Objective The amniotic carrier complex membrane, which contains bFGF and vitamin C (VitC) and is loaded with BMSCs, is planted into the deeply-partial wounds of rabbits. To explore its influence on the epidermis renascence and regenerating speed in the process of the dermis restore. Methods BMSCs were isolated from the marrows of 24 healthy3-month-old New Zealand rabbits, male or female, weighing 1.0-1.5 kg. The BMSCs were cultured in vitro and purified, and then amniotic carrier complex membrane was prepared, whose size was 4.52 cm2. Three deep-partial wounds, with the area of about 3.14 cm2, were produced on the back of each rabbit. All the wounds were randomly divided into 3 groups: group A, group B and group C. Group A was the experimental group in which the amniotic carrier complex membrane was planted, including 1 ml BMSCs, 10 mL bFGF (0.2 mg/L) and 10 mL VitC (0.02 g/L). In group B, the amniotic carrier complex membrane was planted, including only 1 mL BMSCs. In group C, the amniotic carrier complex membrane alone was planted. After the operation, general observation was conducted. At postoperative 7, 14 and 21 days, respectively, the observation by HE, Masson, Van Giesonr staining and immunohistochemical staining of collagen type I was performed. The ink perfusion method was performed to evaluate the velocity and the qual ity of the wound heal ing after the transplantation. Results All the wounds obtained good heal ing. At 14 days after the operation, the ratio of wound heal ing was 60%, 41% and 23% in groups A, B and C, respectively. At 21 days after the operation, the the ratio of wound heal ing was 99%, 90% and 81% in groups A, B and C, respectively. There were significant differences between any two groups (P lt; 0.05). The depth of the newborn dermis, the number of the active collagen type I mascul ine cells and the number of the blood vessels in group A were better and more than in group B. And those in group B were better and more than in group C. At the exterior area of the newborn dermis, there was lots of regenerated epidermis from the peripheral normal skin, which in group A was better than in group B, and in group B was better than in group C. onclusion The amniotic carrier complex membrane transplanted to deep-partial wounds, which is appended withBMSCs, bFGF and VitC, can accelerate repair and reconstruction of the dermis. There has an optimal time of the renascence and regeneration of the epidermis in the process of dermis repair.
Objective To investigate the effect of bFGF on denervated skeletal muscle in accelerating muscle satell ite cell prol iferation, supplying neurotrophic factors and reducing muscle atrophy. Methods Twenty-eight Wistar male rats weredivided into the experimental group and the control group randomly, whose left lower l imb sciatic nerve was excised to make animal models of denervated skeletal muscle. The sil ia gel tubes containing 0.1 g bFGF and normal sal ine were implanted into gastrocnemius in the experimental and control groups, respectively. After 14 and 30 days of operation, gross appearance was observed; muscle wet weight and potential ampl itude of gastrocnemius fibrillation were measured; histological observation and electron microscope observation were made. Results At 14 and 30 days after operation, gastrocnemius atrophy and adhesion were more obvious in the control group than those in the experimental group. At 30 days after operation, the potential amplitude of gastrocnemius fibrillation and muscle wet weight were experimental group (0.220 6 ± 0.301 0) μm and (2.475 7 ± 0.254 6) g in the experimental group, and (0.155 2 ± 0.050 3) μm and (1.459 1 ± 0.642 5) g in the control group. There was a significant difference between two groups (P lt; 0.05). At 14 and 30 days after operation, HE staining showed more muscle satell ite cell nucleiin gastrocnemius of the experimental group than that of the control group; Mallory staining showed more blue connective tissues in the control group than in the experimental group; PCNA staining showed more PCNA positive cell nuclei in the experimental group than in the control group; and the AgNO3 staining testified more grains of vitamin C and less connective tissue proliferation in the experimental group than in the control group. At 30 days after operation, the fiber diameter and the fiber area were (66.368 6 ± 12.672 7) μm and (2 096.112 9 ± 311.563 9) μm2 in the experimental group, (55.504 0 ± 4.945 0) μm and (1 418.068 0 ± 264.953 7) μm2 in the control group. The PCNA positive cell nuclei number was 116.200 ± 5.357 in the experimental group and 53.000 ± 3.937 in the control group, showing statistically significant difference between the two groups (P lt; 0.05). At 14 and 30 day after operation, ompared with control group, the muscle fiber in the experimental group arrangedly more regularly and had lessatrophy fiber and the connective tissue proliferation. Conclusion bFGF can stimulate the proliferation of muscle satell ite cells in denervated gastrocnemius, delay the muscle fiber atrophy and inhibit connective tissues proliferation in muscle fibers.
Objective To study the outcomes of nerve defect repair with the tissue engineered nerve, which is composed of the complex of SCs, 30% ECM gel, bFGF-PLGA sustained release microspheres, PLGA microfilaments and permeable poly (D, L-lacitic acid) (PDLLA) catheters. Methods SCs were cultured and purified from the sciatic nerves of 1-day-old neonatal SD rats. The 1st passage cells were compounded with bFGF-PLGA sustained release microspheres andECM gel, and then were injected into permeable PDLLA catheters with PLGA microfilaments inside. In this way, the tissueengineered nerve was constructed. Sixty SD rats were included. The model of 15-mm sciatic nerve defects was made, and then the rats were randomly divided into 5 groups, with 12 rats in each. In group A, autograft was adopted. In group B, the blank PDLLA catheters with PBS inside were used. In group C, PDLLA catheters, with PLGA microfilaments and 30% ECM gel inside, were used. In group D, PDLLA catheters, with PLGA microfilaments, SCs and 30% ECM gel inside, were used. In group E, the tissue engineered nerve was appl ied. After the operation, observation was made for general conditions of the rats. The sciatic function index (SFI) analysis was performed at 12, 16, 20 and 24 weeks after the operation, respectively. Eelectrophysiological detection and histological observation were performed at 12 and 24 weeks after the operation, respectively. Results All rats survived to the end of the experiment. At 12 and 16 weeks after the operation, group E was significantly different from group B in SFI (P lt; 0.05). At 20 and 24 weeks after the operation, group E was significantly different from groups B and C in SFI (P lt; 0.05). At 12 weeks after the operation, electrophysiological detection showed nerve conduct velocity (NCV) of group E was bigger than that of groups B and C (P lt; 0.05), and compound ampl itude (AMP) as well as action potential area (AREA） of group E were bigger than those of groups B, C and D (P lt; 0.05). At 24 weeks after the operation, NCV, AMP and AREA of group E were bigger than those of groups B and C (Plt; 0.05). At 12 weeks after the operation, histological observation showed the area of regenerated nerves and the number of myel inated fibers in group E were significantly differents from those in groups A, B and C (Plt; 0.05). The density and diameter of myel inated fibers in group E were smaller than those in group A (Plt; 0.05), but bigger than those in groups B, C and D (P lt; 0.05). At 24 weeks after the operation, the area of regenerative nerves in group E is bigger than those in group B (P lt; 0.05); the number of myel inated fibers in group E was significantly different from those in groups A, B, C (P lt; 0.05); and the density and diameter of myel inated fibers in group E were bigger than those in groups B and C (Plt; 0.05). Conclusion The tissue engineered nerve with the complex of SCs, ECM gel, bFGF-PLGA sustained release microspheres, PLGA microfilaments and permeables PDLLA catheters promote nerve regeneration and has similar effect to autograft in repair of nerve defects.
【Abstract】 Objective To design a novel small-cal iber vascular graft using a decellularized allogeneic vascularscaffold pre-loaded with bFGF. Methods The decellularized canine common carotid were obtained by a detergent-enzymatic procedure, then the scaffolds were covalently l inked with heparin and pre-loaded with bFGF, the amount of binding bFGF and releasing curve were assayed by ELISA. Canine BMSCs expanded in vitro were seed on the scaffolds to observe the effects of binding bFGF on prol iferation. Both bFGF pre-loaded and non-pre-loaded decellularized grafts were implanted in canines as carotid artery interposition for 8 weeks, the patency was examined by digital subtraction angiography and histological method. Results Histology and electron microscopic examination of the decellularized scaffolds showed that cellular components were removed completely and that the extracellular matrix structure remained intact. The amount of binding bFGF positively related to the concentration of bFGF. There was a significant difference in the amount of binding bFGF between two different scaffoldsthroughout all bFGF concentrations(P lt; 0.05), and up to 100 ng/mL, the local and sustained release of bFGF from the heparin treated scaffolds were assayed up to 20 days. Additionally, MTT test showed the bFGF-preloaded scaffolds significantly enhanced the prol iferation of seeded BMSCs in vitro compared with non-bFGF-preloaded scaffolds at 3 days after seeding and thereafter(P lt; 0.01). Furthermore, in vivo canine experiments revealed that all 8 bFGF-pre-loaded scaffolds remained patent after 8 weeks of implantation, and host cell l ined the lumen and populated the wall. Only 1 non-bFGF-pre-loaded scaffold was patent, and the other 7 grafts were occluded because of thrombsus formation. Conclusion This study provides a new strategy to develop a small diameter vascular graft with excellent biocompatibil ity and high patency rate.
Objective To explore the impact of basic fibroblast growth factor (bFGF) and parathyroid hormone-related protein (PTHrP) on early and late chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells (BMSCs) induced by transforming growth factor β1 (TGF-β1). Methods BMSCs were isolated from 3 healthy Japanese rabbits (2-month-old, weighing 1.6-2.1 kg, male or female), and were clutured to passage 3. The cells were put into pellet culture system and were divided into 5 groups according to different induce conditions: TGF-β1 group (group A), TGF-β1/bFGF group (group B), TGF-β1/21 days bFGF group (group C), TGF-β1/PTHrP group (group D), and TGF-β1/21 days PTHrP group (group E). At the beginning, TGF-β1 (10 ng/mL) was added to all groups, then bFGF and PTHrP (10 ng/mL) were added to groups B and D respectively; bFGF and PTHrP (10 ng/mL) were added to groups C and E at 21 days respectively. The gene expressions of collagen type I (Col I), Col II, Col X, matrix metalloproteinases (MMP)-13, and alkaline phosphatase (ALP) activity were detected once every week for 6 weeks. The 1, 9-dimethylmethylene blue (DMMB) staining was used to observe the extracellular matrix secretion at 6 weeks. Results The expression of Col I in groups C and E showed a significant downward trend after 3 weeks; the expression in group A was significantly higher than that in groups C and E at 4 and 5 weeks (P lt; 0.05), and than that in groups B and D at 3-6 weeks (P lt; 0.05); and significant differences were found between groups B and C at 3 and 4 weeks, and between groups D and E at 3 weeks (P lt; 0.05). After 3 weeks, the expressions of Col II and Col X in groups C and E gradually decreased, and were significantly lower than those in group A at 4-6 weeks (P lt; 0.05). Groups B and D showed no significant difference in the expressions of Col II and Col X at all time points, but there was significant difference when compared with group A (P lt; 0.05). MMP-13 had no obvious expression at all time points in group A; significant differences were found between group B and groups A, C at 3 weeks (P lt; 0.05); and the expression was significantly higher in group D than in groups A and E (P lt; 0.05). ALP activity gradually increased with time in group A; after 4 weeks, ALP activity in groups C and E obviously decreased, and was significantly lower than that in group A (P lt; 0.05); there were significant differences between groups B and C, and between groups D and E at 2 and 3 weeks (P lt; 0.05). DMMB staining showed more cartilage lacuna in group A than in the other groups at 6 weeks. Conclusion bFGF and PTHrP can inhibit early and late chondrogenic differentiation of BMSCs by changing synthesis and decomposition of the cartilage extracellular matrix. The inhibition is not only by suppressing Col X expression, but also possibly by suppressing other chondrogenic protein.
【Abstract】 Objective Sonic hedgehog (Shh) signaling pathway is involved in an important part of regulating angiogenesis. To investigate the effects of recombinant Shh N-terminant (rShh-N) on the expression and secretion of angiogenesis-related factor—vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Methods Bone marrow mesenchymal stem scells (BMSCs) were isolated from 3-day-old healthy Sprague Dawley rats and cultured to passage 3 in vitro. rShh-N at the concentrations of 0, 10, 100, and 200 ng/mL were applied to culture BMSCs in groups A, B, C, and D, respectively. At 12, 24, 48, and 72 hours of culture, the expressions of VEGF and bFGF mRNA and the levels of VEGF and bFGF in supernatant were measured with real-time quantitative PCR and ELISA, respectively. Results At the gene level, compared with group A, the expressions of VEGF and bFGF mRNA were enhanced in group D (P lt; 0.05) and the upregulation was more significant at 12 and 48 hours than 24 and 72 hours (P lt; 0.01). In group C, bFGF mRNA expression was substantially promoted at 12-72 hours (P lt; 0.05) and VEGF mRNA level was upregulated at 24-72 hours (P lt; 0.05), and both reached peak at 72 hours (P lt; 0.01). In group B, VEGF mRNA expression was inhibited at 12 hours (P lt; 0.05), but the level increased at 48 and 72 hours (P lt; 0.05); bFGF mRNA expression was obviously promoted at 12-48 hours (P lt; 0.05) and the maximum appeared at 48 hours (P lt; 0.01). At the protein level, the secretion of VEGF and bFGF in group D was significantly increased at 12-72 hours, as compared with group A (P lt; 0.05). In group C, VEGF and bFGF secretion was increased at 24-72 hours (P lt; 0.05). The secretion of VEGF in group B was inhibited at 12 and 48 hours (P lt; 0.05) and was promoted at 24 hours (P lt; 0.05); bFGF secretion was up-regulated at 24 and 48 hours (P lt; 0.05). The secretion of VEGF and bFGF in supernatant at 【Abstract】 Objective Sonic hedgehog (Shh) signaling pathway is involved in an important part of regulating angiogenesis. To investigate the effects of recombinant Shh N-terminant (rShh-N) on the expression and secretion of angiogenesis-related factor—vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Methods Bone marrow mesenchymal stem scells (BMSCs) were isolated from 3-day-old healthy Sprague Dawley rats and cultured to passage 3 in vitro. rShh-N at the concentrations of 0, 10, 100, and 200 ng/mL were applied to culture BMSCs in groups A, B, C, and D, respectively. At 12, 24, 48, and 72 hours of culture, the expressions of VEGF and bFGF mRNA and the levels of VEGF and bFGF in supernatant were measured with real-time quantitative PCR and ELISA, respectively. Results At the gene level, compared with group A, the expressions of VEGF and bFGF mRNA were enhanced in group D (P lt; 0.05) and the upregulation was more significant at 12 and 48 hours than 24 and 72 hours (P lt; 0.01). In group C, bFGF mRNA expression was substantially promoted at 12-72 hours (P lt; 0.05) and VEGF mRNA level was upregulated at 24-72 hours (P lt; 0.05), and both reached peak at 72 hours (P lt; 0.01). In group B, VEGF mRNA expression was inhibited at 12 hours (P lt; 0.05), but the level increased at 48 and 72 hours (P lt; 0.05); bFGF mRNA expression was obviously promoted at 12-48 hours (P lt; 0.05) and the maximum appeared at 48 hours (P lt; 0.01). At the protein level, the secretion of VEGF and bFGF in group D was significantly increased at 12-72 hours, as compared with group A (P lt; 0.05). In group C, VEGF and bFGF secretion was increased at 24-72 hours (P lt; 0.05). The secretion of VEGF in group B was inhibited at 12 and 48 hours (P lt; 0.05) and was promoted at 24 hours (P lt; 0.05); bFGF secretion was up-regulated at 24 and 48 hours (P lt; 0.05). The secretion of VEGF and bFGF in supernatant at
ObjectiveTo observe the expressions of transforming growth factor β1 (TGF-β1) and basic fibroblast growth factor (bFGF) induced membrane by Masquelet technique in rats treated with glycoside of short-horned epimedium Herb, and to explore the effect of glycoside of short-horned epimedium Herb on Masquelet induced membrane.MethodsSixty 3-month-old male Wistar rats were randomly divided into 3 groups with 20 rats in each group; a tibial bone defect (6 mm in length) model was established. The blank group (group A) was not treated; the control group (group B) and the experimental group (group C) were filled with vancomycin antibiotic bone cement in the drawing stage, and the bone cement was completely solidified. Group C was given perfused flavonoids glycoside of short-horned epimedium Herb (10 μmol/L) by gavage once a day (0.3 mL) from 1 day after operation, and groups A and B were given the same amount of normal saline by gavage. After operation, the recovery and wound healing of experimental animals were observed; at 4 weeks after operation, X-ray film was taken to observe the recovery of bone defect of proximal tibia; at 6 weeks after operation, the bone defect was observed, and the morphological changes and vascularization degree of granulation tissue and induction membrane tissue were observed; the expressions of TGF-β1 and bFGF were observed by immunohistochemistry staining and ELISA detection.ResultsThe bone defect models of the 3 groups were established successfully, and there was no abnormality after operation. The incisions healed by first intention after operation. At 4 weeks after operation, X-ray films of proximal tibial defect showed that there was obvious space in group A, while bone cement was filled and Kirschner wire fixation was good in groups B and C. At 6 weeks after operation, the gross observation showed that the granulation tissue was filled in the defect area in group A; transparent membrane was formed in groups B and C, and microvessels were seen in some areas, and the microvessels in group C were significantly more than those in group B. Immunohistochemical staining showed that the expressions of TGF-β1 and bFGF were negative in group A, but they were expressed in groups B and C, and the expressions of TGF-β1 and bFGF in group B were significantly less than those in group C. ELISA detection showed that the expressions of TGF-β1 and bFGF in group C were significantly higher than those in groups A and B (P<0.05), but there was no significant difference between groups A and B (P>0.05). ConclusionGlycoside of short-horned epimedium Herb can significantly increase the expressions of TGF-β1 and bFGF, accelerate the process of osteogenesis, and contribute to bone shaping and reconstruction.
To evaluate the effects of bFGF on wound healing and the side-effects of bFGF, a multi-centers and controlled clinical trial were carried out in 32 hospitals in China. One thousand and twenty-four cases with acute wounds such as burn, donor site or operative wound and chronic wounds such as bed sore, draining sinus, ulcer were treated with bFGF. Another 826 cases with the similar wounds were used as control. The results showed: 1. The duration of wound healing was shorted 3-4 days in trial group when compared with the contorl; 2. The successful rates from bFGF on promoting the wound healing for burns, operative wounds and chronic dermal ulcers was 95.2%, 96.5% and 93.5%, respectively; 3. No adverse reaction was found. CONCLUSION: 1. bEGF can make the "silent" reparative cells dividing and proliferating. 2. bFGF can improve the quality and the velocity of wound healing.
Objective To study the release properties of basic fibroblast growth factor (bFGF) chitosan microspheres prepared by cross-linking-emulsion method using chitosan as a carrier material so as to lay a foundation for further study. Methods Using 0.6% sodium tripolyphosphate solution as a crosslinking agent and 1.5% solution of chitosan as a carrier material, bFGF chitosan microspheres were prepared by cross-linking-emulsion method. Laser particle size analyzer and Zeta electric potential analyzer were used to measure the particle diameter distribution, scanning electronic microscope to observe the morphology, and ELISA to determine the drug loading, the encapsulation rate, and the drug release properties. Results The particle size of bFGF chitosan microspheres ranged 20.312-24.152 μm. The microspheres were round with a smooth surface and uniform distribution, and it had no apparent porosity. The drug loading and encapsulation rate of microspheres were (7.57 ± 0.34) mg/g and 95.14% ± 1.58%, respectively. The bFGF chitosan microspheres could continuously release bFGF for 24 days; the bFGF level increased gradually with time and reached (820.45 ± 21.34) ng/mL at 24 days; and the microspheres had a burst effect, and the burst rate was 18.08%, and the accumulative release rate of the microspheres reached 82.05% during 24 days. Conclusion It is easy-to-operate to prepare the bFGF chitosan microspheres with the cross-linking-emulsion method. The bFGF chitosan microspheres have smooth surface, uniform distribution, and no apparent porosity.
Objective The biological effects of fibroblast growth factor (FGF) may be different under different intensities and durations. To investigate the impact of sustained increasing FGF signal upon the development of epiphyseal plate. Methods Epiphyseal plates cultured in vitro were obtained from embryonic C57BL/6J mice, and were divided into control group (0.1% DMSO), basic FGF (bFGF) group (100 μg/L bFGF and 0.1% DMSO), and PD98059 group (100 μg/L bFGF and 50 μmol/L PD98059 with 0.1% DMSO). The total length (TL) and ossified tissue length (OSL) of the cultured bones weremeasured with Calcein staining 6 days after culture. The expressions of Indian hedgehog (Ihh), collagen type II (Col II), and Col X genes were detected by real-time fluorescent quantative PCR 7 days after culture. Results The embryonic bones cultured in vitro continued growth. At 6 days after culture, there was no significant difference in increased percentage of TL between bFGF group and control group (P gt; 0.05), the increased percentage of OSL in bFGF group was significantly less than that in control group (P lt; 0.05). There was no significant difference in the increased percentage of TL and OSL between PD98059 group and control group (P gt; 0.05), but they were significantly higher than those of bFGF group (P lt; 0.05). At 7 days after culture, the gene expressions of Ihh, Col II, and Col X in bFGF group significantly decreased when compared with those in control group (P lt; 0.05). There was no significant difference in the gene expressions of Col II and Col X between PD98059 group and control group (P gt; 0.05), but the gene expressions were significantly higher than those of bFGF group (P lt; 0.05); the expression of Ihh in PD98059 group was significantly higher than that in control group and bFGF group (P lt; 0.05). Conclusion Sustained increasing FGF signal may affect the Col II and Col X expressions by down-regulating Ihh, which may lead to the development retardation of epiphyseal plate cultured in vitro. The external signal regulated kinase pathway may play an important role in the process.