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 review and summarize the latest development of the therapy for the Duchenne muscular dystrophy (DMD). Methods Therecentlypublished articles related to the therapies for DMD were extensively reviewed and briefly summarized. Results The therapeutic approaches for DMD included the gene therapy, the cell therapy, and the pharmacological therapy. The gene therapy and the cell therapy were focused on the treatment for the cause of DMD by the delivery of the missing gene, the modification of the mutated gene, and the transfer of the normal cells including the stem cells, while the pharmacological therapy dealt with the downstream events caused by the dystrophin gene defect, slowed down the pathologic progress of DMD, and improved the DMD patient’s life quality and life span, by medication and other factor treatments. Conclusion There is still no cure for DMD because of various difficulties in replacing or repairing thedefected gene and of the multifaceted nature of the severe symptoms. Therefore,it is imperative for us to find out a more effective treatment that can solve these problems.
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 explore the SV40-mediated immortalization, the related factors and their roles in cell immortalization. METHODS: The original articles about cell immortalization and replicative senescence in recent decade were reviewed. RESULTS: Cell immortalization was a multifaceted phenomenon, it was involved in viral DNA integration, activation of telomerase, inactivation of growth suppressors, and so on, and their roles were closely related. CONCLUSION: The research on cell immortalization may be expected to provide important insights into a broad range of cellular biological phenomenon, and the immortalized cells can play important roles in the research of cell engineering and tissue engineering as standard cells.
OBJECTIVE To investigate possibility of cartilage cultured in centrifuge tube as graft materials. METHODS: Articular chondrocytes isolated from a 3-week-old rabbit formed cartilage after cultivation for 2 weeks. Articular cartilage of humeral head, growth plate of proximal tibia and meniscus were collected from a 6-week-old rabbit. The ultrastructure of chondrocytes and extracellular matrix in the three kinds of cartilages and cultured cartilage were observed by transmission electronic microscopy. RESULTS: Cartilage cultured in centrifuge tube possessed unique ultrastructure and was similar to articular cartilage and growth plate, but it was markedly different from meniscus. The four kinds of cartilages were characteristic of respectively different chondrocytes and extracellular matrix. Cultured cartilage showed typical apoptosis of chondrocytes and "dark chondrocytes" appeared in growth plate. Condrocyte apoptosis was not seen in articular cartilage and meniscus. CONCLUSION: Cartilage cultured in centrifuge tube has unique ultrastructure and may be used as graft materials for articular cartilage and growth plate.
ObjectiveTo prepare the small intestinal submucosa (SIS)-silk composite scaffold for anterior cruciate ligament (ACL) reconstruction, and to evaluate its properties of biomechanics, biocompatibility, and the influence on synovial fluid leaking into tibia tunnel so as to provide a better choice in the clinical application of ACL reconstruction.
MethodsThe silk was used to remove sericin and then weaved as silk scaffold, which was surrounded cylindrically by SIS to prepare a composite scaffold. The property of biomechanics was evaluated by biomechanical testing system. The cell biocompatibility of scaffolds was evaluated by live/dead staining and the cell counting kit 8 (CCK- 8). Thirty 6-week-old Sprague Dawley rats were randomly assigned to 2 groups (n=15). The silk scaffold (S group) and composite scaffold (SS group) were subcutaneously implanted. At 2, 4, and 8 weeks after implanted, the specimen were harvested for HE staining to observe the biocompatibility. Another 20 28-week-old New Zealand white rabbits were randomly assigned to the S group and SS group (n=20), and the silk scaffold and composite scaffold were used for ACL reconstruction respectively in 2 groups. Furthermore, a bone window was made on the tibia tunnel. At last, the electric resistance of tendon graft in the bone window was measured and recorded at different time points after 5 mL of 10% NaCl or 5 mL of ink solution was irrigated into the joint cavity recspectively.
ResultsThe gross observation showed that the composite scaffold consisted of the helical silk bundle inside which was surrounded by SIS. The maximal load of silk scaffold and composite scaffold was respectively (138.62±11.41) N and (137.05±16.95) N, showing no significant difference (P>0.05); the stiffness was respectively (24.65±2.62) N/mm and (24.21±2.39) N/mm, showing no significant difference (P>0.05). The live/dead staining showed that the cells had good activity on both scaffolds. However, the cells on the composite scaffold had better extensibility. In addition, the cell proliferation curve indicated that no significant difference in the absorbance (A) values was founded between groups at various time points (P>0.05). HE staining showed less inflammatory cells and much more angiogenesis in SS group than in S group at 2, 4, and 8 weeks after subcutaneously implanted (P<0.05), indicating good biocompatibility. Additionally, the starting time points of electric resistance decrease and the ink leakage were both significantly later in SS group than in S group (P<0.05). The duration of ink leakage was significantly longer in SS group than in S group (P<0.05).
ConclusionThe SIS-silk composite scaffold has excellent biomechanical properties and biocompatibility and early vacularization after in vivo implantation. Moreover, it can reducing the leakage of synovial fluid into tibia tunnel at the early stage of ACL reconstruction. So it is promising to be an ideal ACL scaffold.
Objective
To review the application of genipin for the modification of natural biomaterials as a crosslinking agent and progress in research.
Methods
Domestic and foreign literature on application of genipin for the modification of natural biomaterials as a crosslinking agent was thoroughly reviewed.
Results
Genipin is an effective natural crosslinking agent with a very low level of cytotoxicity compared with conventional synthetic crosslinking agents. Tissues fixed with genipin can maintain a high level of stability as well as resistance to enzymatic degradation.
Conclusion
Genipin is a promising substitute for conventional synthetic crosslinking agents, which has offered an alternative for modification of natural biomaterials for tissue engineering.
