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 evaluate the effects of composite bone in strategy of tissue engineering on bone defect repair in rats. Methods Sixteen matured Wistar rats (male or female, weighing 250-300 g) were used to prepare platelet lysate (PL). PL/allogeneic decalcified bone granules (ADBG)/Col I (PAC) and ADBG/Col I (AC) were prepared by mixing Col Igel ADBG with or without PL. BMSCs of 8 Wistar rats (male or female, weighing 250-300 g) were isolated and cultured. The 5th passage of BMSCs were co-cultured with PAC at the density of 1 × 106 cells/mL to fabricate the tissue engineered composite PACB in vitro. Forty healthy Wistar rats were made bilateral bone defects in femoral condyles and divided into 4 groups (A, B, C and D, n=10). The defects were filled with equivalent PACB, PAC, AC and Col I in groups A, B, C and D respectively. At 4 weeks, the defect repair was evaluated with radiology, histology, ALP biochemical tests. Results At 4 weeks, the bone density measurement was (7.31 ± 0.54), (4.36 ± 0.67), (2.12 ± 0.47), and (1.09 ± 0.55) pixels in groups A, B, C, and D, respectively. The area of new bone formation in defect area under single view was (412.82 ± 22.31), (266.57 ± 17.22), (94.34 ± 20.22), and (26.12 ± 12.51) pixels in groups A, B, C and D respectively. The ALP contents in femoral condyles were (94.31 ± 7.54), (69.88 ± 4.12), (41.33 ± 3.46), and (21.03 ± 3.11) U/L, respectively. The above indexes of group A were significantly higher than those of groups B, C or D (P lt; 0.05). Three-color flow cytometry assay showed that the T lymphocyte subsets of CD3+CD4+CD8-, CD3+CD8+CD4-, and the ratio of CD4/CD8 displayed no significant difference among four groups (P gt; 0.05). Conclusion Tissue engineered bone PACB is capable to promote the bone defect repair.
Objective To evaluate the cl inical results of allogeneic bone graft for interbody fusion in cervical tuberculosis. Methods Between January 2000 and January 2008, 30 cases of cervical tuberculosis were treated with allogeneic (group A, n=15) or autologous (group B, n=15) il iac crest bone graft combined with anterior fixation after radical debridement. In group A, there were 8 males and 7 females with an average age of 38 years; the disease duration was 6 to 14 months; the preoperative kyphosis Cobb angle was (8.6 ± 11.3)°; the preoperative Japanese Orthopaedic Association (JOA) score was 13.0 ± 3.1 for neurological function; and the length of bone graft was 32 mm on average. In group B, there were 9males and 6 females with an average age of 42 years; the disease duration was 4 to 17 months; the preoperative kyphosis Cobb angle was (4.9 ± 7.4)°; the preoperative JOA score 12.3 ± 4.2; and the length of bone graft was 34 mm on average. There was no significant difference in general data between 2 groups (P gt; 0.05). Results The operation time and bleeding volume in group A were significantly less than those in group B (P lt; 0.05). Wound effusion were found in 2 cases of group A, and the other incisions healed by first intention. No infection occurred in group B. In group A, 13 cases were followed up 12-48 months; in group B, 14 cases were followed up 13-46 months. The time of bone graft heal ing in group A [(7.6 ± 2.1) months] was significantly longer than that in group B [(4.2 ± 1.1) months] (t=2.773, P=0.005). The kyphosis Cobb angles were significantly improved at 6 months and last follow-up after operation in 2 groups when compared with that before operation (P lt; 0.05), but no significant difference was found between 2 groups at different time after operation (P gt; 0.05). There was no significant difference in JOA score at 6 months after operation between group A (14.1 ± 2.6) and group B (14.3 ± 2.4) (t=1.655, P=0.162). The improvement rate for neural function were 83.7% in group A and 87.8% in group B, showing no significant difference (χ2=3.150, P=0.071). There was no loosening of internal fixation and recurrence of tuberculosis in 2 groups during follow-up. Five cases had chronic pain at il iac donor sites in group B. According to Bridwell et al. evaluation standard, the bone fusion was satisfactory in 11 cases (84.6%) and unsatisfactory in 2 cases (15.4%) in group A, and was satisfactory for all in 14 cases (100%) in group B. The satisfactory rate of bone fusion showed no significant difference between 2 groups (χ2=2.680, P=0.115).Conclusion Allogeneic bone grafting has a good cl inical result for spinal fusion in cervical tuberculosis surgery, which can treat tuberculosis bone defect effectively.
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 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.
Objective To explore the clinical application of allogeneic bone transplantation. Methods Related literatures on clinical application of various allograft bone materials of the past 5 years were extensively reviewed. Results The allografts could be processed into various kinds of transplanting materials, such as small blocks, large segments, morselised or granular material, and bone ring, so that they could be applied to bone defect filling, prosthesis revision, limb preservation after tumor resection, and joint fusion. All these applications ended up with good clinical results. However, complications resulted in thestructural transplantation of large block bones could be overlooked. Conclusion The main advantage of bone allograft is that it can be integrated with host bone and be remodelled, so as to reconstruct bone structure and function. How to accelerate their integration and remodelling while exerting normal functions is the problem that requires solution.
Objective To investigate the adhesiveness of osteoblasts and vascular endothel ial cells from rat BMSCs co-cultured on allogeneic freeze-dried partially bone in vitro. Methods The BMSCs were isolated from 4-week-old SD rats (weighing 100-110 g) and cultured in vitro. The third generation of BMSCs were induced into osteoblasts and vascular endothel ial cells. The osteoblasts and vascular endothel ial cells after being induced for 7 days in a ratio of 1 to 1 were directlyco-cultured (experimental group), while the second generation of uninduced BMSCs was used as a control (control group). The growth and prol iferation abil ity were analyzed by MTT examination and the growth curve was drawn at 1-8 days. The osteoblasts and vascular endothel ial cells after being induced for 14 days were implanted in the allogeneic freeze-dried partially bone coated by 20% Col I or not at different densities (0.25 × 106/mL、0.50 × 106/mL、1.00 × 106/mL、2.00 × 106/mL、4.00 × 106/mL), as modified group and unmodified group, the cell adherence rate was calculated after 24 hours. These two kinds of cells were implanted in the pre-disposal treated allogeneic freeze-dried partially bone and observed by scanning electron microscope. Results ALP staining of osteoblasts showed that there were blue grains in cytoplasm at 7 days. CD31 and CD34 immunocytochemical staining of vascular endothelial cell showed that there were positive signals in the cytoplasm at 14 days. The MTT test showed that the prol iferation level of the experimental group was lower than those of the control group. There were significant differences in absorbance value between two group from 3 days to 8 days (P lt; 0.05). The cell adherence rate increased with increasing seeding density when the seeding density was (0.25-1.00) × 106/mL. The cell adherence rate reached the peak when the seeding density was 1.00 × 106/mL. The cell adherence rate decreased when the seeding density was more than 2.00 × 106/mL. There were significant differences in cell adherence rate between modified group and unmodified group at different seeding densities (P lt; 0.05). The prol iferation of the osteoblasts and endothel ial cells presented better growth and histocompatibil ity under scanning electron microscope. Conclusion The growing behavior of two kinds of cells is good in the allogeneic freezedried partially bone coated by 20% Col I , which can be used in reconstrction of vascularized tissue engineered bone.
Objective To investigate the clinical application and complicationof the lyophilized small-segment allogeneic bone used in repairing bone defectscaused by benign bone tumor and tumor-like lesions after resection and curettage. Methods From December 1999 to December 2005, 230 patients (156 males, 74 females; age, 5-56years, averaged 32.8 years), who had bone defects caused by benign bone tumors and tumor-like lesions after surgical resection and curettage, were treated by the lyophilized small-segment allogeneic bones. The cavities left by the tumor curettage ranged in size from 1.0 cm×0.8 cm to 10.0 cm×2.0 cm, andthe bone defects were about 1.0 to 3.5 cm in diameter after the localized resection of the bones. According to the bone defect degrees, the autogenous nonvascular iliac bone and the bone allograft (0.5-30.0 g) were implanted, followed by the drainage for 2-3 days and the use of antibiotics to prevent infection. The postoperative systemic and local reactions were observed, and the regular X-ray examinations were performed to observe the bone union. ResultsThere was no significant difference in the allergic reactions, such as postoperative temperature, drained amount, and body swelling, between this kind of transplantation and the autogenous bonetransplantation. The wounds in 196 patients were healed by the first intension.The wounds in 34 patients had extravasate. Among them, the wound was healed by changing dressing in 30cases; the wound had delayed healing in 4, including 3 whose wounds were healedby changing dressing for 3-4 weeks,and 1 whose wound was healed by taking out the implants. The follow-up for 6-60 months (average, 38 months) revealed that all the patients had the allograft unions of the bones within 6-18 months after the transplantation, and only 6patients had recurrence of the tumor (3.0%). Evaluated by the Mankin,Komender and WANG Zhiqiang’s standards, 196 (85.22%) patients were satisfied with their outcomes while the other 34 (14.78%) patients were not satisfied. Conclusion The lyophilized small-segment allogeneic bone has a good compatibility and osteogenesis, when it is used in repairing bone defects caused by benign bone tumor and tumor-like lesions after resection and curettage. So, this kind of bone is a good, convenient and safe material for the bone transplantation. The important factors affecting the allograft union are as follows: the mechanical stability in the recipient region, local blood supply, and management of the bone cavity left by resection and curettage of the bone tumor.
Objective To investigate the efficacy of the pedicle screw fixation and allograft bone in posterior spinal fusion for the treatment of thoracolumbar vertebral fractures. Methods From September 2006 to March 2008, 105 cases of thoracolumbar vertebral fractures were treated with allograft bone in posterior spinal fusion after the pedicle screw fixation,including 75 males and 30 males aged 15-65 years (mean 37 years). The mixture which consisted of spinous process and vertebral plate sclerotin and homogeneity variant bone was used as bone graft to implant into articular process and processus transversus space or vertebral plate space. The time from injury to surgery varied from 8 hours to 21 days, with an average of 3 days. There were 52 cases of fall ing injuries from height, 35 cases of traffic accident injuries, 11 cases of bruise injuries and 7 cases of tumbl ing injuries. Before operation, the primary cl inical symptoms of patients included local pain combined with l imitation of activity, 30 cases compl icated by various degrees of spinal cord and nerve root functional disturbance. According to Mcaffee classification, there were 7 cases of flexion depressed fractures, 86 cases of blow-out fractures, 9 cases of Chance fractures and 3 cases of dislocation-fracture. According to Frankel grade, there were 11 cases of grade A, 2 cases of grade B, 7 cases of grade C, 10 cases of grade D and 75 cases of grade E. The X-ray examination of all patients denoted that the bodies of injuryed vertebra were compressed and wedge-shaped, and the CT scan showed that 98 cases had spinal stenosis. After 2 weeeks and 3, 6, 12 months, the X-ray films were taken to evaluate bone graft fusion. The Cobb angle was measured. The recovery of nerve function was analyzed. Results The operation time was 55-180 minutes (mean 90 minutes) and the blood loss was 100-900 mL (mean 200 mL). All patients achieved heal ing by first intention with no compl ication. After operation, 93 cases were followed up for 6-15 months with an average of 11 months. Except for 11 patients who were at grade A before operation, one to two grade recover was observed in other patients. The average Cobb angle of injury segment was improved from preoperative 32.1° to postoperative 5.2°. The height of anterior border of injuried vertebral body was recovered from the preoperative average compressed remaining height 61.5% to postoperative 96.8%. The vestigial degree of canal is spinal is anteroposterior diameter was recovered from preoperative 65.7% to postoperative 89.9%. Imageology examination showed that all the patient achieved bone union within 6 months. The fusion rate of bone graft in spinal fusion was 100%. No loosening and breaking of nails occurred. Conclusion Pedicle fixators can restore and fix the thoracolumbar fractures, and the combination of autograft and allograft bone transplantation is a safe, rel iable and effective method.
【Abstract】 Objective To investigate the impact of dermal papillary cells on vascularization of tissue engineered skinsubstitutes consisting of epidermal stem cells and allogeneic acellular dermal matrix. Methods Human foreskins from routinecircumcisions were collected to separate epidermal cells by using dispase with trypsogen. Collagen type IV was used to isolateepidermal stem cells from the 2nd and 3rd passage keratinocytes. Dermal papilla was isolated by the digestion method of collagenaseI from fetus scalp and cultured in routine fibroblast medium. Tissue engineered skin substitutes were reconstructed by seedingepidermal stem cells on the papillary side of allogeneic acellular dermis with (the experimental group) or without (the controlgroup) seeding dermal papillary cells on the reticular side. The two kinds of composite skin substitutes were employed to cover skindefects (1 cm × 1 cm in size) on the back of the BALB/C-nu nude mice (n=30). The grafting survival rate was recorded 2 weeks aftergrafting. HE staining and immunohistochemistry method were employed to determine the expression of CD31 and calculate themicrovessel density at 2 and 4 weeks after grafting. Results Those adhesion cells by collagen type IV coexpressed Keratin 19 andβ1 integrin, indicating that the cells were epidermal stem cells. The cultivated dermal papillary cells were identified by expressinghigh levels of α-smooth muscle actin. The grafting survival rate was significantly higher in experimental group (28/30, 93.3%), thanthat in control group (24/30, 80.0%). HE staining showed that the epithelial layer in experimental group was 12-layered with largeepithelial cells in the grafted composite skin, and that the epithelial layer in control group was 4-6-layered with small epithelial cells.At 2 and 4 weeks after grafting, the microvessel density was (38.56 ± 2.49)/mm2 and (49.12 ± 2.39)/mm2 in experimental group andwas (25.16 ± 3.73)/mm2 and (36.26 ± 3.24)/mm2 in control group respectively, showing significant differences between 2 groups(P lt; 0.01). Conclusion Addition of dermal papillary cells to the tissue engineered skin substitutes can enhance vascularization,which promotes epidermis formation and improves the grafting survival rate.