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 investigate bio characteristics of bone stromal cells (MSC) in different concentrations of alginate combined with xenograft. Methods The configuration and secretion of MSC in different concentrations of alginate combined with xenograft were observed by scanning electron microscope and inverted microscope. Results When the concentration of alginate was 0.25% or 1%, alginate was equally combined in xenograft, 4% and 8% only on the surface of xenograft. After cultured for 4 days, alginate of 0.25# came off from xenograft. But alginate of 1% was equally combined in xenograft with cell secreting well in alginate. The growth of cells in alginate of 4% was restricted and no cell was seen in alginate of 8%. Conclusion Alginate of 1% is suitable fro constructing carrier of tissue engineering bone.
ObjectiveTo investigate the bone regeneration potential of cell-tissue engineered bone constructed by human bone marrow mesenchymal stem cells (hBMSCs) expressing the transduced human bone morphogenetic protein 2 (hBMP-2) gene stably. MethodsThe full-length hBMP-2 gene was cloned from human muscle tissues by RT-PCR and connected into a vector to consturct a eukaryotic expression system. And then the gene expression system was transduced to hBMSCs with lipidosome. hBMSCs were transfected by hBMP-2 gene (experimental group) and by empty plasmid (negative control group), untransfected hBMP-2 served as blank control group. RT-PCR, dot-ELISA, immunohistochemical analysis and ALP activity were performed to compare and evaluate the situation of hBMP-2 expression and secretion after transfection. hBMSCs transfected by hBMP-2 gene were seeded on hydroxyapatite (HA) and incubated for 4 days to construct the hBMP-2 gene modified tissue engineered bone, and then the tissue engineered bone was observed by the inverted phase contrast microscope and scanning electron microscope. Then the hBMP-2 gene modified tissue engineered bone (group A, n=3), empty plasmid transfected hBMSCs seeded on HA (group B, n=3), hBMSCs suspension transfected by hBMP-2 gene (group C, n=3), and hBMP-2 plasmids and lipidosome (group D, n=3) were implanted into bilateral back muscles of nude mice. The osteogenic activity was detected by HE staining and alcian blue staining after 4 weeks. ResultsAt 48 hours and 3 weeks after transfection, RT-PCR and dot-ELISA results indicated that the transfected hBMSCs could express and secrete active and exogenous hBMP-2 stably. The immunohistochemical staining was positive, and the ALP activity in the transfected hBMSCs was significantly higher than that in two control groups (P < 0.05). The transfected hBMSCs had a good attaching and growing on the three-demension suface of HA under inverted phase contrast microscope and scanning electron microscope. In vivo study indicated that a lot of new bone formation was obviously found at 4 out of 6 sides of back muscles in group A. Some new bone formation at both sides of back muscles was observed in 1 of 3 mice in group B. No new bone formation was found in group C. A few new bone formation was observed at one side of back muscles in group D. ConclusionThe tissue engineered bone constructed by hBMP-2 gene modified hBMSCs and HA is able to express and secrete active hBMP2 stably and can promote new bone formation effectively in muscles of nude mice.
Objective To study the properties of the xenogeneic deproteinized cancellous bone used as a scaffold in the bone tissue engineering andits application to the spinal fusion of the lumbar intertransverse process in agoat. Methods The deproteinized bone was derived from an adult pig’s femoral cancellous bone through the physical and chemical treatments. Its morphological features, constituting components, and biomechanical properties were examined by the scanning electron microscopy, X-ray diffraction analysis, and mechanical experimental instrument. The cell-material complex was observed under the inverted phase contrast microscope to evaluate the adhesion and the growth of the osteoblasts. The experimental model of the spinal fusion of the lumbar intertransverse process was produced in 12 male goats aged 6-8 months, which were divided into two groups. In Group A, the tissue engineered bone constructed by thexenogeneic deproteinized cancellous bone, the recombinant human bone morphogenetic protein 2, and the mesenchymal stem cells was used for the spinal fusion; however, in Group B the autoilium was used. The samples were harvested at 4, 8 and 12 weeks postoperatively, and a series of examinations were performed, including the radiography and the histomorphological assay. Results The deproteinized cancellous bone had a natural pore network system, with an aperture ranging in size from 200 to 500 μm, containing a main organic material ofcollagen and the inorganic material of hydroxyapatite. So, the deproteinized cancellous bone had a good mechanical strength and a good histocompatibility. In Group A, the X-ray examination at different timepoints postoperatively showed that at 4 weeks,the bridging areas of all the fusion sites were not clear, especially on the internal side; at 8 weeks, the upper and lower bridged parts had a narrowed gap, with formation of much continuous bony callus; at 12 weeks, a complete fusion occurred. In the early stage, the material density was slightly lowerin Group A than in Group B, but at 12 weeks the density was almost the same in both the groups. Histological examination in the transplant area showed that at 4 weeks in Group A there was a new bone formation in a multipoint way; at 8 weeks, a “sandwichshaped” new bone wascrossed with the transplanting materials; and at 12 weeks, a medullary cavity was remodeled and a new cancellous bone was formed. The osteogenic process of thetissue engineered bone constructed by the xenogeneic deproteinized cancellous bone scaffold was almost the same as the autoilium osteogenesis. Conclusion The xenogeneic deproteinized cancellous bone is a good material in the bone tissue engineering, which can be used as an osteogenesis scaffold andprovide a stable environment for revascularization and osteoblastic differentiation.
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 explore a new method of preparing the composite of DL-polylactic acid (PDLLA), hydroxyapatite(HA), decalcium bone matrix (DBM), and to observe the degradation characteristics of PDLLA/HA/DBM in vitro. METHODS: An emulsion blend method was developed to prepare the composite of PDLLA/HA/DBM based on the weight rate of PDLLA:HA:DBM = 1.5-2:1-1.5:1. The characteristics of the particles was observed by scanning electron microscope. In vitro, PDLLA/HA/DBM and PDLLA were put into PBS(pH7.4) respectively; the pH value, weight and biomechanics of them were determined during the degradation. RESULTS: Without heating, the emulsion blend method could be developed to prepare PDLLA/HA/DBM. Scanning electron microscope showed that the gap diameter in the compound material was 100 to 400 microns, and the porosity was 71.3%; During degradation, the pH value of PDLLA decreased little within 2 weeks, then decreased obviously and decreased to 4.0 at the end of the 4th week; while the pH value of PDLLA/HA/DBM kept quite steady and was 6.4 at the end of the 12th week. The weight of PDLLA decreased little within 4 weeks, then decreased obviously and remained 50% of its prime weight at the end of the 12th week; while the weight of PDLLA/HA/DBM decreased little within 5 weeks, then decreased obviously and remained 60% of the prime at the end of the 12th week. The prime biomechanical strength was 1.33 MPa in PDLLA and 1.71 MPa in PDLLA/HA/DBM. There was significant difference between them (P lt; 0.05). The strength of PDLLA decreased little within 3 weeks, then decrease obviously and was 0.11 MPa at the end of the 12th week; the strength of PDLLA/HA/DBM decreased little within 4 weeks, then decrease obviously and was 0.21 MPa at the end of the 12th week. CONCLUSION: The emulsion blend method is a new method to prepare bone repair materials. As a new bone repair material, PDLLA/HA/DBM is suitable for bone tissue engineering for its good characteristics of porosity and degeneration.
Objective To explore the osteogenesis and angiogenesis effect of bone marrow mesenchymal stem cells (BMSCs) derived osteoblasts and endothelial cells compound with chitosan/hydroxyapatite (CS/HA) scaffold in repairing radialdefect in rats. Methods The BMSCs were isolated from Sprague Dawley rats and the 3rd generation of BMSCs were induced into osteoblasts and endothelial cells. The endothelial cells, osteoblasts, and mixed osteoblasts and endothelial cells (1 ∶ 1) were compound with CS/HA scaffold in groups A, B, and C respectively to prepare the cell-scaffold composites. The cell proliferation was detected by MTT. The rat radial segmental defect model was made and the 3 cell-scaffolds were implanted, respectively. At 4, 8, and 12 weeks after transplantation, the graft was harvested to perform HE staining and CD34 immunohistochemistry staining. The mRNA expressions of osteopontin (OPN) and osteoprotegerin (OPG) were detected by RT-PCR. Results Alkal ine phosphatase staining of osteoblasts showed that there were blue grains in cytoplasm at 7 days after osteogenic induction and the nuclei were stained red. CD34 immunocytochemical staining of the endothelial cells showed that there were brown grains in the cytoplasm at 14 days after angiogenesis induction. MTT test showed that the proliferation level of the cells in 3 groups increased with the time. HE staining showed that no obvious osteoid formation, denser microvessel, and more fibrous tissue were seen at 12 weeks in group A; homogeneous osteoid which distributed with cord or island, and many osteoblast-l ike cells were seen in groups B and C. The microvessel density was significantly higher in groups A and C than group B at 3 time points (P lt; 0.05), and in group A than in group C at 12 weeks (P lt; 0.05). The OPN and OPG mRNA expressions of group A were significantly lower than those of groups B and C at 3 time points (P lt; 0.05). In groups B and C, the OPN mRNA expressions reached peak t8 and 12 weeks, respectively, and OPG mRNA expressions reached peak at 4 weeks. Conclusion BMSCs derived steoblasts and endothelial cells (1 ∶ 1) compound with CS/HA porous scaffold can promote bone formation and vascularization in bone defect and accelerate the healing of bone defect.
OBJECTIVE: To compare the clinical results of repairing bone defect of limbs with tissue engineering technique and with autogeneic iliac bone graft. METHODS: From July 1999 to September 2001, 52 cases of bone fracture were randomly divided into two groups (group A and B). Open reduction and internal fixation were performed in all cases as routine operation technique. Autogeneic iliac bone was implanted in group A, while tissue engineered bone was implanted in group B. Routine postoperative treatment in orthopedic surgery was taken. The operation time, bleeding volume, wound healing and drainage volume were compared. The bone union was observed by the X-ray 1, 2, 3, and 5 months after operation. RESULTS: The sex, age and disease type had no obvious difference between groups A and B. all the wounds healed with first intention. The swelling degree of wound and drainage volume had no obvious difference. The operation time in group A was longer than that in group B (25 minutes on average) and bleeding volume in group A was larger than that in group B (150 ml on average). Bone union completed within 3 to 7 months in both groups. But there were 2 cases of delayed union in group A and 1 case in group B. CONCLUSION: Repair of bone defect with tissue engineered bone has as good clinical results as that with autogeneic iliac bone graft. In aspect of operation time and bleeding volume, tissue engineered bone graft is superior to autogeneic iliac bone.
ObjectiveTo investigate the ability of autologous peripheral blood endothelial progenitor cells (EPCs) in promoting neovascularization of tissue engineered bone and osteogenesis of bone marrow mesenchymal stem cells (BMSCs). MethodThe peripheral blood EPCs and BMSCs from No. 1-9 New Zealand rabbits were isolated, cultured, and identified. According to the cell types, the third generation of cells were divided into 3 groups:EPCs (group A), BMSCs (group B), and co-cultured cells of EPCs and BMSCs (group C, EPCs:BMSCs=1:2) . Then cells were seeded on the partially deproteinised bone (PDPB) packaged with fibronectin to construct tissue engineered bone. After 4 days, autologous heterotopic transplantation of tissue engineered bone was performed in the rabbit's muscles bag of groups A, B, and C (the right arm, left arm, right lower limb respectively, 2 pieces each part). At 2, 4, and 8 weeks after transplantation, the growth of tissue engineered bone was observed, and the rate of bone ingrowth was calculated by HE staining; the expressions of CD34, CD105, and zonula occludens protein 1(ZO-1) were compared by immunohistochemical staining at each time point in tissue engineered bone among 3 groups. ResultsThe EPCs and BMSCs were isolated and identified successfully; immunofluorescent staining showed that EPCs were positive for CD34, CD133, and von Willebrand factor (vWF), and BMSCs were positive for CD29 and CD90 and were negative for CD34. The tissue engineered bone constructed in 3 groups was transplanted successfully. At 2, 4, and 8 weeks after autologous heterotopic transplantation, the general observations showed that the soft tissue around the tissue engineered bone increased and thickened gradually in each group with time passing; the boundary between bone and soft tissue was not clear; the pore space of tissue engineered bone gradually was filled, especially in group C, the circuitous vascular network could be seen in the tissue engineered bone. HE staining showed capillaries and collagen fibers increased gradually, tissue engineered bone ingrowth rate was significantly higher in group C than groups A and B at 4 and 8 weeks (P<0.05) , and group B was significantly higher than group A (P<0.05) . Immunohistochemical staining showed that the expressions of CD34, CD105, and ZO-1 in tissue engineered bone of 3 groups all increased with the extension of time, showing significant differences between groups at each time point (P<0.05) . At 2 weeks after transplantation, the expression of CD105 in group C was significantly higher than that in groups A and B (P<0.05) ; at 4 and 8 weeks, CD34, CD105, and ZO-1 expressions showed significant differences between 2 groups (P<0.05) ; the expression was the highest in group C, and was the lowest in group B. ConclusionsAutologous peripheral blood EPCs and BMSCs have synergistic effect, and can promote neovascularization and osteogenesis of tissue engineered bone in vivo.
Objective To investigate the osteogenesis effects of angiopoietin 1 (Ang-1) gene transfected bone marrow mesenchymal stem cells (BMSCs) seeded on β tricalcium phosphate (β-TCP) scaffolds (tissue engineered bone) with platelet-rich plasma (PRP). Methods BMSCs were isolated from bone marrow tissue of rabbits. The Ang-1 gene was transfected into the BMSCs at passage 2 by lentivector, which were seeded on β-TCP scaffolds with PRP (0.5 mL) after 48 hours of transfection. Bilateral radial segmental bone defects (15 mm in length) were created in 20 3-month-old New Zealand rabbits. Then the tissue engineered bone with the Ang-1 gene transfected BMSCs (experimental group) and untransfected BMSCs (control group) were implanted into the defects in the right and left radius, respectively. X-ray, histology, immunohistochemistry, and biomechanics observations were done at 2, 4, 8, and 12 weeks after operation. Results In vitro, the transfected rate was over 90% and RT-PCR showed that the Ang-1 expression were significantly increased after transfection. The X-ray films showed that some callus formed at 4 weeks, partial bony union was observed at 8 weeks, and complete union at 12 weeks in experimental group; and bone union was not observed at 12 weeks in control group. HE staining showed that capillary appeared at 8 weeks and more capillaries were observed in new bone at 12 weeks in experimental group; only a few capillaries were observed at 12 weeks in control group. At 8 and 12 weeks, the microvascular density were (50.1 ± 7.8) /mm2 and (66.1 ± 3.5) /mm2 in experimental group and were 0 and (30.3 ± 7.2)/mm2 in control group, showing significant differences between 2 groups at 12 weeks (Z= —2.107, P=0.031). Immunohistochemistry examination showed that the positive cells can be found at 8 weeks in experimental group. And the biomechanical analysis showed that maximum loads of experimental group were significantly higher than those of control group in three-point bending test and compression test at 12 weeks (P lt; 0.05). Conclusion The tissue engineered bone with PRP and Ang-1 can increase the osteogenic properties by enhancing capillary regeneration, thus it can be used to repair radial segmental bone defects of rabbit.