OBJECTIVE: To construct tissue engineering bone with bio-derived materials and bone marrow stromal cells (MSCs), and to investigate the effect of allogeneic engineering bone implants on healing of segmental bone defects. METHODS: MSCs being aspirated aseptically from tibial tuberosities of young rhesus monkeys were induced into osteoblasts in vitro and then were cultured and marked with 5-bromo-2-deoxyuridine (BrdU). Tissue engineering bones were constructed with these labeled osteoblasts being seeded onto bio-derived materials made from fresh human bones which were treated physically and chemically, Then the constructs were implanted in 15 allogeneic monkeys to bridge 2.5 cm segmental bone defects of left radius as experimental groups, bio-derived materials only were implanted to bridge same size defects of right radius as control group. and, 2.5 cm segmental bone defects of both sides of radius were left empty in two rhesus monkeys as blank group. Every 3 monkeys were sacrificed in the 1st, 2nd, 3rd, 6th and 12th weeks postoperatively and both sides of the implants samples were examined macroscopically, histologicaly, and immunohistochemicaly. The two monkeys in blank group were sacrificed in the 12th week postoperatively. RESULTS: Apparent inflammatory reactions were seen around both sides of the implants samples in the 1st, 2nd, 3rd weeks, but it weakened in the 6th week and disappeared at the 12th week. The labeled osteoblasts existed at the 6th week but disappeared at the 12th week. The bone defects in experimental group were repaired and the new bone formed in multipoint way, and osteoid tissue, cartilage, woven bone and lamellar bone occurred earlier when compared with control group in which the bone defects were repaired in ’creep substitution’ way. The bone defects in blank group remained same size at the 12th week. CONCLUSIONS: Engineering bones constructed with bio-derived materials and MSCs were capable of repairing segmental bone defects in allogeneic monkeys beyond ’creep substitution’ way and making it healed earlier. Bio-derived materials being constituted with allogeneic MSCs may be a good option in construction of bone tissue engineering.
Objective To give a prel iminary experimental evidence and to prove chitosan and allogeneic morsel ized bone as potential bone substitutions in repairing rabbit radius segmental defect. Methods Chitosan and allogeneic morsel ized bone were mixed with various ratios (1 ∶ 5, 1 ∶ 10, 1 ∶ 25, 1 ∶ 50, and 1 ∶ 100). After preparation, the physicaland chemical properties of the composites were prel iminary detected; the composites at the ratios of 1 ∶ 50 and 1 ∶ 25 had good physical and chemical properties and were used for the animal experiment. The radius segmental defects of 15 mm in length were made in 50 adult New Zealand white rabbits (weighing 2.5-3.0 kg), then the animals were divided into 2 groups. In groups A and B, chitosan/allogeneic morsel ized bone composites were implanted at the ratio of 1 ∶ 50 and 1 ∶ 25, respectively. After 1, 2, 4, 8, and 12 weeks of operation, the gross, histological, immunohistochemical observations were performed. Before the rabbits were sacrified, X-ray films were taken; the serum calcium and alkal ine phosphatase (ALP) concentration were measured; and the biomechanical measurement was carried out at 12 weeks. Results The results of gross observation were essentially consistent with those of the X-ray films. The histological observation showed that the bone formation was earl ier in group A than in group B; the amount of new bone formation in group A was more than that in group B; and the bone forming area in group A was bigger than that in group B (P lt; 0.05) at 4 and 8 weeks after operation. The immunohistochemical staining showed that vascular endothel ial growth factor and insul in-l ike growth factor receptor II proteins expressed in the cytoplasm of 2 groups after 4 and 8 weeks, and the expression in group A was higher than that in group B (P lt; 0.05). There was no significant difference in the serum calcium concentration between 2 groups at each time point (P gt; 0.05). After 4 and 8 weeks, the ALP concentration in group A was significantly higher than that in group B (P lt; 0.05). After 12 weeks, the radius maximum bending loads of groups A and B were (299.75 ± 27.69) N and (278.54 ± 17.09) N, respectively, showing significant difference (t=4.045,P=0.002). Conclusion The composite of chitosan and allogeneic morsel ized bone has good osteogeneic activity and can beused as a bone tissue engineering scaffold, and the optimum ratio of chitosan to allogeneic morsel ized bone was 1 ∶ 50.
Objective To explore the feasibility of allogeneic marrow stromal stem cells(MSCs) as seed cells to construct tissue engineered bone bydetecting the expressions of interleukin 2(IL-2) and IL-2 receptor in rhesus monkeys after implanting these tissue engineered bones.Methods Engineered bones were constructed with osteoblasts which derived from allogeneic MSCs and bio-derived materials in vitro, and then were implanted to bridge 2.5 cm segmental bone defects of left radius in 15 rhesus monkeys as experimental group, bioderived materials only were implanted to bridge same size defects of right radius as control group. Every 3 monkeys were sacrificed in the 1st, the 2nd, the 3rd, the 6th andthe 12th weeks postoperatively and the expressions of IL-2 and IL-2 receptor in blood and graft samples were detected quantitatively by enzymelinked immuneosorbent assay (ELISA).Results There was no significant difference in the contents of IL-2 and its receptor between 2 groups（P＞0.05）. The contents ofIL-2 and its receptor increased from the 2nd week and maintained high level from the 2nd to the 6th week, but decreased after 6 weeks.ConclusionTissue engineered bones constructed with allogeneic MSCs and bio-derived materials show low immunogenicity. Allogeneic MSCs may be used as seed cells to construct tissue engineered bone.
OBJECTIVE: To observe the difference of the fracture reparation using autogeneic-iliac bone and allogenic bone. METHODS: Comminuted fracture of humerus in two sides were made in rabbits. Autogeneic-iliac bone was implanted in one side, while allogenic bone of equal capacity was implanted in the other side. General observation, X-ray, and HE histologic section were done when the rabbits were put to death in different stages. RESULTS: One week after implantation, the graft had been enclosed by connective tissue without infiltration of the inflammatory cells. At the 2nd week, the graft had been enclosed in osteoplastic granulation tissue, and the cartilage callus had formed. At the 3rd week, there had been broken sequestrum among the callus; the cartilage had actively formed the bone; and the medulla had been making. At the 4th week, the sequestrum had disappeared, and the mature callus had appeared; the osteoblasts had arranged in a line around the edge of the mature callus. At the 5th week, the callus was b, compact and approached mature bones. At the 6th week, there had been the compact lamellar structures and the complete haversian’s systems. There was no significant difference between callus of two sides by using image quantitative analysis in the 3rd, 4th week (P gt; 0.05). CONCLUSION: The allogenic bone has good histocompatibility and bone conduction effect, and can be used for bone transplantation substitute with autogenous-iliac bone.
Objective To study the repair and function reconstruction of complex soft tissue defect of posterior of hand and forearm. Methods From May 2001 to November 2003, 8 cases of soft tissue defect of posterior of hand and forearm were repaired with thoracico abdominal flaps with hilum for primary stage. The tendon transplantation and allogeneic tendon function reconstruction of hand were performed for secondary stage. The range of the flap was 9 cm×15 cm to 12cm×38 cm. Allogeneic tendon amounted to 6.Results All the flaps survived. The flap countour was good. The results of allogeneic tendon transplantation were satisfactory and the function of hand was good. Conclusion Repairing complex soft tissue defect of posterior of hand and forearm and reconstructing hand function by use of thoracico abdominal flaps with hilum and transplantation of allogeneic tendon have the satisfactory clinical results.
Objective To study the effect of allogeneic canine cord blood mesenchymal stem cells（cbMSC）transplantation on the distribution of CD4+T and CD8+T in infracted area of hearts. Methods Mononuclear cells of cord blood were isolated by density gradient centrifugation and amplified by adherent culture. 36 adult male dogs were divided into experimental group and control group. Animal models of acute myocardial infarction were established by ligating anterior descending coronary artery. The fourth generations of mesenchymal stem cells (MSC) were transplanted into infarcted area of hearts by left anterior descending coronary artery after 72h induced by 5-aza and transfected by LacZ. The survival of transplanted cells in hearts can be confirmed by βgal expression. CD4+T and CD8+T cells distributed in infarcted area were detected by immunohistochemical staining method. The ImagePlus 5.1 software was used to analyze the images. Results Cells transplanted into infarcted area could survive for a long time. 2, 4, 8 weeks after transplantation, the IOD of CD4+T in experimental group were 44.35±7.03, 19.29±4.11 and 20.27±3.51 respectively, and the CD4+T/CD8+T ratios were 0.63±0.12, 0.51±0.15 and 0.66±0.08. In control group, the IOD of CD4+T at 2, 4, 8 weeks after transplantation were 65.78±10.27, 28.02±2.59, 29.79±6.83, and the CD4+T/CD8+T ratios were 1.28±0.20, 1.34±0.09 and 1.50±0.16. The IOD of CD4+T and CD4+T/CD8+T ratio in experimental group were significantly lower than that in control group. In experimental group the IOD of CD8+T at 2, 4, 8 weeks after transplantation were 69.88±7.84 , 37.80±8.83 and 30.81±7.42, higher than that in control group which were 51.28±10.01, 20.87±4.50 and 19.91±2.87. Conclusion The preliminary results indicated that allogeneic cbMSC transplanted in infarcted area can escape from immune rejection, its mechanism may be associated withdecreasing the amount of CD4+T cells infiltrated in periphery of infarcted area and maintaining CD4+T/CD8+T ratios at a lower level.
Objective To investigate the clinical results of allograft and sural neurovascular flap in repairing calcaneus and skin defects.Methods From February 1996 to December 2002, allograft and sural neurovascular flap were used to repair calcaneusand skin defects in 6 cases. The causes included road accident in 3 cases, strangulation in 2 cases and crashing object in 1 case. The defect locations were at theback of the calcaneus( 1/3, 1/2 and 2/3 of calcaneus in 3 cases, 2 cases and 1case respectively). The flap area ranged from 6 cm×7 cm to 12 cm×17 cm. Results The flaps survived completely in 4 cases; the distal flaps necrosed partly in 2 cases and the wound healed by dressing. The postoperative X-ray films showed that the repaired bone and joint had normal position and the arcus plantaris recovered. After a follow upof 6 months to 3 years all the patients were achieved bone union in allograft and had no complications of absorption, infection and repulsion. The weightbearing and walking functions were restored and the injured foot obtained a satisfactory contour. After 36 months of operation, the sensory recovery of foot occurred. Conclusion The used-allograft iseasy to be obtained and arcus plantaris is easy to recover. The reversesural neurovascular- flap in repairing calcaneus and skin defects has the following advantages: the maintenance of blood supply for injured foot, the less dangerous operation, the simple procedure, the recovery of walking function, and the good appearance and sensation.
Objective To investigate the expression levels and significance of vascular endothel ial growth factor (VEGF) and microvessel density (MVD) in rabbit radius defects repaired with allogeneic and autogenic bone. Methods Forty adult New Zealand rabbits were chosen, and 10 mm bone defect model was created in the bilateral radii of 28 experimental rabbits. The other 12 rabbits provided allogeneic bone under the standard of American Association of Tissue Bank. In the left side, allogeneic bone were used to repair bone defect (experimental group), equal capacity autogenous il iac bone was used in the right side (control group). Animals were sacrificed at 2, 4, 8, and 12 weeks postoperatively. Immunohistochemical method was used to determine the expression of VEGF, CD34 protein and MVD counting. Bone histomorphometric parameters, including percent trabecular area (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) were measured by von Kossa staining undecalcified sl ices. The relation was analyzed between VEGF and MVD, histomorphometric parameters. Results The positive signals of VEGF protein were detected in cytoplasm of vascular endothel ial cells, chondrocytes, osteoblasts, fibroblasts and osteoclasts. At 2 weeks, there was no significant difference in VEGF protein expression between experimental group and control group (P gt; 0.05); at 4 and 8 weeks, the expression of VEGF in control group was significantly higher than that in experimental group (P lt; 0.05); and at 12 weeks, there was no significant difference between two groups (P gt; 0.05). There was a positive correlation (P lt; 0.01) between VEGF expression and MVD value in two groups at 2, 4, 8, and 12 weeks postoperatively. There was no significant difference in bone histomorphometric parameters (BV/TV, Tb.Th, Tb.N, Tb.Sp) between two groups at 12 weeks postoperatively (P gt; 0.05), but there was a positive correlation between VEGF expression and parameters of BV/TV, Tb.Th, and Tb.N (P lt; 0.01); and a negative correlation between VEGF and Tb.Sp (P lt; 0.01). Conclusion VEGF can express diversity at different time and positions, and the different expressions indicated various biology significances in the process of the bone heal ing. It can coordinate growth of cartilage and bone and profit vascular reconstruction of allogeneic bone. VEGF may participate in promoting osteogenesis in the course of allogeneic bone transplantation.
Objective To evaluate the immunological reaction and the outcome of allogeneic chondrocyte transplantation in repairing articular cartilage defects in porcins. Methods Full articular cartilage from the knee of two Shanghai white porcins about one-month-old was removed and cut mechanically, digested by 0.25% trypsin and 0.2% type Ⅱ collagenase and cultured in 10％ DMEM medium. Defects of 0.5 cm×0.5 cm involving the subchodral bone were created in both the left and right femur condyloid in 8 two-month-old Yunnai bama porcins. Allogeneic chondrocyte transplantation were implanted in defects at a density of (1.0-2.0)×106,0.2 ml. The lymphocytes from the receivers’ blood were collected before transplantation and after 3, 5, 7 and 12 weeks of transplantation, then mixed with allogeneic chondrocytes to determin the lymphocyte stimulation index(SI) in vitro. The histological observation in vivo was made after 5, 7 and 24 weeks of transplantation. Results Lymphocyte SI at 3, 5, 7 and 12 weeks(1.457±0.062,1.739±0.142,1.548±0.047，1.216±0.028) after transplantation was higher than that before transplantation(1.102±0.034，Plt;0.05). SI began to increase in the 3rd week and reached the peak value in the 5th week, then gradually declined at the 7th and 12th weeks, showing significant differences when compared with in the 5th week (Plt;0.05). Inflammation and lymphocytes infiltration could be seen in subchondral bone and the intergration area between repair tissue and normal cartilage in the 5th week, and then decreased and limited in subchondral bone in the 7th week. Defects were filled with cartilage tissue, which had good intergration with subchondral bone at 24 weeks after transplantation. Conclusion Immunological reactions can be found at early stage of allogeneic chondrocyte transplantation and then decreased with the time, the fullthickness articular cartilage defects could be repaired mainlywith hyaline cartilage by the allogeneic chondrocyte transplantation. This may provide a new method to repair articular cartilage defects clinically.
Objective To investigate the value of computer-aided design (CAD) in defining the resection boundary, reconstructing the pelvis and hip in patients with pelvis tumors. Methods Between November 2006 and April 2009, 5 cases of pelvis tumors were treated surgically using CAD technology. There were 3 males and 2 females with an average age of 36.4 years (range, 24-62 years). The cause was osteosarcoma, giant cell tumor of bone, and angiosarcoma in 1 case, respectively,and chondrosarcoma in 2 cases. According to the Enneking system for staging benign and mal ignant musculoskeletal tumors, regions I, I + II, III, IV, and I + IV is in 1 case, respectively. According to the principle of reverse engineering, 5 patients with pelvis tumors were checked with lamellar CT/MRI scanning, whose two-dimensional data were obtained in disease area. The three-dimensional reconstruction of pelvic anatomical model, precise resection boundary of tumor, individual surgical template, individual prosthesis, and surgical simulation were precisely made by computer with CAD software. Based on the proposal of CAD, the bone tumor was resected accurately, and allograft il ium with internal fixation instrument or allogeneic il ium with personal ized prosthetic replacement were used to reconstruct the bone defect after tumor was resected. Results The operation was successfully performed in 5 cases. The average operation time was 7.9 hours, and the average blood loss was 3 125 mL. Hemorrhage and cerebrospinal fluid leakage occurred in 1 case, respectively, and were cured after debridement. Five patients were followed up from 24 to 50 months (mean, 34.5 months). All patients began non-weight bearing walk with double crutches at 4-6 weeks after operation, and began walk at 3-6 months after operation. Local recurrence developed in 2 patients at 18 months after operation, and resection and radiotherapy were performed. According to International Society of Limb Salvage criteria for curative effectiveness of bone tumor l imb salvage, the results were excellent in 2 and good in 3. Conclusion The individual surgical template, individual prosthesis, and surgical simulation by CAD ensure the precision and rel iabil ity of pelvis tumors resection. The CAD technology promotes pelvis tumor resection and the reconstruction of pelvis to individual treatment stage, and good curative effectiveness can be obtained.