Objective To observe the heterotopic osteogenes is of the autogenou s marrow stromal cells (MSCs) on the ceramic bovine bone(CBB)/hydrogel scaffold (HG) and t he effects of the recombinant human bone morphogenetic protein2 (rhBMP-2) and the transforming growth factor β (TGF-β) on osteogenesis. Methods The auto genous marrow stromal cells were cultured by the mineralized condition medium (1 0%FBS, dexamethasone 10 nmol, L-vitamin C 50 mg/L, βsodium glycerophosph ate D MEM culture medium 10 mmol). At 5 days, the MSCs differentiation was observed b y TypeⅠcollagen, the Mend calcium-cobalt staining, and the Von-Kossa staining. The cell suspension of 5×106/ml was obtained. There were three groups: Group A: added in rhBMP-2（10 μg）TGF-β（0.05 μg）;Group B: added in TGF-β(0.05 μg); and Group C (the control group): without the growth factor. Then, the MSCs loading on CBB/HG were embedded in the autogenous subcutaneous area at 4 and 8 weeks, and the osteogenesis was observed by the HE staining and the modified Mallory’s trichrome staining, with an image analysis. TypeⅠcollagen and the bone m orphogenetic synthesis were examined by the immunohistochemistry stains. Results Most MSCs induced by the mineralized condition medium at 5 da ys became smalle r and polygon-shaped, and the cytodendrite became shorter. The MSCs were observ e d by the Mend calciumcobalt staining. Some brown and black grains were found in the cytochylema. The MSCs were positive for the TypeⅠcollagen immunohistochemi stry stains. At 20 days, the mineralized nubs were found by the Von Kossas stain s. At 4 weeks, some strips of the new bone were observed by the HE staining an d the modified Mallory’s trichrome staining in all the groups. The bone matrix a rea was significantly larger in Group A than in Group B（P＜0.01). The av erag e gray degrees of TypeⅠcollagen were lower in Groups A and B than in Group C. However, there was no significant difference in the bone morphogenesis among the three groups. At 8 weeks, there- were significantly more snatchy strips and macula mature bone formation in Groups A and B than in Group C. The Type Ⅰcollage n and the bone morphogenesis were not significantly different among the three groups. Conclusion The autogenous marrow stromal cells on the ce ramic bovine bon e /hydrogel scaffold can promote the heterotopic osteogenesis, and the combined use of rhBMP-2 and TGF-β is better than the only use of rhBMP-2 or TGF-β i n promoting osteogenesis.
Objective To investigate the effect of machine-enzyme digestion method on the residual quantity of small intestinal submucosa (SIS) cell and the content of growth factors. Methods Fresh jejunum of pig within 4 hours after harvesting was prepared into SIS after machine digestion (removing placenta percreta, mucosa, and muscular layer), degrease,trypsinization, abstergent processing, and freeze drying. Samples were kept after every preparation step serving as groups A, B, C, D, and E, respectively (n=4 per group). And the fresh jejunum served as control group (group F, n=4). The histological alteration in each preparation process was reviewed with HE staining and scanning electron microscope (SEM). Nest-polymerase chain reaction (PCR) was used to determine the content of death associated protein 12 (DAP12), and enzyme-linked immunosorbent assay (ELISA) was appl ied to detect the content of vascular endothel ial growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor β (TGF-β), tumor necrosis factor α (TNF-α). Results HE staining and SEM observation showed that there were residual cells in groups A and B, and there were no residual cells in groups C, D, and E. Nest-PCR test revealed the occurrence of DAP12 in each group. The contents of DAP12 in groups A, B, C, D, E, and F were (18.01 ± 9.53), (11.87 ± 2.35), (0.59 ± 0.27), (0.29 ± 0.05), (0.19 ± 0.04), and (183.50 ± 120.13) copy × 106/cm2. The content of DAP12 in group F was significant higher than that of other groups (P lt; 0.05), groups A and B was higher than groups C, D, and E (P lt; 0.05), there were significantdifferences among groups C, D, and E (P lt; 0.05), and there was no significant difference between groups A and B (P gt; 0.05). The ELISA test showed the content of VEGF, bFGF, TGF-β, and TNF-α in group A was significantly higher than that of groups B, C, D, and E (P lt; 0.05), and there was no significant difference among groups B, C, D, and E (P gt; 0.05). Conclusion SIS prepared by simple mechanical method has more residual cells, while the machine-enzyme digestion method can effectively remove the cells and significantly reduce the DAP12 content. This approach can not obviously reduce the growth factor content in SIS.
Objective To review the recent progress of the researches in the field of cartilage tissue engineering, and to discuss the challenges in construction of tissue engineered cartilage. Methods Literature related with cartilage tissue engineering was reviewed and analyzed. Results Some techniques have been appl ied in cl inical. As far as the seeding cells, induced pluripotent stem cells have attracted much more attention. Current strategies of scaffold designing are trying to imitate both component and structure of natural extracellular matrix. Cartilage regeneration through the autologous cell homing technique el iminate the transplantation of exotic cells and has become the hot topic. Conclusion Successful treatment of the damaged cartilage using tissue engineering method will depend on the advances of stem cell technology development, biomimetic scaffolds fabrication and proper appl ication of growth factors.
Objective To investigate the mechanism of vascular stromal fraction (SVF) at the early stage after aspirated fat transplantation. Methods Fat was harvested from 5 cases of women undergoing abdominal liposuction operation, and SVF was isolated. Aspirated fat with (group B) or without (group A) SVF was injected subcutaneously into the back of nude mice, and the grafts were harvested at 1, 3, 5, and 7 days. Graft wet weight was measured; and immunohistochemical method (CD31) was performed and the secretion of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) were qnantified by Western blot assay. Results The wet weight of transplanted adipose tissue showed an increasing tendency in groups A and B with time, and no significant difference was found between groups A and B (P gt; 0.05). At 1 and 3 days after transplantation, no CD31 positive cells was seen in 2 groups; the CD31 positive cells of group B were significantly more than those of group A at 5 and 7 days (P lt; 0.05), and the CD31 positive cells at 7 days were significantly more than those at 5 days in 2 groups (P lt; 0.05). Western blot test showed that VEGF expression reached peak at 3 days , then decreased gradually; the expression of VEGF protein in group B was significantly higher than that in group A at 1, 3, and 5 days (P lt; 0.05). The expression of HGF protein in groups A and B remained at a high level within 5 days, but it tended to decrease at 7 days, which was significantly higher in group B than that in group A (P lt; 0.05). Conclusion SVF can enhance angiogenesis by secretion of growth factors at the early stage after aspirated fat transplantation.
Objective To study the effect of platelet-rich plasma (PRP) on repairing chronic wounds of lower l imbs. Methods From May 2007 to November 2007, 47 patients suffering from chronic wounds of lower l imbs were treated. There were 41 males and 6 females, aged from 15 to 68 years (43.2 years on average). The disease was caused by tibiofibulafracture in 20 cases, calcaneus fracture in 4 cases, metatarsal fracture in 1 case, multiple open fracture of lower l imbs in 3 cases, tibia osteomyel itis in 10 cases, femur osteomyel itis in 1 case, soft tissue injury of ankle in 4 cases, infection after amputation in 2 cases, infection after foot orthomorphia in 1 case, and infection after calcaneus tendon neoplasty in 1 case. Their chronic wounds did not healed after 2 to 4 months of therapy. Among them, chronic wounds compl icated with fracture nonunion in 23 cases and positive bacterial culture result in 38 cases. Debridement and autogenous PRP gel injection were appl ied every 2 months and for twice. Results The patients were followed up for 4 months after the first PRP injection. Two months after the first PRP injection, chronic wounds contracted significantly in 34 patients with purulence and necrosis tissue cleaned up, circulation of soft tissue improved and exposed bone or muscle tissue covered by neogenetic granulation. No patient was completely cured. Two months after the second PRP injection, the average coverage rate was 79.3% ± 18.0%, the total cure rate was 29.8%. The volume of the chronic wounds decreased by (9.3 ± 4.9) mL after PRP therapy (2.5 ± 2.7) mL when compared with (11.8 ± 5.6) mL of before therapy, showing significant difference (P lt; 0.05). X-ray photograph showed that among the 23 cases of fracture nonunion, fracture healed completely in 9 cases; bony callus formation increased obviously in 12 cases; no significant change was observed in 2 cases. No aggravated sign of osteomyel itis was notified. Positive results of bacterial culture reduced to 15 cases. Conclusion PRP efficiently enhances the recovery of soft tissue defect and speeds up the chronic wounds heal ing oflower l imbs.
Objective To review the latest research progress of heme oxygenase 1 (HO-1), to thoroughly understand different functions of HO-1 and its influence on osteogenesis and angiogenesis of stem cells, and to analyze HO-1 application in bone tissue engineering. Methods Domestic and international literature on HO-1 in recent years was extensively reviewed and analyzed. Results The activity of HO-1 and its enzymatic products not only have the properties of anti-inflammatory, anti-apoptosis, and cytoprotection, but also can promote angiogenesis combined with other growth factors and protect the vessel which already exist. Moreover, HO-1 has an effect on the proliferation, paracrine signaling, osteoblastic differentiation, and anti-apoptosis of stem cells. Conclusion HO-1 can be used as a multi-function growth factor in bone tissue engineering, but more investigation should emphasis on synergistic effect of each function so as to improve bone repair.
Objective To review research progress of corneal tissueengineering.Methods The recent articles on corneal tissue engineering focus on source and selection of corneal cells, the effects of growth factors on culture of corneal cells in vitro. The preparation and selection of three-dimensional biomaterial scaffolds and their b and weak points were discussed. Results The corneal tissue engineering cells come from normal human corneal cells. The embryo corneal cell was excellent. Several kinds of growth factors play important roles in culture, growth and proliferation of corneal cell, and incroporated into matrix.Growth factors including basic fibroblast growth factor, keratinocyte growth factor, transforming growth factor β1 and epidermal growth factor was favor to corneal cell. Collagen, chitosan and glycosaninoglycans were chosen as biomaterial scaffolds. Conclusion Human tissue engineering cornea can be reconstructed and transplanted. It has good tissue compatibility and can be used as human corneal equivalents.
Objective To summarize the research progress of controlled release of angiogenic factors and osteogenic factors in bone tissue engineering. Methods The domestic and abroad literature on the controlled release structure of growth factors during bone regeneration in recent years was extensively reviewed and summarized. Results The sustained-release structure includes direct binding, microsphere-three-dimensional scaffold structure, core-shell structure, layer self-assembly, hydrogel, and gene carrier. A sustained-release system composed of different sustained-release structures combined with different growth factors can promote bone regeneration and angiogenesis. Conclusion Due to its controllability and persistence, the growth factor sustained-release system has become a research hotspot in bone tissue engineering and has broad application prospects.
OBJECTIVE: To investigate protection of biological activity and controlled release of growth factor by means of drug controlled release technique in tissue engineering. METHODS: Using drug controlled release technique that to embed or microcapsulate the biological drug with biodegradable polymer. RESULTS: The aliphatic polylactone could be used as drug carrier for each drug including the biological matter. And the release behavior of the drug could be controlled by adjusting the molecular structure of the carrier and the controlled release method. The successful example, that to realize regeneration of rat’s sciatic nerve with 5, 10, 15 and 20 mm of gap by using polylactide as nerve guide and the embedding growth factor, had been obtained. CONCLUSION: It is possible to realize protection of biological activity and sustained release of growth factor by using aliphatic polylactone as drug carrier.
OBJECTIVE: From the point of view of material science, the methods of tissue repair and defect reconstruct were discussed, including mesenchymal stem cells (MSCs), growth factors, gene therapy and tissue engineered tissue. METHODS: The advances in tissue engineering technologies were introduced based on the recent literature. RESULTS: Tissue engineering should solve the design and preparation of molecular scaffold, tissue vascularization and dynamic culture of cell on the scaffolds in vitro. CONCLUSION: Biomaterials play an important role in the tissue engineering. They can be used as the matrices of MSCs, the delivery carrier of growth factor, the culture scaffold of cell in bioreactors and delivery carrier of gene encoding growth factors.