We investigated the development of an injectable, biodegradable hydrogel composite of poly(trimethylene carbonate)-F127-poly(trimethylene carbonate)(PTMC11-F127-PTMC11)loaded with bone morphogenetic protein-2 (BMP-2) derived peptide P24 for ectopic bone formation in vivo and evaluated its release kinetics in vitro. Then we evaluated P24 peptide release kinetics from different concentration of PTMC11-F127-PTMC11 hydrogel in vitro using bicinchoninic acid (BCA)assay. P24/PTMC11-F127-PTMC11 hydrogel was implanted into each rat's erector muscle of spine and ectopic bone formation of the implanted gel in vivo was detected by hematoxylin and eosin stain (HE). PTMC11-F127-PTMC11 hydrogel with concentration more than 20 percent showed sustained slow release for one month after the initial burst release. Bone trabeculae surround the P24/PTMC11-F127-PTMC11 hydrogel was shown at the end of six weeks by hematoxylin and eosin stain. These results indicated that encapsulated bone morphogenetic protein (BMP-2) derived peptide P24 remained viable in vivo, thus suggesting the potential of PTMC11-F127-PTMC11 composite hydrogels as part of a novel strategy for localized delivery of bioactive molecules.
This study aims to explore the vascularization of hydroxyapatite/tricalcium phosphate (HA/TCP) biomaterials implanted in mice during osteoinduction. The HA/TCP biomaterials were implanted in muscle of mice, and 2, 4, 6, 8, 10 and 12 weeks after the implantation, the materials were harvested to prepare serial sections and hematoxylin-eosin (HE) staining. The process of vascularization was dynamically described, and the area percentage of neovascularization was quantitatively analyzed. The results showed that neovascularization formation was a continuous and dynamic process. The neovascularization appeared largely in the first two weeks, with a rising trend in week 4, reached peak in week 6, and gradually reduced in week 8. The results provide ideas for improving the success rate of bone tissue engineering, and indicate the mechanism of osteoinduction.
ObjectiveTo detect the difference in the osteogenesis ability of biphasic calcium phosphate (BCP) ceramic granular materials with different mesoporous diameters prepared at different sintering temperatures through in vivo and in vitro experiments, so as to provide evidence for screening BCP materials with better clinical application parameters.MethodsThree kinds of BCP (materials 1, 2, 3) were prepared by mixing hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) at a ratio of 8∶2 and sintered at 1 050, 1 150, and 1 250℃ for 3 hours, respectively. The internal porosity and the diameter, volume, and area of the mesopore were measured by Brunauer-Emmett-Teller test (BET); the composition of the material was evaluated by X-ray diffraction (XRD); the microscopic surface morphology of the material was observed by scanning electron microscopy (SEM). The 3rd generation bone marrow mesenchymal stem cells (BMSCs) from Sprague-Dawley rats were co-cultured with the materials 1, 2, and 3 for 7 days in vitro respectively (groups A, B, and C), and the cells adhesion on the materials was observed by SEM and phalloidine staining, respectively. Cell proliferation activity was measured by cell counting kit 8 method. In vivo, 9 muscle bags were made in dorsal muscles of 9 beagles, respectively. The muscle bags were randomly divided into 3 groups (3 per beagle in each group) and materials 1, 2, and 3 were placed into the muscle bags of groups A, B, and C, respectively. After 1, 2, and 3 months of operation, 3 beagles were anesthetized and the samples were stained with HE, Masson, and Safranin, and the bone formation area ratio in the BCP gap was calculated. Real-time fluorescence quantitative PCR (qRT-PCR) was performed to detect the expressions of bone-related genes [including alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OC)].ResultsThe BET test showed that with the increase of sintering temperature, the internal porosity of the particles did not change significantly, but the diameter, volume, and area of the mesopores gradually decreased. The XRD detection showed that the XRD waves of HA and β-TCP could be seen in all 3 kinds of materials; SEM showed that there were widely distributed macropores on the surface of 3 kinds of BCPs, and the interpores connected with the others. In vitro, BMSCs adhered and proliferated on the surfaces of 3 kinds of BCPs, and the cell biocompatibility of the materials in groups B and C was better than that in group A. In vivo, obvious osteoid tissue deposition could be observed in the intergranular space of 3 kinds of BCPs from 2 months after implantation. The bone formation area ratio of each group increased with time. The bone formation area ratio in group A was significantly higher than that in groups B and C at 2 and 3 months after implantation, and in group A than in group B at 1 month (P<0.05). qRT-PCR showed that the expressions of osteogenic related genes peaked at 2 months in group A, and gradually increased with time in groups B and C. The relative expressions of ALP and OPN mRNAs in group A were significantly higher than those in groups B and C at 1 month after implantation, the relative expression of OC mRNA in group A was significantly higher than that in groups B and C at 2 months after operation, the relative expression of ALP mRNA in groups B and C and the relative expression of OPN mRNA in group B were significantly higher than those in group A, all showing significant differences (P<0.05); there was no significant difference in the relative expression of each gene among the other groups at each time point (P>0.05).ConclusionThe mesoporous diameter of BCP decreases with the increase of sintering temperature. Different mesoporous diameters lead to different ectopic osteogenesis of BCP materials. BCP material with mesoporous diameter of 12.57 nm has better osteogenic ability which can activate the osteogenic gene earlier. The mesoporous diameter is expected to be an adjustable index for optimizing the osteogenic capacity of BCP materials.
Objective To prepare a new plastic bone filler material with adhesive carrier and matrix particles derived from human bone, and evaluate its safety and osteoinductive ability through animal tests. MethodsThe human long bones donated voluntarily were prepared into decalcified bone matrix (DBM) by crushing, cleaning, and demineralization, and then the DBM was prepared into bone matrix gelatin (BMG) by warm bath method, and the BMG and DBM were mixed to prepare the experimental group’s plastic bone filler material; DBM was used as control group. Fifteen healthy male thymus-free nude mice aged 6-9 weeks were used to prepare intermuscular space between gluteus medius and gluteus maximus muscles, and all of them were implanted with experimental group materials. The animals were sacrificed at 1, 4, and 6 weeks after operation, and the ectopic osteogenic effect was evaluated by HE staining. Eight 9-month-old Japanese large-ear rabbits were selected to prepare 6-mm-diameter defects at the condyles of both hind legs, and the left and right sides were filled with the materials of the experimental group and the control group respectively. The animals were sacrificed at 12 and 26 weeks after operation, and the effect of bone defect repair were evaluated by Micro-CT and HE staining. Results In ectopic osteogenesis experiment, HE staining showed that a large number of chondrocytes could be observed at 1 week after operation, and obvious newly formed cartilage tissue could be observed at 4 and 6 weeks after operation. For the rabbit condyle bone filling experiment, HE staining showed that at 12 weeks after operation, part of the materials were absorbed, and new cartilage could be observed in both experimental and control groups; at 26 weeks after operation, the most of the materials were absorbed, and large amount of new bone could be observed in the 2 groups, while new bone unit structure could be observed in the experimental group. Micro-CT observation showed that the bone formation rate and area of the experimental group were better than those of the control group. The measurement of bone morphometric parameters showed that the parameters at 26 weeks after operation in both groups were significantly higher than those at 12 weeks after operation (P<0.05). At 12 weeks after operation, the bone mineral density and bone volume fraction in the experimental group were significantly higher than those in the control group (P<0.05), and there was no significant difference between the two groups in trabecular thickness (P>0.05). At 26 weeks after operation, the bone mineral density of the experimental group was significantly higher than that of the control group (P<0.05). There was no significant difference in bone volume fraction and trabecular thickness between the two groups (P>0.05). Conclusion The new plastic bone filler material is an excellent bone filler material with good biosafety and osteoinductive activity.