Objective To study the effect of local appl ication of different concentrations of nerve growth factor (NGF) on fracture heal ing, and to further search for the appropriate concentration gradient of NGF to promote fracture heal ing. Methods Seventy-five adult male Sprague Dawley rats, weighing (220.0 ± 2.5) g, were made the right tibia fracture model at 1 cm distal from the tibial tubercle and randomly divided into 5 groups (groups A, B, C, D, and E, n=15). Fractures were treated with 0.3 mL normal sal ine containing different concentration of NGF (0.006 48 × 10-2, 0.032 40 × 10-2, 0.162 00 ×10-2, and 0.810 00 × 10-2 μg/g) in groups A, B, C, and D, respectively, and the same amount of normal sal ine in group E. After2, 4, and 6 weeks, the specimens were harvested from 5 rats of each group to perform the biochemical test and histological observation. Before the rats were sacrificed, the arteriovenous blood was taken from the eye-ball to test the alkal ine phosphatase (ALP) activity. Results After 2, 4, and 6 weeks, the gross observation showed that the size and hardness of bone tissue and callus tissue growth gradually increased in groups A, B, C, and D, and group D was higher than groups A, B, C, and E. The X-ray films showed that the calcified area gradually increased in groups A, B, C, and D, and group D was higher than groups A, B, C, and E. The histological observation showed that the trabecular qual ity and maturity in group D were better than those in groups A, B, C, and E. Group D was significantly higher than groups A, B, C, and E (P lt; 0.05) in the gray values of callus tissue and the calcium content of callus tissue at 4 and 6 weeks, in the wet weight of callus tissue at 2 and 4 weeks, and in the ALP content of serum at 2 weeks. The trabecula surface index of osteoblast, the trabecular volume, and the trabecular width decreased as time in the order of groups A, B, C, and D, which were higher than those of group E; group D was the highest, showing significant differences when compared with the other groups (P lt; 0.05). Conclusion The local appl ication of NGF can promote fracture heal ing in rats. The high concentration gradient of NGF (0.810 00 × 10-2 μg/g) has an obvious promotion role on fracture heal ing.
Objective Series of compl icated molecule signal pathway are involved in the bone regeneration. To explore the possibil ity of nuclear factore kappa B (NF-κB) which is taken as the “key activation” during the fracture healing and provide the laboratory evidence for the gene therapy of nonunion or delayed union of fractures. Methods Thirtythree adult male Wistar rats (weighing 180-220 g) were selected and divided randomly into 4 groups: group A (the control group, n=3), the rigth lower segments of radius were injected with normal sal ine 0.3 mL for 7 days, once per day; group B (Bay 11-7082 injection group, n=6), the middle and distal radius were injected with normal sal ine containing 50 μmol/L NF- κB inhibitor Bay 11-7082 0.3 mL for 7 days, once per day; group C (fracture group, n=12), the right middle and distal radius were cut by a sharp scissors to form per fracture model; and group D (Bay 11-7082 treatment group, n=12), based on group C, 0.3 mL of 50 μmol/L Bay 11-7082 were injected into the fracture site for 7 days, once per day. The callus tissues were harvested at 3, 7, 14, and 28 days after fracture for Western blot analysis, alkal ine phosphatase (ALP) activity assessment, prostaglandins E2 (PGE2) production assay, and histological observation. Results The rats of all groups were survivaltill the experiment completion. At 3 and 7 days after injection, there was no significant difference in the ALP activity and PGE2 production between group B and group A (P gt; 0.05); but group C was significantly higher than group A (P lt; 0.01) and group D was significantly lower than group A (P lt; 0.01). The expressions of NF-κB p65, bone morphogenetic protein 7 (BMP-7), and inhibitor of DNA binding 2 (Id2) were observed at fracture sites of 4 groups. There was no significant difference in the expressions of NF-κB p65, BMP-7, and Id2 between group B and group A (P gt; 0.05); the expressions of NF-κB p65 and BMP-7 were significantly higher and the expression of Id2 was significantly lower in group C than group A (P lt; 0.01); and the expressions of NF-κB p65 and BMP-7 were significantly lower and the expression of Id2 was significantly higher in group D than group A (P lt; 0.01). The histological observation showed that a lot of osseous callus formed in group C at 14 and 28 days, but osseous callus just began to form in group D at 28 days. Conclusion NF-κB p65 can facil itate early fracture heal ing of rat radius by elevating the PGE2 production and regulating BMP-7 and Id2 expression.
To evaluate the effects of XiangLingGuBao (XLGB) on femoral fracture heal ing in ovariectomized (OVX) rats. Methods Forty 12-week-old female SD rats weighing (258 ± 14) g were divided randomly into 4 groups (n=10 per group): group A, sham operation by opening the abdominal cavity; group B, bilateral ovariectomy; group C, bilateral ovariectomy, transverse midshaft fracture of the right femur with intramedullary nail fixation, and normal sal ine by gavage; group D, bilateral ovariectomy, transverse midshaft fracture of the right femur with intramedullary nail fixation, and 250 mg/(kg•d) XLGB by gavage. The weight of rabbits in groups A and B was measured 0, 1, 2, 3, 4 and 5 weeks after operation. The right femur of each rat was obtained 5 weeks after operation. Total femur bone mineral density (tBMD), distal femur bone mineral density (dBMD) and middle femur bone mineral density (mBMD) were measured by double energy X-ray absorptiometry CR filming, HE staining and immunohistochemistry staining of groups C and D were performed. Results The weight of rats in group B was obviously higher than that of group A at 3, 4 and 5 weeks after operation (P lt; 0.05), indicating the animal model of postmenopausal osteoporosis was establ ished successfully. CR films showed more callus and obscure fracture l ine in group D, while less callus and distinct fracture l ine in group C. The tBMD and the dBMD of group B were far less than that of group A, the mBMD of group D was significantly higher than that of group C(P lt; 0.05), the tBMD and the dBMD of group D were higher than that of group C, but no significant difference was evidentbetween two groups (P gt; 0.05). Histology observation showed, when compared with group C, most fracture ends in groupD reached bone union, and the introduction of capillaries was evident in the marrow cavity. Immunohistochemistry staining demonstrated that the BMP-2 integrated absorbance (IA) value in groups C and D was 2.236 6 ± 0.181 8 and 3.727 3 ± 0.874 2, respectively, the VEGF IA value in groups C and D was 2.835 5 ± 0.537 0 and 3.839 6 ± 0.223 0, respectively, indicating there were significant differences between two groups (P lt; 0.05). Conclusion XLGB can obviously promote the femoral fracture heal ing in OVX rats, and speed the transformation of woven bone into lamellar bone, which may rely on its role of enhancing expression of BMP-2 and VEGF.
To detect the influence of raloxifene (RLX) on fracture heal ing in rabbit. Methods Eighty healthy New Zealand white rabbits (44 females and 36 males) weighing 1.9-2.1 kg were used. A 0.5-cm bone defect model in the mid-diaphysis of the left forel imb radius was establ ished in 72 rabbits, which thereafter were divided into 4 groups (n=18 per group, 10 females and 8 males): groups A, B and C received 7.5, 15.0 and 30.0 mg/ (kg• d) RLX, respectively, from the 2nd to the 50th postoperative day; group D received no further treatment. The rest untreated 8 rabbits (4 females and 4 males) served as normal control for serum osteocalcin detection. At different postoperative time points, bone mineral density detection, X-ray scanning, biomechanics measurement, histology and immunohistochemistry observations were conducted; serum estradiol, plasma cholesterol, serum osteocalcin and the ratio of uterine weight to body weight were detected. Results The bone mineral density of each group reached a peak 20 days after operation, showing a significant difference between groups A, B and C and group D (P lt; 0.05), and no significant differences among groups A, B and C (P gt; 0.05). On the 30th and 50th postoperative day, the maximum failure load and the maximum displacement of groups A, B andC were greater than those of group D (P lt; 0.05), but no significant differences among groups A, B and C were evident (P gt;0.05). On the 7th, 20th and 30th postoperative day, the X-ray score of fracture heal ing of groups A, B and C was greater than group D (P lt; 0.05); on the 50th postoperative day, there was significant difference between groups B and C and group D, and between group A and group C (Plt; 0.05), and no significant difference was evident between group B and group C (P gt; 0.05). The percentage of new bone formation in the fractured area of groups A, B and C was greater than that of group D on the 30th and 50th postoperative day (P lt; 0.05). For the type II collagen protein secretion in the fractured area, groups B and C were superior to group D on the 30th postoperative day (P lt; 0.05), and there was no significant difference between group A and group D (P gt; 0.05); no significant differences among four groups were evident on the 50th postoperative day (P gt; 0.05). On the 10th, 30th and 50th postoperative day, the serum osteocalcin of groups A, B, C and D was higher than that of normal control (P lt; 0.05), groups B and C were higher than group D (P lt; 0.05), and there was no significant difference between groups A, B and C, and between group A and group D (P gt; 0.05). For the plasma cholesterol, on the 30th postoperative day, no significant change was detected in each group (P gt; 0.05); on the 50th postoperative day, obvious decrease was observed in groups A, B and C, showing a significant difference compared with group D (P lt; 0.05). On the 30th and 50th postoperative day, there was significant difference between groups B and C and group D in serum estradiol (P lt; 0.05), and no significant differences were evident among other groups (P gt; 0.05). On the 30th and 50th postoperative day, the ratio of uterine weight to body weight in groups B and C was less than that of group D (P lt; 0.05), and no significant difference was evident between group A and group C (P gt; 0.05). Conclusion Oral administration of 7.5 mg/ (kg• d) RLX can promote the fracture heal ing of rabbit radius defect models safely and effectively.
Objective To investigate the changes in the expression level of bone morphogenetic protein 2 (BMP2) in the bone callus of rats with femoral fracture and brain injury to explore the effect of the brain injury on the fracture healing and to explore the related mechanism. Methods Thirty-two 12 week old SD rats weighing 368±25 g were randomly divided into 4 groups of 8 rats in each. The rats in Group A had a femoral fracture and a brain injury for 1 week; the rats in Group B had a femoral fracture but without brain injury for 1 week; the rats in Group C had a fracture and a brain injury for 2 weeks; and the rats in Group D had a fracture but without brain injury for 2 weeks. Thus, Groups A and C were used as the femoral fracture and brain injury models, and Groups B and D as the pure femoral fracture models for the controlled study. After the X-ray films were taken, the bone callus was obtained 1 week and 2 weeks after operation, respectively. Then, the bone callus growth and its histology were examined with theHE staining, the expression and changes in the level of BMP-2 were examined with the immunohistochemical staining, and the level of BMP-2 mRNA was measured with the RT-PCR. Results The X-ray films showed that less bone callus formation was found in Group A, and the fracture line in Group B was clearer than that in Group A. There was a greater amount of callus in Group C, and the fracture line was blurred. Only a little bone callus formation was found in Group D. The HE staining indicated that more fibroblasts and early-stage chondrocytes were found in Group A; some fibroblasts in the fracture interspace and fewer early-stage chondrocytes in Group B; some newly-formed trabecular bone at the end of the fracture in Group C; but no trabecular bone formation in Group D. The immunohistochemical staining indicated that the positive expression of BMP2 was b in the cytoplasms of the fibroblasts, the mesenchymal cells, the vascular endothelial cells, the early-stage chondrocytes, and the osteoblasts. The number of the positive cells was greater in Group A than in Group B, with a higher color intensity. The number of the positive cells was greater in Group C than in Group D, with a higher color intensity. The percentages of the cells positive for BMP-2 in the callus were greater in Groups A and C (0.762%±0.052%，0.756%±0.079%)than in Groups B and D (0.702%±0.052%，0.672%±0.044%) at the same time point, ith a statistically significant difference (Plt;0.05). The RT-PCR analysis showed that the expression of BMP-2 mRNA in the callus in Groups A-D was decreased in sequence. There was a significantly higher level of the expression in Groups A and C(1.07±0.13，0.78±0.11) than in Groups B and D(0.91±0.12，0.61±0.08) at the same time point (Plt;0.05). Conclusion The brain injury can promote the fracture healing process, which is probably related to an increase in the expression level of BMP-2 after the brain injury.
OBJECTIVE: To observe the difference of the fracture reparation using autogeneic-iliac bone and allogenic bone. METHODS: Comminuted fracture of humerus in two sides were made in rabbits. Autogeneic-iliac bone was implanted in one side, while allogenic bone of equal capacity was implanted in the other side. General observation, X-ray, and HE histologic section were done when the rabbits were put to death in different stages. RESULTS: One week after implantation, the graft had been enclosed by connective tissue without infiltration of the inflammatory cells. At the 2nd week, the graft had been enclosed in osteoplastic granulation tissue, and the cartilage callus had formed. At the 3rd week, there had been broken sequestrum among the callus; the cartilage had actively formed the bone; and the medulla had been making. At the 4th week, the sequestrum had disappeared, and the mature callus had appeared; the osteoblasts had arranged in a line around the edge of the mature callus. At the 5th week, the callus was b, compact and approached mature bones. At the 6th week, there had been the compact lamellar structures and the complete haversian’s systems. There was no significant difference between callus of two sides by using image quantitative analysis in the 3rd, 4th week (P gt; 0.05). CONCLUSION: The allogenic bone has good histocompatibility and bone conduction effect, and can be used for bone transplantation substitute with autogenous-iliac bone.
OBJECTIVE: To observe the promoting effect of phenytoin on fracture healing. METHODS: Fourty cases with close fractures of tibia and fibula were included and divided into two groups randomly: the experimental group was administrated with phenytoin and Chinese traditional drug-Jiegudan orally, while the control group was given Jiegudan only. Longitudinal percussion pain, clinical healing time of fracture, growth of calus in X-ray film were detected to evaluate the clinical result. RESULTS: All the fractures were healed in 3 months. But the experimental group was superior to the control group in all indexes. CONCLUSION: Administration of phenytoin orally can markedly promote fracture healing.
Thirty native goats were equally divided into two groups at random.A transverse fracture wasmade at the middle of the femur and tibia on the same side .The c;pver-shaped pin was used to fix thefemur and the Kirshners wire for the tibia.The experimental animals were given L-Dopu tablers.The animals were undergone the gross examination,roentgenographic eXamination, histolgical study,electron microseopic scanning and the examination of blood chemistry at 2,4, 6,8and 12 weeks afteroperatio...
ObjectiveTo investigate the mechanism of Semaphorin 3A (Sema3A) in fracture healing after nerve injury by observing the expression of Sema3A in the tibia fracture healing after traumatic brain injury (TBI). MethodsA total of 192 Wistar female rats, 8-10 weeks old and weighing 220-250 g, were randomly divided into tibia fracture group (group A, n=48), TBI group (group B, n=48), TBI with tibia fracture group (group C, n=48), and control group (group D, n=48). The tibia fracture model was established at the right side of group A; TBI model was made in group B by the improved Feeney method; the TBI and tibia fracture model was made in group C; no treatment was given in group D. The tissue samples were respectively collected at 3, 5, 7, 14, 21, and 28 days after operation; HE staining, immunohistochemistry staining, and Western blot method were used for the location and quantitative detection of Sema3A in callus tissue. ResultsHE staining showed that no obvious changes were observed at each time point in groups B and D. At 3 and 5 days, there was no obvious callus growth at fracture site with inflammatory cells and fibrous tissue filling in groups A and C. At 7 and 14 days, fibrous tissue grew from periosteum to fracture site in groups A and C; the proliferation of chondrocytes in exterior periosteum gradually formed osteoid callus at fracture site in groups A and C. The chondrocyte had bigger size, looser arrangement, and more osteoid in group C than group A. Group B had disorder periosteum, slight subperiosteal bone hyperplasia, and no obvious change of bone trabecula in group B when compared with group D. At 21 and 28 days, cartilage callus was gradually replaced by new bone trabecula in groups A and C. Group C had loose arrange, disorder structure, and low density of bone trabecula, big callus area and few chondrocyte and osteoid when compared with group A; group B was similar to Group D. Immunohistochemistry staining showed that Sema3A expression in chondrocytes in group C was higher than that in group A, particularly at 7, 14, and 21 day. Sema3A was significantly higher in osteoblasts of new bone trabecula in group A than group C, especially at 14 and 21 days (P<0.05). Western blot results showed that the Sema3A had the same expression trend during fracture healing in groups A and C. However, the expression of Sema3A protein was significantly higher in group C than group A (P<0.05) and in group B than group D (P<0.05) at 7, 14, 21, and 28 days. ConclusionAbnormal expression of Sema3A may play a role in fracture healing after nerve injury by promoting the chondrocytes proliferation and reducing the distribution of sensory nerve fibers and osteoblast differentiation.