Various Concentrations Of bFGF Research Articles

Culture supplementation of bFGF, GDNF, and LIF alters in vitro proliferation, colony formation, and pluripotency of neonatal porcine germ cells.

Gonocytes in the neonatal testis have male germline stem cell properties and as such have important potential applications in fertility preservation and regenerative medicine. Such applications require further studies aimed at increasing gonocyte numbers and evaluating their pluripotency in vitro. The objective of the present study was to test the effects of basic fibroblast growth factor (bFGF), glialcell line-derived neurotrophic factor (GDNF), and leukemia inhibitory factor (LIF) on in vitro propagation, colony formation, and expression of pluripotency markers of neonatal porcine gonocytes. Testis cells from 1-week-old piglets were cultured in basic media (DMEM + 15% FBS), supplemented with various concentrations of bFGF, GDNF, and LIF, either individually or in combinations, in a stepwise experimental design. Gonocytes and/or their colonies were evaluated every 7days and the gonocyte- (DBA) and pluripotency-specific markers (POU5F1, SSEA-1, E-cadherin, and NANOG) assessed on day 28. Greatest gonocyte numbers and largest colonies were found in media supplemented with 10ng/mL bFGF and 10ng/mL bFGF + 100ng/mL GDNF + 1500 U/mL LIF, respectively. The resultant gonocytes and colonies expressed both germ cell- and pluripotency-specific markers. These results shed light on thegrowth hormone requirements of porcine gonocytes for in vitro proliferation and colony formation.

Changes in Expression of Nestin, CD44, Vascular Endothelial Growth Factor, and Glutamine Synthetase by Mature Müller Cells After Dedifferentiation.

Müller cells are dedifferentiated after retinal injuries and are transformed into nestin-positive progenitor cells that play crucial roles in remodeling. The purpose of this study was to determine the changes in the expression of nestin, CD44 (a receptor of hyaluronan), vascular endothelial growth factor (VEGF), and glutamine sythetase in cultured Müller cells after dedifferentiation by basic fibroblast growth factor (bFGF) and insulin. Cells from a rat retinal Müller cell line (TR-MUL5) and from primary rat retinal Müller cells were grown in culture. The cells were incubated in various concentrations of bFGF (1.0, 10, 100 ng/mL) with or without insulin (5 μM) for 48 h. Changes in the expression of nestin, CD44, VEGF, and glutamine synthetase were determined by immunoblot and immunohistochemistry. Exposure of TR-MUL5 cells to 10 ng/mL of bFGF led to the maximum increase in nestin by 1.5-fold, whereas the exposure had no effects on the expression of CD44. Addition of insulin (5 μM) to the bFGF significantly increased the CD44 level in TR-MUL5 cells by 1.4-fold. Immunohistochemistry showed that the combined treatments also upregulated the expression of nestin and CD44 in primary retinal Müller cells. Immunoblot analyses showed that exposure to bFGF and insulin caused significant increases of nestin (4.9-fold), CD44 (3.4-fold), and VEGF (1.44-fold) and decreases in glutamine synthetase (0.7-fold). The inflammation and angiogenesis that develop after retinal injuries may be due to an upregulation of CD44 and VEGF by the dedifferentiated Müller cells.

Enhanced Proliferation and Chondrogenic Differentiation of Human Synovium-Derived Stem Cells Expanded with Basic Fibroblast Growth Factor

Synovium-derived stem cell (SDSC) is one of valuable sources for cartilage regeneration. Basic fibroblast growth factor (bFGF) was reported to augment the differentiation potential of mesenchymal stem cells originating from a variety of sources. In this study, we applied various concentrations of bFGF to monolayer cultures of SDSCs and evaluated its effects on proliferation and chondrogenesis. SDSCs expressed mRNAs of FGF receptor 1, 2, 3, and 4, but produced only FGF receptor 3 protein. The SDSCs were expanded as monolayer supplemented with various concentrations of bFGF (0, 0.1, 1, 10, and 100 ng/mL) before chondrogenesis. Cell shrinkage and increased actin expression was noted as well as enhanced proliferation by bFGF treatment in monolayer cultures. Cell surface marker CD34 and CD49a expression of SDSCs was decreased with 10 and 100 ng/mL of bFGF. In micromass pellet cultures, bFGF-treated SDSCs showed augmented sizes, weights, and glycosaminoglycan accumulation of pellets by bFGF supplementation. Messenger RNA and protein expression of type II and type X collagen were upregulated in pellets cultured bFGF. These results demonstrated that bFGF was an effective agent for the enhancement of SDSC proliferation and chondrogenesis. From the results in this study, we could elect the 10 ng/mL of bFGF as an optimal concentration for pretreatment of SDSCs before chondrogenic differentiation.

Effect of basic fibroblast growth factor on the cellular repopulation of decellularized anterior cruciate ligament allografts

The use of decellularized anterior cruciate ligament (ACL) allografts in ACL replacement surgery may allow for the native structure of the ligament to be retained, thereby recapturing the function of the ligament post-injury. Our previous work has focused on repopulating decellularized allograft ACL tissue with ACL fibroblasts in order to prevent destructive remodelling of the implanted tissue by extrinsic host cells. In this study, the use of basic fibroblast growth factor (bFGF) to improve the cellular repopulation of decellularized ACL tissue was assessed. A concentration of 6 ng/ml bFGF was demonstrated to be effective in increasing cellular growth in the absence of tissue; however, this concentration, as well as reduced and increased levels of bFGF (0.1 and 60 ng/ml, respectively), failed to increase cellular repopulation of ACL fibroblast-seeded decellularized tissue after 28 days of culture. Mean repopulation levels of 11-19% of fresh tissue [3200-5300 cells/mg dry weight (dwt) tissue] were achieved after 28 days in culture. Qualitative observation of histological samples suggested that different repopulation characteristics exist at various concentrations of bFGF and, in particular, that bFGF may be stimulating a catabolic pathway resulting in matrix destruction. Significant differences in the effects of bFGF observed between cell-only and cell-and-tissue studies serve to reinforce the concept that cells respond to stimuli in a different manner, depending on the surrounding environment. As a result, caution should be used when information obtained from studies utilizing cells alone is applied to the development of tissue-engineered constructs.

Increase in the Biocompatibility of the Neutralized Chitosan Dermal Scaffold by Reconstruction of Wound Healing Microenvironment: In Vitro Study

The porous neutralized chitosan scaffold (NCS) was prepared by freeze-dry method. Its poor cell binding capacity was improved approximately five folds by mixing or coating of atelomeric type I collagen. In order to recreate wound-healing microenvironment within the NCS for the better wound healing effect, various concentrations of bFGF and fibronectin (FN) were supplied in the secondary freeze-dry process of the scaffold. NCS+ bFGF and NCS+FN improved the cell binding capacity by four folds and three folds respectively. Therefore supplementation of collagen, b-FGF and/or fibronectin in the NCS can improve the biocompatibility of the chitosanbased scaffold which itself revealed poor cell binding capacity.

Modifying the Proliferative State of Target Cells to Control DNA Expression and Identifying Cell Types Transfected In Vivo

Although the majority of current gene transfer techniques have focused on increasing the ability of the DNA to enter the cell, it is possible that changing the proliferative and migratory state of cells will influence the cells ability to take up and express plasmid DNA. This study was designed to test the hypothesis that growth factors (basic fibroblast growth factor (bFGF) and hepatocyte growth factor/scatter factor (HGF/SF)) used to alter the proliferative and migratory state of cells can alter plasmid DNA uptake and expression. In vitro studies indicate that enhancing cell proliferation with growth factor exposure enhances plasmid DNA uptake and expression. Furthermore, dual localized delivery of bFGF and plasmid DNA in vivo increases the expression, 3-6 times over control, as compared to plasmid delivery alone. Dual delivery of a factor promoting cell proliferation and a plasmid led to a further increase in the expression of the plasmid encoding bone morphogenetic protein-2 in a rat cranial defect by specific cell populations. The results of these studies suggest that increasing the proliferative state of target cell populations can enhance non-viral gene transfer.

The inhibiting effects of suramin on bFGF induced proliferation of cultured human RPE cells

To study the inhibiting effects of suramin on FGF induced proliferation of cultured RPE cells and discuss the effects and mechanisms of suramin in the prevention of proliferative vitreoretinopathy (PVR). Human RPE cells at passage 6 were seeded into 96-well plates. In the 5 control groups, different concentrations of bFGF (1, 10, 100 and 500 ng/ml) or suramin (31.25 microg/ml) were added into the culture medium. In the 4 treatment groups, 31.25 microg/ml suramin with different concentrations of bFGF (1, 10, 100 and 500 ng/ml) were added to the medium. The blank group was incubated with serum-free DMEM only. After incubated for 48 hours, the proliferation of cells in all groups was measured by the MTT assay and expressed as light absorption values. The separate and combine effects of suramin and bFGF were analysed. Under the phase-contrast microscope, no changes of cell morphology could be detected between various groups. Suramin showed an inhibitory effects on bFGF induced proliferation of RPE cells (F=6.73, P < 0.01). Light absorption value (A value) in bFGF-treated control groups was higher than that of the blank group and reached to the peak in cells treated with 10 ng/ml of bFGF; with no further increase in cells treated with higher levels of bFGF. The A value in suramin-treated control group was lower than that of the blank group. The A-value in all treated groups was lower than that of the control groups treated with various concentrations of bFGF. In the treated group, the highest A value was detected in cells treated with 100 ng/ml bFGF. With further increase of bFGF concentration to 500 ng/ml, the A-value was not statistically different from that of cells treated with 100 ng/ml bFGF. Suramin can inhibit the bFGF induced proliferation of RPE cells. The mechanisms of the effects of suramin in the inhibition of proliferation of RPE cells and in the prevention of PVR at least partly due to its competition effects with the growth factors.

Bladder Regeneration by Bladder Acellular Matrix Combined with Sustained Release of Exogenous Growth Factor

We investigate the use of bladder acellular matrix (BAM) as a carrier of exogenous growth factor. Basic fibroblast growth factor (bFGF) protein was loaded in a lyophilized BAM by rehydrating the matrix with the growth factor solution. In vitro and in vivo release of bFGF from BAM was traced by radiolabeled bFGF. To assess biological effect of the released bFGF, BAMs incorporating bFGF were implanted into back subcutis of mice and vascular endothelial growth factor levels in the local tissue were determined. Furthermore, bladder augmentation was performed in rats with BAM grafts containing various concentrations of bFGF. At 4 and 12 weeks the graft size at maximal cystometric capacity was evaluated, followed by histological and physiological evaluation. Sustained release of bFGF from BAM was observed in vitro and in vivo. In vivo biological activity of bFGF in BAM as determined by vascular endothelial growth factor levels was preserved for more than 3 weeks. In the bladder augmentation model angiogenesis into the matrix was promoted and graft shrinkage was significantly inhibited by incorporated bFGF in a dose dependent manner at 4 weeks. BAM can function as a platform for sustained release of exogenous bFGF. This delivery method of growth factor could be a useful tool for bladder reconstruction.

Effect of Growth Factors on Epithelial Restitution of Human Colonic Mucosa in Vitro

Background: Epithelial restitution enables resurfacing of epithelial discontinuities by enterocyte migration. This study investigated the effect of basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF-1), and epidermal growth factor (EGF) on restitution of human colonic mucosa in vitro. Methods: After base-line incubation human colonic mucosal strips, mounted in Ussing chambers, were luminally exposed to 0.5 mM sodium deoxycholate (NaDOC) for lOmin. Thereafter tissues were incubated with buffer alone or luminal buffer containing various concentrations of bFGF, IGF-1, and EGF for 3 h. Resistance (R) was calculated from potential difference (PD) and short-circuit current (Isc). All tissues were processed for light microscopy. Extent of damage was measured by morphometry. Results: Luminal 0.5 mM NaDOC for 10 min caused R to drop by 43% (n = 4; P < 0.05). Compared with controls 50 ng/ml EGF induced an approximately 30% R increase until the end of the experiments (P < 0.05, n = 4, paired). Ten minutes after injury 50.2±4% of the mucosa was damaged (n = 6), and after 3 h damage was significantly reduced by EGF (17.2±3% versus 31.7±4%, 50 ng/ml EGF versus controls) (P <0.05, n = 6 per group). Histology showed that EGF stimulated enterocyte migration over the basal lamina. Various doses of bFGF and IGF-1 did not impair restitution when compared with controls. Conclusion: In contrast to bFGF and IGF-1, EGF was shown to promote epithelial restitution of human colonic mucosa in vitro.

Basic fibroblast growth factor promotes in vivo muscle regeneration in murine muscular dystrophy

Due to the lack of dystrophin, a subsarcolemmal protein, mdx mutant mice undergo spontaneous rounds of myofiber necrosisregeneration from the age of weaning. Muscle regeneration is likely to be controlled by basic fibroblast growth factor (bFGF) which is detectable in regenerating areas of mdx muscles. Moreover, the proliferation of mdx satellite cells seems to be particularly sensitive to bFGF in culture. We injected various concentrations of bFGF in the tibialis anterior muscle of 4-week-old mice at the time of the first round of muscle necrosis-regeneration, and we evaluated the in vivo effects of bFGF on mdx muscle regeneration by quantitative histology. Seven days after bFGF injection, the number of regenerated myofibers was significantly increased proportionally to the injected bFGF concentration. This effect was due to an enhanced replication of muscle satellite cells as shown by labeling the proliferating cells with 5-bromo-2′-deoxyuridine (BrdU). These results may provide a possibility of improving dystrophin-deficient muscle regeneration by increasing the availability of bFGF.

Basic fibroblast growth factor (bFGF) increases the survival of embryonic and postnatal basal forebrain cholinergic neurons in primary culture

Basic fibroblast growth factor (bFGF) is found in high concentrations in the mammalian central nervous system. It is a mitogen for glia and it influences the development and survival of specific populations of neurons. In this study, we investigated the effect of various concentrations of bFGF on the survival of embryonic and postnatal cholinergic basal forebrain neurons plated at low and high density in the presence and absence of glia. We observed that 50 and 100 ng/ml of bFGF increased the survival of embryonic cholinergic neurons plated at high density. This effect was observed only in the presence of glia. Lower concentrations of 10 and 20 ng/ml had no effect on cholinergic neuronal survival. The number of GFAP (glial fibrillary acidic protein)-positive cells in high-density embryonic cultures was increased by all concentrations of bFGF. In low-density embryonic cultures, an increase in cholinergic neuron survival was observed at concentrations ranging from 20 to 100 ng/ml. The number of GFAP-positive cells in low-density cultures was also increased by all concentrations of bFGF. Similar to low-density embryonic cultures, the survival of cholinergic neurons from postnatal day 2 cultures was significantly increased in the presence of glia at concentrations of 20, 50 and 100 ng/ml of bFGF. Postnatal glia was affected by all concentrations of bFGF, as was observed in embryonic cultures. This study indicates that high concentrations of bFGF can influence cholinergic neuronal survival by stimulating and increasing glia, which may produce factor(s) that are necessary for cholinergic neuron survival.