OBJECTIVE To explore the effect of basic fibroblast growth factor (bFGF) combined with autogenous vein graft conduit on peripheral nerve regeneration. METHODS Fifty four New Zealand rabbits were divided into three groups. The main trunk of sciatic nerve of rabbit in one side was severed and bridged by autogenous vein. 0.2 ml bFGF solution (4,000 U/ml) was intravenously injected to the vein graft conduit as group A, the same amount of saline solution as group B, and no solution injection as group C. Microscopic examination, axon video analysis and nerve conduct velocity were performed at the 10th, 30th, and 100th day after operation. RESULTS The nerve fibers were grown into vein graft conduit in all groups at 30th after operation, they were more and regular in group A than that of group B and C, and the axon regeneration rate in group A was more than that of group B and C. CONCLUSION bFGF combined with autogenous vein graft conduit can markedly promote nerve regeneration.
Objective To review the recent researches of basic fibroblast growth factor (bFGF) in tendon tissue engineering. Methods Recentoriginal related literature was extensively reviewed and analyzed. Results bFGF played an important role in establishing standard tendon tissue engineering cell lines, inducing the compound and analysis of extracellular matrix, enhancing interactions between cells and extracellular matrix and accelerating tissue engineering materials’ neovascularization. Conclusion The progresses in increasing endogenetic bFGF expression, controlling the release of exogenous bFGF and improving the bioutilization of bFGF has laid foundation for wider use of bFGF in tendon tissue engineering.
Objective To investigate the preparation of decellularized Achilles tendons and the effect of co-culture of human fibroblasts on the scaffold so as to provide a scaffold for the tissue engineered ligament reconstruction. Methods Achilles tendons of both hind limbs were harvested from 10 male New Zealand white rabbits (5-month-old; weighing, 4-5 kg). The Achilles tendons were decellularized using trypsin, Triton X-100, and sodium dodecyl sulfate (SDS), and then gross observation, histological examination, and scanning electron microscope (SEM) observation were performed; the human fibroblasts were seeded on the decellularized Achilles tendon, and then cytocompatibility was tested using the cell counting kit 8 method at 1, 3, 5, 7, and 9 days after co-culture. At 4 weeks after co-culture, SEM, HE staining, and biomechanical test were performed for observing cell-scaffold composite, and a comparison was made with before and after decellularization. ResultsAfter decellularization, the tendons had integrated aponeurosis and enlarged volume with soft texture and good toughness; there was no loose connective tissue and tendon cells between tendon bundles, the collagen fibers arranged loosely with three-dimensional network structure and more pores between tendon bundles; and it had good cytocompatibility. At 4 weeks after co-culture, cells migrated into the pores, and three-dimensional network structure disappeared. By biomechanical test, the tensile strength and Young’s elastic modulus of the decellularized Achilles tendon group decreased significantly when compared with normal Achilles tendons group and cell-scaffold composite group (P lt; 0.05), but no significant difference was found between normal Achilles tendons group and cell-scaffold composite group (P gt; 0.05). There was no significant difference in elongation at break among 3 groups (P gt; 0.05). ConclusionThe decellularized Achilles tendon is biocompatible to fibroblasts. It is suit for the scaffold for tissue engineered ligament reconstruction.
ObjectiveTo investigate the effect of Wnt5a derived from tumor-associated fibroblasts （CAFs） on the migration and invasion of gastric cancer cells. MethodsThe differentially expressed genes Wnt5a between CAFs and normal gastric fibroblasts (NGFs) in gastric cancer tissues and their corresponding normal gastric tissues using the GEO database GSE194261 dataset were screened. Immunohistochemical method was used to detect the expression of Wnt5a protein in tissue samples of clinical gastric cancer patients, and the relationship between Wnt5a protein expression and clinicopathological features of gastric cancer was analyzed. CAFs and NGFs were extracted from fresh surgical specimens of gastric cancer patients, and the expression of Wnt5a in CAFs was detected by real-time fluorescence quantitative-polymerase chain reaction and Western blot experiment. Transwell invasion and migration experiment was used to observe the effects of CAFs, inhibition of Wnt5a expression in CAFs and different concentrations of recombinant Wnt5a protein on the migration and invasion ability of gastric cancer MGC-803 and MKN-28 cell lines in vitro. ResultsThrough the screening of GEO database GSE194261 data set, it was found that Wnt5a was more expressed in CAFs than NGFs (P<0.05). Immunohistochemical results showed that the expression of Wnt5a protein in gastric cancer tissues was significantly stronger than that in normal gastric tissues (P<0.05), and the expression of Wnt5a protein was related to T stage of tumor (χ2=5.035, P<0.05), but not related to gender, age, degree of tumor differentiation, lymph node metastasis, vascular invasion and nerve invasion (P>0.05). Inhibiting Wnt5a derived from CAFs could inhibit the invasion and migration of gastric cancer cells. By stimulating gastric cancer cells with different concentrations of human recombinant Wnt5a protein, it was found that when the concentration of human recombinant Wnt5a protein was greater than 100 ng/mL, the invasion and migration abilities of MGC-803 and MKN-28 gastric cancer cells were significantly increased (P<0.05). ConclusionWnt5a is highly expressed in CAFs derived from the interstitial tissue of gastric cancer, which is related to the invasion depth of gastric cancer and can promote the invasion and migration of gastric cancer cells.
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.
OBJECTIVE: To investigate the influence of basic fibroblast growth factor (bFGF) on adhesion characteristics of osteoblasts, aimed at the important problem in bone tissue engineering of how to promote the adherence of osteoblasts to extracellular matrix materials. METHODS: 5 ng/ml, 10 ng/ml, 50 ng/ml, 100 ng/ml, 200 ng/ml bFGF were used to induce bone marrow stromal-derived osteoblasts of rabbit for 24 hours before incubation, and the common culture medium as the control. The attached cells were calculated with stereology method at 0.5 hour, 1st hour, 2nd hour, 4th hour, 8th hour after seeding. RESULTS: The number of attached cells was significant higher in the experimental group when induced by 10 ng/ml bFGF than that in the control group (P lt; 0.01); the number did not increase with the increase of bFGF concentration and there was no significant difference between the experimental group induced by 100 ng/ml bFGF and control group, and the number was even obviously lower in the experimental group when induced by 200 ng/ml than the control group (P lt; 0.01). CONCLUSION: bFGF can influence the adhesion characteristics of osteoblasts, 10 ng/ml bFGF can promote the adherence of osteoblasts to matrix materials, but 200 ng/ml bFGF may inhibit cell adhesion.
Objective To study the biocompatibility of tendon mixedextraction of bovine collagen(tMEBC) and to explore the feasibility of using the threedimensional framework as periodontal tissue engineering scaffold. Methods After being prepared, the tMEBC were cultured with the P4P6 of human periodontal ligament fibroblasts (HPDLFs) in vitro. Threedimensional framework was prepared from bovine tendon. The P4-P6 of HPDLFs (with an initial density of 5×106 cells／ml) were cultured in vitro. Cell attachment andproliferation were measured by cell counting 1 day, 3,5, and 10 days after cell seeding. Histological examination was performed with light microscope and scanning electron microscope 5 and 10 days after cell seeding. Results Porous structure, which supported the proliferation and attachment of HPDLFs, was found in tMEBC. The density of cell increased from 0.556×104 cells／ml 24 hours after cell seeding to 3.944×104 cells／ml 10 days after seeding. Light and scanning electron microscope examinationindicated that HPDLFs were attached and extended on the three-dimensional scaffolds and were well embedded in the newly formed tissue matrix. ConclusiontMEBC has good biocompatibility with the HPDLFs, and can be used as scaffold for cell transplantation in periodontal tissue engineering.
ObjectiveTo study the effects of leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF) on the proliferation and differentiation of human bone marrow mesenchymal stem cells (hBMSCs). MethodshBMSCs at passage 4 were divided into 4 groups according to different culture conditions:cells were treated with complete medium (α-MEM containing 10%FBS, group A), with complete medium containing 10 ng/mL LIF (group B), with complete medium containing 10 ng/mL bFGF (group C), and with complete medium containing 10 ng/mL LIF and 10 ng/mL bFGF (group D). The growth curves of hBMSCs at passage 4 in different groups were assayed by cell counting kit 8; cellular morphologic changes were observed under inverted phase contrast microscope; the surface markers of hBMSCs at passage 8 including CD44, CD90, CD19, and CD34 were detected by flow cytometry. ResultsThe cell growth curves of each group were similar to the S-shape; the cell proliferation rates in 4 groups were in sequence of group D > group C > group B > group A. Obvious senescence and differentiation were observed very early in group A, cells in group B maintained good cellular morphology at the early stage, with slow proliferation and late senescence; a few cells in group C differentiated into nerve-like cells, with quick proliferation; and the cells in group D grew quickly and maintained cellular morphology of hBMSCs. The expressions of CD44 and CD90 in groups A and C at passage 8 cells were lower than those of groups B and D; the expressions of CD19 and CD34 were negative in 4 groups, exhibiting no obvious difference between groups. ConclusionLIF combined with bFGF can not only maintain multiple differentiation potential of hBMSCs, but also promote proliferation of hBMSCs.
Objective To select the appropriate media to culture the epidermal stem cells in vitro,and to observe the biological characteristics of the epidermal stem cells. Methods The epidermal stem cells were cultured in five different media, including FAD, FAD+1 ng/ml bFGF, FAD+5 ng/ml bFGF, FAD+10 ng/ml bFGF and K-SFM, and the same fetous fibroblasts were used as the nutrient cells. The proliferation ability was investigated by cell growth curve and MTT detection. Then the biological characteristics of epidermal stem cells were observed through phasecontrast microscope, cell growth curve, BrdU detection and FBM analysis. Results The epidermal stem cells grew best in FAD with bFGF and nutrient cells. And the epidermal stem cells retained proliferative capacity, and formed larger and more expandable clones in vitro. And 80.2% of the cells show a G0/G1 cycle, and the cells had long cell proliferation cycle. Conclusion The above results demonstrate that the media with bFGF and the use of nutrient layer were appropriate to culture epidermal stem cell in vitro. And the epidermal stem cells have a slow cell cycle, characteristics of immaturity.
OBJECTIVE To study the early protective effects of basic fibroblast growth factor(bFGF) on the experimental acute spinal cord injury. METHODS Thirty-four SD rats were randomly divided into three groups, and were subjected to contusion of thoracolumbar spinal cord. A thin plastic tube was placed in subarachnoid space below the injury level for perfusion. The bFGF-treated rats were received 20 microliters bFGF(containing bFGF 100 U) at once, 30 min, 1, 2, 3, 4, 6, 12, 24 and 48 hours after injury, and an equal volume of normal saline was given to the control group at the same time. The injured spinal cord was detected by morphological observation and biochemical index after injury. RESULTS The degree of ionic disorder in bFGF-treated rats was significantly ameliorated and the contents of H2O were also markedly decreased. The morphological finding showed that the damages of gray and white matter in bFGF-treated rats were slighter than those of saline-treated rats. CONCLUSION bFGF has some protective effects on the secondary lesion of early spinal cord injury in rats.