral head

SummaryTo explore a new method for the therapy of the avascular necrosis of the femoral head, the recombinant plasmid pCD-hVEGF165 was mixed with collagen and was implanted in the necrotic femoral head. The expression of vascular endothelial growth factor (VEGF) was detected by RNA dot hybridization and immunohistochemical method. The repair of the femoral head was observed by histological method. The results showed that the expression of VEGF was detectable in the femoral head treated with VEGF gene. Angiogenesis in these femoral heads was more abundant than the control. Bone repairing was augmented in the femoral head treated with VEGF gene. The results suggest that angiogenesis in bone tissue could be augmented by gene transfection of VEGF and bone repairing would be accelerated accordingly.

Molecular tissue engineering: Concepts, status and challenge

Tissue engineering has confronted many difficulties mainly as follows: 1) How to modulate the adherence, proliferation, and oriented differentiation of seed cells, especially that of stemcells. 2) Massive preparation and sustained controllable delivery of tissue inducing factors or plasmid DNA, such as growth factors, angiogenesis stimulators, and so on. 3) Development of “intelligent biomimetic materials” as extracellular matrix with a good superficial and structural compatibility as well as biological activity to stimulate predictable, controllable and desirable responses under defined conditions. Molecular biology is currently one of the most exciting fields of research across life sciences, and the advances in it also bring a bright future for tissue engineering to overcome these difficulties. In recent years, tissue engineering benefits a lot from molecular biology. Only a comprehensive understanding of the involved ingredients of tissue engineering (cells tissue inducing factors, genes, biomaterials) and the subtle relationships between them at molecular level can lead to a successful manipulation of reparative processes and a better biological substitute. Molecular tissue engineering, the offspring of the tissue engineering and molecular biology, has gained an increasing importance in recent years. It offers the promise of not simply replacing tissue, but improving the restoration. The studies presented in this article put forward this new concept for the first time and provide an insight into the basic principles, status and challenges of this emerging technology.

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

SummaryThe 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 transfected MSCsin 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 susceptible toin vitro lipofectamine mediated TGF-β1 gene transfer and that transgene expression persisted 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 engineering, 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 articular cartilage defects that occur as a result of injury and osteoarthritis.

Molecular tissue engineering: Applications for modulation of mesenchymal stem cells proliferation by transforming growth factor β1 gene transfer

SummaryThe effect of transforming growth factor β1 (TGF-β1) gene transfection on the proliferation of bone marrow-derived mesenchymal stem cells (MSCs) and the mechanism was investigated to provide basis for accelerating articular cartilage repairing using molecular tissue engineering technology. TGF-β1 gene at different doses was transduced into the rat bone marrow-derived MSCs to examine the effects of TGF-β1 gene transfection on MSCs DNA synthesis, cell cycle kinetics and the expression of proliferating cell nuclear antigen (PCNA). The results showed that 3 μl lipofectaminemediated 1 μg TGF-β1 gene transfection could effectively promote the proliferation of MSCs best; Under this condition (DNA/Lipofectamine= 1μg/3μl) flow cytometry and immunohistochemical analyses revealed a significant increase in the3H incorporation, DNA content in S phase and the expression of PCNA. Transfection of gene encoding TGF-β1 could induce the cells at G0/G1 phase to S1 phase, modulate the replication of DNA through the enhancement of the PCNA expression, increase the content of DNA at S1 phase and promote the proliferation of MSCs. This new molecular tissue engineering approach could be of potential benefit to enhance the repair of damaged articular cartilage, especially those caused by degenerative joint diseases.

The experimental study on the effect calcitonin gene-related peptide on bone resorption mediated by interleukin-1

SummaryTo investigate the effect of calcitonin gene-related peptide (CGRP) on bone resorption mediated by interleukin-1β(IL-1β)in vitro, the osteoclasts isolated from the long bones of newborn SD rats were co-cultured with osteoblasts on ivory slices placed in 24-well plates. 24 h later, conditioned media containing CGRP and/or IL-1β were added to the wells respectively, and continued culturing for 48 h. After the cells were stripped off by ultrasonication, the ivory slices were stained in toludine blue. The number and the total area of resorption lacunae on each slice were measured by computer imaging analysis system. Our results showed that IL-1β significantly stimulated bone resorption, but CGRP inhibited the effect mediated by IL-1β in a dose-dependent manner, It is suggested that CGRP may inhibit osteoclastic bone resorption through two ways: One is that CGRP functions directly on osteoclasts to block their activation; the other is that CGRP regulates the release of cytokines by osteoblasts and indirectly affects the function of osteoclasts.

Biodegradation of Absorbable Hydroxyapatite/Poly-DL-lactide Composites in Different Environment

To develop a new generation of absorbable fracture fixation devices with enhanced biocompatibility, the biodegradation mechanism and its influence on the cellular response at the tissue/implant interface of hydroxyapatite/poly-DL-lactide (HA/PDLIA) composites were investigated in vitro and in vivo. HA/PDLIA rods were immersed in phosphate-buffered saline, or implanted in muscle and bony tissue for 52 weeks. Scanning electron microscopic and histological studies were done. The degradation rate was the slowest in vitro, slower in muscle tissue and fast in bone. In vitro, the composites degraded heterogeneously and a hollow structure was formed. In bone, the limited clearing capacity leads to the accumulation of oligomeric debris, which contribute totally to the autocatalytic effect. So, the fastest degradation and intense tissue response were seen. In muscle tissue, oligomeric debris migrated into vicinal fibers over a long distance from the original implant cavity and the tissue reactions were, however, quite moderate. For the same size organic/inorganic composite, the environment where it was placed is the major factor in determining its biodegradation process and cellular reaction. In living tissue, factors such as cells, enzymes and mechanical stress have an obvious influence on the biodegradation and biological process at the tissue/implant interface. The biocompatibility of the HA/PDLIA composites is enhanced with the incorporating of the resorbable HA microparticles.

Treatment of floating knee injury in children.

SummaryThe necessity and superiority of the surgical operation on children with floating knee injury and the fracture union and complications were investigated. Twenty-eight children with floating knee injury were subjected to open reduction and internal fixation or external fixator. The patients were followed up for 18 months to 7 years. The curative effectiveness was scored by Karlstrom criteria. The results showed that no nonunion or deformity was found. The affected limb was 1.2 cm to 1.5 cm longer in 2 cases, 0.8 to 1.2 cm shorter in 3 cases than the contralateral. No severe dysfunction of knee joint occurred. The excellent-good rate was 92.8% and the curative rate 71.4% respectively. So for children whose age is older than 5 years, its a good way to treat the fractures of femur and tibia with open reduction and internal fixation or external fixator. The method can be advantageous for the nursing care, early function recovery, shortening of the hospital stay and avoidance of severe complications.

Biocompatibility studies on bone marrow stromal cells with chitosan-gelatin blends

To study the effect of chitosan-gelatin blends on the growth and proliferation of in vitro cultured bone marrow stromal cells (BMSCs) and explore a new carrier for the application of tissure engineering, cells from long bones of young rabbitsaged less than two weeks were expanded in vitro for one week and seeded onto the surface of pure chitosan and chitosan-gelatin blends. Cells attached to and proliferated on both pure chitosan and chitosan-gelatin blends were monitored with the aid of an inverted light microscope and a scanning electron microscope. The cell viability was monitored by MTT after 2, 4, 6, 8 days seeding. BMSCs could be attached to and proliferated on both pure chitosan and chitosan-gelatin blends and remain their morphologies seen in vivo. Chitosan-gelatin blends could promote BMSCs to proliferate (P<0.01). It is confirmed that chitosan-gelatin blends maintain the bioactivity feature of chitosan and even enhance the growth and proliferation of in vitro cultured BMSCs because of the adding of gelatin. It is a potential carrier for the delivery of cells tissue engineering.

Construction of antisense transforming growth factorβ1 gene and its effect on the proliferation by expression in osteosarcoma cells

SummaryTo construct the antisense transforming growth factorβ1 (TGFβ1) gene and investigate the effect of TGFβ1 autocrine loop blockage on the proliferation of osteosarcoma cells. TGFβ1 cDNA was cloned by RT-PCR from human osteosarcoma cells (MG-63) and inserted into pcDNA3 to construct an antisense expression vector, which was dubbed pcDNA3-TGFβ1(−). MTT was used to detect the proliferation of osteosarcoma cells transfected by antisense TGFβ1 gene. Our results showed that the proliferation of the transfected osteosarcoma cells was suppressed markedly. It is concluded that TGFβ1 autocrine loop blockage in osteosarcoma cells could inhibit cell proliferation, which might be helpful for gene therapy of osteosarcoma.

Transfection of articular chondrocytes with rhBMP7 gene and its expression

SummaryIn order to investigate the possibility of expression of exogenous gene in transduced articular chondrocytes, plasmid pcDNA3-rhBMP7 was delivered to cultured chondrocytes. Through immunohistochemical staining and RT-PCR assay, the expression of rhBMP7 gene was detected. And the bioactivity of transgene expression product was detected through MTT assay as well. It was confirmed that exogenous gene could be expressed efficiently in transduced chondrocytes and the transgene therapy on the problems of articular cartilge.