Half-life modeling of basic fibroblast growth factor released from growth factor-eluting polyelectrolyte multilayers.

Growth factor-eluting polymer systems have been widely reported to improve cell and tissue outcomes; however, measurements of actual growth factor concentration in cell culture conditions are limited. The problem is compounded by a lack of knowledge of growth factor half-lives, which impedes efforts to determine real-time growth factor concentrations. In this work, the half-life of basic fibroblast growth factor (FGF2) was determined using enzyme linked immunosorbent assay (ELISA). FGF2 release from polyelectrolyte multilayers (PEMs) was measured and the data was fit to a simple degradation model, allowing for the determination of FGF2 concentrations between 2 and 4 days of culture time. After the first hour, the FGF2 concentration for PEMs assembled at pH = 4 ranged from 2.67 ng/mL to 5.76 ng/mL, while for PEMs assembled at pH = 5, the concentration ranged from 0.62 ng/mL to 2.12 ng/mL. CRL-2352 fibroblasts were cultured on PEMs assembled at pH = 4 and pH = 5. After 2 days, the FGF2-eluting PEM conditions showed improved cell count and spreading. After 4 days, only the pH = 4 assembly condition had higher cells counts, while the PEM assembled at pH = 5 and PEM with no FGF2 showed increased spreading. Overall, the half-life model and cell culture study provide optimal concentration ranges for fibroblast proliferation and a framework for understanding how temporal FGF2 concentration may affect other cell types.

Hydrophobically-modified gelatin hydrogel as a carrier for charged hydrophilic drugs and hydrophobic drugs.

Gelatin molecules have been chemically crosslinked using potentially cytotoxic reagents to prepare stable hydrogels. Hydrophobic interaction is a means of forming physical crosslinks that is a good candidate for enhancing the stability of gelatin hydrogels without using cytotoxic chemicals. In this study, we proposed a new method to fabricate hydrogels from hydrophobically-modified gelatin (HMG) with high content of hydrophobic segments. HMG was first dissolved in dimethyl sulfoxide and poured into a vial with the desired shape. After the solution was freeze-dried, the solid construct was hydrated. The HMG hydrogel containing basic fibroblast growth factor promoted angiogenesis in vivo, indicating that the positively charged hydrophilic growth factor formed an electrostatic complex with negatively charged HMG hydrogel and was gradually released in vivo with the degradation of the hydrogel. In addition, we showed that the hydrophobic segments of HMG enhanced the adsorption of fluorescein sodium, a model for hydrophobic therapeutic agents, to the hydrogel through hydrophobic interaction. Furthermore, in vitro experiments indicated that the hydrophobic agents would be released from the hydrogel in a controlled manner in vivo. These results show that the HMG hydrogel has significant potential as a carrier for both charged hydrophilic drugs and hydrophobic drugs.

Structure-Based Site-Specific PEGylation of Fibroblast Growth Factor 2 Facilitates Rational Selection of Conjugate Sites.

Polyethylene glycol modification (PEGylation) can enhance the pharmacokinetic properties of therapeutic proteins by the attachment of polyethylene glycol (PEG) to the surface of a protein to shield the protein surface from proteolytic degradation and limit aggregation. However, current PEGylation strategies often reduce biological activity, potentially as a result of steric hindrance of PEG. Overall, there are no structure-based guidelines for selection of conjugate sites that retain optimal biological activity with improved pharmacokinetic properties. In this study, site-specific PEGylation based on the FGF2-FGFR1-heparin complex structure is performed. The effects of the conjugate sites on protein function are investigated by measuring the receptor/heparin binding affinities of the modified proteins and performing assays to measure cell-based bio-activity and in vivo stability. Comprehensive analysis of these data demonstrates that PEGylation of FGF2 that avoids the binding sites for fibroblast growth factor receptor 1 (FGFR1) and heparin provides optimal pharmacokinetic enhancement with minimal losses to biological activity. Animal experiments demonstrate that PEGylated FGF2 exhibits greater efficacy in protecting against traumatic brain injury-induced brain damage and neurological functions than the non-modified FGF2. This rational structure-based PEGylation strategy for protein modification is expected to have a major impact in the area of protein-based therapeutics.

In vivo characterization of RC28-E, a fusion protein targeting VEGF and bFGF: Pharmacokinetics and ocular distribution in primates.

Administration of RC28-E, a VEGF/bFGF dual decoy receptor (IgG1 Fc-fusion protein), have shown relative therapeutic value in ocular in vivo models, including laser-induced choroidal neovascularization (CNV) in monkeys and streptozotocin (STZ)-induced diabetic retinopathy (DR) in rats. In the present study, we have elucidated the pharmacokinetics profiles of RC28-E at the systemic, vitreous and aqueous humor after administration in a primate model (Macaca fascicularis). Moreover, here we tease out the ocular tissue distribution of RC28-E after intravitreal administration, and we also determine the systemic bioavailability after both intravitreal and intravenous administration. Our results show that RC28-E is rapidly and well-distributed into ocular tissues after intravitreal administration. Drug exposure in choroid and retina was approximately one-quarter and one-twelfth of that in vitreous humor, while its half-life in vitreous and aqueous humor were well-sustained (3.3 and 3.0 days). Remarkably, RC28-E could cross the blood-ocular barrier, and the systemic bioavailability of RC28-E was ~25%. No drug accumulation after multiple administration was noticed, but low titers of antibody produce against RC28-E were detected. Overall, RC28-E exhibited high clinical value due to adequate pharmacokinetic profiling, safety and efficacy.

LED-Based Photoacoustic Imaging for Monitoring Angiogenesis in Fibrin Scaffolds.

IMPACT STATEMENT: Noninvasive imaging techniques provide insight into physiology that is complementary to tissue morphology obtained by invasive histology. Optical imaging techniques, such as laser speckle contrast analysis, are used in vivo to longitudinally evaluate vascularization. Despite their high spatial resolution, these techniques have a limited imaging depth. In this study, we demonstrate how a dual LED-based photoacoustic (PA) and ultrasound system can delineate changes in perfusion at depth within scaffolds containing basic fibroblast growth factor. Perfusion changes detected by PA corroborated with vessel density. PA imaging could be a noninvasive and sensitive method for evaluating vascularization at depth in larger constructs.

Injectable basic fibroblast growth factor-loaded alginate/hyaluronic acid hydrogel for rejuvenation of geriatric larynx.

Increase in the geriatric population has led to an increase in the number of elderly patients with laryngeal atrophy and dysfunction. Symptoms of voice change, dysphagia, and aspiration pneumonia negatively influence patient's health status, quality of life, and life span. Injection laryngoplasty used to treat laryngeal dysfunctions does not recover intrinsic functions of the larynx. Thus, we fabricated an injectable basic fibroblast growth factor (bFGF)-loaded alginate (ALG)/hyaluronic acid (HA) hydrogel for inducing rejuvenation of geriatric laryngeal muscles. Optimal in situ-forming bFGF-loaded ALG/HA hydrogel for injection laryngoplasty was prepared and the release profile of bFGF was analyzed. For in vivo analysis, the bFGF-loaded ALG/HA hydrogel was injected into the laryngeal muscles of 18-month-old Sprague-Dawley rats. The rejuvenation efficacy of bFGF-loaded ALG/HA hydrogel in geriatric laryngeal muscle tissues 4- and 12-weeks post-injection was evaluated by quantitative polymerase chain reaction (qPCR), histology, immune-fluorescence staining and functionality analysis. The bFGF-loaded ALG/HA hydrogel induced an increase in the expression of myogenic regulatory factor-related genes, hypertrophy of muscle fiber, proliferation of muscle satellite cells, and angiogenesis and decreased interstitial fibrosis. Administration of the bFGF-loaded ALG/HA hydrogel caused successful glottal gap closure. Thus, the bFGF-loaded ALG/HA hydrogel could be a promising candidate for laryngoplasty aimed at rejuvenating geriatric larynx. STATEMENT OF SIGNIFICANCE: In this manuscript, optimal in situ-forming bFGF-loaded ALG/HA hydrogel for injection laryngoplasty was prepared and the release profile of bFGF was analyzed. Herein, we introduced the materials and methods of injection laryngoplasty for geriatric rat experiment. In addition, we studied effects of bFGF-loaded ALG/HA hydrogel on the therapeutic rejuvenation of geriatric rat larynx. The bFGF-loaded ALG/HA hydrogel induced an increase in the expression of myogenic regulatory factor-related genes, hypertrophy of muscle fiber, proliferation of muscle satellite cells, and angiogenesis and decreased interstitial fibrosis. Furthermore, our functional analysis through the high-speed camera setup demonstrated that the administration of the bFGF-loaded ALG/HA hydrogel induced successful glottal gap closure. Thus, the bFGF-loaded ALG/HA hydrogel could be a promising candidate for injection laryngoplasty with therapeutic effects.

Collagen/Gelatin Sponges (CGSs) Provide Both Protection and Release of bFGF: An In Vitro Study.

It has been reported that collagen/gelatin sponges (CGSs) are able to sustain the release of basic fibroblast growth factor (bFGF) for approximately 10 days via the formation of ion complexes between bFGF and gelatin. CGSs impregnated with bFGF have been proven to promote dermis-like tissue formation in various in vivo studies and clinical trials. However, the bioactivities of bFGF released from CGSs have not been explored in vitro. In this study, we explored the ability of CGS impregnated with bFGF, stored at 37°C for up to 14 days, to promote fibroblast proliferation and the sustained release of bFGF. We analyzed the cellular viability and proliferation in 2D and in 3D cell cultures, by a CCK-8 assay. Furthermore, in order to characterize the morphological alteration of fibroblasts, we studied 3D cultures by microscopy with a scanning electron microscope (SEM) and a confocal microscope. Our analyses revealed that the fibroblasts were elongated and flanked each other. They infiltrated and migrated inside the CGSs and were oriented along the CGS structure. Thus, these data prove that CGSs protect and sustain the efficient release of growth factor for more than 7 days.

Prevention of tooth extraction-triggered bisphosphonate-related osteonecrosis of the jaws with basic fibroblast growth factor: An experimental study in rats.

Osteonecrosis of the jaw induced by administration of bisphosphonates (BPs), BP-related osteonecrosis (BRONJ), typically develops after tooth extraction and is medically challenging. As BPs inhibit oral mucosal cell growth, we hypothesized that suppression of the wound healing-inhibiting effects could prevent BRONJ onset after tooth extraction. Since basic fibroblast growth factor (bFGF) promotes wound healing, but has a short half-life, we examined whether the initiation of BRONJ could be prevented by applying a bFGF-containing gelatin hydrogel over the extraction sockets of BRONJ model rats. Forty-three rats, received two intravenous injections of zoledronic acid 60 µg/kg, once per week for a period of 2 weeks, underwent extraction of a unilateral lower first molar. The rats here were randomly assigned to the bFGF group (n = 15 rats, gelatin hydrogel sheets with incorporated bFGF applied over the sockets); the phosphate-buffered saline (PBS) group (n = 14 rats, gelatin hydrogel sheets without bFGF applied over the sockets); or the control group (n = 14 rats, nothing applied over the sockets). One rat in the bFGF group was sacrificed immediately after tooth extraction. Twenty-one rats were sacrificed at 3 weeks, and the remaining 21 rats were sacrificed at 8 weeks after tooth extractions. The harvested mandibles were analyzed using micro-computed tomography and sections were evaluated qualitatively for mucosal disruption and osteonecrosis. The incidence of osteonecrosis at 8 weeks after tooth extraction was 0% in the bFGF group, 100% in the PBS group, and 85.7% in the control group. The frequency of complete coverage of the extraction socket by mucosal tissue was significantly greater in the bFGF group than in the other groups. These results suggest that application of bFGF in the extraction socket promoted socket healing, which prevented BRONJ development. The growth-stimulating effects of bFGF may have offset the inhibition of wound healing by BP.

Development of a stent capable of the controlled release of basic fibroblast growth factor and argatroban to treat cerebral aneurysms: In vitro experiment and evaluation in a rabbit aneurysm model.

An ideal stent to treat cerebral aneurysms should have an antithrombotic effect on the inner stent blood-facing side and a tissue organization effect on the outer aneurysmal side of the stent. The objective of this study is to evaluate the feasibility of a drug containing stent in the in vivo treatment of cerebral aneurysms. Argatroban, an antithrombotic drug, is encapsulated in biodegradable poly (d,l-lactide-co-glycolide) (PLGA) microspheres for the controlled release with an in vitro study conducted to evaluate the drug release and anticoagulation behavior of released drug. Basic fibroblast growth factor (bFGF), an organization drug, is released from gelatin hydrogels. The stents are coated with gelatin hydrogels incorporating bFGF and PLGA microspheres containing argatroban, and applied to the carotid artery aneurysm of an elastase-induced rabbit model. Most of the aneurysm cavity is occupied by loose connective tissues in the group treated with drug-coated stents, whereas extensive massive hematomas are observed in the group treated with drug-free stents. The occurrence rate of in-stent thrombus is small in the drug-coated stents. The stent incorporating bFGF and PLGA microspheres containing argatroban is an effective device for cerebral aneurysm treatment. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2185-2194, 2019.

The use of heparin, bFGF, and VEGF 145 grafted acellular vascular scaffold in small diameter vascular graft.

We aim to test the application of heparin, bFGF, and VEGF 145 grafted acellular vascular scaffold in small diameter vascular graft. The amount of bFGF and VEGF 145 were determined by ELISA. Femoral artery transplantation was performed. Mechanical strength of acellular vascular scaffolds was determined. Angiography was performed for blood vessel patency. Factor VIII and α2-actin expression was detected by immunohistochemistry. bFGF and VEGF 145 had stable release at 60 and 70 days in vitro, and the release rate of VEGF 145 was slightly slower than that of bFGF. After transplantation, 9 months of the vascular patency rate was 100% at 1, 3, and 9 months, and, was up to 90% at 18 months, while the patency rate in group with grafted heparin only at 1-month was 60%, at 3-month was 40%, at 9-month was 15%, and at 18-month was 10%. The blood vessels taken after 18 months had no significant difference in the mechanical properties between the transplanted and the natural vessels. Positive expression of factor VIII and α2-actin was observed. The heparinized and bFGF and VEGF 145 grafted allogeneic vascular acellular scaffolds are preliminarily obtained, which show good biocompatibility and patency and are of great importance for small diameter vascular graft. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 00B: 000-000, 2018. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 672-679, 2019.

Modelling optimal delivery of bFGF to chronic wounds using ODEs.

In this paper, we present an ordinary differential equation model depicting the interactions of basic fibroblast growth factor (bFGF) and its binding agents in a chronic wound. The delivery of bFGF was treated as a control variable and is coupled to an objective functional. By optimising the objective functional with respect to the control, predictions for optimal delivery rates of bFGF are proposed. The optimal control is then validated by comparing the cost of the objective functional for the optimal delivery rate and several alternative delivery rates. This paper addresses two objectives of effective drug delivery to chronic wounds. The first is to provide insight for the priority of delivering bFGF: to minimise the quantity of bFGF, or to optimise the distribution of bound bFGF. For effective concentrations of bound bFGF, the optimisation of bound bFGF must be prioritised over the minimisation of bFGF delivered. The second objective is to comment on the effect of the proteolytic environment within the wound, with the concentration of bound bFGF starting to decrease late in the treatment period for highly proteolytic environments. This will lead to long term complications with wound closure after the treatment has been completed. Also, it was found that for highly proteolytic environments, the cost of delivering bFGF increased. The need for optimal drug delivery is made apparent by the burden of chronic wounds on the medical industry across the developed world.

Bioanalytical method development and validation for determination of fibroblast growth factor peptide and its application to pharmacokinetic studies.

Fibroblast growth factor peptide (FGF-P) is a polypeptide analog of FGF-2 that could be a potential mitigation and treatment agent for radiation syndromes. Prior to conducting preclinical pharmacokinetics, we developed and validated the LC-MS/MS bioanalytical method for determination of FGF-P in rat plasma for the first time. FGF-P was extracted from rat plasma using the protein precipitation technique followed liquid-liquid extraction using dichloromethane as a solvent. The mobile phases consisted of two components: (a) 0.1% formic acid in water; and (b) acetonitrile: 0.1% formic acid in water (95:5) under gradient elution. The validated method was also successfully applied to a pharmacokinetic study of FGF-P (10 mg/kg, intravenous) in Wistar rats. The method proved to be specific, accurate, precise, and linear over the concentration range of 2-500 ng/mL with coefficient of determination greater than 0.99 in all validation batches. The within-run and between-run accuracy was 87.97-115.00% with a precision of less than 14%. The mean recoveries ranged from 88.14% to 101.73%. The stability of the compound in plasma samples was proven under various storage conditions. After intravenous administration of FGF-P (10 mg/kg) the C0 was 70.4 µg/mL and the AUC was 86.2 µg*min/mL.

Fabrication and Characterization of Core-Shell Nanofibers Using a Next-Generation Airbrush for Biomedical Applications.

The core-shell polymeric nanofiber, owing to its better controlled release of embedded or encapsulated drugs in contrast with the single-compartment nanofibers, has been extensively studied for biomedical applications such as tissue engineering and wound healing. Electrospinning with co-axial needles is the dominant technique to fabricate nanofiber mat, however, associated with potential limitations such as high voltage requirement, costly equipment, slow deposition rate, required trained personal, not suitable in situ fabrication, and direct deposition of core-shell nanofibers on the wound at patient bedside. To address the above limitations, the work aims to introduce a novel co-axial airbrushing method to fabricate core-shell nanofibers using a simple setup and low-cost equipment, yet having a unique ability for fabrication at patient bedside and direct deposition on wound bed. Air-brush with a coaxial needle is designed to flow two different polymers solution with model biomolecules through core [PEO (polyethylene oxide)/poly-dl-lactide/PCL (polycaprolactone)] and shell (PCL/PEO) needle for the fabrication of the model core-shell nanofiber. Various processing parameters such as flow rate, air pressure, working distance, and concentration of polymer solution which affect the morphology of core-shell nanofibers were studied and found to have a prominent effect. The PCL-PEO nanofiber possesses a defined shell and core structure, tunable sustained release behavior of model proteins (bovine serum albumin and basic fibroblast growth factor; bFGF), and improved mechanical strength. In vitro interaction of human bone marrow-derived mesenchymal stem cells with core-shell fibers demonstrated the cytocompatibility and proliferative and differentiative (for bFGF loaded) properties of the core-shell nanofiber mat. Co-axial airbrushing can be used as a superior less-expensive technique for the fabrication of biomolecules/drug encapsulated core-shell fibers scaffold at patient bedside, which can mimic complex in vivo environment and could modulate cells behavior close to their in vivo condition for tissue regeneration and wound healing.

Collagen/Heparin Bi-Affinity Multilayer Modified Collagen Scaffolds for Controlled bFGF Release to Improve Angiogenesis In Vivo.

Basic fibroblast growth factor (bFGF) is an important protein for wound healing and angiogenesis in tissue engineering, but the lack of a viable delivery system hampers its clinical application. This study aims to maintain the long-term controlled release of bFGF by utilizing a collagen/heparin bi-affinity multilayer delivery system (CHBMDS), which is fabricated by the alternate deposition of negatively charged heparin, positively charged collagen, and CBD-bFGF (a collagen-binding domain [CBD] was fused into the native bFGF) via specific or electrostatic interaction. The results show that CHBMDS not only support localized and prolonged release of CBD-bFGF(over 35 days) but also lead to enhanced angiogenesis (higher density and larger diameter (≈70 µm) of newly formed blood vessels in subcutaneous tissue of SD rat after 5 weeks). This system could act as a versatile approach for bFGF delivery and further improve therapeutic efficacy for injured tissues.

Therapeutic Effect of Controlled Release of Dual Growth Factor Using Heparin-Pluronic Hydrogel/Gelatin-Poly (Ethylene Glycol)-Tyramine Hydrogel System in a Rat Model of Cavernous Nerve Injury.

The number of cases of erectile dysfunction (ED) caused after radical prostatectomy (RP) prostate cancer treatment is increasing steadily. Although various studies have been conducted for treatment of post-RP ED, there is still a need for more effective methods. A dual growth factor incorporated heparin-pluronic/gelatin-poly(ethylene glycol)-tyramine (HP/GPT) hydrogel, which consists of a basic fibroblast growth factor (bFGF)-loaded HP hydrogel and nerve growth factor (NGF)-loaded GPT hydrogel, can control dose and rate of growth factor release. In this study, we demonstrated that dual growth factor incorporated HP/GPT hydrogel could further improve erectile function in a rat model of bilateral cavernous nerve injury (BCNI). We showed that erectile function was decreased after BCNI, but it was further improved by treatment with a dual growth factor incorporated HP/GPT hydrogel compared with groups treated with single growth factor in a rat model of cavernous nerve injury. Also, we observed an increase in cyclic guanosine monophosphate (cGMP) levels in the dual growth factor group when compared with the groups treated with single growth factor. This effect was associated with greater upregulation of nitric oxide synthase and endothelial nitric oxide synthase expression in the penile tissue of the group treated with dual growth factor incorporated HP/GPT than in the other experimental groups. Apoptosis in the penile tissue treated with the dual growth factor incorporated HP/GPT hydrogel was lower than those treated singly with either bFGF or NGF incorporated GPT hydrogel. Both α-smooth muscle actin and CD31 expression increased in the group treated with dual growth factor incorporated HP/GPT hydrogel when compared to in the other experimental groups. Altogether, our results proved that the sequential and continuous release of growth factors from dual growth factor incorporated HP/GPT hydrogel prevented fibrosis and nerve damage induced by BCNI in the corpus cavernosum, and promoted the recovery of erectile function. Dual growth factor incorporated HP/GPT hydrogel may be a potent clinical application for the treatment of post-RP ED and could potentially be used various biomedical application in tissue regnerative medicine.

Sustained-release of FGF-2 from a hybrid hydrogel of heparin-poloxamer and decellular matrix promotes the neuroprotective effects of proteins after spinal injury.

INTRODUCTION: The short lifetime of protein-based therapies has largely limited their therapeutic efficacy in injured nervous post-spinal cord injury (post-SCI). METHODS: In this study, an affinity-based hydrogel delivery system provided sustained-release of proteins, thereby extending the efficacy of such therapies. The affinity-based hydrogel was constructed using a novel polymer, heparin-poloxamer (HP), as a temperature-sensitive bulk matrix and decellular spinal cord extracellular matrix (dscECM) as an affinity depot of drug. By tuning the concentration of HP in formulation, the cold ternary fibroblast growth factor-2 (FGF2)-dscECM-HP solution could rapidly gelatinize into a hydrogel at body temperature. Due to the strong affinity for FGF2, hybrid FGF2-dscECM-HP hydrogel enabled sustained-release of encapsulated FGF2 over an extended period in vitro. RESULTS: Compared to free FGF2, it was observed that both neuron functions and tissue morphology after SCI were clearly recovered in rats treated with FGF2-dscECM-HP hydrogel. Moreover, the expression of neurofilament protein and the density of axons were increased after treatment with hybrid FGF2-dscECM-HP. In addition, the neuroprotective effects of FGF2-dscECM-HP were related to inhibition of chronic endoplasmic reticulum stress-induced apoptosis. CONCLUSION: The results revealed that a hybrid hydrogel system may be a potential carrier to deliver macromolecular proteins to the injured site and enhance the therapeutic effects of proteins.

Targeted delivery of FGF2 to subchondral bone enhanced the repair of articular cartilage defect.

It is reported that growth factor (GF) is able to enhance the repair of articular cartilage (AC) defect, however underlying mechanisms of which are not fully elucidated yet. Moreover, the strategy for delivering GF needs to be optimized. The crosstalk between AC and subchondral bone (SB) play important role in the homeostasis and integrity of AC, therefore SB targeted delivery of GF represents one promising way to facilitate the repair of AC defect. In this study, we firstly investigated the effects and mechanism of FGF2 on surrounding SB and cartilage of detect defects in rabbits by using a homogenous collagen-based membranes. It was found that FGF2 had a modulating effect on the defect-surrounding SB via upregulation of bone morphogenetic protein (BMP)-2, BMP4 and SOX9 at the early stage. Low dose FGF2 improved the repair upon directly injected to SB. Inhibition of BMP signaling pathway compromised the beneficial effects of FGF2, which indicated the pivotal roles of BMP in the process. To facilitate SB targeted FGF2 delivery, a double-layered inhomogeneous collagen membrane was prepared and it induced increase of BMP2 and BMP4 in the synovial fluid, and subsequent successful repair of AC defect. Taken together, this targeted delivery of FGF2 to SB provides a promising strategy for AC repair owing to the relatively clear mechanism, less amount of it, and short duration of delivery. STATEMENT OF SIGNIFICANCE: Articular cartilage (AC) and subchondral bone (SB) form an integral functional unit. The homeostasis and integrity of AC depend on its crosstalk with the SB. However, the function of the SB in AC defect repair is not completely understood. The application of growth factors to promote the repair articular cartilage defect is a promising strategy, but still under the optimization. Our study demonstrate that SB plays important roles in the repair of AC defect. Particularly, SB is the effective target of fibroblast growth factor 2 (FGF2), and targeted delivery of FGF2 can modulate SB and thus significantly enhances the repair of AC defect. Therefore, targeted delivery of growth factor to SB is a novel promising strategy to improve the repair of AC defect.

Structure of a Multilayer Nanofilm To Increase the Encapsulation Efficiency of Basic Fibroblast Growth Factor.

In this study, we established the structure of a multilayer nanofilm that more efficiently encapsulates basic fibroblast growth factor (bFGF). First, a positively charged layer material was selected from biocompatible polymers such as collagen (Col), poly(beta-amino ester) (Poly2), and chitosan (Chi), while considering the film thickness. We then investigated the change in bFGF encapsulation efficiency when the multilayer structure was changed from a tetralayer to a trilayer. As a result, we obtained a highly improved bFGF encapsulation efficiency in the nanofilm using a positively charged layer formed by a blend of Col and Poly2 and a negatively charged poly(acrylic acid) (PAA) layer within a trilayered structure. In particular, we found that a significant amount of adsorbed bFGF was desorbed again during the film fabrication process of a tetralayered nanofilm. In the conventional nanofilm, bFGF was regarded as a polycation and formed a multilayer nanofilm that was composed of a tetralayered structure and was represented as (polycation/polyanion/bFGF/polyanion) n where n = number of repeated tetralayers. Here, we suggested that bFGF should not be considered a polycation, rather it should be considered as a small quantity of molecule that exists between the polyanion and polycation layers. In this case, the nanofilm is composed of repeating units of (polycation/polyanion/bFGF/polycation/polyanion), because the amount of adsorbed bFGF is considerably lower than that of other building blocks.

A pro-angiogenic degradable Mg-poly(lactic-co-glycolic acid) implant combined with rhbFGF in a rat limb ischemia model.

Site-specific controlled release of exogenous angiogenic growth factors, such as recombinant human basic fibroblast growth factor (rhbFGF), has become a promising approach to improve peripheral vascular disease. Here, we have developed an implant composed of spiral magnesium (Mg) and a coating made using poly(lactic-co-glycolic acid) (PLGA) with encapsulated rhbFGF (Mg-PLGA-rhbFGF). The encapsulated protein could release continually for 4weeks with well preserved bioactivity. We compared the angiogenic effect produced by Mg-PLGA-rhbFGF with that of a PLGA implant loaded with rhbFGF (PLGA-rhbFGF). The incorporation of Mg in the implant raised the microclimate pH in the polymer, which preserved the stability of rhbFGF. Mg-PLGA-rhbFGF exhibited advantages over PLGA-rhbFGF implant in terms of a cytocompatibility evaluation. An in vivo angiogenesis test further confirmed the efficacy of released rhbFGF. HE, CD31 and α-SMA staining revealed that the controlled release of rhbFGF from the Mg-PLGA-rhbFGF implant was superior in promoting angiogenesis compared with that of the PLGA-rhbFGF implant. Four weeks post-implantation, the capillary density of the Mg-PLGA-rhbFGF group was significantly higher than that of the PLGA-rhbFGF, control and the normal group (p<0.05, p<0.01 and p<0.01, respectively). Furthermore, the limb blood perfusion ratios of the Mg-PLGA-rhbFGF and PLGA-rhbFGF groups were dramatically increased, at 99.1±2.9% and 80.7±3.2%, respectively, whereas the ischemic limb did not recover in the control group. The biocompatibility of the implants was also evaluated. In conclusion, Mg-PLGA-based, sustained local delivery of rhbFGF promotes post-ischemic angiogenesis and blood flow recovery. The results suggest potential therapeutic usefulness of Mg-PLGA-rhbFGF for tissue ischemia. STATEMENT OF SIGNIFICANCE: Magnesium (Mg)-based implant has been already used in patients with critical limb ischemia. Site-specific controlled release of recombinant human basic fibroblast growth factor (rhbFGF), has become a promising approach to improve peripheral vascular disease. We report here on a novel combination implant composed of spiral magnesium and a coating made using poly(lactic-co-glycolic acid) (PLGA) with encapsulated rhbFGF (Mg-PLGA-rhbFGF). The preparation method does not involve any complex processes and results in a high encapsulation efficiency (approximately 100%). The degradation of metal Mg raise the microclimate pH in the PLGA polymer, which could well preserve the bioactivity of rhbFGF incorporated in the implant. Mg-PLGA-based, sustained local delivery of rhbFGF promotes post-ischemic angiogenesis and blood flow recovery in rat limb ischemic model. This work marks the first report for controlled release of rhbFGF in combination with metal Mg, and suggests potential therapeutic usefulness of Mg-PLGA-rhbFGF for tissue ischemia.

Dual-delivery of FGF-2/CTGF from Silk Fibroin/PLCL-PEO Coaxial Fibers Enhances MSC Proliferation and Fibrogenesis.

The success of mesenchymal stem cell transplantation is highly dependent on their survival and controlled fate regulation. This study demonstrates that dual-delivery of connective tissue growth factor (CTGF) and fibroblast growth factor 2 (FGF-2) from a core-shell fiber of Silk Fibroin/poly(L-lactic acid-co-ε-caprolactone)-polyethylene oxide (SF/PLCL-PEO) enhanced fibrogenic lineage differentiation of MSCs. The core-shell structure was confirmed by transmission electron microscopy (TEM), fluorescence microscopy and attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopy. A sequential release of FGF-2 and CTGF was successfully achieved in this manner. FGF-2 plays an important role in stem cell proliferation and, meanwhile when accompanied with CTGF, has a slightly additive effect on fibrogenic differentiation of MSCs, whereas CTGF promotes fibrogenesis and alleviates osteogenesis, chondrogenesis and adipogenesis.