Acceleration of bone repairation by BMSCs overexpressing NGF combined with NSA and allograft bone scaffolds.

BACKGROUND: Repairation of bone defects remains a major clinical problem. Constructing bone tissue engineering containing growth factors, stem cells, and material scaffolds to repair bone defects has recently become a hot research topic. Nerve growth factor (NGF) can promote osteogenesis of bone marrow mesenchymal stem cells (BMSCs), but the low survival rate of the BMSCs during transplantation remains an unresolved issue. In this study, we investigated the therapeutic effect of BMSCs overexpression of NGF on bone defect by inhibiting pyroptosis. METHODS: The relationship between the low survival rate and pyroptosis of BMSCs overexpressing NGF in localized inflammation of fractures was explored by detecting pyroptosis protein levels. Then, the NGF+/BMSCs-NSA-Sca bone tissue engineering was constructed by seeding BMSCs overexpressing NGF on the allograft bone scaffold and adding the pyroptosis inhibitor necrosulfonamide(NSA). The femoral condylar defect model in the Sprague-Dawley (SD) rat was studied by micro-CT, histological, WB and PCR analyses in vitro and in vivo to evaluate the regenerative effect of bone repair. RESULTS: The pyroptosis that occurs in BMSCs overexpressing NGF is associated with the nerve growth factor receptor (P75NTR) during osteogenic differentiation. Furthermore, NSA can block pyroptosis in BMSCs overexpression NGF. Notably, the analyses using the critical-size femoral condylar defect model indicated that the NGF+/BMSCs-NSA-Sca group inhibited pyroptosis significantly and had higher osteogenesis in defects. CONCLUSION: NGF+/BMSCs-NSA had strong osteogenic properties in repairing bone defects. Moreover, NGF+/BMSCs-NSA-Sca mixture developed in this study opens new horizons for developing novel tissue engineering constructs.

A novel adhesive dual-sensitive hydrogel for sustained release of exosomes derived from M2 macrophages promotes repair of bone defects.

The repair of bone defects remains a huge clinical challenge. M2 macrophage-derived exosomes (M2-Exos) can act as immunomodulators to promote fracture healing; however, how to retain the sustained release of exosomes to the target area remains a challenge. Here, we report a composite hydrogel loaded with M2-Exos aiming to accelerate bone defect healing. It was verified that the F127/HA-NB hydrogel had a dense network structure, tissue adhesiveness, and dual sensitivity to temperature and light. F127/HA-NB loaded with M2-Exos (M2-Exos@F127/HA-NB) exhibited good biocompatibility and achieved sustained release of exosomes for up to two weeks. The study showed that both M0-Exos and M2-Exos@F127/HA-NB significantly promoted osteogenic differentiation of rat bone marrow mesenchymal stem cells. The mechanism study implied that M2-Exos activates the Wnt/ß-catenin signaling pathway to promote osteogenic differentiation of BMSCs. Finally, we evaluated the osteogenetic effects of M2-Exos@F127/HA-NB in a rat cranial defect model, and the results showed that M2-Exos@F127/HA-NB had superior bone regeneration-promoting effects. This study provides a new strategy for cell-free treatment of bone defects.

[Learning curve analysis of unilateral biportal endoscopic lumbar interbody fusion].

Objective: To analyze the learning curve of unilateral biportal endoscopic lumbar interbody fusion (UBE-LIF). Methods: Fifty-five patients with single-segment lumbar degenerative disease treated with UBE-LIF between December 2020 and February 2022 were selected as the research subjects. The patients were grouped according to the operation sequence, the first 27 cases were in the early group, and the last 28 cases were in the late group. There was no significant difference between the two groups in age, gender, disease type, and surgical segment distribution ( P>0.05). The operation time, the amount of hemoglobin loss (the difference between 1 day before operation and 3 days after operation), the hospital stay after operation, and the incidence of perioperative complications were recorded; the learning curve of UBE-LIF was analyzed by log-curve regression analysis. Results: All the operations were successfully completed without changing to other operations. The operation time, the amount of hemoglobin loss, and hospital stay in the early group were significantly more than those in the late group ( P<0.05). Complications occurred in 2 cases (7.4%) in the early group, including 1 case of dural tear during operation and 1 case of epidural hematoma after operation, and 1 case (3.6%) with transient radiculitis in the late group. There was no significant difference in the incidence of complications between the two groups ( P=0.518) . The log-curve regression analysis showed that the operation time decreased significantly with the increase of the number of patients ( P<0.05). The operation time tended to be stable after the surgeon completed 17 cases. Conclusion: For single-level lumbar degenerative disease, the operation time of UBE-LIF can decrease gradually with the increase of the number of patients, and tend to be stable after 17 cases.

Puerarin suppresses inflammation and ECM degradation through Nrf2/HO-1 axis in chondrocytes and alleviates pain symptom in osteoarthritic mice.

Osteoarthritis (OA) is the most common degenerative joint disorder with no effective drugs. Puerarin is a dietary supplement that has wide-ranging pharmacological effects. This study aimed to investigate the effects of Puerarin on OA. The effects of Puerarin on apoptosis, extracellular matrix (ECM) metabolism, and inflammation-related factors were assessed; also, the nuclear factor-κB (NF-κB) signaling pathway and Nrf2/HO-1 (nuclear factor (erythroid-derived 2)-like 2/heme oxygenase-1) axis were evaluated to elucidate the working mechanism of Puerarin. Mice were fed with Puerarin to evaluate the therapeutic effect of Puerarin on Osteoarthritis in vivo. The results showed that Puerarin suppressed inflammatory mediators and apoptosis induced by IL-1ß treatment in chondrocytes, it may also suppress ECM degradation in IL-1ß treated chondrocytes. The mechanism study revealed that Nrf2/HO-1 pathway is involved in Puerarin induced inhibition of NF-κB signaling pathway. Finally, in vivo study demonstrated that Puerarin could postpone the progression of OA in mice and relieve the symptoms of pain. In conclusion, Puerarin may potentially alleviate OA progression, and the mechanism may relate to the Nrf2/HO-1 pathway regulation.

Xanthohumol suppresses inflammation in chondrocytes and ameliorates osteoarthritis in mice.

Osteoarthritis (OA), manifested as degeneration and damage of the articular cartilage is a progressive disease of joints. Previous studies have shown that extracellular matrix degradation and inflammation have quite a significant performance in the occurrence and development of OA. In various maladies, an anti-inflammatory effect has been demonstrated for Xanthohumol (XN); while OA is an inflammation related disease. The current in vivo and in vitro study aimed to investigate the therapeutic effect of XN on OA as well as its working mechanism. The results showed that XN has the capability to hinder the expression of nitric oxide synthase (INOS), IL-1ß-promoted inducible nitric oxide (NO), necrosis factor-α of tumor (TNF-α), interleukin-6 (IL-6), and cyclooxygenase-2 (COX-2) in vitro. In addition, XN has been found to down-regulate the expression of matrix metalloproteinase-13 and prothrombin stimulated by IL-1ß and up-regulates type II collagen and Aggrecan expression. At the same time, it was discovered that XN activates nuclear factor (Nrf2) in chondrocytes stimulated by IL-1ß and inhibits nuclear factor B (NF-кB) signal transduction. The DMM model manifests that XN has an inhibitory impact on the progression of osteoarthritis and thus may be a candidate drug to slow down and delay the development of OA.

[Lentivirus-mediated silencing of P75 neurotrophin receptor combined with nerve growth factor overexpression and transfection of bone marrow mesenchymal stem cells combined with demineralized bone matrix for heterotopic osteogenesis].

OBJECTIVE: To investigate the effects of silencing P75 neurotrophin receptor (P75NTR) and nerve growth factor (NGF) overexpression on the proliferative activity and ectopic osteogenesis ability of bone marrow mesenchymal stem cells (BMSCs) combined with demineralized bone matrix for heterotopic osteogenesis. METHODS: BMSCs of Sprague Dawley (SD) rats were cultured and passaged by adherent isolation method. The third generation BMSCs were transfected with lentivirus mediated P75NTR gene silencing (group B), NGF overexpression gene (group C), P75NTR silencing and NGF overexpression double genes (group D), respectively, and untransfected cells as control (group A). After 7 days of transfection, the expression of fluorescent protein of the target gene was observed by fluorescence microscope; cell counting kit 8 method was used to detect the cells activity for 8 days after transfection; the expressions of P75NTR and NGF proteins in each group were detected by Western blot. The adhesion of BMSCs to demineralized bone matrix (DBM) was observed by inverted phase contrast microscope and scanning electron microscope after transfection of p75NTR silencing and NGF overexpression double genes. After transfection, BMSCs and DBM were co-cultured to prepare 4 groups of tissue engineered bone, which were respectively placed in the dorsal subcutaneous tissue of 8-week-old SD rats to construct subcutaneous ectopic osteogenesis model ( n=6). HE staining was performed at 4 and 8 weeks after operation. ALP staining was used to observe the formation of calcium nodules at 8 weeks after operation. The expressions of Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and osteocalcin (OCN) were detected by real-time fluorescent quantitative PCR. RESULTS: At 7 days after transfection, there was no fluorescence expression in group A, red fluorescence expression was seen in group B, green fluorescence expression in group C, and red-green compound fluorescence expression in group D. The fluorescence expression rate of target gene was about 70%. Western blot detection showed that the relative expression of P75NTR protein in groups A and C was significantly higher than that in groups B and D, and the relative expression of NGF protein in groups C and D was significantly higher than that in groups A and B ( P<0.05). With the passage of time, the cell proliferation activity increased in all groups, especially in group D, which was significantly higher than that in group A at 3-8 days ( P<0.05). The results of inverted phase contrast microscope and scanning electron microscope showed that BMSCs could adhere well to DBM. In the subcutaneous ectopic osteogenesis experiment, HE staining showed that at 4 and 8 weeks after operation, the more bone tissue was formed in group D than in the other 3 groups. ALP staining showed that group D had the highest ALP activity and better osteogenic expression. Compared with group A, the relative expressions of Runx2, ALP, and OCN mRNAs in group D were significantly higher than those in group A ( P<0.05). CONCLUSION: Silencing P75NTR and NGF overexpression double genes co-transfected BMSCs with DBM to construct tissue engineered bone has good ectopic osteogenic ability. By increasing NGF level and closing P75NTR apoptosis channel, it can not only improve cell activity, but also promote bone tissue regeneration.

ß-Hydroxyisovalerylshikonin inhibits IL-1ß-induced chondrocyte inflammation via Nrf2 and retards osteoarthritis in mice.

Osteoarthritis is a chronic degenerative disease characterized by cartilage destruction. It is the fourth most disabling disease worldwide and is currently incurable. Inflammation and extracellular matrix (ECM) degradation are considered to be substantial reasons for accelerating the progression of OA. ß-Hydroxyisoamylshikonin (ß-HIVS) is a natural naphthoquinone compound with anti-inflammatory and antioxidant activity. However, the effect of ß-HIVS on OA is still unclear. In this study, we found that ß-HIVS can down-regulate the expression of NO, PEG2, IL-6, TNF-α, COX-2, and iNOS, suggesting its anti-inflammatory effects in chondrocytes; we also found that ß-HIVS may down-regulate the expression of ADAMTS5 and MMP13 and up-regulate the expression of aggrecan and collagen II to inhibit the degradation of ECM. Mechanistically, ß-HIVS inhibited the NFκB pathway by activating the Nrf2/HO-1 axis, thereby exerting its anti-inflammatory and inhibitory effects on ECM degradation. In vivo experiments also proved the therapeutic effects of ß-HIVS on OA in mice, and Nrf2 is the target of ß-HIVS. These findings indicate that ß-HIVS may become a new drug for the treatment of OA.

[Effect of lentivirus-mediated silencing of P75 neurotrophin receptor gene on osteogenic differentiation of bone marrow mesenchymal stem cells in rats].

OBJECTIVE: To investigate the effect of small interfering RNA (siRNA) lentivirus-mediated silencing of P75 neurotrophin receptor (P75NTR) gene on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in rats. METHODS: Three lentivirus-mediated P75NTR gene siRNA sequences (P75NTR-siRNA-1, 2, 3) and negative control (NC)-siRNA were designed and transfected into the 3rd generation Sprague Dawley (SD) rat BMSCs. The cells morphological changes were observed under an inverted microscope, and the expressions of P75NTR gene and protein in cells were detected by real-time fluorescence quantitative PCR and Western blot. Then the best silencing P75NTR-siRNA for subsequent osteogenic differentiation experiments was screened out. The 3rd generation SD rat BMSCs were randomly divided into experimental group, negative control group, and blank control group (normal BMSCs). The BMSCs of negative control group and experimental group were transfected with NC-siRNA and the selected P75NTR-siRNA lentiviral vector, respectively. The cells of each group were cultured by osteogenic induction. The expressions of osteogenic related proteins [osteocalcin (OCN) and Runx related transcription factor 2 (Runx2)] were detected by Western blot; the collagen type Ⅰ expression was observed by immunohistochemical staining; the osteogenesis of BMSCs was observed by alkaline phosphatase (ALP) detection and alizarin red staining. RESULTS: After lentivirus-mediated P75NTR transfected into BMSCs, the expressions of P75NTR mRNA and protein significantly reduced ( P<0.05), and the best silencing P75NTR-siRNA was P75NTR-siRNA-3. After P75NTR gene was silenced, MTT test showed that the cell proliferation in the experimental group was significantly faster than those in the two control groups ( P<0.05). After osteogenic induction, the relative expressions of OCN and Runx2 proteins, collagen type Ⅰ expression, and ALP activity were significantly higher in the experimental group than in the two control groups, the differences were significant ( P<0.05). With the prolongation of osteogenic induction, the mineralized nodules in the experimental group gradually increased. CONCLUSION: Silencing the P75NTR gene with siRNA lentivirus can promote the osteogenic differentiation of rat BMSCs and provide a new idea for the treatment of bone defects.

[Research progress of P75 neurotrophin receptor and new idea of nonunion treatment].

Objective: To review the research progress of P75 neurotrophin receptor (P75NTR) so as to clarify its mechanism, and to explore its relationship with nonunion so as to provide a new idea for the treatment of nonunion. Methods: The related domestic and foreign literature of P75NTR in recent years was extensively reviewed, summarized, and analyzed to find out the mechanism of action of P75NTR and the pathological factors of nonunion formation. Results: P75NTR can express in nonunion tissues and lead to defect of fibrin degradation and inhibition of angiogenesis, which play an important role in the pathogenesis of nonunion. Conclusion: It needs to be confirmed by further study whether the purpose of treating nonunion can be achieved by blocking the effects described above of P75NTR.

Dual plating with bone block arthrodesis of the first metatarsophalangeal joint: A clinical retrospective review.

BACKGROUND: First metatarsal phalangeal joint (MTP) arthrodesis is challenging in the setting of bone loss. The purpose of this study was to describe the results of interpositional grafting and arthrodesis of the first MTP joint using two plates in a 90/90 configuration. MATERIALS AND METHODS: Eleven patients had an MTP arthrodesis with 90-90 plating with an interpositional allograft. We analyzed the fusion rate, restoration of first ray length, patient satisfaction, and complication rates. RESULTS: The overall union rate was 90.9%, with an average restoration of 11 ± 4.5mm in length to the first ray. The average time to fusion was 10.7 ± 1 weeks. The mean preoperative AOFAS score improved significantly. The complication rate was 18.2% and included one superficial wound infection and one non-union who underwent a successful fusion after revision. CONCLUSION: Arthrodesis of the first MTP joint with two 90/90 plates and restoration of length using an interpositional graft has excellent patient satisfaction and functional outcomes.

A Short-Term Retrospective of First Metatarsophalangeal Joint Arthrodesis Using a Plate With PocketLock Fixation.

BACKGROUND: Various techniques have been described for arthrodesis of the first metatarsophalangeal (MTP) joint. The purpose of this study was to retrospectively review the results of fixation for the first MTP arthrodesis of patients treated using dome-shaped reamers to prepare the joint surfaces and a novel MTP Plate with PocketLock fixation. METHODS: Between July 2012 and November 2013, 16 feet in 16 patients were treated with a first MTP arthrodesis with a MTP Plate with PocketLock fixation. The mean patient age was 58.8 years (range, 46-82 years). Physical and radiographic examinations were performed at follow-up visits. The average follow-up period was 17.3 months. The radiographs were examined for union (3 bridging cortices), time to union, hardware failure, or other radiographic complications. The charts were reviewed to assess AOFAS-MTP-IP (American Orthopaedic Foot & Ankle Society metatarsophalangeal-interphalangeal) scores and postoperative complications. RESULTS: Fusion was seen in 11 of 16 feet (68.8%) and partial union in 1 patient (6.3%). Five nonunions (31.2%) were noted in the sample group: All were symptomatic and required revision surgery. No malunions were identified in our sample. One hardware failure was documented in a nonunion patient. The mean time to osseous union was 81.7 ± 15.9 days. The preoperative AOFAS MTP-IP score was 55.6 and the postoperative score was 64.7. CONCLUSION: The high nonunion and revision surgery rates demonstrate that this particular plate should be used with caution for a first MTP joint arthrodesis. LEVEL OF EVIDENCE: Therapeutic, Level IV: Case series.

[Progress of induced osteogenesis of bone marrow mesenchymal stem cells transfected by double-gene].

OBJECTIVE: To review the research progress of induced osteogenesis of bone marrow mesenchymal stem cells (BMSCs) transfected by double-gene. METHODS: The recent literature concerning the comparative research of induced osteogenesis of BMSCs transfected by double-gene was extensively reviewed. The characteristics of BMSCs, the advantage and effect of synergistic inductive osteogenesis, the application prospect and problems of BMSCs transfected by double-gene were summarized. RESULTS: The effect of induced osteogenesis concerning BMSCs transfected by double-gene is far superior to single gene transfection and the activity of osteoblast is also significantly increased. The research used in bone tissue engineering experiment also obtain good effect. CONCLUSION: Induced osteogenesis of BMSCs transfected by double-gene is able to make up for the lack of a single gene transfection and has great development prospects in the orthopaedic field.

Comparing the chondrogenic potential in vivo of autogeneic mesenchymal stem cells derived from different tissues.

OBJECTIVE: to compare the chondrogenic ability of mesenchymal stem cells (MSCs) derived from different tissues in rabbits' full-thickness articular cartilage defects. METHODS: sixty New Zealand white rabbits of ordinary grade with a body weight of 2.5 approximately 3.5kg were selected for this study. Six were sacrificed for preparation of deminerized bone matrix (DBM) as scaffold. Fifty-four were used for cartilage defects model. Full-thickness cartilage defect of knee joint was created on trochlear groove at two sides of the femur with a diameter of 4 mm and thickness of 3 mm. All 54 rabbits were randomly divided into 6 groups and treated by autogeneic MSCs isolated from bone marrow, periosteum, synovium, adipose tissue and muscle, respectively. The 6th group was a control group with nothing plugged into the defects. Every three rabbits were killed at three time points, which were 4, 8, and 12 weeks after the operation in each group. The reparative tissue samples were evaluated grossly, histologically, immunohistochemically, and graded according to gross and histological scales 12 weeks postoperatively. We input the scores into SPSS 11.5 software and the analysis of variance (one-way-ANOVA) and student-newman-keuls (SNK-q) test were used to process statistical analysis and find out if the differences between each group had statistical significance. RESULTS: fifty-four rabbits are included in the final analysis. The defects are all repaired by hyaline-like tissue except the control group. The bone-marrow-MSCs produced much more cartilage matrix than that of other groups. Gross and histological grading scale indicates that the defects repaired by MSCs isolated from bone marrow are superior to that repaired by MSCs isolated from periosteum, synovium, adipose tissue, and muscle (p < 0.05). In adipose-MSCs and muscle-MSCs group, some defects are even repaired by fibrous tissue. CONCLUSION: bone-marrow-MSCs have greater in vivo chondrogenic potential than periosteum-, synovium-, adipose- and muscle-MSCs.

In vitro and in vivo biocompatibility investigation of diamond-like carbon coated nickel-titanium shape memory alloy.

OBJECTIVE: To investigate the biocompatibility of diamond-like carbon (DLC) coated nickel-titanium shape memory alloy (NiTi SMA) in vitro and in vivo. METHODS: The in vitro study was carried out by co-culturing the DLC coated and uncoated NiTi SMA with bone marrow mesenchymal stem cells (MSCs), respectively, and the in vivo study was carried out by fixing the rabbits' femoral fracture model by DLC coated and uncoated NiTi SMA embracing fixator for 4 weeks, respectively. The concentration of the cells, alkaline phosphatase (AKP), and nickel ion in culture media were detected, respectively, at the first to fifth day after co-culturing. The inorganic substance, osteocalcin, alkaline phosphatase (ALP), and tumor necrosis factor (TNF) in callus surrounding fracture and the Ni(+) in muscles surrounding fracture site, liver and brain were detected 4 weeks postoperatively. RESULTS: The in vitro study showed that the proliferation of MSCs and the expression of AKP in the DLC-coated group were higher than the uncoated group (P < 0.05), while the uncoated group released more Ni(2+) into the culture media than that in the coated group (P < 0.05). The in vivo study revealed that the inorganic substance and AKP, osteocalcin, and TNF expression were significantly higher in the DLC coated NiTi SMA embracing fixator than that in the uncoated group (P < 0.05). Ni(2+) in liver, brain, and muscles surrounding the fracture were significantly lower in the DLC coated groups than that in the uncoated group (P < 0.05). CONCLUSION: Nickel-titanium shape memory alloy coated by diamond-like carbon appears to have better biocompatibility in vitro and in vivo compared to the uncoated one.

[Effect factors analysis of knee function recovery after distal femoral fracture operation].

OBJECTIVE: To investigate the effect factors of knee function recovery after operation in distal femoral fractures. METHODS: From January 2001 to May 2007, 92 cases of distal femoral fracture were treated. There were 50 males and 42 females, aged 20-77 years old (average 46.7 years old). Fracture was caused by traffic accident in 48 cases, by falling from height in 26 cases, by bruise in 12 cases and by tumble in 6 cases. According to Müller's Fracture classification, there were 29 cases of type A, 12 cases of type B and 51 cases of type C. According to American Society of Anesthesiologists (ASA) classification, there were 21 cases of grade I, 39 cases of grade II, 24 cases of grade III, and 8 cases of grade IV. The time from injury to operation was 4 hours to 24 days with an average of 7 days. Anatomical plate was used in 43 cases, retrograde interlocking intramedullary nail in 37 cases, and bone screws, bolts and internal fixation with Kirschner pins in 12 cases. After operation, the HSS knee function score was used to evaluate efficacy. Ten related factors were applied for statistical analysis, to knee function recovery after operation in distal femoral fractures, such as age, sex, preoperative ASA classification, injury to surgery time, fracture type, treatment, reduction quality, functional exercise after operation, whether or not CPM functional training and postoperative complications. RESULTS: Wound healed by first intention in 88 cases, infection occurred in 4 cases. All patients followed up 16-32 months with an average of 23.1 months. Clinical union of fracture was achieved within 3-7 months after operation. Extensor device adhesions and the scope of activities of <80 degrees occurred in 29 cases, traumatic arthritis in 25 cases, postoperative fracture displacement in 6 cases, mild knee varus or valgus in 7 cases and implant loosening in 6 cases. According to HSS knee function score, the results were excellent in 52 cases, good in 15 cases, fair in 10 cases and poor in 15 cases with an excellent and good rate of 72.83%. Single factor analysis showed that age, preoperative ASA classification, fracture type, reduction quality, whether or not CPM functional exercise, and postoperative complications were significantly in knee function recovery (P < 0.05). logistic regression analysis showed that the fracture type, quality of reduction, whether or not CPM functional exercise, and age were major factors in the knee joint function recovery. CONCLUSION: Age, preoperative ASA classification, fracture type, reduction quality, and whether or not CPM functional training, postoperative complications factors may affect the knee joint function recovery. Adjustment to the patient's preoperative physical status, fractures anatomic reduction and firm fixation, early postoperative active and passive functional exercises, less postoperative complications can maximize the restoration of knee joint function.

[Study on effect of NGF on fracture healing].

OBJECTIVE: To investigate the effect of NGF on fracture healing, 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 saline, BMP-2, BMP-2/NGF/normal saline, and normal saline 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. Before the 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 < 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 < 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 < 0.05). CONCLUSION: NGF promotes the healing of fractures. NGF possesses synergistic effect on ectopic bone formation induced by BMP-2.