OBJECTIVE: To sum up the clinical results of bio-derived bone transplantation in orthopedics with tissue engineering technique. METHODS: From January 2000 to May 2002, 52 cases with various types of bone defect were treated with tissue engineered bone, which was constructed in vitro by allogeneous osteoblasts from periosteum (1 x 10(6)/ml) with bio-derived bone scaffold following 3 to 7 days co-culture. Among them, there were 7 cases of bone cyst, 22 cases of non-union or malunion of old fracture, 15 cases of fresh comminuted fracture of bone defect, 4 cases of spinal fracture and posterior route spinal fusion, 3 cases of bone implant of alveolar bone, 1 case of fusion of tarsotarsal joint. The total weight of tissue engineered bone was 349 g in all the cases, averaged 6.7 g in each case. RESULTS: All the cases were followed up after operation, averaged in 18.5 months. The wound in all the case healed by first intention, but 1 case with second intention. Bone union was completed within 3 to 4.5 months in 50 cases, but 2 cases of delayed union. Six cases were performed analysis of CD3, CD4, CD8, ICAM-1 and VCAM-1 before and after operation, and no obvious abnormities were observed. CONCLUSION: Bio-derived tissue engineered bone has good osteogenesis. No obvious rejection and other complications are observed in the clinical application.
Objective To study the ectopic osteogenesis and vascularization ofthe tissue engineered bone promoted by an artificial bone composite that consists of coral hydroxyapatite (CHA), 1,25-(OH)2 D3, human marrow stromal osteoblast (hMSO), and human umbilical vein endothelial cell (hUVEC).Methods After the isolation and the culture in vitro, hMSO and hUVEC were obtained. Then, hMSO (5×105/ml) and hUVEC (2.5×105/ml) were seeded at a ratio of 2∶1 onto the CHA scaffolds coated with 1,25-(OH)2 D3 (the experimental group) or onto the CHA scaffolds without 1,25-(OH)2 D3 (the control group). The scaffolds were culturedin vitro for 3 days, and then the scaffolds were implanted into the pockets that had beenmade on the backs of 18 nude mice. Then, 6 of the mice were implanted with one experimental engineered bone bilaterally; another 6 mice were implanted with onecontrol engineered bone bilaterally; the remaining 6 mice were implanted with one experimental engineered bone and one control engineered bone on each side. At4, 8 and 12 weeks after operation, the retrieved scaffolds and cells were examined by the nake eye and histology as well as by the scanning electron microscopy. The quantitative assessment of the newly-formed bone and the quantitative analysis of the newly-formed blood vessels were performed. Results The evaluationsby the histology revealed that at 4 weeks the original bone tissues grew into the scaffolds in all the groups, but significantly more newly-formed bone tissuesand newly-formed blood vessels were found in the experimental group. At 12 weeks the newly-formed bone tissues were found in all the groups, but there was a typical bone unit found in the experimental group. There was a significantly smaller amount of capillary vessels in the control group than in the experimental group at all the time points. The evaluations by the scanning electron microscopy revealed that at 4 weeks in the experimental group there were great amounts of extracelluar matrix that embedded the cells, and plenty of capillary vessels were found on the surface of the implanted bone materials and some of them grew into the materials; however, in the control group there was a smaller amount of capillary vessels although much extracelluar matrix was still found there. At 8 weeks sarciniform osteoids were found on some of the implanted materials, with much extracelluar matrix and many newly-formed capillary vessels in the experimental group; however, in the control group there were fewer capillary vessels and lower degrees of the bone maturity. The quantitative assessment of the newly-formed bone showed that the newformed bones were 3.1±0.52 in the experimental group but2.30±0.59 in the control group at 8 weeks (Plt;0.05), and 4.63±0.55 vs. 3.53±0.62 at 12 weeks. There was a significant difference at these two time points between the two groups (Plt;0.05). The quantitative analysis of the newly-formed blood vessels showed that the vascular areas were 28.74％±7.81％i n the experimental group but 19.52％±4.57% in the control group at 4 weeks (Plt;0.05), and 24.66％±7.38％ vs. 1784%±5.22% at 12 weeks. There was a significant difference at these two time points between the two groups (Plt;0.05). Conclusion 1,25-(OH)2 D3 as an active factor can increase the interaction between hMSO and hUVEC, and thus promote the ectopic osteogenesis and vascularization in the tissue engineered bone.
Objective To study the potential of a bioderived material combined with Pluronic F-127 in vitro as a delivery vehicle for WO-1 in the bone repair therapy. Methods Bio-derived materials were fabricated and loaded with WO-1 by Pluronic F-127. Micromorphology and porosity were detected by the scanning electron microscope and the digital image analysis system respectively. The WO-1 release from the system in vitro was studied by the high performance liquid chromatography. Results Bio-derived material-WO-1 drug delivery systems were created with the interconnected pore network. Theporosity and pore size of the system were 55% and 522.43±16.75 μm respectively, compared with those of bio-derived materials, which were 75% and 623.67±12.31 μm respectively. And the main composition of the system was HA. The in vitrorelease kinetics of WO-1 revealedthat an effective therapeutic concentration(0.2-0.8 μg/ml) of WO-1 was maintained for 6 days after a high initial burst release. Conclusion The bio-derived material-WO-1 drug delivery system can be used in the bone repair therapy. However, the in vivostudy on it is still needed.
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 investigate the morphology and biomechanics of in vivo osteogensis after repairing rabbit skull defects with plastic engineered bone which was prefabricated with alginate gel, osteoblasts and bone granules. Methods Twenty-eight rabbits were divided into group A (n=16), group B(n=8) and group C(n=4).The bilateral skull defects of 1 cm in diameter were made. Left skull defects filled with alginate gel-osteoblasts-bone granules(group A1) and right skull defects filled withalginate gel-bone granules(group A2).The defects of group B was left, as blank control and group C had no defect as normal control. The morphological change and bone formation were observed by methods of gross, histology and biomechanics. Results In group A1, the skull defects were almost entirely repaired by hard tissue 12 weeks after operation. The alginate gel-osteoblasts-bone granule material had changed into bone tissue with fewbone granules and some residuary alginate gel. The percentage of bone formation area was 40.92%±19.36%. The maximum compression loading on repairing tissue ofdefects was 37.33±2.95 N/mm; the maximum strain was 1.05±0.20 mm; andloading/strain ratio was 35.82±6.48 N/mm. In group A2, the alginate and bone granules material partially changed into bone tissue 12 weeks after operation. The percentage of bone formation area was 18.51%±6.01%. The maximum compression loading was 30.59±4.65 N; the maximum strain was 1.35±0.44 mm; and the loading/strainratio was 24.95±12.40 N/mm. In group B, the skull defects were mainly repaired bymembrane-like soft tissue with only few bone in marginal area；the percentage of bone formation area was 12.72%±9.46%. The maximum compression loading was 29.5±2.05 N; the maximum strain was 1.57±0.31mm；and the loading/strainratio was 19.90±5.47 N/mm.In group C, the maximum compression loading was 41.55±2.52 N; the maximum strain was 095±017 mm; and the l oading/strain ratio was 47.57±11.22 N/mm. 〖 WTHZ〗Conclusion〓〖WTBZ〗The plastic engineered bone prefabricated with algina te gelosteoblastsbone granule may shape according to the bone defects and ha s good ability to form bone tissue, whose maximum compression loading can reach 89 % of normal skull and the hardness at 12 weeks after operation is similar to that of normal skull.
Objective To study the vascularization of the compositeof bio-derived bone and marrow stromal stem cells(MSCs) in repairing goat tibial shaft defect.Methods Bio-derived bone was processed as scaffold material. MSCs were harvested and cultured in vitro. The multiplied and induced cells were seeded onto the scaffold to construct tissue engineered bone. A 20 mm segmental bone defect inlength was made in the middle of the tibia shaft in 20 mature goats and fixed with plate. The right tibia defect was repaired by tissue engineered bone (experimental side), and the left one was repaired by scaffold material (control side).The vascularization and osteogenesis of the implants were evaluated by transparent thick slide, image analysis of the vessels, and histology with Chinese ink perfusion 2, 4, 6, and 8 weeks after operation.Results More new vessels were found in control side than in experimental side 2 and 4 weeks after implantation (Plt;0.05). After 8 weeks, there was no significant difference in number of vessels between two sides(Pgt;0.05), and the implants were vascularized completely. New bone tissue was formed gradually as the time and the scaffold material degraded quickly after 6 and 8 weeks in the experimental side. However, no new bone tissue was formed andthe scaffold degraded slowly in control side 8 weeks after operation.Conclusion Bio-derived bone has good quality of vascularization. The ability of tissue-engineered bone to repair bone defect is better than that of bio-derived bone alone.
To summarize the medium-term cl inical result of bio-derived bone transplantation in orthopedics with tissue engineering technique. Methods From December 2000 to June 2001, 10 cases of various types of bone defect were treated with tissue engineered bone, which was constructed in vitro by allogenous osteoblasts from periosteum (1 × 106/ mL) with bio-derived bone scaffold following 3 to 7 days co-culture. Six men and 4 women were involved in this study, aged from 14 to 70 years with a median of 42 years. Among them, there were 2 cases of bone cyst, 1 case of non-union of old fracture, 6 cases of fresh comminuted fracture with bone defect, and 1 case of chronic suppurative ostemyel itis. The total weight of tissue engineered bone was 3-15 g in all the cases, averaged 7.3 g in each case. Results The wound in all the case healed by first intention. For 7 year follow up, bone union was completed within 3.0 to 4.5 months in 9 cases, but loosening occurred and the graft was taken out 1 year after operation in 1 case. The X-ray films showed that 9 cases achieved union except one who received resection of the head of humerus. No obvious abnormities were observed, and the function of affected l imbs met daily l ife and work. Conclusion Bio-derived tissue engineered bone has good osteogenesis. No obvious rejection and other compl ications are observed in the cl inical appl ication.
Objective To evaluate repair of critical-sized cranialdefect with tissue engineered bone fabricated by coral, bone mesenchymal stem cells(MSCs) and sustainedly released recombinant human bone morphogenetic -protein 2 (rhBMP-2) by collagen. Methods Three scaffolds of rhBMP-2+coral,collagen+rhBMP-2+coral and MSCs+collagen+rhBMP-2+coral were fabricated. Forty New Zealand rabbits were made the models of critical-sized defects and divided into5 groups according to different implants: group Ⅰ, auto-ilium; group Ⅱ,coral; group Ⅲ, rhBMP-2+coral; grop Ⅳ, collagen+rhBMP-2+coral; and group Ⅴ,MSCs+collagen+rhBMP-2+coral. Repair of bone defect was evaluated after 8 and 16 weeks of implantation by gross obeservation, X-ray,HE staining and Masson’s trichrome staining. Results Repair ofbone defect in group Ⅴ was similar to that in group Ⅰ, andwas better than that in group Ⅳ; and group Ⅲ was worse. The gross appearance showed that defect region filled with bony tissue which had similar strength to adjacent bone and formed bone union with surrounding bone. The X-ray result displayed high radiopacity(80.45%±2.52% in the 16thweek). Histological observation showed new lamellar bone tissue and with few pore blank area. However, only transpasent fibrous tissue filled the defect in group Ⅱ. Conclusion Collagen may be a suitable sustained release system for rhBMP-2. And MSCs may have important effect on enhancing repair of bone defect. Tissueengineered bone fabricated by MSCs+collagen+rhBMP-2+coral may be a useful material for bone defect repair.
Objective To explore the histological changes of bio-derived bone prepared by different methods after implantation, and to provide the scaffold material from xenogeneic animal for tissue engineering. Methods Theextremities of porcine femur were cut into 0.5 cm×0.5 cm×0.5 cm. Then they were divided into 5 groups according to different preparation methods: group A was fresh bone just repeatedly rinsed by saline; group B was degreased; group C was degreased and decalcificated; group D was degreased, acellular and decalcificated; group E wasdegreased and acellular. All the materials were implantated into femoral muscle pouch of rabbit after 25 kGy irradiation sterilization. The cell counting ofinflammatory cells and osteoclasts, HE and Masson staining, material degradation, collagen and new bone formation were observed at 2, 6, and 12 weeks postoperatively. Results The residue level of trace element in biomaterials prepared by different methods is in line with the standards. All the animals survived well. There were no tissue necrosis, fluid accumulation or inflammation at all implantation sites at each time point. The inflammatory cells counting was most in group A, and there was significant difference compared with other groups(P＜0.05). There was no significant difference in osteoclasts counting among all groups. For the index of HE and Masson staining, collagen and new bone formation, groups C and D were best, group E was better, and groups A and B were worse. Conclusion The degreased, acellular and decalcificated porcine bone is better in degradation,bone formation, and lower inflammatory reaction, it can be used better scaffold material for tissue engineered bone.
Objective To investigate bone regeneration of the cell-biomaterial complex using strategies of tissue engineering based on cells.Methods Hydroxyapatite/collagen (HAC) sandwich composite was produced to mimic the natural extracellular matrix of bone, with type Ⅰ collagen servingas a template for apatite formation. A three-dimensional ploy-porous scaffoldwas developed by mixing HAC with poly(L-lactic acid) (PLA) using a thermally induced phase separation technique (TIPS). The rabbit periosteal cells were treated with 500 ng/ml of recombinant human bone morphogenetic protein 2(rhBMP-2), followed by seeded into pre-wet HAC-PLA scaffolds. Eighteen 3-month nude mice were implanted subcutaneously cell suspension (groupA, n=6), simple HAC-PLA scaffold (group B, n=6) and cell-biomaterial complex(group C, n=6) respectively.Results Using type Icollagen to template mineralization of calcium and phosphate in solution, we get HAC sandwich composite, mimicking the natural bone both in compositionand microstructure. The three dimensional HAC-PLA scaffold synthesized by TIPShad high porosity up to 90%, with pore size ranging from 50 μm to 300 μm. SEMexamination proved that the scaffold supported the adhesion and proliferation of the periosteal cells. Histology results showed new bone formation 8 weeks after implantation in group C. The surface of group A was smooth without neoplasma. Fibrous tissueinvasion occured in group B and no bone and cartilage formations were observed.Conclusion The constructed tissue engineering bone has emerged as another promising alternative for bone repair.