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.
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 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.
【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.
Objective To evaluate the effect of the local del ivery of basic fibroblast growth factor 2 (bFGF-2) on the osseointegration around titanium implant of diabetic rats. Methods The bFGF-2-loaded poly (lactic-co-glycol ic acid) microspheres were prepared by water/oil/water (W/O/W) double-emulsion solvent evaporation method. Thirty-five male SPF level Sprague Dawley rats, weighing 220-250 g and aged 9 weeks, were selected as experimental animals. Ten rats were fedwith the routine diet as normal control group. The other 25 rats were made the diabetic animal model by giving high fat-sugar diet and a low dose streptozotocin (30 mg/ kg) intravenously; 20 rats were made the diabetic animal model successfully. Then 20 rats were randomly divided into diabetic control group (n=10) and bFGF-2 intervention group (n=10). A hole was drilled in the right tibia bone of all rats, and the titanium implant treated by micro-arc oxidation surface was planted into the hole. Simultaneously, the previously prepared microspheres and blood were mixed and were loaded on the surface of the implant before it was implanted into the rats of the bFGF-2 intervention group. At 4 and 8 weeks, the tibia containing implants was harvested, embedded with resin and made undecalcified tissue sl ices to compare the osseointegration. Results At 4 weeks, the implants of the normal control group were surrounded by new lamellar bone with continuity; whereas the tissue around the implants of the diabetic control group contained l ittle woven bone and some fibrous tissue; and obvious new formed bone with continuity was observed in bFGF-2 intervention group. At 8 weeks, the results of 3 groups were similar to those at 4 weeks. At 4 weeks, the percentage of bone-implant contact (BIC) in diabetic control group was significantly less than those in normal control group (P lt; 0.05) and in bFGF-2 intervention group (P lt; 0.05); the BIC in bFGF-2 intervention group was less than in normal control group, but showing no significant difference (P gt; 0.05). After 8 weeks, the BIC in normal control group and in bFGF-2 intervention group were significantly greater than that in diabetic control group (P lt; 0.05), but there was no significant difference between bFGF-2 intervention group and normal control group (P gt; 0.05). Conclusion Local del ivery of bFGF-2 around titanium implants may improve the osseointegration in diabetic rats.
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.
Objective To explore the effect of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and the combination of bFGF and EGF in the neural differentiation of human bone marrow mesenchymal stem cells (hBMSCs), and the role of Wnt/β-catenin signaling pathway in this process. MethodsThe identified 4th-generation hBMSCs were divided into five groups according to different induction conditions, namely control group (group A), EGF induction group (group B), bFGF induction group (group C), EGF and bFGF combined induction group (group D), and EGF, bFGF, and Dickkopf-related protein 1 (DKK-1) combined induction group (group E). After 7 days of continuous induction, the cell morphology was observed by inverted fluorescence phase contrast microscopy, levels of genes that were related to neural cells [Nestin, neuron-specific enolase (NSE), microtubule-associated protein 2 (MAP-2), and glial fibrillary acidic protein (GFAP)] and key components of the Wnt/β-catenin signaling pathway (β-catenin and Cyclin D1) were detected by RT-PCR, and the levels of proteins that were related to neural cells (Nestin and GFAP) as well as genes that were involved in Wnt/β-catenin signaling pathway [β-catenin, phosphorylation β-catenin (P-β-catenin), Cytoplasmic β-catenin, and Nuclear β-catenin] were explored by cellular immunofluorescence staining and Western blot. ResultsWhen compared to groups A and B, the typical neuro-like cell changes were observed in groups C-E, and most obviously in group D. RT-PCR showed that the relative expressions of Nestin, NSE, and MAP-2 genes in groups C-E, the relative expressions of GFAP gene in groups D and E, the relative expression of NSE gene in group B, the relative expressions of β-catenin gene in groups C and D, and the relative expressions of Cyclin D1 gene in groups B-D significantly increased when compared with group A (P<0.05). Compared with group E, the relative expressions of Nestin, NSE, MAP-2, GFAP, β-catenin, and CyclinD1 genes significantly increased in group D (P<0.05); compared with group C, the relative expression of Nestin gene in group D significantly decreased (P<0.05), while NSE, MAP-2, and GFAP genes significantly increased (P<0.05). The cellular immunofluorescence staining showed that the ratio of NSE- and GFAP-positive cells significantly increased in groups C-E than in group A, in group D than in groups C and E (P<0.05). Western blot assay showed that the relative expression of NSE protein was significantly higher in groups C and D than in group A and in group D than in groups C and E (P<0.05). In addition, the relative expression of GFAP protein was significantly higher in groups C-E than in group A and in group D than in group E (P<0.05). Besides, the relative expressions of β-catenin, Cytoplasmic β-catenin, Nuclear β-catenin, and the ratio of Nuclear β-catenin to Cytoplasmic β-catenin were significantly higher in groups C and D than in group A and in group D than in group E (P<0.05), whereas the relative expression of P-β-catenin protein was significantly lower in groups C and D than in group A and in group D than in group E (P<0.05). Conclusion Different from EGF, bFGF can induce neural differentiation of hBMSCs. In addition, EGF can enhance the hBMSCs neural differentiation of bFGF, while the Wnt/β-catenin signaling pathway may play a positive regulatory role in these processes.
Objective To explore the therapeutic effect of basic fibroblast growth factor (bFGF) on spinal cord injury (SCI) in rats and the influence of Notch/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Methods A total of 40 10-week-old male Sprague Dawley (SD) rats were selected to establish T10-segment SCI model by a free falling object. Among them, 32 successful models were randomly divided into model group and bFGF group, with 16 in each group. Another 16 SD rats were selected as sham-operation group, with only T10 processes, dura mater, and spinal cord exposed. After modeling, the rats in bFGF group were intraperitoneally injected with 100 μg/kg bFGF (once a day for 28 days), and the rats in model group and sham-operation group were injected with normal saline in the same way. The survival of rats in each group were observed after modeling. Basso-Beattie-Bresnahan (BBB) scores were performed before modeling and at immediate, 14 days, and 28 days after modeling to evaluate the functional recovery of hind limbs. Then, the spinal cord tissue at the site of injury was taken at 28 days and stained with HE, Nissl, and propidium iodide (PI) to observe the pathological changes, neuronal survival (number of Nissl bodies) and apoptosis (number of PI red stained cells) of the spinal cord tissue; immunohistochemical staining and ELISA were used to detect the levels of astrocyte activation markers [glial fibrillary acidic protein (GFAP)] and inflammatory factors [interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), interferon γ (IFN-γ)] in tissues, respectively. Western blot was used to detect the expressions of Notch/STAT3 signaling pathway related proteins [Notch, STAT3, phosphoryl-STAT3 (p-STAT3), bone morphogenetic protein 2 (BMP-2)] in tissues. Results All rats survived until the experiment was completed. At immediate after modeling, the BBB scores in model group and bFGF group significantly decreased when compared to sham-operation group (P<0.05). At 14 and 28 days after modeling, the BBB scores in model group significantly decreased when compared to sham-operation group (P<0.05); the bFGF group showed an increase compared to model group (P<0.05). Compared with before modeling, the BBB scores of model group and bFGF group decreased at immediate after modeling, and gradually increased at 14 and 28 days, the differences between different time points were significant (P<0.05). The structure of spinal cord tissue in sham-operation group was normal; in model group, there were more necrotic lesions in the spinal cord tissue and fewer Nissl bodies with normal structures; the number of necrotic lesions in the spinal cord tissue of the bFGF group significantly reduced compared to the model group, and some normally structured Nissl bodies were visible. Compared with sham-operation group, the number of Nissl bodies in spinal cord tissue significantly decreased, the number of PI red stained cells, GFAP, IL-1β, TNF-α, IFN-γ, Notch, p-STAT3 /STAT3, BMP-2 protein expression levels significantly increased in model group (P<0.05). The above indexes in bFGF group significantly improved when compared with model group (P<0.05). Conclusion bFGF can improve motor function and pathological injury repair of spinal cord tissue in SCI rats, improve neuronal survival, and inhibit neuronal apoptosis, excessive activation of astrocytes in spinal cord tissue and inflammatory response, the mechanism of which may be related to the decreased activity of Notch/STAT3 signaling pathway.
ObjectiveTo investigate the effect of transforming growth factorβ1 (TGF-β1) and basic fibroblast growth factor 1 (bFGF-1) on the cellular activities, prol iferation, and expressions of ligament-specific mRNA and proteins in bone marrow mesenchymal stem cells (BMSCs) and ligament fibroblasts (LFs) after directly co-cultured. MethodsBMSCs from 3-month-old Sprague Dawley rats were isolated and cultured using intensity gradient centrifugation. LFs were isolated using collagenase. The cells at passage 3 were divided into 6 groups: non-induced BMSCs group (group A), non-induced LFs group (group B), non-induced co-cultured BMSCs and LFs group (group C), induced BMSCs group (group D), induced LFs group (group E), and induced co-cultured BMSCs and LFs group (group F). The cellular activities and prol iferation were examined by inverted contrast microscope and MTT; the concentrations of collagen type Ⅰ and type Ⅲ were determined by ELISA; and mRNA expressions of collagen types I andⅢ, fibronectin, tenascin C, and matrix metalloproteinase 2 (MMP-2) were measured by real-time fluorescent quantitative PCR. ResultsA single cell layer formed in the co-cultured cells under inverted contrast microscope. Group F had fastest cell fusion ( > 90%). The MTT result indicated that group F showed the highest absorbance (A) value, followed by group D, and group B showed the lowest A value at 9 days after culture, showing significant difference (P < 0.05). Moreover, the result of ELISA showed that group F had the highest concentration of collagen type Ⅰ and type Ⅲ (P < 0.05); the concentration of collagen type Ⅲ in group E was significantly higher than that in group D (P < 0.05), but no significant difference was found in the concentration of collagen type Ⅰ between 2 groups (P > 0.05). The ratios of collagen type Ⅰ to type Ⅲ were 1.17, 1.19, 1.10, 1.25, 1.17, and 1.18 in groups A-F; group D was higher than the other groups. The real-time fluorescent quantitative PCR results revealed that the mRNA expressions of collagen type Ⅰ and type Ⅲ and fibronectin were highest in group F; the expression of tenascin C was highest in group D; the expression of MMP-2 was highest in group E; and all differencs were significant (P < 0.05). ConclusionDirectly co-cultured BMSCs and LFs induced by TGF-β1 and bFGF-1 have higher cellular activities, proliferation, and expressions of ligament-specific mRNA and protein, which can be used as a potential source for ligament tissue engineering.