Ceramiclike xenogeneic bone (CXB) was obtained from the fresh bone of pig ribs being treated by physical and chemical methods to deprive of its organic substance. The CXB possessed the same natural porous network system as that of the human. The CXB was cultured with the bone marrow stromal cells of rabit. When the marrow cells had integrated with the CXB, thus a new material was obtained. (CXB-BM), and was implanted sacro-spinal muscle of rabbit. The specimens were observed under phase microscope, light microscope and electronic scanning microscope. The results showed that: at the 2nd week after the implantation of CBX-BM composite material there began the new bone formation, and the rate of bone formation was increased with time. There was evident new bone formation after 24 weeks. The process of the new bone formation were quite similar to the composite graft of HAP red autogenous and marrow, but the former degraded faster and formed typical cancellous structure earlier. There was no new bone formation when CXB was implanted alone in the control. Both the mechanism of osteogenetic potential and its clinical application were discussed.
Forty-eight cases of multi-structural defects of hands were primarily repaired or reconstructed from July 1989 to 1997. The structural defects included: the defects of radial or ulnar aspect of hands involving fingers and skin, multiple fingers defects and the fingers and skin defects of whole hand. In this series, there were 32 males and 16 females with age ranged from 17 to 46 years old. The composite tissue grafts were obtained from wrap-around flap or 2nd toe skin flap of the foot. The result showed that composite 108 tissues transplantations, or 48 cases, were all survived. After a follow-up of 38.5 months (ranged from 5 months to 6 years), the grasp, pinch and opposition function of the reconstructed finger were restored, the two-point discrimination sensation was 4 mm-12 mm. Most of the patients had resumed their original works. So that the primary repair of multi-structural defects of hands by composite tissues transplantation was feasible and valuable, but thorough debridement and skilled microsurgecal technique were required.
Objective To investigate the influence of collagen on the biomechanics strength of tissue engineering tendon. Methods All of 75 nude mice were madethe defect models of calcaneous tendons, and were divided into 5 groups randomly. Five different materials including human hair, carbon fibre (CF), polyglycolic acid (PGA), human hair and PGA, and CF and PGA with exogenous collagen were cocultured with exogenous tenocytes to construct the tissue engineering tendons.These tendons were implanted to repair defect of calcaneous tendons of right hind limb in nude mice as experimental groups, while the materials without collagenwere implanted to repair the contralateral calcaneous tendons as control groups. In the 2nd, 4th, 6th, 8th and 12th weeks after implantation, the biomechanicalcharacteristics of the tissue engineering tendon was measured, meanwhile, the changes of the biomechanics strength were observed and compared. Results From the 2nd week to the 4th week after implantation, the experimental groups were ber than the control groups in biomechanics, there was statistically significantdifference (Plt;0.05). From the 6th to 12th weeks, there was no statisticallysignificant difference between the experiment and control groups (Pgt;0.05). Positivecorrelation existed between time and intensity, there was statistically significant difference (Plt;0.05). The strength of materials was good in human hair,followed by CF, and PGA was poor. Conclusion Exogenous collagen can enhance the mechanics strength of tissue engineering tendon, and is of a certain effect on affected limb rehabilitation in early repair stages.
ObjectiveTo evaluate the effects and mechanism of indoleamine 2, 3-dioxygenase (IDO) modified rat bone marrow mesenchymal stem cells (BMSCs) in composite tissue allograft rejection. MethodsBMSCs isolated from Brown Norway (BN) rats (aged, 4-6 weeks) were infected by IDO[green fluorescent protein (GFP)]-lentivirus. The high expression target gene and biological activity cell line (IDO-BMSCs) were screened. IDO mRNA and protein expressions were detected by RT-PCR and Western blot. The biological activity of IDO in supernatant was detected by measuring the amount of kynurenine generation. In mixed lymphocyte reaction system, different numbers of IDO-BMSCs mixed with responding cells (peripheral blood mononuclear cell isolated from 4-6-week-old LEWIS rats, as recipient) and stimulating cells (peripheral blood mononuclear cell isolated from BN rats, as donor), with the cells ratios of 1:5:5, 1:10:10, 1:50:50, and 1:100:100 (as experimental groups 1, 2, 3, and 4, respectively). Each reaction system was blocked by 1 mmol/L 1-methyl-tryptophan (1-MT) (IDO specific inhibitor). IDO-BMSCs mixed with responding cells (1:5) as the negative control group, responding cells mixed with stimulating cells (1:1) as positive control group; and IDO-BMSCs were cultured in RPMI 1640 medium alone as blank control group. MTT assay was used to detect the T lymphocytes proliferation at 5 days. Furthermore, GFP-BMSCs (group A), IDO-BMSCs (group B), and normal saline (group C) were infused via the tail vein of allogeneic limb transplantation rats, and graft survival time and rejection were observed in each group. ResultsThe IDO expression of BMSCs after genetic modification was higher than that before genetic modification. IDO-BMSCs could significantly improved kynurenine concentration in culture medium supernatant when compared with GFP-BMSCs (P<0.05). Before adding 1-MT, with the ratio of IDO-BMSCs to responding cells decreased, T lymphocytes proliferation rate increased in experimental groups 1, 2, and 3, showing significant differences between groups (P<0.05); there was no significant difference between experimental group 4 and the positive control group (P>0.05). After adding 1-MT, T lymphocytes proliferation rate was significantly higher than that before adding 1-MT in the other experimental groups (P<0.05) except experimental group 4 (P>0.05). In vivo, IDO-BMSCs could promote colonization in allograft, inhibit transplantation rejection, and prolong survival time of composite tissue allograft; the survival time of composite tissue allograft was (11.5±0.6) days in group A, (14.5±0.8) days in group B, and (9.0±0.3) days in group C, and it was significantly longer in group B than in groups A and C, and in group A than in group C (P<0.05). ConclusionIDO-BMSCs can promote the survival of allogeneic composite tissue grafts in rats, and its mechanism may involve in inhibition of T lymphocytes proliferation and promotion their own colonization in allograft.
Objective To evaluate the cl inical effect and the pathological characteristics of acellular allogeneic dermal matrix in repairing unstable burn scar. Methods From January 2007 to June 2008, 19 cases of unstable burn scars (24 parts) were treated, including 16 males (20 parts) and 3 females (4 parts) with a median age of 27 years (range, 3-58 years). Theinjury was caused by flame (14 cases, 18 parts), electricity (4 cases, 5 parts), and hot water (1 case, 1 part). The unstable burn scars located on hands (8 cases), forearms (2 cases), thighs (3 cases), legs (2 cases), feet (2 cases), chest (1 case), and abdomen (1 case). Scar formed for 3 months to 1 year. The area of defect varied from 7 cm × 5 cm to 22 cm × 15 cm after scar removal. Defects were covered with acellular allogeneic dermal matrix and autogenous spl it-thickness skin graft. At 6-18 months after operation, the pathological observations of the epidermis, the basal membrane, and structural components of the dermis were done. Results All wounds healed by first intention. Scar ulcer disappeared completely in 18 cases and the composite skin grafts all survived. Some bl isters occurred in 1 case and were cured after dressing changing. All patients were followed up 10 months to 2 years (18 months on average). The grafted-skin was excellent in the appearance, texture, and elasticity. The function recovered well. Only superficial scar was observed at skin donor sites. Pathological observation showed that the epidermis and the basal membrane of the skin grafts were similar to that of normal skin, and no significant difference was found in newly capillaries between them. Collagen fibers arranged regularly, and there were few inflammatory cells in the matrix. Conclusion Acellular allogeneic dermal matrix with autogenous spl it-thickness skin graft may effectivly repair the wound after removing the unstable burn scar, and its structure is similar to that of normal skin.
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 evaluate the advantages and disadvantages of vascularized free peroneal composite flaps for reconstruction of oral and maxillofacial defects. Methods From November 1999 to December 2002, 28 cases of oral maxillofacial defects were reconstructed with vascularized free peroneal composite flaps, with fibulacutaneous flap in 21 cases and with fibulamyocutaneous flap in 7 cases. Three cases received insertion of dental implants into the fibula flap. The flap size was 3.0 cm×5.5 cm to 8.0 cm ×12.0 cm; the fibula length was 5.5 cm to 16.0 cm. Results Of the 28 flaps reconstructed, 24 survived,3 necrosed partially and 1 necrosed completely. All the 5 implants survived andachieved good bone integration in 3 cases. Twenty-six cases were followed up 1-36 months with an average of 18.5 months, the facial appearance and the vocal function were satisfactory in 23 cases. Conclusion Vascularized peroneal flap has many advantages and is one of the optimal flaps for reconstruction of oral maxillofacial defects.
Objective To develop a novel porous three-dimensional scaffold and to investigate its physico-chemical properties for tissue engineering cartilage.Methods Refined 88% deacetylation degree chitosan was prepared and dissolved in 0.2 mol/L acetate acid and fully mixed with highly purified porcine type Ⅱcollagen in 0.5 mol/L acetate acid solution in a ratio of 4 to 1 (wt/wt). Freeze-drying process was employed to fabricate the composite scaffold. The construct wascross-linked by use of 1-ethyl-3(3-dimethyl aminopropyl) carbodiimide (EDC) and Nhydroxysuccinimide (NHS). A mechanical tester was utilized to determine the tensilestrength change before and after cross-linking. The microstructure was observed via scanning electron microscopy (SEM). The lysozyme degradation was performedto evaluate the degradability of the scaffold in vitro. Results A bulk scaffold with desired configuration was obtained. The mechanical test showed that the crosslinking treatment could enhance the mechanical strength of the scaffold. The SEM results revealed that the two constituents evenly distributed in the scaffold and that the matrix was porous, sponge-like with interconnected pore sizing 100250 μm. In vitro lysozyme degradation indicated that crosslinked or uncross-linked composite scaffolds had faster degradation rate than the chitosan matrix. Conclusion Chitosan and typeⅡcollagen can be developed into a porous three-dimensional scaffold. The related physico-chemical tests suggest that the composite socaffold meets requirements for tissue engineered scaffold and may serve as an alternative cellcarrier for tissue engineering cartilage.
Objective To evaluate the ability of inductive osteogenesis of allgraft demineralized bone containing basic fibroblast growth factor (bFGF/ALB) in repairing bone defect. Methods Thirty-two New Zealand white rabbits were randomly divided into four groups (groups A,B,C and D, n=8). A segmental bone defect of15 mm inlength was made on the bilateral radius respectively and the defects filled with ALB/bFGF in group A, with ALB in group B, with bFGF in group C and without any materials in group D serving as blank control. At 2, 4, 6 and 8 weeks after operation, all restored bones were evaluated by roentgenography, histological observation and Ca2+detection of osteotylus. Results The X-ray films showed that groups A and B had a little shadow of bone formation at 2 weeks, while groups C and D had transparent shadow; that group A had denser shadow and new bone formation at 4 weeks and 6 weeks, groups B and C had a little increase of shadow and group D had little shadow at fractured ends; and that group A had formation of bone bridge at 8 weeks, the new formed bone in fractured ends of group B closed with each other, the gap still existed in group C, and the defects filled with the soft tissue in group D. The Ca2+content of group A was higher than that of groups B, C and D at 4 weeks (Plt;0.05) and 8 weeks (Plt;0.01). The histological observaton showed that the degree of bone restoration of group A was superior to that of groups B, C and D. Conclusion bFGF/ALB is a good material to improve bone restoration.
Objective To compare and evaluate the capability of pure autogenous bone and the enhanced autogenous bone combined with bone morphogenetic protein in bone repair of femoral head. Methods Eighteen femoral heads of 9 dogs weredrilled by trephine, 4 mm in diameter, followed by respective implantations of autogenous bone grafting (group B) and of the enhanced autogenous bone composite, combined with bone morphogenetic protein (group C), with the selfrepair of bone defect as the control (group A). Three, six, nine weeks after the operation, radiological examination, computerized tomography, light and electronic microscopes were performed to investigate the bone healing of the defect in the femoral head. Results In group A, it could be observed that there washematoma organization and delayed woven bone formation in the 3rd week after operation, and therewas little replacement of woven bone by bone trabecula in the 9th week; in group B, the autogenous bone implanted were dead in the 3rd week and maintained in situ in the 9th week; in group C, active new bone formation, either endochondral or intramembranous ossification, was found in the 3rd week and entire repair of the bone defect by bone trabecula in the 9th week after operation. Conclusion The enhanced autogenous bone combined with bone morphogenetic protein could promote reconstruction of the bone defect in femoral head, superior to pure autogenous bone which could provide a framework for the new bone formation.