【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 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 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.
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 expression changes of vascular endothel ial growth factor (VEGF), basic fibroblast growth factor (bFGF), and bone morphogenetic protein 2 (BMP-2) in femoral neck fracture, traumatic, and non-traumatic avascular necrosis of femoral head (ANFH), and to study the relationshi p between the expressions of VEGF, bFGF, BMP-2mRNA and bone mass so as to explore the pathogenesis of ANFH and provide the exprimental basis for individual treatment of ANFH. Methods Femoral head specimens were obtained from 59 donors undergoing total hip replacement, including 22 cases of traumatic ANFH (group A, 13 cases of Ficat stage III and 9 cases of Ficat stage IV), 19 cases of non-traumatic ANFH (group B, 11 cases of Ficat stage III and 8 cases of Ficat stage IV; 10 cases of steroid-induced ANFH, 7 cases of alcohol ic ANFH, and 2 cases of unexplained ANFH), and 18 cases of fresh femoral neck fracture (group C). There was no significant difference in the general data among 3 groups (P gt; 0.05). The bone mineral density (BMD) at weight-bearing area of the femoral head was measured with dual energy X-ray absorptiometry. The pathological changes were observed by using optical microscope and scanning electron microscope. The percentage of empty bone lacuna and the percentage of trabecular bone area were calculated. The expressions of VEGF, bFGF, and BMP-2 mRNA in femoral head were detected by use of in-situ hybridization technique. Results The BMD in groups A and B were significantly lower than that in group C (P lt; 0.05), and there was significant difference between group A and group B (P lt; 0.05). In the necrosis area of groups A and B, the bone trabecula was rarefactive and not of integrity, with a great number of empty bone lacuna. In healthy area, more fiber hyperplasia was observed in group A, the prol iferated and hypertrophic fat cells in the medullary cavity in group B. Scanning electron microscope showed that many osteocytes underwent fatty degeneration and necrosis, and that the prol iferation of fat cells in bone matrix was observed in groups A and B. While in group C, the femoral head had intact articular cartilage and intact bone trabeculae, and osteocytes were clearly seen. The percentage of empty bone lacuna was significantly higher (P lt; 0.05) and the percentage of trabecular bone area was significantly lower (P lt; 0.05) in groups A and B than group C; and there was significant difference in the percentage of empty bone lacuna between groups A and B (P lt; 0.05). The expressions of VEGF, bFGF, and BMP-2 mRNAwere significantly lower in groups A and B than group C (P lt; 0.05), and the expressions of BMP-2 and bFGF mRNA in group A were significantly higher than those in group B (P lt; 0.05). There were positive l inear correlation between the expressions of VEGF mRNA, bFGF mRNA, BMP-2 mRNA and the BMD and percentage of trabecular bone area, respectively. While there were significantly negative correlation between the expressions of VEGF mRNA, bFGF mRNA, BMP-2 mRNA and percentage of empty bone lacuna. Conclusion The repair capacity of local femoral head in traumatic ANFH is ber than that in non-traumatic ANFH. The expressions of VEGF mRNA, bFGF mRNA, and BMP-2 mRNA decl ine in traumatic and nontraumatic ANFH.
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.
ObjectiveTo observe the effect of vascular endothelial growth factor/polylactide-polyethyleneglycol-polylactic acid copolymer/basic fibroblast growth factor (VEGF/PELA/bFGF) mixed microcapsules in promoting the angiogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) in vitro. MethodsThe BMSCs were isolated by the method of whole bone marrow adherent, and sub-cultured. The passage 3 BMSCs were identified by Wright-Giemsa staining and flow cytometry, and used for subsequent experiments. VEGF/PELA/bFGF (group A), PELA/bFGF (group B), VEGF/PELA (group C), and PELA (group D) microcapsules were prepared. The biodegradable ability and cytotoxicity of PELA microcapsule were determined, and the slow-released ability of VEGF/PELA/bFGF mixed microcapsules was measured. The passage 3 BMSCs were co-cultured with the extracts of groups A, B, C, and D, separately. At 1, 3, 7, 14, and 20 days after being cultured, the morphological changes of induced BMSCs were recorded. At 21 days, the induced BMSCs were tested for DiI-labeled acetylated low density lipoprotein (Dil-ac-LDL) and FITC-labeled ulex europaeus agglutinin I (FITC-UEA-I) uptake ability. The tube-forming ability of the induced cells on Matrigel was also verified. The differences of the vascularize indexes in nodes, master junctions, master segments, and tot.master segments length in 4 groups were summarized and analyzed. ResultsThe isolated and cultured cells were identified as BMSCs. The degradation time of PELA was more than 20 days. There was no significant effect on cell viability under co-culture conditions. At 20 days, the cumulative release of VEGF in the mixed microcapsules exceeded 95%, and the quantity of bFGF exceeded 80%. The morphology of cells in groups A, B, and C were changed. The cells in groups A and B showed the typical change of cobble-stone morphology. The numbers of double fluorescent labeled cells observed by fluorescence microscope were the most in group A, and decreases from group B and group C, with the lowest in group D. The cells in groups A and B formed a grid-like structure on Matrigel. Quantitative analysis showed that the differences in the number of nodes, master junctions, master segments, and tot.master segments length between groups A, B and groups C, D were significant (P<0.05). The number of nodes and the tot.master segments length of group A were more than those of group B (P<0.05). There was no significant differences in the number of master junctions and master segments between group A and group B (P>0.05). ConclusionVEGF/PELA/bFGF mixed microcapsules have significantly ability to promote the angiogenic differentiation of rat BMSCs in vitro.
Objective To investigate the efficacy of basic fibroblast growth factor (bFGF) combined with topical oxygen therapy for deep II degree burn wounds, by comparing the effects of bFGF combined with topical oxygen therapy and bFGF with routine therapy. Methods From February 2004 to July 2009, 85 patients with deep II degree burn wounds (117 wounds) were enrolled and divided into 4 groups randomly according to different treatments. There was no significant difference in sex, age, disease course, wound size, and wound treatment size among 4 groups (P gt; 0.05). In group A, 18 patients (28 wounds) were treated routinely; in group B, 23 patients (30 wounds) were treated with routine methods and topical oxygen therapy; in group C, 19 patients (25 wounds) were treated with routine methods and bFGF therapy; and in group D, 25 patients (34 wounds) were treated with routine methods and bFGF/topical oxygen therapy. Topical oxygen therapy was administered to the wound for 90 minutes per day for 3 weeks. The bFGF therapy was appl ied everyday (150 U/ cm2) for 3 weeks. Results All cases were followed up 6-12 months (9 months on average). The wound heal ing times in groups A, B, C, and D were (27.3 ± 6.6), (24.2 ± 5.8), (22.2 ± 6.8), and (18.2 ± 4.8) days, respectively; showing significant difference between group A and group D (P lt; 0.05). The wound heal ing rates in groups A, B, C, and Dwere 67.8% ± 12.1%, 85.1% ± 7.5%, 89.2% ± 8.3%, and 96.1% ± 5.6%, respectively; showing significant differences between group A and groups B, C, D (P lt; 0.05). The therapic effective rates in groups A, B, C, and D were 75%, 90%, 92%, and 100%, respectively; showing significant difference between group A and group D (P lt; 0.05). The Vancouver scar scale scoring of group D 6 months after treatment was better than that of group A (P lt; 0.05). Conclusion The bFGF combined with topical oxygen therapy can enhance deep II degree burn wound heal ing. Furthermore, the therapy method is simple and convenient.
ObjectiveTo discuss whether human amniotic mesenchymal stem cells (hAMSCs) possesses the characteristic of mesenchymal stem cells, and could differentiate into ligament cells in vitro after induction. MethodsThe hAMSCs were separated through enzyme digestion, and the phenotypic characteristics of hAMSCs were tested through flow cytometry. The cells at passage 3 were cultured with L-DMEM/F12 medium containing transforming growth factor β1 (TGF-β1)+basic fibroblast growth factor (bFGF) (group A), containing hyaluronic acid (HA) (group B), containing TGF-β1+bFGF+HA (group C), and simple L-DMEM/F12 medium (group D) as control group. The morphology changes of cells in each group were observed by inverted phase contrast microscope at 21 days after induction; the cellular activities and proliferation were examined by sulforhodamine (SRB) colorimetric method; and specific mRNA and protein expressions of ligament including collagen type I, collagen type III, and tenascin C (TNC) were measured by real-time fluorescence quantitative PCR and immunohistochemical staining. ResultsThe flow cytometry result indicated that hAMSCs expressed mesenchymal stem cell phenotype. After 21 days of induction, the cells in groups A, B, and C grew like spindle-shaped fibroblasts under inverted phase contrast microscope, and cells showed single shape, obvious directivity, and compact arrangement in group C. The SRB result indicated that the cells in each group reached the peak of growth curve at 6 days; the cellular activities of groups A, B, and C were significantly higher than that of group D at 6 days after induction. Also, the immunohistochemical staining results showed that no expressions of TNC were detected in 4 groups at 7 days; expressions of collagen type I in groups A, B, and C were significantly higher than that in group D at 7, 14, and 21 days (P<0.001); the expressions of collagen type III in groups A, B, and C were significantly higher than that in group D at 14 and 21 days (P<0.001). There was an increasing tendency with time in collagen type I of group B, in collagen type III and TNC of groups A and C, showing significant difference among different time points (P<0.001). The real-time fluorescence quantitative PCR results revealed that the mRNA expressions of collagen type I and TNC in group C were significantly higher than those in groups A and B (P<0.05), and the mRNA expression of collagen type III in group B were significantly higher than that in groups A and C at 21 days (P<0.05). The mRNA expressions of collagen type I and TNC in groups A and C and mRNA expression of collagen type III in group C had an increasing tendency with time, showing significant difference among different time points (P<0.001). ConclusionThe hAMSCs possesses the characteristics of mesenchymal stem cells and excellent proliferation capacity. After in vitro induction, the expressions of ligament specific genes can be up-regulated and the synthesis of ligament specific proteins can be also strengthened. As a result, it can be used as one of ligament tissue engineering seed cell sources.