【Abstract】 Objective To investigate the in vivo osteogenic feasibil ity of tissue engineered periosteum constructedby porcine SIS and BMSCs in allogenic New Zealand rabbit. Methods The tissue engineered periosteum constructed by SIS scaffold and BMSCs was prepared in vitro .Twelve 2-month-old New Zealand rabbits were used in the experiments. The 1.5-2.0 cm critical bone defects were made in the both sides of radius of the animals. The tissue engineered periosteum was grafted into one side defect randomly, while the other side defect was only grafted SIS. Four weeks after operation, the forearms of all animals were checked by X-ray. Then, animals were sacrificed to harvest the specimen which were treated promptly for HE and Masson staining.The X-ray film and the morphological tissue staining outcome were evaluated qual itatively. Results After operation,all animals had a normal behavior and diet; the incision healed normally; the forearm could move normally for bearing weight.The tissue engineered periosteum constructed by allogenic BMSCs and heterogeneic SIS scaffold could form new bone tissue, andbridged the bone defect which could be confirmed either in X-ray film or histological staining. The newly formed bone tissue had similar bone density to normal bone. A lot of irregular newly formed vessels and medullary cavity inserted in the newly borned tissue. No lymphocytes infiltrated in histological examination. While the control side had no any osteogenesis neithter in X-ray, nor in HE and Masson staining inspecting; the defect space only occupied with some connective tissue. Conc lu sion Tissue engineered periosteum can form new bone in allogenic rabbit and has the feasibil ity to repair the segmental diaphysis defect.
Objective To study the differentiation of the human osteoblasts during the construction of the tissue engineered periosteum with the human acellular amniotic membrane(HAAM).Methods To construct the tissue engineered periosteum (n=60) with HAAM, the human fetal osteoblasts were used. The fetal osteoblasts were cultured for 2, 4, 6, 8, and10 days, and then their total RNA was extracted, which were reversely transcripted to cDNA. The realtime PCR analysis was used to reveal Cbfal and Osterix, and the cycle threshold (Ct) was also measured. The simplycultured osteoblasts were used as the control group (n=20).Results The expression of Cbfa1 was higher in the experimental group on the 2nd day when compared with that on the 4th, 6th, and 8th day(P＜0.05). The same result existed on the 10th day when compared with that on the 4th and 8th day. The expression of Osterix increased and was highest on the 8th day when compared with the other results(P＜0.05). Both of the 2 gene expressions were decreased in the control group when compared with those in the experimental group, but with no significant difference(P＞0.05). Conclusion Cbfa1 and Osterix can be normally expressed by the osteoblasts after their integration with HAAM. As a scaffold, HAAM can be used to keep the osteoblast phenotype and differentiation with an osteoconductive ability. Such a cell-scaffold complex may provide a basis for the osteogenesis.
ObjectiveTo investigate the feasibility of tissue engineered periosteum (TEP) constructed by porcine small intestinal submucosa (SIS) and bone marrow mesenchymal stem cells (BMSCs) of rabbit to repair the large irregular bone defects in allogenic rabbits. MethodsThe BMSCs were cultivated from the bone marrow of New Zealand white rabbits (aged, 2 weeks-1 month). SIS was fabricated by porcine proximal jejunum. The TEP constructed by SIS scaffold and BMSCs was prepared in vitro. Eighteen 6-month-old New Zealand white rabbits whose scapula was incompletely resected to establish one side large irregular bone defects (3 cm×3 cm) model. The bone defects were repaired with TEP (experimental group,n=9) and SIS (control group,n=9), respectively. At 8 weeks after operation, the rabbits were sacrificed, and the implants were harvested. The general condition of the rabbits was observed; X-ray radiography and score according to Lane-Sandhu criteria, and histological examination (HE staining and Masson staining) were performed. ResultsAfter operation, all animals had normal behavior and diet; the incision healed normally. The X-ray results showed new bone formation with normal bone density in the defect area of experimental group; but no bone formation was observed in control group. The X-ray score was 6.67±0.32 in experimental group and was 0.32±0.04 in control group, showing significant difference (t=19.871,P=0.001). The general observation of the specimens showed bone healing at both ends of the defect, and the defect was filled by new bone in experimental group; no new bone formed in the control group. The histological staining showed new bone tissue where there were a lot of new vessels and medullary cavity, and no macrophages or lymphocytes infiltration was observed in the defect area of experimental group; only some connective tissue was found in the control group. ConclusionTEP constructed by porcine SIS and BMSCs of rabbit can form new bone in allogenic rabbit and has the feasibility to repair the large irregular bone defects.
ObjectiveTo evaluate the effect of tissue engineered periosteum on the repair of large diaphysis defect in rabbit radius, and the effect of deproteinized bone (DPB) as supporting scaffolds of tissue engineering periosteum. MethodsBone marrow mesenchymal stem cells (BMSCs) were cultured from 1-month-old New Zealand Rabbit and osteogenetically induced into osteoblasts. Porcine small intestinal submucosa (SIS) scaffold was produced by decellular and a series mechanical and physiochemical procedures. Then tissue engineered periosteum was constructed by combining osteogenic BMSCs and SIS, and then the adhesion of cells to scaffolds was observed by scanning electron microscope (SEM). Fresh allogeneic bone was drilled and deproteinized as DPB scaffold. Tissue engineered periosteum/DPB complex was constructed by tissue engineered periosteum and DPB. Tissue engineered periosteum was "coat-like" package the DPB, and bundled with absorbable sutures. Forty-eight New Zealand white rabbits (4-month-old) were randomly divided into 4 groups (groups A, B, C, and D, n=12). The bone defect model of 3.5 cm in length in the left radius was created. Defect was repaired with tissue engineered periosteum in group A, with DPB in group B, with tissue engineered periosteum/DPB in group C; defect was untreated in group D. At 4, 8, and 12 weeks after operation, 4 rabbits in each group were observed by X-ray. At 8 weeks after operation, 4 rabbits of each group were randomly sacrificed for histological examination. ResultsSEM observation showed that abundant seeding cells adhered to tissue engineered periosteum. At 4, 8, and 12 weeks after operation, X-ray films showed the newly formed bone was much more in groups A and C than groups B and D. The X-ray film score were significantly higher in groups A and C than in groups B and D, in group A than in group C, and in group B than in group D (P<0.05). Histological staining indicated that there was a lot of newly formed bone in the defect space in group A, with abundant newly formed vessels and medullary cavity. While in group B, the defect space filled with the DPB, the degradation of DPB was not obvious. In group C, there was a lot of newly formed bone in the defect space, island-like DPB and obvious DPB degradation were seen in newly formed bone. In group D, the defect space only replaced by some connective tissue. ConclusionTissue engineered periosteum constructed by SIS and BMSCs has the feasibility to repair the large diaphysis defect in rabbit. DPB isn't an ideal support scaffold of tissue engineering periosteum, the supporting scaffolds of tissue engineered periosteum need further exploration.