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 construct adenovirus expressing NGF (Ad-NGF) and to investigate its promotive effect on the reparation and regeneration of sciatic nerve injury in rats. Methods NGF gene sequence was cloned into shuttle plasmid pCA13 of adenovirus type 5. After packed in HEK-293 cells, the recombinant adenoviruses-Ad-NGF underwent sequence identification. Thirty-two male SD rats weighing 180-200 g were randomly divided into 4 groups (n=8 rats per group). Sciatic nerve injury model was establ ished by disconnecting and direct suturing the right sciatic nerve in the rat. Theright gastrocnemius muscle of group A and C received Ad-NGF injection and adenovirus vector without NGF gene sequence injection, respectively, and 1 × 108 PFU/per time was given every other day for three times. Group B and D received NGF injection (200 U/d) and normal sal ine (100 ?L/d), respectively, for 3 weeks. The effect of various treatments on injured sciatic nerve was evaluated by performing sciatic nerve function index and nerve electrophysiology detections 31 days after operation. Meanwhile, the sciatic nerve in the anastomosis and at the site 1 cm distal to the anastomosis were obtained, and underwent RTPCR and Western blot analysis for detecting NGF mRNA and protein expression level in the injured sciatic nerve in the rats. Histology, immunohistochemistry, and transmission electron microscope observations were conducted. Results Ad-NGF carrying NGF gene sequence was constructed successfully and confirmed by sequence analysis. The sciatic nerve function index, nerve conduction velocity, evoked potential ampl itude, and latent period of group A was better than those of other groups (P lt; 0.05), and there were no significant differences among group B, C, and D (P gt; 0.05). RT-PCR and Western blot detection: the expression levels of NGF mRNA and protein in group A were greater than those of group B, C, and D (P lt; 0.05), and no significant differences were noted among group B, C, and D (P gt; 0.05). Histology and immunohistochemistry observation showed that the regeneration of the sciatic nerve in group A was obvious superior to that of other groups. Transmission electron microscopy observation suggested there was significant difference between group A and groups B, C, and D in terms of axonal diameter of sciatic nerve cross-section, myel in sheath thickness and nerve fiber number (P lt; 0.05), and there were no significant differences among group B, C, and D (P gt; 0.05). Conclusion Ad-NGF can effectively promote the repair of sciatic nerveinjury in rats, and is a new method for obtaining large amounts of NGF in the area of injured peripheral nerve.
Objective To investigate the effects of NGF on the prol iferation, mitotic cycle, collagen synthesis and migration of human dermal fibroblasts (HDFs), and to explore the function of NGF on the wound heal ing. Methods The 3rd generation of HDFs were incubated with various concentrations of NGF (0, 25, 50, 100, 200 and 400 ng/mL), the cell prol iferation was measured with MTT assay. After treated with NGF at 0, 100 ng/mL, the cell cycle of HDFs was determined by flow cytometry (FCM). Hydroxyprol ine and real-time fluorescence quantitative PCR (FQ-PCR) were used to measure collagen synthesis at protein level and mRNA level respectively. The in vitro cell scratch wound model was set up to observe the effect of NGF (0, 50, 100 and 200 ng/mL) on the migration of HDFs after 24 hours of culture. Results Absorbance value of HDFs for different concentrations of NGF (0, 25, 50, 100, 200, and 400 ng/ mL) showed that NGF did not influence the prol iferation of HDFs (P gt; 0.05). When HDFs were treated with NGF at 0 and 100 ng/mL, the result of FCM analysis showed that percentage of HDFs in G0/G1, S, G2/M phases were not changed (P gt; 0.05). Compared with control group, the expression of Col I and Col III were not significantly different, measured by both hydroxyprol ine and FQ-PCR (P gt; 0.05). The rates of HDFs’ migration at various concentrations of NGF (0, 50, 100, 200 ng/ mL) were 52.12% ± 6.50%, 80.67% ± 8.51%, 66.33% ± 3.58%, and 61.19% ± 0.97%, respectively, indicating that NGF could significantly enhanced the migration of HDFs at 50 and 100 ng/mL (P lt; 0.05). Conclusion NGF does not influence prol iferation, mitotic cycle and collagen synthesis of HDFs, but significantly enhanced migration in an in vitro model of wounded fibroblasts.
Objective To prepare nerve growth factor (NGF)-insulin composite gel and observe the effects of NGF-insulin composite gel on deep second degree scald wound healing in diabetic rats. Methods Carbomer 980, NGF (4 000 U), and insulin (800 U) were used to prepare the insulin gel, NGF gel, and NGF-insulin composite gel. The character of NGF-insulin composite gel was observed, and the in vitro drug release was tested. Seventy-five SPF Wistar male rats, weighing 200-250 g, were divided into 5 groups randomly, 15 rats each group: normal control group (group A), diabetes control group (group B), insulin gel treatment group (group C), NGF gel treatment group (group D), and NGF-insulin composite gel treatment group (group E). The type 1 diabetes rat model was established by intraperitoneal injection of Streptozotocin (55 mg/kg) in groups B, C, D, and E, while the rats in group A were injected with the same dose of citric acid and calcium citrate buffer. After modeling success, deep second degree scald wound on the back was made with constant temperature water bath box. Wounds were treated with carbomer blank gel in groups A and B, with insulin composite gel in group C, with NGF gel in group D, and with NGF-insulin composite gel in group E, once a day. At 3, 7, 11, 15, and 21 days after injury, the scald wound healing was observed and healing rate was calculated; the full-thickness skin specimens were harvested from 3 rats of each group for histological and immuohistochemical staining observation. Results The NGF-insulin composite gel was clear and transparent, and had good moisture retention capacity and adhesion; it was easy to apply and clean up. The drug release in vitro lasted more than 24 hours and maintained for 30 days. No rat died during the experiment. At 3 days after injury, wound area did not reduce in all groups; at 7, 11, 15, and 21 days, group E had the highest wound healing rate, and group B had the lowest; significant differences were found between group E and group B and when compared with the other groups (P lt; 0.05). HE staining showed that group E surpassed other groups in the growth of granulation tissue and collagen fiber. Immunohistochemical results showed that the CD34 and proliferating cell nuclear antigen (PCNA) expressed at 3 days, and the number of positive cells increased gradually with time; the microvessel density and PCNA expression were highest in group E and were lowest in group B, showing significant differences when compared with the other groups and between group E and group B (P lt; 0.05). Conclusion NGF-insulin composite gel can improve deep second degree scald wound healing in diabetic rats.
Objective To obtain highly purified and large amount of Schwann cells (SCs) by improved primary culture method, to investigate the biocompatibility of small intestinal submucosa (SIS) and SCs, and to make SIS load nerve growth factor (NGF) through co-culture with SCs. Methods Sciatic nerves were isolated from 2-3 days old Sprague Dawley rats and digested with collagenase II and trypsin. SCs were purified by differential adhesion method for 20 minutes and treated with G418 for 48 hours. Then the fibroblasts were further removed by reducing fetal bovine serum to 2.5% in H-DMEM. MTT assay was used to test the proliferation of SCs and the growth curve of SCs was drawn. The purity of SCs was calculated by immunofluorescence staining for S-100. SIS and SCs at passage 3 were co-cultured in vitro. And then the adhesion, proliferation, and differentiation of SCs were investigated by optical microscope and scanning electron microscope (SEM). The NGF content by SCs was also evaluated at 1, 2, 3, 4, 5, and 7 days by ELISA. SCs were removed from SIS by repeated freeze thawing after 3, 5, 7, 10, 13, and 15 days of co-culture. The NGF content in modified SIS was tested by ELISA. Results The purity of SCs was more than 98%. MTT assay showed that the SCs entered the logarithmic growth phase on the 3rd day, and reached the plateau phase on the 7th day. SCs well adhered to the surface of SIS by HE staining and SEM; SCs were fusiform in shape with obvious prominence and the protein granules secreted on cellular surface were also observed. Furthermore, ELISA measurement revealed that, co-culture with SIS, SCs secreted NGF prosperously without significant difference when compared with the control group (P gt; 0.05). The NGF content increased with increasing time. The concentration of NGF released from SIS which were cultured with SCs for 10 days was (414.29 ± 20.87) pg/cm2, while in simple SIS was (4.92 ± 2.06) pg/cm2, showing significant difference (P lt; 0.05). Conclusion A large number of highly purified SCs can be obtained by digestion with collagenase II and trypsin in combination with 20-minute differential adhesion and selection by G418. SIS possesses good biocompatibility with SCs, providing the basis for further study in vivo to fabricate the artificial nerve conduit.
Objective To explore the possible mechanism of nerve growth factor (NGF) mixed insul in on the angiogenesis of burn wounds and the effect on the expressions of Bcl-2 and Bax in diabetic rats. Methods A total of 75 SPF male Wistar rats, weighing 200-220 g, were selected randomly and divided into nomal control (group A, n=15), the rats with diabetic control (group B, n=15), insul in treatment (group C, n=15), NGF treatment (group D, n=15), NGF and insul in treatment (group E, n=15) groups. In groups B, C, D, and E, streptozotocin was given by intraperitoneal injection at dose of 10 mg/kg on the 1st day and 50 mg/kg on the 3rd day to prepare the diabetic rat models. In group A, citric acid buffer at the samedose was given. After 1 month of diabetic models, second degree scald was made on the back of the rats, and then wounds were treated with 3-layer normal sal ine gauze in groups A and B, with 3-layer gauze containing 5 U Novol in 30R and subcutaneous injection of Novol in 30R (4-6 U/kg) everyday in group C, with 3-layer gauze containing 5 mL NGF (25 U/mL) in group D, and with a combination of groups C and D in group E. At 7, 11, 15, and 21 days, the wound heal ing rate was calculated; at 3, 7, 11, 15, and 21 days, the expressions of Bcl-2, Bax, and CD34 were determined and the microvascular density was measured by immunohistochemistry staining. Results All rats survived till experiment was finished. The area of wounds became smaller gradually with time. Group E was better than other groups in the wound heal ing rate (P lt; 0.05), the skin keratosis, the hair growth, and the granulation tissue and collagen fibers growth. With time, the expressions of CD34 and Bcl-2 increased gradually, reached the peak at 15 days and decreased at 21 days; the expression was ber in group E than in other groups (P lt; 0.05). At 3 days, Bax did not express; at 7 days, Bax began to express in new vascular endothel ial cells and the expression increased gradually with time; the expression was weaker in group E than in other groups (P lt; 0.05). Conclusion A combination of NGF and insul in local appl ication can enhance the angiogenesis of the burn wound in diabetic rats and accelerate wound heal ing by increasing the expression of Bcl-2 and decreasing the expression of Bax and restraining apoptosis of the wounds vascular endothel ial cells of diabetic rats.
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.
Thirty-five SD rats were divided into 3 groups, in which 5 rats were served as control. The other 2 groups, 15 rats in each received either NGF solution or normal saline. The injury at the level of T8 spinal segment of the rat in these two groups were made by dropping a weight of 10 g from a height 2.5 cm after a total laminectomly from T7-11 segments. A thin plastic tube was inserted into the subarachnoid space below the injured segments. NGF was introduced through the tube at intervals of 1, 2, 3, 4, 8, 12, 24, 48 hours in the NGF group, and normal saline in the normal saline group. At 4, 8, 24 hours following surgery, 5 rats in each group were sacrificed and the injured segments were resected for examination. The contents of water and calcium were measured by dry-wet method and atomic absorption spectroscopy. The result showed that total calcium and water contents in normal saline group were markly increased, however, the changes of these two parametere were not so prominent in NGF group. It was suggested that NGF play a role in protecting the spinal cord by maintaining the calcium level of the injured segment.
ObjectiveTo study the effect of the loaded concentration gradient of nerve growth factor (NGF) immobilized conduit on rat peripheral nerve defect repair. MethodsThe peripheral nerve conduits made of poly (ε-caprolactone)-block-poly (L-lactide-co-ε-caprolactone) were prepared with uniform loads or concentration gradient loads by combining differential absorption of NGF/silk fibroin (SF) coating, and the gradient of NGF was immobilized in the nerve conduits. ELISA method was used to exam the NGF release for 12 weeks in vitro. Twenty-four male Sprague Dawley rats (weighing, 220-250 g) were selected to establish the right sciatic nerve defect model (14 mm in length) and randomly divided into 4 groups according to repair methods. The transected nerve was bridged by a blank conduit without NGF in group A, by a conduit containing uniform loads of NGF in group B, by a conduit concentration gradient loads of NGF in group C, and by the autogenous nerve segment in group D. The gross observation, electrophysiological examination, histological observation, and transmission electron microscope observation were carried out to assess the nerve regeneration at 12 weeks after surgery. ResultsThe cumulative release amount of NGF was (14.2±1.4) ng/mg and (13.7±1.3) ng/mg in gradient of NGF loaded conduits and uniform NGF loaded conduits respectively at 12 weeks, showing no significant difference (t=0.564, P=0.570). All the animals survived to completion of the experiment; plantar ulcers occurred at 4 days, which healed at 12 weeks; groups C and D were better than groups A and B in ulcerative healing. At 12 weeks after surgery, the compound muscle action potential of group A was significantly lower than that of groups B, C, and D (P<0.05), and group B was significantly lower than groups C and D (P<0.05), but no significant difference was found between groups C and D (P>0.05). The axon density of group C was significantly higher that of groups A, B, and D (P<0.05); group D was significantly higher than groups A, B, and C, and group C was significantly higher than groups A and B in the axon number, axon diameter, and area of muscle fiber (P<0.05); the thickness of myelin sheath of groups C and D was significantly larger than that of groups A and B (P<0.05), but no significant difference was found between groups C and D (P>0.05). ConclusionGradient of NGF loaded nerve condnits for rat sciatic nerve defect has similar results to autogenous nerve, with a good bridge, which can promote the sciatic nerve regeneration, improve the myelinization of the regenerating nerve, and accelerate the function reconstruction of the regenerating nerve.
ObjectiveTo construct recombinant adenovirus expressing nerve growth factor (NGF) and myelin associated glycoprotein (MAG) (Ad-NGF-MAG) and to investigate its effect on repair and regeneration of sciatic nerve injury in rats. MethodsNGF and MAG gene sequences were cloned into shuttle plasmid pCA13 of adenovirus type 5. After packed in HEK293 cells, the recombinant Ad-NGF-MAG underwent sequence and identification. Thirty-two male Sprague Dawley rats were randomly divided into 4 groups (n=8): control group (normal control), adenovirus vector group (Ad group), Ad-NGF group, and Ad-NGF-MAG group. The sciatic nerve injury model was established by transection of the right sciatic nerve; then, the empty adenovirus vector, Ad-NGF, and Ad-NGF-MAG were injected into the gastrocnemius muscle of the affected limb at a dose of 1×108 PFU every other day for 3 times in Ad group, AdNGF group, and Ad-NGF-MAG group, respectively. The right sciatic nerve was exposed only, and then the incision was closed in the control group. The sciatic nerve function index (SFI) was measured, and neuro-electrophysiology was observed; mRNA and protein expressions of NGF and MAG were detected by RT-PCR and Western blot; and histological examination was performed at 31 days after operation. ResultsRecombinant adenovirus vectors of Ad-NGF and Ad-NGF-MAG were constructed successfully. All rats survived and incision healed by first intension. The SFI, nerve conduction velocity, evoked potential amplitude, and latent period of Ad-NGF-MAG group were significantly better than those of Ad group and Ad-NGF group (P < 0.05). MAG mRNA and protein expressions of Ad-NGF-MAG group were the highest in all the groups (P < 0.05). The expressions of NGF mRNA and protein increased in Ad-NGF group and AdNGF-MAG group when compared with control group and Ad group (P < 0.05). Histological examination showed that the nerve had good continuity in control group; nerve fibers disarranged in Ad group; neurons connections formed in some nerve fibers of Ad-NGF group, but nerve fibers arrange disorderly; and the growth of the nerve were ordered and wellstructured in Ad-NGF-MAG group. ConclusionAd-NGF-MAG can effectively promote the growth of the nerve and inhibit the form of abnormal branches, facilitating the repair of sciatic nerve injury in rats.