Objective To summarize the recent progress of cell-based approaches for promoting bone regeneration in distraction osteogenesis (DO). Methods Recent literature concerning enhancement of bone regeneration following DO using cell-based approaches was reviewed and analyzed. Results An overview of 4 different cell-based approaches was mainly provided: single cell injection, cell scaffold-based strategies/injectable tissue engineered bone, microtissue technology or cell aggregate technology, and stem cell gene therapy. Each has its advantages and disadvantages. Other methods are still in the experimental research except that compound injection of bone marrow mesechymal stem cells and platelet-rich plasma has been applied to clinical practice. Conclusion The cell-based approach is a promising strategy in the field of bone regenerative medicine. These approaches have bright future in promoting bone regeneration and reducing the treatment period in DO in the clinical application. However, well-designed preclinical studies are required to establish safe and effective guidelines for cell-based approaches to promoting bone regeneration during DO.
Objective To investigate whether combining use of platelet-rich plasma (PRP) and decalcified bone matrix (DBM) has synergistic action on promoting bone consol idation and heal ing. Methods Forty male New Zealand rabbits (weighing 2.2-2.8 kg) were randomly divided into 4 groups (n=10). The whole blood was extracted from the central aural artery and PRP was prepared with the Landesberg’s method. An 1 cm-defect was made below the tibiofibular joint of the lefttibia through osteotomy. In group A, defect was repaired by distraction osteogenesis (1 cm); in group B, defect was repaired with 0.5 cm DBM and then by distraction osteogenesis (0.5 cm); in group C, defect was repaired by distraction osteogenesis (1 cm) and local injection of 1 mL PRP; in group D, defect was repaired by 0.5 cm DBM combined with 1 mL PRP and then by distraction osteogenesis (0.5 cm). Then lengthening started at 7 days after operation, at a rate of 1 mm/day and 0.5 mm every time for 10 days (groups A and C) or for 5 days (groups B and D). After the lengthening, the consolidation was performed. The X-ray films were taken at 0, 12, 17, 27, and 37 days after operation. At 37 days after operation, the tibial specimens were harvested for Micro-CT scanning, three-dimensional reconstruction and biomechanical test. Results The X-ray films showed that new bone formation in groups B and C was obviously better than that in groups A and D at 37 days. The bone mineral density (BMD), bone mineral content (BMC), and bone volume fraction (BVF) of groups B and C were significantly higher than those of groups A and D (P lt; 0.05); the BMD and BMC of group C were significantly higher than those of group B (P lt; 0.05); the BVF had no significant difference between groups B and C (P gt; 0.05). There was no significant difference in BMD, BMC, and BVF between groups A and D (P gt; 0.05). The trabecula number (Tb.N) of group C was significantly more than that of other groups (P lt; 0.05), and the trabecula spacing (Tb.Sp) of group C was significantly smaller than that of other groups (P lt; 0.05), but no significant differencewas found among other groups (P gt; 0.05). There was no significant difference in the trabecula thickness among 4 groups (P gt; 0.05). The ultimate angular displacement had no significant difference among 4 groups (P gt; 0.05). The maximum torque of groups B and C was significantly higher than that of groups A and D (P lt; 0.05); the maximum torque of group C was significantly higher than that of group B (P lt; 0.05); no significant difference was found between groups A and D (P gt; 0.05). Conclusion In the rabbit bone defect/lengthening model, local injection of PRP can enhance bone consol idation effectively during consol idation phase. In normal distraction rate, DBM can promote bone consol idation during distraction osteogenesis. In the early stage of distraction osteogenesis, combining use of DBM and PRP can not further promote bone consolidation and healing.
Objective To compare the effect between vascularization osteogenesis and membrane guided osteogenesis in the bone repair by the tissue engineered bone with pedicled fascial flap packing autologous red bone marrow (ARBM), so as to provide a reference for the bone defect repair in cl inic. Methods The tissue engineered bone was constructed with ARBM and the osteoinductive absorbing recombinant human materials with recombinant human bone morphogenetic protein 2. Sixty New Zealand rabbits (aged 4-5 months, weighing 2.0-2.5 kg) were randomly divided into group A (n=16), group B (n=22), and group C (n=22). The complete periosteum defect model of 1.5 cm in length was prepared in right ulnar bone, then the tissue engineered bone was implanted in the bone defect area in group A, the tissue engineered bonewith free fascial flap in group B, and the tissue engineered bone with pedicled fascial flap in group C. At 4, 8, 12, and 16 weeks, the tissue of bone defect area was harvested from 4 rabbits of each group for the general, histological, and immunohistochemical staining observations; at 8, 12, and 16 weeks, 2 rabbits of groups B and C, respectively were selected to perform ink perfusion experiment by axillary artery. Results The general observation showed that the periosteum-l ike tissues formed in the fascial flap of groups B and C, chondroid tissues formed in group B, new bone formed in group C, and the fibrous and connective tissues in group A at 4 and 8 weeks; a few porosis was seen in group A, more new bone in group B, and bone stump formation in group C at 12 and 16 weeks. Histological observation showed that there were few new blood vessels and new bone trabeculae in groups A and B, while there were large amounts of new blood vessels and mature bone trabeculae in group C at 4 and 8 weeks. There were a few new blood vessels and new bone trabeculae in group A; more blood vessels, significantly increased mature trabeculae, and the medullary cavity formation in group B; and gradually decreased blood vessels, the mature bone structure formation, and the re-opened medullary cavity in group C at 12 and 16 weeks. The immunohistochemical staining observation showed that the levels of CD105, CD34, and factor VIII were higher in group C than in groups A and B at different time points.The bone morphometry analysis showed that the trabecular volume increased gradually with time in 3 groups after operation; the trabecular volume in group C was significantly more than those in groups A and B at different time points (P lt; 0.05); and there was significant difference between groups A and B (P lt; 0.05) except the volume at 4 weeks (P gt; 0.05). The vascular image analysis showed that the vascular regenerative area ratio in group C was significantly higher than those in groups A and B at different time points (P lt; 0.05). The ink perfusion experiment showed that the osteogenic zone had sparse ink area with no obvious change in group B, while the osteogenic zone had more intensive ink area and reached the peak at 8 weeks, then decreased in group C. Conclusion The tissue engineered bone with pedicled fascial flap packing ARBM has the vascularization osteogenesis effect at early stage, but the effect disappears at late stage gradually when the membrane guided osteogenesis is main.
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.
【Abstract】 Objective To evaluate the effectiveness of HA mixed with adenovirus mediated rhBMP-2 gene (AdvrhBMP-2) transferred BMSCs of goats on distraction osteogenesis. Methods Nineteen adult goats were used for the experiment,no matter they were male or female, and the weight of the goats were 15-20 kg. The 10 mL marrow was obtained from theil iac crest of each goat. The BMSCs was expanded and passaged conventionally. The 3th BMSCs was infected by Adv-rhBMP-2 at 200 multipl icity of infection (MOI). The 1×108 infected BMSCs were digested by 0.25% trypsin and collected, then mixed with HA. The right tibia lengthening models were developed, and mixture with BMSCs was injected in the osteotomy position. The goats were divided randomly into 4 groups according to the material injected in operation, group A: Adv-rhBMP-2/BMSCs/HA (n=6); group B: Adv-rhBMP-2/BMSCs (n=5); group C: Adv-β-gal/BMSCs/HA (n=4); group D: sham without any injections (n=4). After a seven-day latency period following ostectomy, distraction was carried out at a rate of 1 mm/day for 4 weeks. Roentgenography was practiced in 5, 8 and 12 weeks. After 12 weeks, the goats were sacrificed and dual-energy X-ray absorptiometry (DXA), biomechanical test and histology results were studied. Results After five and eight weeks surgery, X-raytest showed the distraction callus was more in group A and B than group C and D, and the radiographic score was significantly higher in group A and B than in the other two groups(P lt; 0.05); after 12 weeks surgery, the continued callus was formed in the distraction defects in all groups. DXA showed the mean bone mineral content of distraction callus in group A, B, C, D was (4.175 ± 1.921), (2.600 ± 0.638), (2.425 ± 0.826) and (1.175 ± 0.574) g, and the mean bone mineral density was (0.612 ± 0.196), (0.630 ± 0.159), (0.450 ± 0.166) and (0.266 ± 0.113) g/cm2. The group A and B was significantly higher than group C and D (P lt; 0.05).Biomechanical test showed the maximum loading of group A, B, C, D was (490.20 ± 155.08), (350.59 ± 80.48), (221.95 ± 68.79) and (150.65 ± 92.29) N, and elastic modulus was (178.24 ± 105.80), (105.88 ± 27.09), (81.18 ± 48.67) and (50.35 ± 47.64) MPa. The group A was significantly higher than in group C and D (P lt; 0.05). Histology observation revealed abundant bone formation in the distraction defects in group A, and the bone trabecula was arranged longitudinal and netl ike. Histomorphology analysis revealed the bone volume in group A, B, C, D was 72.35% ± 5.68%, 67.58% ± 7.42%, 49.63% ± 4.87% and 38.87% ± 2.35%, and the bone formation was significantly greater in group A compared with group D (P lt; 0.05). Conclusion HA mixed with rhBMP-2 modified BMSCs can accelerate distraction osteogenesis in goats.
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 observe the heterotopic osteogenes is of the autogenou s marrow stromal cells (MSCs) on the ceramic bovine bone(CBB)/hydrogel scaffold (HG) and t he effects of the recombinant human bone morphogenetic protein2 (rhBMP-2) and the transforming growth factor β (TGF-β) on osteogenesis. Methods The auto genous marrow stromal cells were cultured by the mineralized condition medium (1 0%FBS, dexamethasone 10 nmol, L-vitamin C 50 mg/L, βsodium glycerophosph ate D MEM culture medium 10 mmol). At 5 days, the MSCs differentiation was observed b y TypeⅠcollagen, the Mend calcium-cobalt staining, and the Von-Kossa staining. The cell suspension of 5×106/ml was obtained. There were three groups: Group A: added in rhBMP-2（10 μg）TGF-β（0.05 μg）;Group B: added in TGF-β(0.05 μg); and Group C (the control group): without the growth factor. Then, the MSCs loading on CBB/HG were embedded in the autogenous subcutaneous area at 4 and 8 weeks, and the osteogenesis was observed by the HE staining and the modified Mallory’s trichrome staining, with an image analysis. TypeⅠcollagen and the bone m orphogenetic synthesis were examined by the immunohistochemistry stains. Results Most MSCs induced by the mineralized condition medium at 5 da ys became smalle r and polygon-shaped, and the cytodendrite became shorter. The MSCs were observ e d by the Mend calciumcobalt staining. Some brown and black grains were found in the cytochylema. The MSCs were positive for the TypeⅠcollagen immunohistochemi stry stains. At 20 days, the mineralized nubs were found by the Von Kossas stain s. At 4 weeks, some strips of the new bone were observed by the HE staining an d the modified Mallory’s trichrome staining in all the groups. The bone matrix a rea was significantly larger in Group A than in Group B（P＜0.01). The av erag e gray degrees of TypeⅠcollagen were lower in Groups A and B than in Group C. However, there was no significant difference in the bone morphogenesis among the three groups. At 8 weeks, there- were significantly more snatchy strips and macula mature bone formation in Groups A and B than in Group C. The Type Ⅰcollage n and the bone morphogenesis were not significantly different among the three groups. Conclusion The autogenous marrow stromal cells on the ce ramic bovine bon e /hydrogel scaffold can promote the heterotopic osteogenesis, and the combined use of rhBMP-2 and TGF-β is better than the only use of rhBMP-2 or TGF-β i n promoting osteogenesis.
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.
Objective To examine the mRNA expression of activin A(ACT A) and follistatin(FS) during mandibular lengthening and to elucidate the regulating pattern of during mandibular distractionosteogenesis.Methods Skeletally mature-white New Zealand rabbits were established right mandibular distraction osteogenesis models and the mandibles were lengthened 7 days after osteomy. Atthe end of latency period and the end of distraction period, 10,20, 30, 40 and60 days after fixation, the regenerating tissue of animals’ lengthened mandibles and that of the other side normal mandibles were harvested to extract RNA andto analyse ACT A, FS mRNA by RT-PCR.Results The expression of ACT A mRNA was not detectable in normal bone tissue and ACT A mRNA began to express at the end of latency period. The expression of ACT AmRNA increased gradually along with the beginning of distraction and reached the peak on the 10th and 20th days of distraction which was 5.04 and 4.98 times as much as that of the end of latency period, respectively. The trend of expression of FS mRNA during mandibular distraction osteogenesis was the same as expression of ACT A mRNA. Conclusion ACT A/FS play an important role during rabbit mandibular distraction osteogenesis.