OBJECTIVE: To investigate a cryophylactic agent (CPA) to protect tissue engineered tendon (TET) in deep low temperature. METHODS: Sixty-four BALB/C inbred nude mice were chosen, which included 4 as blank control group, left sides of 60 as experimental group and their right sides as control group. Transformed human embryonic tendon cells of the 54th passage and artificial materials of carbon fiber (CF) and polyglycolic acid (PGA) were co-cultured in vitro to construct TET. TET was frozen in liquid nitrogen with four kinds of CPA (groups A, B, C, and D) for 2 months. They were thawed quickly and transplanted into hind limbs of nude mice to repair the defects of Achilles tendon, which was 5 mm in length and 65.7% of total Achilles tendon. In control group, no cryopreservation treatment was taken. The morphological, histological, ultrastructure, and immunohistochemistry examinations were made and short tandem repeat loci were detected 2, 4, 6, 8, and 12 weeks later. RESULTS: In the experimental group, the morphological properties of tendon cells resumed gradually and the capability of synthesizing collagen enhanced by degrees. Tendon cells survived and could secret type I collagen and there was less difference between experimental and control groups 12 weeks after transplantation. In group A, vacuole in mitochondrion of tendon cell decreased, tendon cell arranged in order and abundant collagen fibers were found and linked. CONCLUSION: The cryopreservation agent in group A can protect TET in deep low temperature.
OBJECTIVE To prevent early closure of growth plate and developmental deformities of limbs by allografts of cultured cartilages into growth plate defects of rabbits. METHODS Chondrocytes isolated from articular cartilage of 1-month rabbits formed cartilage after cultivation in centrifuge tubes. The cartilages cultured for two weeks were implanted into growth plate defects of proximal tibiae of 6-weeks rabbits. At 4th and 16th weeks, X-ray, histologic and immunohistochemical examination were performed. RESULTS The tibiae had no marked deformities after 4 weeks of operation. Histologic examinations showed that the defects were filled with cartilage. Immunohistochemical results of type II collagen were positive. The tibiae with allografts of cultured cartilages had no evident deformities after 16 weeks of operation. Histologic examination showed nearly closure of growth plates. On the contrary, the tibiae on control side formed severe deformities and growth plate were closed. CONCLUSION Allograft of cultured cartilages into growth plate defects may replace lost growth plate tissues, maintain normal growth of limbs and prevent developmental deformity.
【Abstract】 Objective To review the recent progress of cell therapy in cl inical appl ications. Methods Therecent l iterature about cell therapy in cl inical appl ications was extensively reviewed. Results Based on the advances in cell biology, especially the rapid progress in stem cell biology, an increasing number of cl inical trials about cell therapy for management of various diseases, such as cardiovascular system diseases, neural system diseases, musculo-skeletal diseases, diabetes, stress urinary incontinence, and others, had been reported with encouraging results. All these showed that cell therapy had great potentials in cl inical appl ication. Conclusion Cell therapy provides a novel approach for the treatment of many human diseases. However, the mechanism remains to be fully elucidated.
ObjectiveTo review the research progress of novel cross-linking methods applied in bio-derived materials. MethodsThe literature about the latest progress in the cross-linking methods of bio-derived materials was reviewed and analyzed. ResultsThe novel cross-linking methods of the bio-derived materials can be divided into chemical methods, physical methods, and biological methods, whose available application and cross-linking properties are greatly depended on their mechanisms. So proper methods should be developed to meet the various application requirements of the materials. A series of studies shows the feasibility and availability of the cross-linked bio-derived materials in the repair and reconstruction of the tissue. ConclusionBio-derived materials modified by novel cross-linking methods are proved to obtain excellent biocompatibility and tissue repair ability, better mechanical properties and degradation properties, and so on. Those methods provide researchers more choices to cross-linking materials, which are help to obtain the clinical tissue engineering products.
Objective To compare biological characteristics between articular chondrocyte and meniscal fibrochondrocyte cultured in vitro andto investigate the possibility of using cultured cartilage as a substitute for meniscus.Methods Chondrocytes isolated from articular cartilage and meniscus of rabbits aged 3 weeks were respectively passaged in monolayer and cultured in centrifuge tube. Cartilages cultured in centrifuge tube and meniscus of rabbit aged 6 weeks were detected by histological examination and transmission electron microscopy. Growth curves of articular chondrocytes and meniscalfibrochondrocytes were compared; meanwhile, cell cycles of articular chondrocytes and meniscal fibrochondrocytes in passage 2and 4 were separately measured by flow cytometry.Results Articular chondrocytes in passage 4 were dedifferentiated. Articular chondrocytes formed cartilage 2 weeks after cultivation in centrifuge tube, but meniscal fibrochondrocytes could not generate cartilage. The differences in ultrastructure and histology obviously existed between cultured cartilage and meniscus; moreover, apoptosis of chondrocytes appeared in cultured cartilage. Proportion of subdiploid cells in articular chondrocytes passage 2 and 4 was markedly higher than that in passage 2 and 4 fibrochondrocytes(Plt;0.05). Conclusion Meniscal fibrochondrocytes can not form cartilage after cultivationin centrifuge tube, while cartilage cultured in centrifuge tube from articular chondrocytes can not be used as graft material for meniscus. Articular cartilage ismarkedly different from meniscus.
OBJECTIVE: To investigate the biological characteristics of continuously subcultured human embryonic skeletal myoblasts, and choose the optimal seeding cells for muscle tissue engineering. METHODS: Human embryonic skeletal myoblasts were subcultured in vitro. The growth curve, rate of myotube formation(RMF) were used to evaluate the proliferative and differentiation ability of myoblasts, and to investigate the influence of fibroblasts contamination on myoblasts. RESULTS: The beginning 6 passages of myoblasts showed b proliferative and differentiation ability. From the 8th to 20th passage, the rate of fibroblasts contamination was increased, it mainly showed the growth characteristics of fibroblasts with increased proliferation and low differentiation. After subcultured to the 20th passage, the degeneration of myoblasts was obvious. CONCLUSION: The myoblasts within 6 passages should be used as the seeding cells of muscle tissue engineering because of b proliferative ability and high rate of myotube formation.
Objective To investigate the effects of NGF on the prol iferation, mitotic cycle, collagen synthesis and migration of human dermal fibroblasts (HDFs), and to explore the function of NGF on the wound heal ing. Methods The 3rd generation of HDFs were incubated with various concentrations of NGF (0, 25, 50, 100, 200 and 400 ng/mL), the cell prol iferation was measured with MTT assay. After treated with NGF at 0, 100 ng/mL, the cell cycle of HDFs was determined by flow cytometry (FCM). Hydroxyprol ine and real-time fluorescence quantitative PCR (FQ-PCR) were used to measure collagen synthesis at protein level and mRNA level respectively. The in vitro cell scratch wound model was set up to observe the effect of NGF (0, 50, 100 and 200 ng/mL) on the migration of HDFs after 24 hours of culture. Results Absorbance value of HDFs for different concentrations of NGF (0, 25, 50, 100, 200, and 400 ng/ mL) showed that NGF did not influence the prol iferation of HDFs (P gt; 0.05). When HDFs were treated with NGF at 0 and 100 ng/mL, the result of FCM analysis showed that percentage of HDFs in G0/G1, S, G2/M phases were not changed (P gt; 0.05). Compared with control group, the expression of Col I and Col III were not significantly different, measured by both hydroxyprol ine and FQ-PCR (P gt; 0.05). The rates of HDFs’ migration at various concentrations of NGF (0, 50, 100, 200 ng/ mL) were 52.12% ± 6.50%, 80.67% ± 8.51%, 66.33% ± 3.58%, and 61.19% ± 0.97%, respectively, indicating that NGF could significantly enhanced the migration of HDFs at 50 and 100 ng/mL (P lt; 0.05). Conclusion NGF does not influence prol iferation, mitotic cycle and collagen synthesis of HDFs, but significantly enhanced migration in an in vitro model of wounded fibroblasts.
Objective To study the gene expressions of human osteoblasts during the construction of tissue engineered bone with the bioderived material. Methods The fetal osteoblasts were used to construct tissue engineered bone with the bio-derived material and then were cultured 2,4,6,8 and 10 days in vitro. Real-time PCR analysis indicated that Cbfa 1, Osterix, Collagen type Ⅰ,osteocalcin(OC) and Integrin α5 and β1 were present in osteoblasts with bio-derived materials.Results The change ofCbfa1 was consistent with the change of Osterix. On 2nd day and 8th day, the expression of Osterix in experimental group was higher than that in control group, P＜0.05. Collagen type Ⅰ’s change was consistent with change of OC expression, and its expression was higher in experimental group than that in control group on 2nd, 4th, 6th and 8th day. The Integrinexpression was high all along. Conclusion The important genes can be expressed normally by integrating osteoblasts with bioderived scaffolds. As skeleton tissue engineering scaffold, the bio-derived bone is conducive to keepthe osteoblast’s phenotype and differentiation with osteoconductive ability. The osteoblast can enter proliferation stage favorably and the scaffold materials exert no effects on it. Bio-derived bone can also supply more space for cellsto proliferate. The bio-derived materials promote osteoblasts adhesion.