Objective Tissue engineered bone (TEB) lacks of an effective and feasible method of storage and transportation. To evaluate the activity of osteogenesis and capabil ity of ectopic osteogenesis for TEB after freeze-dried treatment in vitro and in vivo and to explore a new method of preserving and transporting TEB. Methods Human bone marrow mesenchymal stem cells (hBMSCs) and decalcified bone matrix (DBM) were harvested from bone marrow and bone tissue of the healthy donators. TEB was fabricated with the 3rd passage hBMSCs and DBM, and they were frozen and dried at extremely low temperatures after 3, 5, 7, 9, 12, and 15 days of culture in vitro to obtain freeze-dried tissue engineered bone (FTEB). TEB and FTEB were observed by gross view and scanning electron microscope (SEM). Western blot was used to detect the changes of relative osteogenic cytokines, including bone morphogenetic protein 2 (BMP-2), transforming growth factor β1 (TGF-β1), and insul in-l ike growth factor 1 (IGF-1) between TEB and FTEB. The ectopic osteogenesis was evaluated by the methods of X-ray, CT score, and HE staining after TEB and FTEB were transplanted into hypodermatic space in athymic mouse. Results SEM showed that the cells had normal shape in TEB, and secretion of extracellular matrix increased with culture time; in FTEB, seeding cells were killed by the freeze-dried process, and considerable extracellular matrix were formed in the pore of DBM scaffold. The osteogenic cytokines (BMP-2, TGF-β1, and IGF-1) in TEB were not decreased after freeze-dried procedure, showing no significant difference between TEB and FTEB (P gt; 0.05) except TGF-β1 15 days after culture (P lt; 0.05). The ectopic osteogenesis was observed in TEB and FTEB groups 8 and 12 weeks after transplantation, there was no significant difference in the calcified level of grafts between TEB and FTEB groups by the analysis of X-ray and CT score. On the contrary, there was no ectopic osteogenesis in group DBM 12 weeks after operation. HE staining showed that DBM scaffold degraded and disappeared 12 weeks after operation. Conclusion The osteogenic activity of TEB and FTEB is similar, which provides a new strategy to preserve and transport TEB.
Objective To provide the seed cells for bone tissue engineering, to establ ish immortal ized human bone marrow mesenchymal stem cells (MSCxj) and to investigate the ectopic osteogenesis of MSCxj. Methods MSCxjs of the 35thand 128th generations were maintained and harvested when the cell density reached 2 109. Then, these cells were co-cultured with heterogeneous bone scaffold in groups A (the 35th generation, n=12) and group B (the 128th generation, n=12); heterogeneous bone alone was used in group C (n=12). The cell prol iferation was observed by scanning electron microscopy (SEM) after 48 hours and 18 days of osteogenic induction culture. The complex was implanted subcutaneouly through a 3-mm-incision at both sides of the back in 18 nude mice. Tetracycl ine label ing was performed before the animals were sacrificed. Tetracycl ine fluorescence staining, HE staining, ponceau staining, and immunohistochemistry staining for osteocalcin were performed at 4, 8, and 12 weeks after transplantation; the morphologic quantitative analysis was made. Results After 48 hours, SEM showed that MSCxjs adhered to heterogeneous bone and grew well; after 18 days, a large number of new filamentous extracellular matrix and small granules were found to cover the cells. The results of tetracycl ine fluorescence staining, HE staining, and ponceau staining in groups A and B showed that the osteogenesis was not obvious at 4 weeks after transplantation; osteoid matrix deposition was noted around and in theheterogeneous bone at 8 weeks; and osteogenesis was increased at 12 weeks. There was no significant difference in bone formation between groups A and B. Osteogenesis was not observed in group C. The osteocalcin expressions were positive in groups A and B. The bone ingrow percentages of groups A and B were 5.64% ± 2.68% and 4.92% ± 2.95% at 8 weeks, and 13.94% ± 2.21% and 14.34% ± 3.46% at 12 weeks, showing significant differences between 8 weeks and 12 weeks at the same group (P lt; 0.05) and no significant difference between groups A and B at the same time (P gt; 0.05). Conclusion MSCxj has favorable abil ities of ectopic osteogenesis and can be appl ied as seeded cells in bone tissue engineering.
Objective To observe the release pattern of the microcysts and the effect of ectopic osteogenesis of combined micromorselized bone by optimized preparation of microcysts. Methods Optimized poly-DLlactide-co-glycolide (PLGA) microcysts manufacturing method was performed with the orthogonal design, and the accumulated release amount of microcysts was calculated at 2 h, 4 h, 8 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, 96 h, 120 h, 144 h, 168 h, 192 h, 216 h, 240 h and 264 h. Twentyfour Wistar rats were divided into 4 groups (n=6) and 1 cm length incision was cut in their bilateral thighs skin, forming 48 gluteus maximus muscle sackmodels. In group A,collagen was implanted to bilateral muscle sacks respectively. In group B, collagen and autologous morselized bone were implanted to bilateral muscle sacks. Ingroup C, collagen and rhBMP-2/PLGA delayed release microcysts were implanted to bilateralmuscle sacks respectively. In group D, collagen and morselized bone/rhBMP-2/PLGA delayed release microcysts were implanted to bilateral muscle sacks. Gross and histologic observations were made at 3, 4 and 5 weeks postoperatively.Results Every optimized variance had an effect on particle diameter of microcyst and its encapsulating rate. The microcyst’s surface was smooth and had a fine spheroplast, which released slowly within 11 days in vitro. In thethird week postoperatively, the graft in group A could not be touched, while the graft in all other 3 groups was still found. After 3 weeks, collagen was absorbed completely in group A, the residual collagen could be seen in groups B, C andD. After 4 weeks, collagen could be seen in group A; micromorselized bone continued to be absorbed and became smaller in group B; microsphere became smaller, osteoblasts increased in group C; micromorselized bone and microsphere continuedto be absorbed, oteoblasts and chondroblasts increased. After 5 weeks, implantsbecame small, microsphere was absorbed, osteoblasts and chondroblasts became more in groups B, C and D. Microcysts presented with white granuloshape and were packaged in tissue pieces. Histologic observation showed that the PLGA microcysts in 3 weeks and 4 weeks could be absorbed gradually as the time in vivo, if combining with morselzed bone they could produce abundant induced osteoblasts and chondroblasts. Conclusion Optimizing the preparation technology of microcysts has delayed their release during a long period in vitro. Autologous micromorselized bone can be ectopicly induced to produce large amount of osteoblasts in gluteus maximus muscle sack, where PLGA microcysts can combine organically and bring about the bone formation with less amount of growth factors.
Objective To study the mechanism of ectopic osteogenesis of nacre/Polylactic acid (N/P) artificial bone combined with allogenic osteoblasts, and to explore the possibility as a scaffold material of bone tissue engineering. Methods The allogenic- osteoblasts seeded onto N/P artificial bone were co-cultured in vivo 1 week.The N/P artificial bone with allogenic osteoblasts were implanted subcutaneously into the left back sites of the New Zealand white rabbits in the experimental group and the simple N/P artificial bone into the right ones in the control group. The complexes were harvested and examined by gross observation, histologic analysis and immunohistochemical investigation 2, 4 and 8 weeks after implantation respectively.Results In experimental group, the osteoid formed after 4 weeks, and the mature bone tissue withbone medullary cavities formed after 8 weeks; but in control group there was nonew bone formation instead of abundant fibrous tissue after 4 weeks, and more fibrous tissue after 8 weeks.Conclusion N/P artificial bone can be used as an optical scaffold material of bone tissue engineering.
ObjectiveTo evaluate the ectopic osteogenesis of recombinant human bone morphogenetic protein 2 (rhBMP-2) loaded chitosan (CS)/dextran sulfate (DS) by micro-CT. MethodsrhBMP-2/CS/DS microspheres were prepared by the ionic crosslinking and its shape was observed under the scanning electron microscope. The release of rhBMP-2 was determined from resultant microspheres by ELISA assay. Forty-eight Sprague Dawley male rats were randomly divided into 4 groups (n=12), quadriceps muscle bag model was made, gelatin sponge (group A), CS/DS microspheres (group B), rhBMP-2 (group C), and CS/DS/rhBMP-2 microspheres (group D) were implanted into the bags respectively. The tissue samples with heterotopic ossification were harvested for micro-CT scanning at 4, 8, 12, and 16 weeks. The tissue mineral density (TMD), bone volume fraction (BVF), trabecular thickness (Tb.Th), trabecular number (Tb.N), bone mineral density (BMD), and tissue mineral content (TMC) were measured. ResultsThe prepared rhBMP-2/CS/DS microspheres with smooth surfaces were spherical and evenly disperses without obvious agglomeration. At 2 hours, microsphere started a sudden release period in vitro; the release reached a peak at 2 days; and the release cycle lasted about 20 days. The rats survived to the end of the experiment. At each time point after operation, no radiation developed and no osteogenesis was observed by three dimensional reconstruction in groups A and B. However, radioactive strength and reconstructed bone tissue gradually increased in groups C and D, and group D had more radioautography and more bone tissues than group C. At each time point, TMD, BVF, Tb.Th, Tb.N, BMD, and TMC of groups A and B were zero. Ectopic bone formed with time, the other parameters showed an increasing trend except Tb.N in groups C and D, showing significant difference when compared with groups A and B at each time point (P < 0.05). There was no significant difference between groups C and D at 4 weeks (P>0.05); the parameters of group D were significantly higher than those of group C at 8-16 weeks (P < 0.05). ConclusionrhBMP-2/CS/DS microspheres have stronger ability of ectopic bone formation than single rhBMP-2.