OBJECTIVE To manufacture adriamycin-porous tricalcium phosphate (A-PTCP) ceramic drug delivery system (DDS) as a possible method for bone defect treatment after bone tumor operation. METHODS A-PTCP DDS was made from putting adriamycin into PTCP. Thirty rabbits were divided randomly into group A(24 rabbits) and group B(6 rabbits). A-PTCP was implanted in the greater trochanter of the right femur in group A. Adriamycin were injected into veins in group B. Muscle around A-PTCP and plasma were taken out at different period. Adriamycin concentrations in muscle and plasma were measured by high performance liquid chromatography (HPLC). RESULTS A-PTCP could gradually release adriamycin over 10 weeks. Adriamycin concentrations in the muscle were higher than that in plasma. CONCLUSION A-PTCP may be a new method for repairing bone defects after bone tumor operation.
Objective To investigate the influence of different dose levels of hydroxyapatite/tricalcium phosphate (HA/TCP) on the proliferation and alkalinephosphatase (ALP) activity of rabbit osteoblasts. Methods Three different doselevels of HA/TCP (10%, 40%, 70%) were co-cultivated with rabbit osteoblasts respectively. The proliferation and ALP expression capacity of osteoblasts were examined with MTT method and enzyme histochemistry once every 24 hours until 5 days. Three control groups of other materials were treated and examined in the sameway: rabbit osteoblasts as normal control; polyvinylchloride as positive control; titanium alloy as negative control. Results There was remarkable timeeffect relationship in the proliferation of osteoblasts. Ten percent HA/TCP did not affect osteoblasts growth while 40% HA/TCP could slow the cell growth rate down though time-effect relationship still existed. The proliferation of osteoblasts stagnated when co-cultivated with 70% HA/TCP. On the other hand, 10% HA/TCP could cause reversible damage on ALP activity of osteoblasts, whereas when the dose was40%, and the cultivation lasted 6 days the damage was irreversible. Three different dose levels of titanium alloy (10%, 40%, 70%) had no effect on the proliferation or ALP activity of osteoblasts. Conclusion Dosage is an important factor affecting the biocompatibility evaluation of biomaterial. It suggests that dose choosing should be more specified upon each individual biomaterial. It also indicates that ALP may be a good supplementary index of the cell compatibility of material.
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.
Objective To observe the effect of cationic liposomal ceftazidime (CLC) combined with nano-hydroxyapatite/β-tricalcium phosphate (n-HA/β-TCP) in the treatment of chronic osteomyelitis of rabbits. Methods Thirty healthy New Zealand white rabbits (4-6 months old; weighing, 2-3 kg) were selected to prepare the chronic osteomyelitis models. After 4 weeks, the gross observation, X-ray examination, and bacteriological and histopathological examinations were done; the models were made successfully in 27 rabbits. Of 27 rabbits, 24 were randomly divided into 4 groups (n=6): only debridement was performed in group A; ceftazidime was given (90 mg/kg), twice a day for 8 weeks after debridement in group B; ceftazidime and n-HA/β-TC were implanted after debridement in group C; and CLC and n-HA/β-TCP were implanted after debridement in group D. Before and after treatments, X-ray examination was done, and Norden score was recorded. At 8 weeks after treatment, the specimens were harvested for gross observation and for gross bone pathological score (GBPS) using Rissing standard; half of the specimens was used for histological observation and Smeltzer scoring, the other half for bacteriological examination and calculation of the positive rate of bacteria culture. Results At 8 weeks after treatment, Norden score of group D was significantly lower than that of groups A, B, and C (P lt; 0.05), but no significant difference was found among groups A, B, and C (P gt; 0.05). At 8 weeks after treatment, sinus healed in groups C and D, but sinus was observed in groups A and B; the GBPS scores of groups C and D were significantly lower than those of groups A and B (P lt; 0.05). The Smeltzer scores of groups C and D were significantly lower than those of groups A and B (P lt; 0.05). The positive rates of bacteria culture of groups C (0) and D (0) were significantly lower than those of group A (25.0%) and group B (16.7%) (P lt; 0.05). Conclusion CLC combined with n-HA/β-TCP has good effect in treating chronic osteomyelitis of rabbits, and it has better effect in treating chronic osteomyelitis of rabbits than ceftazidime with n-HA/β-TCP.
ObjectiveTo study the effect of three-dimensional (3D) printed β-tricalcium phosphate (β-TCP) scaffold loaded poly (lactide-co-glycolide) (PLGA) anti-tuberculosis drug sustained release microspheres on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and its cytotoxicity.MethodsIsoniazid and rifampicin/PLGA sustained release microspheres were prepared by W/O/W multiple emulsion method. The β-TCP scaffolds were prepared by 3D printing technique. The microspheres were loaded on the scaffolds by centrifugal oscillation method to prepare composite materials. The BMSCs of Sprague Dawley rat were isolated and cultured by whole bone marrow adherent method, and the third generation cells were used for the following experiments. BMSCs were co-cultured with osteogenic induction medium (group A), PLGA anti-tuberculosis drug sustained release microsphere extract (group B), 3D printed β-TCP scaffold extract (group C), and 3D printed β-TCP scaffold loaded PLGA anti-tuberculosis drug sustained release microsphere composite extract (group D), respectively. Cytotoxicity was detected by cell counting kit 8 (CCK-8) method; the calcium deposition was observed by alizarin red staining; and the mRNA expressions of alkaline phosphatase (ALP), osteocalcin (OCN), and bone sialoprotein (BSP) were detected by real-time fluorescence quantitative PCR (RT-qPCR).ResultsCCK-8 assay showed that the absorbance (A) value of groups A, B, C, and D increased gradually with the culture time prolonging. After cultured for 24, 48, and 72 hours, the A value decreased in the order of groups A, C, B, and D. There was no significant difference between groups B and D (P>0.05), but there were significant differences between other groups (P<0.05). The cytotoxicity was evaluated as grade 0-2, and the toxicity test was qualified. Alizarin red staining showed that red mineralized nodules were formed in all groups at 21 days after osteogenic induction, but the number of mineralized nodules decreased sequentially in groups C, D, A, and B. RT-qPCR test results showed that the relative expressions of OCN and BSP genes in groups A, B, C, and D increased gradually with the culture time prolonging. The relative expression of ALP gene increased at 7 and 14 days, and decreased at 21 days. After cultured for 7, 14, and 21 days, the relative expressions of ALP, OCN, and BSP genes decreased sequentially in groups C, D, A, and B; the differences were significant between groups at different time points (P<0.05).Conclusion3D printed β-TCP loaded PLGA anti-tuberculosis drug sustained release microsphere composites have no obvious cytotoxicity to BMSCs, and can promote BMSCs to differentiate into osteoblasts to a certain extent.
ObjectiveTo evaluate the biological effect on vascularization during bone repair of prevascularized porous β-tricalcium phosphate (β-TCP) tissue engineered bone (hereinafter referred to as prevascularized tissue engineered bone), which was established by co-culture of endothelial progenitor cells (EPCs) and bone marrow mesenchymal stem cells (BMSCs) based on tissue engineering technology. Methods EPCs and BMSCs were isolated from iliac bone marrow of New Zealand white rabbits by density gradient centrifugation and differential adhesion method. The cells were identified by immunophenotypic detection, BMSCs-induced differentiation, and EPCs phagocytosis. After identification, the third-generation cells were selected for subsequent experiments. First, in vitro tubule formation in EPCs/BMSCs direct contact co-culture (EPCs/BMSCs group) was detected by Matrigel tubule formation assay and single EPCs (EPCs group) as control. Then, the prevascularized tissue engineered bone were established by co-culture of EPCs/BMSCs in porous β-TCP scaffolds for 7 days (EPCs/BMSCs group), taking EPCs in porous β-TCP scaffolds as a control (EPCs group). Scanning electron microscopy and laser scanning confocal microscopy were used to observe the adhesion, proliferation, and tube formation of cells. Femoral condyle defect models of 12 New Zealand white rabbits were used for implantation of prevascularized tissue engineered bone as the experimental group (n=6) and porous β-TCP scaffold as the control group (n=6). The process of vascularization of β-TCP scaffolds were observed. The numbers, diameter, and area fraction of neovascularization were quantitatively evaluated by Microfill perfusion, Micro-CT scanning, and vascular imaging under fluorescence at 4 and 8 weeks. ResultsThe isolated cells were BMSCs and EPCs through identification. EPCs/BMSCs co-culture gradually formed tubular structure. The number of tubules and branches, and the total length of tubules formed in the EPCs/BMSCs group were significantly more than those in the EPCs group on Matrigel (P<0.05) after 6 hours. After implanting and culturing in porous β-TCP scaffold for 7 days, EPCs formed cell membrane structure and attached to the material in EPCs group, and the cells attached more tightly, cell layers were thicker, the number of cells and the formation of tubular structures were significantly more in the EPCs/BMSCs group than in the EPCs group. At 4 weeks after implantation, neovascularization was observed in both groups. At 8 weeks, remodeling of neovascularization occurred in both groups. The number, diameter, and area fraction of neovascularization in the experimental group were higher than those in the control group (P<0.05), except for area fraction at 4 weeks after implantation (P>0.05). ConclusionThe prevascularized tissue engineered bone based on direct contact co-culture of BMSCs and EPCs can significantly promote the early vascularization process during bone defects repair.
Objective To explore a novel nanometer biomaterial which could induce the regeneration of tooth tissues intell igently, and to evaluate the feasibil ity of using this kind of biomaterial as the scaffold for tooth tissue engineering by investigating the role it plays in tooth tissue engineering. Methods The scaffold for tooth tissue engineering containing recombinant human bone morphogenetic protein 2 (rhBMP-2) was prepared by mixing nanoscale β tricalcium phosphate (β-TCP)/collagen particles. Forty-six 8-10 weeks old specific pathogen free Sprague Dawley (SD)rats, including 34 females and 12 males, weighing 250-300 g, were involved in this study. Tooth germs were removed under a stereomicroscope from the mandible of newborn SD rat, then digested and suspended. Scanning electronic microscope (SEM), adhesion rate of cells, and MTT assay were used to evaluate the effects of the scaffold on the tooth germ cells cultured in vitro. The tissue engineered tooth germ which was constructed by tooth germ cells and scaffold was transplanted under SD rat’s kidney capsule as the experimental group (n=12); the tooth germ cells (cell-control group, n=12) or scaffold without cells (material-control group, n=4) were transplanted separately as control groups Specimens were harvested to perform general and histological observations at 4 and 8 weeks after transplantation. Results β-TCP/collagen showed a loose and porous appearance with soft texture and excellent hydrophil icity. Tooth germ cells grew well and could attach to the scaffold tightly 3 days after coculture. The adhesion rates of tooth germ cells were 27.20% ± 2.37%, 44.52% ± 1.87%, and 73.81% ± 4.15% when cocultured with scaffold for 4, 8, and 12 hours, respectively. MTT assay showed that the cell prol iferation status of experimental group was similar to that of the control group, showing no significant difference (P gt; 0.05). Some white calcified specimens could be harvested at 4-8 weeks after transplantation. At 4 weeks after transplantation some typical structures of dental cusp and enamel-dentin l ike tissues could be seen in the experimental group. Enamel-dentin l ike tissues also formed in some specimens of cell-control group, but they arranged irregularly. At 8 weeks after transplantation the enamel-dentin l ike tissue of experimental group exhibited a mature appearance and organized structure in comparison with that at 4 weeks. And mature enamel or dentin l ike tissue also could be seen in cell-control group. In contrast, there was no enamel or dentin l ike tissue in material-control group at 4 or 8 weeks after transplantation. Conclusion rhBMP-2 decorated β-TCP/collagen scaffold has good biocompatibil ity and can be used as a novel nanometer biomaterial, so it is a good choice in scaffolds for tooth tissue engineering.
Objective To study an optimal ratio of small intestinal submucosa (SIS) and (hydroxyapatite-tricalcium phosphate,HA-TCP,SIS/HA-TCP) compositions according to the effect of SIS/HA-TCP compositions with different ratios on repairing rabbit femoral condyle defect. Methods Thirty-six rabbits were made into bone defect models of 6 mm in diameter and 10 mm in depth in both sides of femoral condyles. Three different ratios of SIS/HA-TCP compositions (w/w: 1, 0.5, 0.25) were implanted into rabbit femoral condyle defect. After 2, 4, 8 and 12 weeks of operation, the repair effect wasobserved grossly. The histological evaluations were performed by histological scoring system and computer imaging analysis system. Results The amount of new bone formation in SIS/HA-TCP(0.5) group was more than that in SIS/HA-TCP(1) and SIS/HA-TCP(0.25) groups. Histological observation: In SIS/HA-TCP(1) group, few new bone formation was seen and bone defect was repaired in the 12th week. In SIS/HA-TCP(0.5) group, immature woven bone was found in the defect in the 2nd week; more immature woven bone appeared and formed trabeculae in the 4th week; the regenerated bone was vigorously growing into the interspaces of the implanted materials in the 8th week; the implanted materials was basically replaced by bony structure and the lamellar bone appeared in the 12thweek. The results of SIS/HA-TCP (0.25) group were similar to that of SIS/HA-TCP(0.5) group. The histological scoring was higher in SIS/HA-TCP(0.5) and SIS／HA-TCP(0.25) groups than that in SIS/HA-TCP(1) group (Plt;0.05) in the 2nd, 4th, 8th, and 12th weeks. The scoring was higher in SIS/HA-TCP(0.5) roup than that in SIS/HA-TCP(0.25) group in the 2nd and 12th weeks(P＜0.05). In new bone formation and the degradation of HA-TCP, SIS/HA-TCP(0.5) and SIS／HA-TCPC(0.25) groups were superior to SIS/HA-TCP(1) group(Plt;0.05), SIS/HA-TCP(0.5) group was superior to SIS/HA-TCP(0.25) group (Plt;0.05). Conclusion SIS/HA-TCP(0.5) has better effects of repairing bone defect and it can be used as a reference ratio in constructing bone scaffolds.
Objective To review the research progress of strontium (Sr) modified β-tricalcium phosphate composite biomaterials (SrTCP) promoting osteogenesis through immune regulation, and provides reference and theoretical support for the further development and research of SrTCP bone repair materials in bone tissue engineering in the future. Methods The literature about SrTCP promoting osteogenesis through immune regulation at home and abroad in recent years was extensively reviewed, and the preparation methods, immune mechanism and application of promoting osteogenesis were summarized and analyzed. Results The preparation methods of SrTCP include solid-state reaction sintering method, solution combustion quenching method, direct doping method, ion substitution method, etc. SrTCP has immune regulatory effects, which can play an immune regulatory role in inducing macrophage polarization, inducing angiogenesis and anti oxidative stress to promote osteogenesis. ConclusionAt present, studies have shown that SrTCP can promote bone defect repair through immune regulation. Subsequent studies can start from the control of the optimal repair concentration and release rate of Sr, and further clarify the specific mechanism of SrTCP in promoting angiogenesis and anti oxidative stress, which is helpful to develop new materials for bone defect repair.
ObjectiveTo explore the osteogenesis effect of advanced-platelet-rich fibrin (A-PRF) and β-tricalcium phosphate (β-TCP) composite. MethodsThirty-two healthy female New Zealand rabbits were randomly selected. A-PRF was prepared by collecting blood from middle auricular artery. Rabbits were randomly divided into 6 groups: groups A, B, C, D, and E (6 rabbits in each group) and group F (2 rabbits). Bone defects (6 mm in diameter, 8 mm in depth) were drilled into femur condyle of each rabbit’s both back legs. Then A-PRF and β-TCP composites manufactured by different proportion were planted into bone defects of group A (1∶1), group B (2∶1), group C (4∶1), group D (1∶2) and group E (1∶4) (V/V). The bone defect was not repaired in group F. The specimens were collected at 8 and at 12 weeks after operation. Then gross observation, X-ray examination, Micro-CT examination, and biomechanical test were performed. The bone volume/total volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular spacing (Tb.Sp), compressive strength, and modulus of elasticity were calculated. ResultsThe gross observation and X-ray examination showed that the osteogenesis effect at 12 weeks was better than that at 8 weeks. At the same time point, the repair of bone defect and the formation of new bone in group B were better than those in other groups. Micro-CT examination showed that the trabeculae of new bone in group B were the most and the trabeculae arranged closely at 8 and 12 weeks. Besides there were significant differences in BV/TV, Tb.N, and Tb.Sp between group B and the other groups (P<0.05). There were significant differences in Tb.N and Tb.Th in group B, BV/TV and Tb.Sp in group C, Tb.Sp in group D between 8 weeks and 12 weeks (P<0.05). Biomechanical tests showed that the compression strength and elastic modulus of group B were the highest, and the compression strength and elastic modulus of group C were the lowest at 8 and at 12 weeks, showing significant differences (P<0.05). There were significant differences in compression strength and elastic modulus of each group between 8 weeks and 12 weeks (P<0.05). ConclusionThe A-PRF and β-TCP composite can repair femoral condylar defects in rabbits, and the osteogenesis is better in proportion of 2∶1.