Bioactive bFGF Research Articles

Engineered Basic Fibroblast Growth Factor Specifically Bonded with Injectable Extracellular Matrix Hydrogel for the Functional Restoration of Cerebral Ischemia in Rats.

Cerebral ischemia was one of the leading causes of mortality and disability worldwide. Extracellular matrix (ECM) hydrogel held great potential to replace volumetric brain tissue loss following ischemic injury but with limited regenerative effect for functional restoration when implanted alone. In the present study, an engineered basic fibroblast growth factor (EBP-bFGF) was constructed, which fused a specific ECM-binding peptide (EBP peptide) with bFGF. The recombinant EBP-bFGF showed typical binding capacity with ECM without affecting the bioactivity of bFGF both invitro and invivo. Furthermore, the EBP-bFGF was used for bioactive modification of ECM hydrogel to repair cerebral ischemia. The combination of EBP-bFGF and ECM hydrogels could realize the sustained release of bFGF in the ischemic brain and improve the regenerative effect of ECM, which protected the survival of neurons, enhanced angiogenesis, and decreased the permeability of blood-brain barrier, ultimately promoted the recovery of motor function. In addition, transcriptome analysis revealed neuregulin-1/AKT pathway involved in this process. Therefore, EBP-bFGF/ECM hydrogel would be a promising therapeutic strategy for cerebral ischemia.

Spinal cord decellularized matrix scaffold loaded with engineered basic fibroblast growth factor-overexpressed human umbilical cord mesenchymal stromal cells promoted the recovery of spinal cord injury.

Spinal cord injury (SCI) will lead to irreversible damage of sensory and motor function of central nervous system, which seriously affects patient's quality of life. A variety of nerve engineering materials carrying various stem cells and cell growth factors had used to promote the repair of SCI, but they could not mimic the actual matric niche at spinal cord to promote cell proliferation and differentiation. Thus, developing novel biomaterial providing better niche of spinal cord is a new strategy to treat the severe SCI. In this study, we constructed porcine spinal cord decellularized matrix scaffold (SC-DM) with biocompatibility to load engineered basic fibroblast growth factor-overexpressing human umbilical cord mesenchymal stromal cells (bFGF-HUCMSCs) for treating SCI. The continuously released bioactive bFGF factors from grafted bFGF-HUCMSCs and three-dimensional niche by SC-DM promoted the differentiation of endogenous stem cells into neurons with nerve conduction function, leading a markedly motor function recovery of SCI. These results indicated that the functional bFGF-HUCMSCs/SC-DM scaffold provided more suitable matric niche for nerve cells, that would be a promising strategy for the clinical application of SCI.

Double layer composite membrane for preventing tendon adhesion and promoting tendon healing

Electrospun membranes and hydrogels are widely used to prevent tendon adhesion. Hydrophobic anti-inflammatory drugs could be fully loaded on the electrospinning membrane through the electrospinning process, which can better prevent tendon adhesion. Basic fibroblast growth factor (bFGF) could promote tendon healing. However, the bioactivity of free bFGF is easily inactivated, therefore, a suitable carrier is needed. As a carrier, hydrogel has little effect on the bioactivity of the protein drugs. In this work, a poly(lactic-co-glycolic) acid (PLGA) electrospun membrane loaded with ibuprofen (IBU) was prepared and named EMI. Additionally, Methoxy poly(ethylene glycol)-block-poly(L-valine) (PEG-PLV) was synthesized. bFGF was added to the PEG-PLV solution, a hydrogel containing bFGF (PLVB) was obtained after gelling. PLVB was applied to the surface of EMI, a double-layer composite membrane named EMI-PLVB was obtained. This membrane was used to prevent Achilles tendon adhesion and promote healing. IBU and bFGF in EMI-PLVB were continuously released in vitro. The inflammatory factors at the tendon healing site were significantly reduced, and the production of type I collagen (Col- I) and type III Collagen (Col-III) at the tendon healing site was also increased in vivo. In conclusion, this double-layer composite membrane drug release system can effectively prevent tendon adhesion and promote tendon healing.

Bioactive ECM Mimic Hyaluronic Acid Dressing via Sustained Releasing of bFGF for Enhancing Skin Wound Healing.

Successful dermal wound regeneration requires the coordination of repair cells and cellular signals with the extracellular matrix (ECM), which serves as an indispensable mechanical and biological supporter for cell functions and communications with varied cytokines during healing processes. Here, we developed an injectable bioactive wound dressing, methacrylated hyaluronic acid (Me-HA)-based hydrogel loading with basic fibroblast growth factor (bFGF), endowing the dressing with the pleiotropic bioactivity to mimic natural ECM. This bFGF@Me-HA dressing was applied to a mouse with full-thickness excisional wounds to investigate its positive roles in wound repair owing to the complementary functions of HA with sustained release of bioactive bFGF. Compared with the single Me-HA and bFGF group, bFGF@Me-HA hydrogel dressings significantly enhanced wound healing with accelerated re-epithelialization, granulation formation, collagen, deposition and skin appendage regeneration. Further investigations showed significantly promoted cell proliferation and vascularization in the bFGF@Me-HA group, which was mediated by the upregulation of transforming growth factor-β (TGF-β) and vascular endothelial growth factor (VEGF) expressions. In conclusion, this bFGF@Me-HA hydrogel realized the optimization of simple ECM mimic dressing via introducing the bioactive effector, bFGF, and has the potential to be widely used as an effective bioactive ECM-based wound dressing in future wound care.

Silver crosslinked injectable bFGF-eluting supramolecular hydrogels speed up infected wound healing.

Topical wound dressings with various silver compositions that exhibit effective bacterial inhibition properties are often used to treat infected wounds. However, a silver dressing with no bioactive functionality will typically delay subsequent wound repair processes. Therefore, development of a simple wound dressing containing silver and loaded with a bioactive drug is a very attractive solution. Herein, we developed a silver crosslinked injectable chitosan-silver hydrogel as a silver immobilization matrix, loaded with basic fibroblast growth factor (bFGF) as its cargo (namely, bFGF@CS-Ag) for treatment of both acute and infected wounds. The in vivo results showed that bFGF@CS-Ag significantly enhanced infectious wound regeneration compared to that of acute wounds. Further investigation demonstrated that the improved wound repair by bFGF@CS-Ag was ascribed to the effectiveness of bacterial inhibition, the promotion of granulation formation, collagen deposition, neovascularization and re-epithelization, and to the reduction of the inflammatory response through promotion of M2 macrophage polarization. These results proved that the immobilization of silver in the hydrogel not only reduced the side effects of silver on the bioactivity of bFGF but also allowed elution of bFGF in a controlled release manner. Thus, this novel system has promising therapeutic potential for topical treatment of wounds.

Spatiotemporal delivery of basic fibroblast growth factor to directly and simultaneously attenuate cardiac fibrosis and promote cardiac tissue vascularization following myocardial infarction

Following myocardial infarction (MI), the destruction of vasculature in the infarcted heart muscle and progression of cardiac fibrosis lead to cardiac function deterioration. Vascularization of the damaged tissue and prevention of cardiac fibrosis represent promising strategies to improve cardiac function. Herein we have developed a bFGF release system with suitable release kinetics to simultaneously achieve the two goals. The release system was based on an injectable, thermosensitive, and fast gelation hydrogel and bFGF. The hydrogel had gelation time <7 s. It can quickly solidify upon injection into tissue so as to increase drug retention in the tissue. Hydrogel complex modulus can be tuned by hydrogel solution concentration. The complex modulus of 176.6 Pa and lower allowed cardiac fibroblast to maintain its phenotype. Bioactive bFGF was able to gradually release from the hydrogel for 4 weeks. The released bFGF promoted cardiac fibroblast survival under ischemic conditions mimicking those of the infarcted hearts. It also attenuated cardiac fibroblasts from differentiating into myofibroblasts in the presence of TGFβ when tested in 3D collagen model mimicking the scenario when the bFGF release system was injected into hearts. Furthermore, the released bFGF stimulated human umbilical endothelial cells to form endothelial lumen. After 4 weeks of implantation into infarcted hearts, the bFGF release system significantly increased blood vessel density, decreased myofibroblast density and collagen content, augmented cardiac cell survival/proliferation, and reduced macrophage density. In addition, the bFGF release system significantly increased cardiac function. These results demonstrate that delivery of bFGF with appropriate release kinetics alone may represent an efficient approach to control cardiac remodeling after MI.

Heparan sulfate proteoglycan promotes fibroblast growth factor-2 function for ischemic heart repair.

It is well known that the basic fibroblast growth factor (bFGF) promotes angiogenesis after myocardial infarction (MI), but its biological functions decrease in the event of diffusion, enzymolysis, and weak binding with co-receptors in vivo. Heparan sulfate proteoglycans (HSPG) are a major component of extracellular matrices and have been shown to regulate a wide range of cellular functions and bioprocesses by acting as a co-receptor for bFGF and affecting its bioactivities. However, the influence of HSPG on the function of bFGF after myocardial infarction is unknown. Here, exogenous HSPG along with bFGF was injected into the hearts of rats to deliver the angiogenic growth factor for ischemic heart repair following induced MI. The specific binding of HSPG with bFGF protein was demonstrated, which was about 6-fold stronger than the binding of bFGF with heparin. The biological mechanisms of HSPG binding with bFGF were further studied by cell adhesion assay, and assays of bFGF and matrix metalloproteinase 2 (MMP2) activities demonstrated that HSPG enhances cell adhesion, promotes the bioactivity of bFGF in angiogenesis, and protects bFGF from enzymolysis. Our results indicate that HSPG has potential clinical utility as a delivery agent for heparin-binding growth factors. Additionally, HSPG shows high binding affinities with different ECM proteins which also help to anchor bFGF to heart tissue. Therefore, extracellular proteins that mimic the bio-scaffold of the extracellular matrix could promote the activities of bFGF to facilitate ischemic heart repair.

Lung endothelial cell-targeted peptide-guided bFGF promotes the regeneration after radiation induced lung injury

Basic fibroblast growth factor (bFGF) can protect the lung against radiation-induced pulmonary vascular endothelial apoptosis and subsequent radiation-induced lung injury (RILI). However, guiding bFGF to pulmonary vascular endothelial cells is a key determinant for the success of bFGF therapy. To improve the lung-targeting ability of bFGF, a lung endothelial cell-targeting peptide was fused to bFGF (LET-bFGF). An in vitro biological activity assay indicated that fusion of LET did not affect the bioactivity of bFGF. In addition, the fused protein showed superior lung-targeting ability following intravenous injection. Upon injecting LET-bFGF intravenously after thorax radiation, LET-bFGF could better protect against pulmonary vascular endothelial cell apoptosis as early as 4 h post-radiation. Compared with native bFGF, enhanced therapeutic effects of LET-bFGF were also observed in terms of decreased vascular abnormalities, disorganized lung structure, inflammatory cell migration, and lung density at 2 months post-radiation. Therefore, lung endothelial cell-targeted bFGF may represent a promising remedy for RILI.

Porous Silk Fibroin Microspheres Sustainably Releasing Bioactive Basic Fibroblast Growth Factor.

Basic fibroblast growth factor (bFGF) plays a significant role in stimulating cell proliferation. It remains a challenge in the field of biomaterials to develop a carrier with the capacity of continuously releasing bioactive bFGF. In this study, porous bFGF-loaded silk fibroin (SF) microspheres, with inside-out channels, were fabricated by high-voltage electrostatic differentiation, and followed by lyophilization. The embedded bFGF exhibited a slow release mode for over 13 days without suffering burst release. SEM observations showed that incubated L929 cells could fully spread and produce collagen-like fibrous matrix on the surface of SF microspheres. CLSM observations and the results of cell viability assay indicated that bFGF-loaded microspheres could significantly promote cell proliferation during five to nine days of culture, compared to bFGF-unloaded microspheres. This reveals that the bFGF released from SF microspheres retained obvious bioactivity to stimulate cell growth. Such microspheres sustainably releasing bioactive bFGF might be applied to massive cell culture and tissue engineering as a matrix directly, or after being combined with three-dimensional scaffolds.

Controlled basic fibroblast growth factor release device made of poly(ethyleneglycol) dimethacrylates for creating a subcutaneous neovascular bed for cell transplantation.

Subcutaneous space is a potential site for the transplantation of cells such as islets for treatment of type 1 diabetes. To enhance engraftment, an optimal space for the growth of the transplanted cells is needed along with neovascularization. In this study, we developed a device using a photocurable resin, poly(ethyleneglycol) dimethacrylates (PEGDM), for controlled release of basic fibroblast growth factor (bFGF) to create a subcutaneous neovascular bed in rats. The device consists of a disk-shaped capsule with micropores and is composed of tri(ethyleneglycol) dimethacrylate (TEGDM) and a drug formulation of PEGDM. The release rate was tuned by changing the number of pores and the composition of water and PEGDM in the drug formulation. bFGF released from devices incubated in phosphate-buffered saline (PBS) enhanced the growth of fibroblasts, indicating bioactivity of bFGF after release. Histological evaluation showed a significant increase in the extent of vasculature that was dependent on the amount of bFGF loaded into the device. A perfusion study using fluorescein isothiocyanate dextran 2000 kDa showed linear and capillary staining patterns, indicating potent functional vasculature. In conclusion, the controlled bFGF releasing device could provide a neovascular bed with the required vascularization in the subcutaneous space. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3017-3024, 2017.

Inkjet-based multilayered growth factor-releasing nanofilms for enhancing proliferation of mesenchymal stem cells in vitro

We report the preparation and characterization of inkjet-based basic fibroblast growth factor (bFGF)-containing nanofilms on a flexible PET substrate. bFGF and heparin (HEP) were assembled by inkjet-based layer-by-layer (LbL) assembly driven by electrostatic interactions. The bFGF/HEP nano-assembly surface coatings were formed via alternating printing adsorption of positively charged bFGF and negatively charged HEP; the process was monitored by UV–vis spectroscopy and quartz crystal microbalance. The bFGF release profile could be controlled by altering the number of layers of printed LbL films. Mesenchymal stem cells, which are capable of extended proliferation in vitro, require a continuous supply of bFGF for proliferation. However, enhancing mesenchymal stem cell proliferation by continuous supplying bFGF is difficult, even with medium replacement, because of the instability of bFGF. Here, we established a novel system for releasing bioactive bFGF from a modified surface by using an inkjet-based nanofilm fabrication method.

The addition of the collagen binding domain of fibronectin potentiates the biochemical availability of basic fibroblast growth factor for the culture of induced pluripotent stem cells

Event Abstract Back to Event The addition of the collagen binding domain of fibronectin potentiates the biochemical availability of basic fibroblast growth factor for the culture of induced pluripotent stem cells Cyril Addi1, 2, Frederic Murschel1, Benoit Liberelle1, Nesrine Riahi1, Olivier Feraud2 and Gregory De Crescenzo1 1 Ecole Polytechnique de Montreal, Department of Chemical Engineering, Canada 2 Université Paris-Sud, Hôpital Paul Brousse, INSERM U935, ESTeam Paris-Sud, France Introduction: The culture of various cell types including induced pluripotent stem cells (iPSC), requires the use of growth factors such as bFGF (basic Fibroblast Growth Factor)[1],[2]. These factors are generally added to the medium in their diffusive form and have a very short half-life[3]: they disappear quickly from the culture medium and must be replaced at high cost. We here explore the stable and oriented tethering of bFGF on a cell culture substrate, namely gelatin (denatured collagen). Our strategy relies on the expression of two fusion proteins being labelled with two distinct peptides (the E and Kcoils) that bind to each other with great affinity and specificity[4]. That is, a chimera corresponding to bFGF fused to the Ecoil peptide on the one hand, and the collagen-binding domain (CBD) of fibronectin fused to the Kcoil peptide on the other hand (Fig. 1). Materials and Methods: The Ecoil-bFGF fusion protein was produced in E. coli and purified by Ni-NTA affinity chromatography followed by TEV protease cleavage and ion exchange chromatography. The CBD-Kcoil fusion protein was produced in E. coli and purified by solubilizing inclusions bodies in an 8 M urea buffer, dialyzing against PBS, and then followed by a Ni-NTA affinity chromatography. The ability of Ecoil-bFGF protein to bind to Kcoil was assessed by surface plasmon resonance (SPR), using a Kcoil-coated chip while its ability to bind to its cognate receptor was tested by subsequent injections of a recombinant chimera of the FGF Receptor (FGFR1-Fc). Finally, the suitability of the proposed approach for cell culture will be soon assessed by cultivating iPSCs on a gelatin-coated substrate to be functionalized with CBD-Kcoil/Ecoil-bFGF. Results and Discussion: Both fusion proteins were produced and purified with high purity. The yield of the purification of Ecoil-bFGF was assessed by ELISA and reached 1.1 mg per liter of bacteria. On the one hand, SPR analyses (Fig. 2) demonstrated that Ecoil-bFGF specifically bound to a Kcoil-coated chip, and subsequently interacted with FGFR1-Fc with high affinity (KD = 1.8 nM), in good agreement with the literature[5]. On the other hand, the bioactivity of CBD-Kcoil was assessed via an Enzyme-Linked Immunosorbent Assay (ELISA) against bFGF: gelatin-coated substrates were incubated with CBD-Kcoil then Ecoil-bFGF, and the formation of the gelatin/CBD-Kcoil/Ecoil-bFGF complex was demonstrated by the specific recruitment of anti-bFGF antibodies. Altogether, the data indicated that our novel strategy enabled the specific capture of bioactive bFGF via collagen/CBD and coiled-coil interactions. Conclusion: Providing iPSCs with bioactive bFGF in a non-diffusible form may significantly decrease expenses related to cell culture. Furthermore, the modular approach we undertook may make this strategy amenable to the development of more complex scaffolds combining several growth factors (bFGF, EGF, VEGF ...), and thus pave the way to the development of novel approaches in the field of tissue engineering. This project was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC), CREATE program - MEDITIS scholarship.

Functional and pathological improvements of the hearts in diabetes model by the combined therapy of bFGF-loaded nanoparticles with ultrasound-targeted microbubble destruction

Diabetic cardiomyopathy (DCM) is the leading cause of morbidity and mortality among the diabetic patients and currently there is no effective means to reverse its pathological progress. Basic fibroblast growth factor (bFGF) has shown promise as a molecular therapy for DCM, but its delivery is inefficient and non-specific. In the present study, a therapy combining nanoparticle (NP) carrier and ultrasound-targeted microbubble destruction (UTMD) was reported the first time for bFGF delivery to the heart of diabetic rats. bFGF-loaded NP (bFGF-NP) were prepared with Poloxamer 188-grafted heparin copolymer using water-in-water technique, and the morphology, encapsulation efficiency, and bioactivity of bFGF-NP were studied. The cellular uptake and cytotoxicity of bFGF-NP were evaluated with primary cultures of the left ventricular (LV) cardiomyocytes in vitro. Therapeutic effects of bFGF-NP/UTMD on the heart of DCM rats were studied by measuring LV systolic and diastolic functions, hemodynamic characteristics and indicators of cardiac remodeling including myocardial collagen volume fraction and capillary density. Results demonstrated that bFGF-NP showed good round morphology, efficient bFGF encapsulation and stable bioactivity of bFGF in vitro. bFGF-NP/UTMD combined treatment significantly enhanced the efficiency of bFGF cellular uptake (P<0.05) without obvious cytotoxicity. Significant improvements (P<0.05) in both cardiac functions and tissue morphology in the DCM rats were observed in bFGF-NP/UTMD group. These were not achievable using free bFGF, bFGF-NP or UTMD treatment alone. Our results show that combining a non-viral vector with UTMD technique is an effective strategy to deliver bFGF to the heart, and the resulting growth factor therapy has demonstrated potential to reverse the progress of DCM by restoring the cardiac functions and even the structure of damaged cardiac tissues.

Biological augmentation of rotator cuff repair using bFGF-loaded electrospun poly(lactide-co-glycolide) fibrous membranes.

Clinically, rotator cuff tear (RCT) is among the most common shoulder pathologies. Despite significant advances in surgical techniques, the re-tear rate after rotator cuff (RC) repair remains high. Insufficient healing capacity is likely the main factor for reconstruction failure. This study reports on a basic fibroblast growth factor (bFGF)-loaded electrospun poly(lactide-co-glycolide) (PLGA) fibrous membrane for repairing RCT. Implantable biodegradable bFGF–PLGA fibrous membranes were successfully fabricated using emulsion electrospinning technology and then characterized and evaluated with in vitro and in vivo cell proliferation assays and repairs of rat chronic RCTs. Emulsion electrospinning fabricated ultrafine fibers with a core-sheath structure which secured the bioactivity of bFGF in a sustained manner for 3 weeks. Histological observations showed that electrospun fibrous membranes have excellent biocompatibility and biodegradability. At 2, 4, and 8 weeks after in vivo RCT repair surgery, electrospun fibrous membranes significantly increased the area of glycosaminoglycan staining at the tendon–bone interface compared with the control group, and bFGF–PLGA significantly improved collagen organization, as measured by birefringence under polarized light at the healing enthesis compared with the control and PLGA groups. Biomechanical testing showed that the electrospun fibrous membrane groups had a greater ultimate load-to-failure and stiffness than the control group at 4 and 8 weeks. The bFGF–PLGA membranes had the highest ultimate load-to-failure, stiffness, and stress of the healing enthesis, and their superiority compared to PLGA alone was significant. These results demonstrated that electrospun fibrous membranes aid in cell attachment and proliferation, as well as accelerating tendon–bone remodeling, and bFGF-loaded PLGA fibrous membranes have a more pronounced effect on tendon–bone healing. Therefore, augmentation using bFGF–PLGA electrospun fibrous membranes is a promising treatment for RCT.

Intramyocardial Injection of a Synthetic Hydrogel with Delivery of bFGF and IGF1 in a Rat Model of Ischemic Cardiomyopathy

It is increasingly appreciated that the properties of a biomaterial used in intramyocardial injection therapy influence the outcomes of infarcted hearts that are treated. In this report the extended in vivo efficacy of a thermally responsive material that can deliver dual growth factors while providing a slow degradation time and high mechanical stiffness is examined. Copolymers consisting of N-isopropylacrylamide, 2-hydroxyethyl methacrylate, and degradable methacrylate polylactide were synthesized. The release of bioactive basic fibroblast growth factor (bFGF) and insulin-like growth factor 1 (IGF1) from the gel and loaded poly(lactide-co-glycolide) microparticles was assessed. Hydrogel with or without loaded growth factors was injected into 2 week-old infarcts in Lewis rats and animals were followed for 16 weeks. The hydrogel released bioactive bFGF and IGF1 as shown by mitogenic effects on rat smooth muscle cells in vitro. Cardiac function and geometry were improved for 16 weeks after hydrogel injection compared to saline injection. Despite demonstrating that left ventricular levels of bFGF and IGF1 were elevated for two weeks after injection of growth factor loaded gels, both functional and histological assessment showed no added benefit to inclusion of these proteins. This result points to the complexity of designing appropriate materials for this application and suggests that the nature of the material alone, without exogenous growth factors, has a direct ability to influence cardiac remodeling.

BFGF-grafted electrospun fibrous scaffolds via poly(dopamine) for skin wound healing.

Electrospun fibrous membranes coated with basic fibroblast growth factor (bFGF) are effective medical devices to promote wound healing. However, the current strategies of adding bFGF generally cause degradation of electrospun materials or damage to the bioactivity of the biomolecules. Here, we have developed a simple strategy for surface bFGF-functionalization of electrospun fibers in an aqueous solution, which maintained original fiber properties and growth factor bioactivity. Polydopamine (PDA) forming the mussel foot protein was chosen as an adhesive polymeric bridge-layer between substrate poly(lactide-co-glycolide) (PLGA) fibers and bFGF. The bFGF-grafted PDA was analyzed using scanning electron microscopy, water contact angle measurements, and X-ray photoelectron spectroscopy. Improved hydrophilicity together with a stable fibrous structure and biodegradable fibrous matrix suggested that the PLGA/PDA-bFGF electrospun fibrous scaffolds have great potential for promoting wound healing. In vitro experiments showed that the bFGF-grafted PLGA electrospun fibrous scaffolds have highly enhanced adhesion, viability, and proliferation of human dermal fibroblasts. In vivo results showed that such scaffolds shortened wound healing time, accelerated epithelialization and promoted skin remodeling. Therefore, this PDA modification method can be a useful tool to graft biomolecules onto polymeric electrospun fibrous scaffolds which are potential scaffold candidates for repairing skin tissue.

Tendon healing and anti-adhesion properties of electrospun fibrous membranes containing bFGF loaded nanoparticles

The ideal scaffolds should contain growth factors and thus can regulate cellular behaviors and tissue assembly. Electrospun fibrous membranes are widely-used scaffolds but growth factors are highly susceptible to losing their bioactivity during the electrospinning process. In this study, pre-formulated dextran glassy nanoparticles (DGNs) loaded with basic fibroblast growth factor (bFGF) were electrospun into a poly-l-lactic acid (PLLA) copolymer fiber to secure the bioactivity of bFGF in a sustained manner and then bioactivity retention was certificated by promoting cell proliferation and tendon healing. Meanwhile, the barrier effect of electrospun membrane was evaluated for clinic concern. In the in vitro release study, the protein encapsulation efficiency of the bFGF/DGNs-PLLA membrane reached 48.71 ± 13.53%, with a release kinetic of nearly 30 days. The enhanced cell proliferation and intrinsic tendon healing show that the bFGF/DGNs-loaded PLLA fibrous membrane can release bFGF sustainably and secure the bioactivity of bFGF better than the emulsion electrospun bFGF-loaded PLLA and PLLA fibrous membranes. Meanwhile, the anti-adhesion effect of electrospun membrane as barrier was fortunately combined as clinic concern.

Creating 3D Angiogenic Growth Factor Gradients in Fibrous Constructs to Guide Fast Angiogenesis

Fast angiogenesis in 3D fibrous constructs that mimic the morphology of the extracellular matrix remains challenging due to limited porosity in the densely packed constructs. We investigated whether mimicking the in vivo chemotaxis microenvironment for native blood vessel formation would stimulate angiogenesis in the fibrous constructs. The chemotaxis microenvironment was created by introducing 3D angiogenic growth factor gradients into the constructs. We have developed a technique that can quickly fabricate (∼40 min) such 3D gradients by simultaneously electrospinning polycaprolactone (PCL) fibers, encapsulating gradient amount of bFGF (stabilized by heparin) into poly(lactide-co-glycolide) (PLGA) microspheres, and electrospraying the microspheres into PCL fibers. Gradient formation was confirmed by fluorescence microscopy. Gradients with different steepnesses were obtained by modulating the initial concentration of the bFGF solution. All of the constructs were able to sustainedly release bioactive bFGF over a 28 day period. The release kinetics was dependent on the bFGF loading and steepness of the gradient. In vitro cell migration study demonstrated that bFGF gradients significantly increased the depth of cell migration. To assess the efficacy of bFGF gradients in inducing angiogenesis, we implanted constructs subcutaneously using mouse model. bFGF gradients significantly promoted cell penetration into the constructs. After 10 days of implantation, a high density of mature blood vessels (positive to both CD31 and α-SMA) were formed in the constructs. Vessel density was increased with the increase in steepness of the bFGF gradient. These gradient constructs may have potential to engineer vascularized tissues for various applications.

Bioactive nanofibers for fibroblastic differentiation of mesenchymal precursor cells for ligament/tendon tissue engineering applications

Mesenchymal stem cells and precursor cells are ideal candidates for tendon and ligament tissue engineering; however, for the stem cell-based approach to succeed, these cells would be required to proliferate and differentiate into tendon/ligament fibroblasts on the tissue engineering scaffold. Among the various fiber-based scaffolds that have been used in tendon/ligament tissue engineering, hybrid fibrous scaffolds comprising both microfibers and nanofibers have been recently shown to be particularly promising. With the nanofibrous coating presenting a biomimetic surface, the scaffolds can also potentially mimic the natural extracellular matrix in function by acting as a depot for sustained release of growth factors. In this study, we demonstrate that basic fibroblast growth factor (bFGF) could be successfully incorporated, randomly dispersed within blend-electrospun nanofibers and released in a bioactive form over 1 week. The released bioactive bFGF activated tyrosine phosphorylation signaling within seeded BMSCs. The bFGF-releasing nanofibrous scaffolds facilitated BMSC proliferation, upregulated gene expression of tendon/ligament-specific ECM proteins, increased production and deposition of collagen and tenascin-C, reduced multipotency of the BMSCs and induced tendon/ligament-like fibroblastic differentiation, indicating their potential in tendon/ligament tissue engineering applications.

Development of inhalable dry powder formulation of basic fibroblast growth factor

Basic fibroblast growth factor (bFGF) is a promising agent for therapy of asthma or chronic obstructive pulmonary disease. We aim to develop an inhalable powder formulation of bFGF, which may provide a safe, effective, and convenient way of delivering bFGF to the disease-ridden lungs. Development of a bFGF dry powder formulation is constrained by the poor stability of bFGF and the uncertainty in compatibility of the protein with carrier excipients. With these constraints in mind, we prepared dry powders containing bFGF in combinations of albumin, phospholipid, lactose, and/or leucine, by spray drying, and evaluated the aerodynamic properties of the powders and the stability of bFGF loaded in the powders. While an ethanolic solution of phospholipid, albumin, and lactose produced dispersible powder, bFGF was unstable in ethanol. The stability of bFGF was preserved when spray-dried with lactose in an aqueous solution. Leucine was required to obtain dry powder with good dispersibility; however, increase in the leucine content more than 50% (w/w) negatively influenced the bFGF stability with no additional benefit to the aerodynamic properties of the powders. Dry powders containing 20% (w/w) leucine provided desirable aerodynamic properties (fine particle fraction of 25.2 ± 5.4% and mass median aerodynamic diameter of 4.7 ± 0.9 μm) and 98.1 ± 7% recovery of bioactive bFGF. This result warrants further investigation of the biological activity of the inhaled bFGF in a disease model.