Objective To evaluate the histocompatibil ity of porous hydroxyapatite (HAP) coating NiTi shape memory alloy and to provide a theoretical basis for its cl inical appl ication in bone defect repair. Methods Twenty-four Chinchilla rabbits weighing 2.0-2.5 kg were randomized into experimental group and control group (n=12). HAP coating NiTi shape memory alloy was implanted into the distal part of left femur of 12 rabbits in the experimental group, while holes without alloy implantation were performed on the control group. At 7, 14, 28 and 56 days after implantation, the animals werekilled (3 rabbits in each group at a time). Gross observation, histology observation, BMP-2 immunohistochemistry observation and image grey scale analysis were performed. And the histology observation was evaluated by GB/T16886.6-1997 in terms of inflammation, capsule wall of fibrous tissue, materials degradation and the response of peripheral tissue. Results All of the animals survived until being killed. The implants reached a peak embedded in bone tissue wholly, without loosening and bone absorption. The inflammatory cell infiltration and fibrous hyperplasia were at 7 days after implantation, with the formation of cyst wall of fibrous tissue and the implant wrapped by the cyst wall. The response of connective tissue proliferation was still obvious in partial samples of experimental group at 56 days after implantation, which was wrose than the control group but consistent with the in vivo implantation standard of GB/T16886.6-1997. Immunohistochemistry observation displayed the endogenous BMP-2 were in the cytoplasm of MSCs and osteoblast. The result of image analysis showed the expression of BMP-2 were staged in line with the repair of bone defect, two groups witnessed the peak expression of the BMP-2 at 14 days after implantation. There wereno significant differences among different time points in the staining gray scale of BMP-2 (P gt; 0.05). Conclusion HAP coating NiTi shape memory alloy, as a biomedical material, has excellent histocompatibility with bone.
Objective To investigate the effect of rhBMP-2 combined with porous CPC on spine fusion in rabbits. Methods rhBMP-2 (1 mg) was loaded with 1 g CPC and 6.0 cm × 2.0 cm × 0.5 cm absorbable gelatin sponge (AGS), respectively, and thereafter frozen to prepare the biomaterial of rhBMP-2/CPC and rhBMP-2/AGS. Forty-five 24-week-old New Zealand rabbits (weight 2.5-3.5 kg) were randomly divided into 3 groups: group A (n=17), group B (n=11) and group C (n=17).With the exposure and removal of L5, 6 transverse process’s posterior bone cortex in all the rabbits, the corresponding cancellous bones were exposed and the posterior bilateral intertransverse bone grafting of L5, 6 were performed on the three groups, then the rhBMP-2/CPC, rhBMP-2/AGS and CPC was implanted into the rabbits of group A, B and C, respectively. Gross observation, histology assay and image examination were conducted 4, 8 and 24 weeks after operation. Results Decalcified hard tissue section demonstrated obvious callus connections in group A, small pieces of callus in group B, and fibrous connection and few cartilage in group C at 4 and 8 weeks after operation. By Kacena measurement standard, the score of group A, B and C at 4 weeks after operation was (7.30 ± 0.76), (3.68 ± 1.60) and (1.75 ± 0.54) points, respectively, and their score at 8 weeks after operation were (8.32 ± 1.11), (3.75 ± 1.23) and (1.47 ± 0.23) points, respectively, indicating there were significant differences between group A and group B as well as between group A and group C at different time points (P lt; 0.05). Undecalcified hard tissue section demonstrated that there was cancellous bone-l ike tissue regeneration in group A, and fiber connection around the implants and l ittle ossification in group C at 4 and 8 weeks after operation. By three dimensions reconstructed CT, group A, B and C scored (2.50 ± 0.57), (1.00 ± 0.00) and (1.00 ± 0.00) points respectively, indicating there was a significant difference between group C and groups A and B as well as between group A and group B (P lt; 0.05). Conclusion As a carrier of rhBMP-2, the CPC is capable of promoting spine bone fusion in rabbits and is a new type of artificial bone repair material.
Objective To investigate the cl inical appl ication of self-setting CPC loading rhBMP-2 for repair of bone defects and to evaluate the cl inical effect and safety. Methods From June 2006 to September 2007, 112 bone defects patients were treated by CPC loading rhBMP-2 (rhBMP-2/CPC group) or CPC (control group). The range of bone defect was from1 cm × 1 cm × 1 cm to 4 cm × 3 cm × 3 cm. In the control group, 63 patients included 31 males and 32 females, aging from 17 to 70 years with an average of 47.4 years. The bone defects were located as follows: calcaneus in 19 patients, tibial plateau in 20 patients, proximal humerus in 8 patients, distal radius in 9 patients and thoracolumbar vertebrae in 7 patients. In the rhBMP-2/CPC group, 49 patients included 31 males and 18 females, aging from 16 to 68 years with an average of 45.6 years. The bone defects were located as follows: calcaneus in 11 patients, tibial plateau in 16 patients, proximal humerus in 7 patients, distal radius in 2 patients, distal tibia in 2 patients and thoracolumbar vertebrae in 11 patients. All defects were repaired with rhBMP-2/CPC (2-5 g) and CPC (2-50 g) in the rhBMP-2/CPC group and the control group, respectively. Results A total of 108 patients got primary heal ing after operation. Incisions oozing l ight yellow fluids were found in 4 patients (control group in 1, rhBMP-2/CPC group in 3), and then healed through dressing changes and taking glucocorticoid. There were no allergic or toxic reaction, no rush or high fever, no fluctuation of hepatic and renal function, blood routine, CRP and urine routine. All patients were followed up for 12 to 24 months (mean 13.2 months). The X-ray examination showed that the implanted material was firmly bonded to the bone at the interface and the anatomic contour of the bone at the sites of defects was successfully restored, and no ablation occurred. All patients got bone union after 3 months of operation. The movement and function of flexion and extension of affected l imbs recovered to the normal level. Conclusion Repairing bone defects with rhBMP-2/CPC is safe and effective. Using rhBMP-2/CPC is a promising therapy to deal with bone defects.
Objective To investigate the effect of NGF on fracture heal ing, and to study the role of BMP-2 induced osteoblast. Methods Sixty cleaned male Kunming mice (aging 6-8 weeks and weighing 23-25 g) were made fracture models in the middle of femoral shaft and randomly divided into four groups (groups A, B, C and D, n=15). Fracture was treated with NGF/ normal sal ine, BMP-2, BMP-2 /NGF/normal sal ine, and normal sal ine in groups A, B, C and D, respectively. After 14, 21 and 28 days, the specimens were selected from 5 mice each group to do the biochemical and histological analysis. Beforethe mice were killed, the arteriovenous blood was taken from their eye-ball to test the ALP activity. Results After 14 days,21 days and 28 days, the gross observation showed that the size and hardness of bone tissue, and callus tissue growth increased in groups A, B and C order and were higher than those in group D; the X-ray films showed that the calcified area increased in groups A, B and C order and were higher than those in group D; the histological observation showed that the trabecular maturity increased in groups A, B and C order and were higher than those in group D. The osteoblast area, the gray degree value of the radiographs in callus tissue, the ALP contents of serum and callus tissue, calcium content of callus tissue and net weight of callus were higher in groups A, B and C than in group D. There were significant differences (P lt; 0.05) in osteoblast area and gray degree values of the radiographs at 14, 21 and 28 days; in ALP contents of serum at 14 days; in ALP contents of callus tissue at 14 days and 21 days; in calcium content of callus tissue at 21 days and 28 days among 4 groups. There were significant differences in net weight of callus between groups B, C and groups A, D at 14 days (P lt; 0.05). At 21 days and 28 days, the trabecular surface index of osteoblast, the average trabecular volume and the mean trabecular width decreased as time went on, having an increase order of groups A, B, C and was higher in groups A, B, C than in group D, showing significant differences among 4 groups (P lt; 0.05). Conclusion NGF promotes the heal ing of fractures. NGF possesses synergistic effect on ectopic bone formation induced by BMP-2.
Objective To investigate the effects of the recombinant plasmid pIRES-hBMP-2-hVEGF165 on differentiation and maturation of hBMSCs in vitro. Methods The co-expressing vector of hBMP-2 and hVEGF165 was constructed. The BMSCs were isolated and cultured from health adult human denoted marrow. By the l ipofection method, the reconstructed plasmids pIRES-hBMP-2-hVEGF165, pIRES-hBMP-2, pIRES-hVEGF165 and pIRES neo empty vector, weretransfected to hBMSCs (groups A, B, C and D). The untransfected cells were harvested as control group (group E). After4 weeks of culture, RT-PCR was employed to assay the hBMP-2, hVEGF165 and osteocalcin mRNA expression in hBMSCs. The expressions of hBMP-2 and hVEGF165 of BMSCs were assayed by Western blot. The level of ALP activities of BMSCs was determined. Col I was also determined by immunohistochemical staining. Results Compared to group E, the hBMSCs in group A secreted high level of hBMP-2, hVEGF165, Col I and osteocalcin; osteocalcin and Col I expressed at high level in group B, and hVEGF165 expressed at high level in group C. Otherwise, the expression of hVEGF165 in group B and the expressions of hBMP-2 and Col I in group C resemble to that of groups D and E, no expression or few expression was observed. The activities of ALP in groups A, B, C, D and E were 0.91 ± 0.03, 0.90 ± 0.02, 0.64 ± 0.03, 0.67 ± 0.01 and 0.66 ± 0.02, respectively. The activity of ALP of groups A and B were significantly increased compared with that of group E (P lt; 0.05); there was no significant difference among groups C, D and E (P gt; 0.05). Conclusion The recombinant plasmid pIRES-hBMP-2-hVEGF165 can be successfully transfected into BMSCs with cation l iposome-mediated transfection method, the exogenous hBMP-2 and hVEGF165 genes can be expressed constitutively in the transfected BMSCs, and it can enhance the differentiation abil ities of BMSCs.
【Abstract】 Objective To compare the effect of PLGA and collagen sponge combined with rhBMP-2 on repairing ofarticular cartilage defect in rabbits respectively. Methods PLGA and collagen sponge were made into cyl inders which were 4 mm in diameter and 3 mm in thickness, and compounded with rhBMP-2 (0.5 mg). Defect 4 mm in diameter were made in both of femoral condyles of 24 two-month-old New Zealand white rabbits. The defects in right 18 knees were treated with PLGA/rhBMP-2 composites (experimental group 1), and the left 18 knees were treated with collagen sponge/rhBMP-2 composites (experimental group 2), the other 12 knees were left untreated as control group. At 4, 12 and 24 weeks after operation, the animals were sacrificed and the newly formed tissues were observed macroscopically and microscopically, graded histologically and analyzed statistically. Results From the results of macroscopical and microscopical observation, in the experimental group 1, the defects were filled with smooth and translucent cartilage; while in the experimental group 2, the white translucent tissues did notfill the defects completely; and in the two experimental groups, the new cartilage tissues demarcated from the surrounding cartilage,chondrocytes distributed uniformly but without direction; a l ittle fibrous tissue formed in the control group 4 weeks postoperatively. In the experimental group 1, the defects were filled completely with white, smooth and translucent cartilage tissue without clear l imit with normal cartilage; while in the experimental group 2, white translucent tissues formed, the boundary still could be recognized; in the two experimental groups, the thickness was similar to that of the normal cartilage; the cells paralleled to articular surface in the surface layer, but in the deep layer, the cells distributed confusedly, the staining of matrix was positive but a l ittle weak; subchondral bone and tide mark recovered and the new tissue finely incorporated with normal cartilage;however, in the control group, there was a l ittle of discontinuous fibrous tissue, chondrocytes maldistributed in the border andthe bottom of the defects 12 weeks postoperatively. In the experimental group 1, white translucent cartilage tissues formed, the boundary disappeared; in the experimental group 2, the color and the qual ity of new cartilage were similar to those of 12 weeks; in the two experimental groups, the thickness of the new cartilage, which appeared smooth, was similar to that of the normal cartilage, the chondrocytes arranged uniformly but confusedly; the staining of matrix was positive and subchondral bone and tide mark recovered, the new tissue finely incorporated with normal cartilage; in the control group, a layer of discontinuous fibrous tissue formed in the bottom of the defects 24 weeks postoperatively. Results of histological grade showed that there were significantdifference between experimental group (1 and 2) and control group at any time point (P lt; 0.01); the scores of 12 weeks and 24 weeks in experimental group 1 and 2 had a significant difference compared with that of 4 weeks (P lt; 0.01), there was no significant difference between 12 weeks and 24 weeks (P gt; 0.05), and there were no significant difference between the two experimental groups at the same time point (P gt; 0.05). Conclusion Both PLGA and collagen sponge as a carrier compounded with rhBMP-2 can repair articular cartilage defects.
【Abstract】 Objective To evaluate the effect on repairing composite defect of mandible and skin by pre fabricatedmusculocutaneous flap including ectopic bone induced by BMP-2 and collagen in rabbits’ latissimus dorsimuscle. Meth ods Twenty-four rabbits (4-6 weeks old) were randomly divided into 3 groups: experimental, control and blank control group (n=8 in each group). Composite carriers composed of BMP-2 and collagen I sponge were implanted into latissimus dorsi muscle pouches of rabbits. The bone formation was evaluated with roentgenography, ALP staining, Von Kossa staining, HE staining, toluidine blue staining and CD31 immunohistochemical labell ing of microvessels. After 6 weeks, the mandibular defect of 8 mm in diameter with local skin defect of 2 cm × 3 cm was made in experimental group, and a musculocutaneous flap including ectopic-induced bone was prefabricated to transfer and repair the composite defect. The mandibular defect of 8 mm in diameter without local skin defect was made in control and blank control group. Free ectopic-induced bone was used for the repair of mandibular defect in control group, but repairing was not performed in blank control group. All the samples were detected 6 weeks after operation for tetracycl ine fluorescent staining, X-ray, histological examination and bone quantity analysis to evaluate the effect. Results Bone formation induced by BMP-2/collagen composites were found as woven bone between 4 to 6 weeks. It showed that cartilaginous osteogenesis was the mainly type of bone formation. Microvessels could beseen in the bony tissues. The composite defects of mandible and skin were healed well in the experimental group. Major bony tissue were seen in the control group, while it still remained bony defect in the blank control group. The bone quantity analysis in the experimental, control, and blank control group were (1.594 ± 0.674), (0.801 ± 0.036), and (0.079 ± 0.010) mm2, there were significant differences between each groups (P lt; 0.05). Conclusion Prefabrication of musculocutaneous flap including boneinduced by the composite of BMP-2 and collagen is feasible and prevalent. It can be regarded as vascularized bone graft and used in repairing composite defect of bone and skin.
【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.
【Abstract】 Objective To investigate the manufacture and biocompatibil ity of a bioabsorbable poly-D,L-lactic acid(PDLLA) plate containing rhBMP-2. Methods rhBMP-2 was composited with PDLLA (0.05 mg/plate) through vacuum to repare PDLLA plate containing rhBMP-2. Thirty-two New Zealand rabbits (male or female) weighted (3.0 ± 0.5) kg were used in he study. A 2.5 mm middle ulna osteotomy was made bilaterally. The bones as well as periosteum were removed. The right side of all the animals was experimental side (n=32), was fixed internally by PDLLA plate containing rhBMP-2.The left side of all the animals was control side (n=32), was fixed by common PDLLA plate. After a follow-up of 2, 4, 8 and 12 weeks, the ulnas were examined visually, radiographically, histologically, and by computer graph analysis to compare the biocompatibil ity. Re sults Po rosity of PDLLA plate containing rhBMP-2 was 90%, aperture was 150 μm, tensile strength was higher than 50 MPa, three point flexural strength was higher than 90 MPa and intrinsic viscosity was 1.6 dL/g (chloroform solvent). All animals had a good heal ing 1 or 2 weeks after operation. All the animal’s diet and movement were normal. All the fractures were stable. The plates in the experimental group degraded faster than those in the control group. Relative values of callus density evaluated by X-ray at 2, 4, 8 and 12 weeks after operation in the experimental group were 39.22 ± 2.48, 48.79 ± 1.26, 63.78 ± 1.78 and 78.60 ± 1.25 respectively. Those in the control defects were 33.83 ± 1.13, 41.28 ± 1.25, 55.23 ± 0.68 and 66.54 ± 1.33. At each time point, the experimental side produced better and more trabeculae than the control side did (P ＜ 0.01). Histological examination showed that the newbone formation in the experimental side at 2, 4, 8 and 12 weeks after operation was 0.106% ± 0.015%, 0.292% ± 0.019%, 0.457% ± 0.048% and 0.601% ± 0.037%, while those in the control side was 0, 0.193% ± 0.019%, 0.339% ± 0.029% and 0.574% ± 0.047%respectively. At each time point, the experimental side produced better new bone formation than the control side did (P lt; 0.05). The experimental side showed better compatibil ity between plates and surrounding tissue, faster bone formation, more bone regeneration mass and better medullary canal structure than the control side. Conclusion PDLLA plate containing rhBMP-2 has good biocompatibil ity and osteoinducibil ity to enhance fracture heal ing.
Objective To construct recombinant lentiviral vectors of porcine bone morphogenetic protein 2 (BMP-2) gene and to detect BMP-2 gene activity and bone marrow mesenchymal stem cells (BMSCs) osteogenetic differentiation so as to lay a foundation of the further study of osteochondral tissue engineering. Methods BMSCs were isolated from bone marrow of 2-month-old Bama miniature porcines (weighing, 15 kg), and the 2nd generation of BMSCs were harvested for experiments. The porcine BMP-2 gene lentiviral vector was constructed by recombinant DNA technology and was used to transfect BMSCs at multiplicity of infection (MOI) of 10, 25, 50, 100, and 200, then the optimal value of MOI was determined by fluorescent microscope and inverted phase contrast microscope. BMSCs transfected by BMP-2 recombinant lentiviral vectors served as experimental group (BMP-2 vector group); BMSCs transfected by empty vector (empty vector group), and non-transfected BMSCs (non-transfection group) were used as control groups. RT-PCR, immunohistochemistry staining, and Western blot were performed to detect the expressions of BMP-2 mRNA and protein. Then the BMSCs osteogenesis was detected by alkaline phosphatase (ALP) staining, ALP activities, and Alizarin red staining. Results The recombinant lentiviral vectors of porcine BMP-2 gene was successfully constructed and identified by RT-PCR and gene sequencing, and BMSCs were successfully transfected by BMP-2 recombinant lentiviral vectors. Green fluorescent protein could be seen in the transfected BMSCs, especially at MOI of 100 with best expression. The immunohistochemistry staining and Western blot showed that BMSCs transfected by BMP-2 recombinant lentiviral vectors could express BMP-2 protein continuously and stably at a high level. After cultivation of 2 weeks, the expression of ALP and the form of calcium nodules were observed. Conclusion The porcine BMP- 2 gene lentiviral vector is successfully constructed and transfected into the BMSCs, which can express BMP-2 gene and protein continuously and stably at a high level and induce BMSCs differentiation into osteoblasts.