The effect of collagen-binding NGF-β on the promotion of sciatic nerve regeneration in a rat sciatic nerve crush injury model

Nerve growth factor plays a critical role in peripheral nerve regeneration. However, the lack of efficient NGF delivery approach limits its clinical application. It has demonstrated in our previous work that the native human NGF-β (NAT-NGF) fused with a collagen-binding domain (CBD) could bind to collagen specifically. Since collagen is the major component of nerve extracellular matrix, we speculated that the collagen-binding NGF would target to nerve cells and improve their regeneration. In this report, we found that the fusion protein could specifically bind to endogenous collagen of the rat sciatic nerves and maintain NGF activity both in vitro and in vivo. In the rat sciatic nerve crush injury model, we found that collagen-binding NGF could be retained and concentrated at the nerve injured site to promote nerve repair and enhance function recovery following nerve damage. Thus, the collagen-binding NGF could improve the repair of peripheral nerve injury.     

A myocardial patch made of collagen membranes loaded with collagen-binding human vascular endothelial growth factor accelerates healing of the injured rabbit heart

Tissue-engineered myocardial patches could be useful in the repair of myocardial injuries. The aim of the present study was to evaluate a collagen targeting delivery system for myocardial repair. A specific peptide collagen-binding domain (CBD) was fused to human vascular endothelial growth factor (VEGF) to enhance the binding of VEGF to collagen. In this study, collagen membranes loaded with CBD-VEGF, natural VEGF, or phosphate-buffered saline are used as cardiac patches to repair the infarcted myocardium in a rabbit model. CBD-VEGF/collagen group could effectively induce more cells to penetrate into the collagen membrane after 4 weeks and promote more vascularization in infarcted myocardium after 12 weeks compared with the other two control groups. Echocardiography and hemodynamic studies both show cardiac function improvement in the CBD-VEGF/collagen group. These results reveal that implantation of CBD-VEGF collagen membrane patch into the infarcted myocardium could effectively improve left ventricle cardiac function and increase the vascular density.     

Release rate controls biological activity of nerve growth factor released from fibrin matrices containing affinity-based delivery systems

Previously, combinatorial techniques were used to identify peptide sequences exhibiting high, medium, and low affinity for heparin. Bidomain peptides were synthesized containing a transglutaminase sequence for one domain and one of the heparin-affinity sequences for the other domain. A delivery system was made consisting of bi-domain peptides, heparin, and nerve growth factor (NGF), which binds to heparin with moderate affinity. The goal of this research was to determine whether peptide affinity for heparin and the molar ratio of peptide to heparin affected the release rate of NGF from the delivery system and the biological activity of NGF released. This study also explored whether peptide affinity modulated biological activity independent of release rate. Mathematically modeling the delivery system confirmed that release could be controlled by both peptide affinity and molar ratio of peptide to heparin. Experimentally, the rate of NGF release from the delivery system was found to be affected by peptide affinity and molar ratio. The delivery system presented biologically active NGF as assayed by embryonic chick dorsal root ganglia (DRGs) neurite extension, where extension was similar to or increased for DRGs grown in fibrin matrices containing the delivery system compared to DRGs grown with NGF in the culture media. Furthermore, by modulating the molar ratio of peptide to heparin in the delivery system, similar release rates of NGF were obtained for different affinity peptides and these conditions promoted similar levels of neurite extension, demonstrating that release rate appears to be the main mechanism controlling the biological activity of released NGF.     

Rationally designed peptides for controlled release of nerve growth factor from fibrin matrices

The purpose of this research was to identify peptide sequences with varying affinity for nerve growth factor (NGF) and use them in the rational design of affinity-based drug delivery systems. A phage display library (12 amino acid random peptide sequence) was screened against NGF-conjugated chromatography resin three times and fractions containing phage of varying affinity were eluted by decreasing the pH of the eluent. These phages were isolated, amplified; then their DNA was purified and sequenced to determine the identity of the random peptide domain. Consensus peptides based on these sequences were synthesized and screened for their ability to bind NGF and release it at different rates from fibrin matrices. The ability of fibrin matrices containing these peptides and NGF to deliver to biologically active NGF was tested using a chick dorsal root ganglia model. A mathematical model was developed to further understand how the affinity of a peptide can modulate release of NGF and to aid in design optimization for the delivery system. The peptides identified in this study were determined to have varying affinities for NGF suggesting that this approach can serve as a model for tailoring the affinity of a drug delivery system for a target protein drug.     

Peripheral nerve regeneration with sustained release of poly(phosphoester) microencapsulated nerve growth factor within nerve guide conduits

Prolonged delivery of neurotrophic proteins to the target tissue is valuable in the treatment of various disorders of the nervous system. We have tested in this study whether sustained release of nerve growth factor (NGF) within nerve guide conduits (NGCs), a device used to repair injured nerves, would augment peripheral nerve regeneration. NGF-containing polymeric microspheres fabricated from a biodegradable poly(phosphoester) (PPE) polymer were loaded into silicone or PPE conduits to provide for prolonged, site-specific delivery of NGF. The conduits were used to bridge a 10 mm gap in a rat sciatic nerve model. Three months after implantation, morphological analysis revealed higher values of fiber diameter, fiber population and fiber density and lower G-ratio at the distal end of regenerated nerve cables collected from NGF microsphere-loaded silicone conduits, as compared with those from control conduits loaded with either saline alone, BSA microspheres, or NGF protein without microencapsulation. Beneficial effects on fiber diameter, G-ratio and fiber density were also observed in the permeable PPE NGCs. Thus, the results confirm a long-term promoting effect of exogenous NGF on morphological regeneration of peripheral nerves. The tissue-engineering approach reported in this study of incorporation of a microsphere protein release system into NGCs holds potential for improved functional recovery in patients whose injured nerves are reconstructed by entubulation.     

Silk fibroin matrices for the controlled release of nerve growth factor (NGF)

Nerve conduits (NC) for peripheral nerve repair should guide the sprouting axons and physically protect the axonal cone from any damage. The NC should also degrade after completion of its function to obviate the need of subsequent explanation and should optionally be suitable for controlled drug release of embedded growth factors to enhance nerve regeneration. Silk fibroin (SF) is a biocompatible and slowly biodegradable biomaterial with excellent mechanical properties that could meet the above stated requirements. SF material (films) supported the adherence and metabolic activity of PC12 cells, and, in combination with nerve growth factor (NGF), supported neurite outgrowth during PC12 cell differentiation. NGF-loaded SF-NC were prepared from aqueous solutions of NGF and SF (20%, w/w), which were air-dried or freeze-dried (freezing at -20 or -196 degrees C) in suitable molds. NGF release from the three differently prepared SF-NC was prolonged over at least 3 weeks, but the total amount released depended on the drying procedure of the NC. The potency of released NGF was retained within all formulations. Control experiments with differently dried NGF-lactose solutions did not evidence marked protein aggregation (SEC, HPLC), loss of ELISA-reactivity or PC12 cell bioactivity. This study encourages the further exploitation of SF-NC for growth factor delivery and evaluation in peripheral nerve repair.     

The effect of collagen-targeting platelet-derived growth factor on cellularization and vascularization of collagen scaffolds

Platelet-derived growth factor BB (PDGF-BB) was an important inductive factor during wound healing, but the lack of efficient delivery system limited its clinical application. Here, a peptide of seven amino acids was firstly utilized to engineer PDGF-BB to construct a collagen-targeting system. This peptide functioned as collagen-binding domain (CBD) to specially target the PDGF-BB to the collagen and restrict its diffusion. In our study, in vitro activity assay showed that the CBD-fused PDGF (CBD-PDGF) and native PDGF (NAT-PDGF) possessed similar activity to stimulate the human fibroblast proliferation. As expected, this peptide promoted the binding of PDGF to collagen scaffolds, and CBD-PDGF caused more cells to proliferate on the collagen gel than NAT-PDGF when the same amounts of PDGF were added. In the in vivo experiment, CBD-PDGF-loaded collagen scaffolds were uniformly cellularized and vascularized compared to that of NAT-PDGF-loaded scaffolds. Our study demonstrated that the CBD targeted PDGF to the collagen scaffold to exert its functions, and it suggested this could be an effective system for targeting tissue regeneration and wound repair.     

Impact of cell type and density on nerve growth factor distribution and bioactivity in 3-dimensional collagen gel cultures

Local delivery of protein agents is potentially important in many tissue engineering systems. In this report, we evaluate an experimental system for measuring the rate of nerve growth factor (NGF) transport and biological activity within a 3-dimensional, tissue-like environment. Fetal brain cells or PC12 cells were suspended throughout collagen gel cultures; controlled-release matrices were used to control the spatial and temporal pattern of NGF release. Experimentally measured concentration profiles were compared to profiles predicted by a mathematical model encompassing diffusion and first-order elimination. Our results suggest that NGF moves through gels by diffusion while being eliminated at a rate that depends on cell density. Since diffusion and elimination also govern protein transport in brain tissue, the collagen gel serves as a model system that replicates the main features of transport in the brain and, therefore, can be used to identify new strategies that enhance NGF distribution in the central nervous system. As an example of the utility of this biophysical model, we demonstrate that implantation of multiple controlled-release matrices can broaden NGF distribution in gel cultures; this broadening was accompanied by a significant increase in cellular biological activity. This approach may be useful in customizing NGF distribution throughout degenerating or damaged central nervous system tissue while minimizing toxicity to surrounding healthy tissue.     

In vivo induction and delivery of nerve growth factor, using HEK-293 cells

Tissue-engineering strategies offer hope to patients facing functional impairment after nerve injury. We have previously demonstrated that HEK-293 cells can release nerve growth factor (NGF) in vitro, using an inducible system of expression. In this study, our objective was to assess the efficacy of the NGF delivery system in vivo, using nude rats. HEK-293 cells were transfected with human NGF cDNA. Ponasterone A (PonA) was used as the inducing agent. NGF collection chambers were implanted subcutaneously in nude rats. Sealed chambers were filled with one of the following: (1) DMEM, (2) untransfected 293 cells (EcR-293) plus PonA, (3) untransfected EcR-293 without PonA, (4) transfected 293 cells (hNGF-EcR-293) plus PonA, or (5) transfected hNGF-EcR-293 without PonA. Chambers were aspirated 24, 48, and 120 h postimplantation. NGF secretion was analyzed in the following ways: (1) NGF protein expression bioactivity was assessed in a PC-12 cell bioassay, and (2) the concentration of secreted NGF was quantified by NGF ELISA. NGF quantification by ELISA reached a maximal release of 12.9 +/- 3.57 ng/mL at 120 h. PC-12 cells exposed to media from induced transfected HEK-293 cell chambers demonstrated higher levels of differentiation compared with controls. We conclude that hNGF-EcR-293 cells can inducibly secrete bioactive NGF when exposed to the induction agent PonA. This regulated delivery system can secrete bioactive NGF for up to 5 days in vivo. We believe this regulated delivery system will be useful for tissue-engineered nerve constructs.     

The effect of the controlled release of nerve growth factor from collagen gel on the efficiency of neural cell culture

In this study, we tested the hypothesis that the amount of nerve growth factor (NGF) required for pheochromocytoma (PC12) cell culture can be dramatically reduced by controlled release of NGF from a collagen gel coating on the culture surface. Cells were cultured on collagen gels loaded with various amounts of NGF. As a control, PC12 cells were cultured on collagen gels with daily addition of various amounts of NGF to the culture medium. After an initial 12h burst, NGF was steadily released from the gels for 4 days. Apoptotic activity and cell viability were determined using terminal uridine nick end labeling and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, respectively. Neuronal differentiation was determined using immunocytochemistry and Western blot analysis. Compared to 100 ng NGF daily addition (300 ng over 3 days), 1 ng NGF daily addition showed dramatically decreased cell viability and neuronal differentiation and increased apoptotic activity. In contrast, collagen gels loaded with 10 ng NGF yielded cell viability, apoptotic activity, and neuronal differentiation similar to those of culture with 100 ng NGF daily addition. Our method reduced the amount of NGF required for PC12 cell culture to 1/3th of that used in daily addition without affecting cell viability, apoptosis, or differentiation. This method could economize large-scale culture of stem cells by reducing the amount of costly growth factors needed.     

Gene-modified mesenchymal stem cells express functionally active nerve growth factor on an engineered poly lactic glycolic acid (PLGA) substrate

Delivery of cellular and/or trophic factors to the site of injury may promote neural repair or regeneration and return of function after peripheral nerve or spinal cord injury. Engineered scaffolds provide a platform to deliver therapeutic cells and neurotrophic molecules. We have genetically engineered mesenchymal stem cells (MSCs) from the green rat (CZ-004 [SD TgN(act-EGFP)OsbCZ-004]) to express nerve growth factor (NGF) using an adenoviral vector. Cells maintained their stem cell phenotype as judged by expression of CD71 and CD172 markers, and absence of the hematopoietic marker CD45. Cells continued to express green fluorescent protein (GFP) on a long-term basis. Morphology, viability, and growth kinetics were maintained when cells were grown on a poly-lactic-co-glycolic acid (PLGA) polymer scaffold. Under appropriate growth conditions, they differentiated into chondrogenic, osteogenic, and adipogenic phenotypes, demonstrating that they retained their characteristics as MSCs. NGF was secreted from transduced MSCs at physiologically relevant levels ( approximately 25 ng/mL) measured by enzyme-linked immunoabsorbent assay (ELISA). Secreted NGF was functionally active in a neurite growth assay with PC12 cells. We conclude that MSCs are a good candidate for delivery of therapeutic factors into the injured nervous system. They are autologous, may be genetically modified to express neurotrophins, and are compatible with polymer surfaces that may be used as a potential delivery system.     

Peripheral nerve regeneration using composite poly(lactic acid-caprolactone)/nerve growth factor conduits prepared by coaxial electrospinning

Many neurotrophic factors have been shown to promote neurite outgrowth by improving the microenvironment that is required for nerve regeneration. However, the delivery of these bioactive agents to the nerve injury site, as well as effective and local release, remains a challenging problem. We have developed a novel composite nerve conduit comprised of poly(lactic acid-caprolactone) (P(LLA-CL)) and nerve growth factor (NGF). This was developed from core-shell structured biodegradable nanofibers, which were fabricated by coaxial electrospinning of P(LLA-CL) for the shell and bovine serum albumin (BSA) or BSA/NGF for the core. In rats, gaps of 10-mm long sciatic nerves were bridged using an autograft, an empty P(LLA-CL) conduit, a NGF injection P(LLA-CL) conduit, a P(LLA-CL)/NGF composite conduit, respectively. Regenerated nerve fibers were harvested and morphological and functional evaluation of nerve regeneration was performed at 12 weeks postsurgery. Although partial biodegradation and small cracks in the conduits were observed, the conduit outlines remained intact for 12 weeks after surgery. Based on functional and histological observations, the number and arrangement of regenerated nerve fibers, myelination, and nerve function reconstruction was similar in the P(LLA-CL)/NGF conduit group to that of the nerve autograft group (p > 0.05), but was significantly greater to the empty P(LLA-CL) and injection NGF P(LLA-CL) conduit groups (both p < 0.05). Therefore, the composite P(LLA-CL)/NGF conduit, which exhibited favorable mechanical properties and biocompatibility, could effectively promote sciatic nerve regeneration in rats.     

神经生长因子不同给药方式对坐骨神经吻合后修复与再生的影响*

背景：神经生长因子对神经损伤后的修复有促进作用,但目前对不同用药方式的优劣性尚有争议。 目的：观察鞘膜内与局部应用神经生长因子对兔坐骨神经吻合后修复与再生的影响。 方法：将24只新西兰大白兔坐骨神经切断后再缝合,分别向兔鞘膜内或损伤局部注射30 μg神经生长因子或等量生理盐水。 结果与结论：坐骨神经损伤后12周,鞘膜内注射神经生长因子组损伤坐骨神经鞘膜增厚,神经纤维排列较整齐,与正常神经纤维差异不大;且其神经干传导速度、有髓神经纤维数目、髓鞘厚度也明显高于其他组(P < 0.05),损伤局部注射神经生长因子组次之。说明神经生长因子具有促进外周神经吻合后修复与再生的功能,鞘膜内应用优于局部注射。     

Regeneration of uterine horns in rats by collagen scaffolds loaded with collagen-binding human basic fibroblast growth factor

Severe damages of uterine endometrium which prevent embryos from implantation and placentation finally often result in infertility or pregnant complications. There is lack of effective treatments due to the limitation of native materials available and complexity of the function and internal environment of uterus. In the present study, a collagen targeting basic fibroblast growth factor (bFGF) delivery system was constructed by a collagen membrane loaded with bFGF fused a collagen-binding domain (CBD) to the N-terminal which limits the diffusion of bFGF from collagen. We tested the bFGF delivery system in rats under the severe uterine damage model (partial rat uterine horn excision/reconstruction), and found this delivery system improved regeneration abilities of uterine endometrium and muscular cells, improved vascularization, as well as better pregnancy outcomes in rats. Therefore, this targeting delivery system may be an effective strategy for uterine tissue regeneration.     

The use of laminin modified linear ordered collagen scaffolds loaded with laminin-binding ciliary neurotrophic factor for sciatic nerve regeneration in rats

Nerve conduit provides a promising strategy for nerve injury repair in the peripheral nervous system (PNS). However, simply bridging the transected nerve with an empty conduit is hard to satisfy functional recovery. The regenerated axons may disperse during regeneration in the empty lumen, limiting the functional recovery. Our previous work had reported that linear ordered collagen scaffold (LOCS) could be used as a nerve guidance material. Here we cross-linked LOCS fibers with laminin which was a major component of the extracellular matrix in nervous system. Ciliary neurotrophic factor (CNTF) plays a critical role in peripheral nerve regeneration. But the lack of efficient CNTF delivery approach limits its clinical applications. To retain CNTF on the scaffold, a laminin binding domain (LBD) was fused to the N-terminal of CNTF. Compared with NAT-CNTF, LBD-CNTF exhibited specific laminin-binding ability and comparable neurotrophic bioactivity. We combined LBD-CNTF with the laminin modified LOCS fibers to construct a double-functional bio-scaffold. The functional scaffold was filled in silicon conduit and tested in the rat sciatic nerve transection model. Results showed that this functional biomaterial could guide the axon growth, retain more CNTF on the scaffolds and enhance the nerve regeneration as well as functional recovery.     

Controlled release of nerve growth factor from fibrin gel

Nerve growth factor (NGF) is known to promote the axonal regeneration in injured nerve system. Delivery of NGF for a long period in a controlled manner may enhance the regeneration efficacy. In this study, we investigated whether NGF can be released from fibrin gel for a long period in a controlled manner. We also investigated whether sustained delivery of NGF using fibrin gel can enhance the efficacy of NGF in vitro. The addition of heparin to fibrin gel decreased the rate of NGF release from the fibrin gel. As the concentrations of thrombin and fibrinogen in fibrin gel increased, the NGF release rate decreased significantly, and the initial release burst decreased. NGF was released for up to 14 days in vitro. The bioactivity of NGF released from fibrin gel was assessed by morphological changes of pheochromocytoma (PC12) cells cultured in the presence of NGF-containing fibrin gel. NGF released from fibrin gel exhibited significantly higher degrees of PC12 cell viability and differentiation than NGF added in a free form daily into the culture medium. This study demonstrates that fibrin gel can release NGF in a sustained, controlled manner and in a bioactive form.     

Polyphosphoester microspheres for sustained release of biologically active nerve growth factor

Controlled delivery of neurotrophic proteins to a target tissue by biodegradable polymer microspheres has been explored widely for its potential applications in the treatment of various disorders in the nervous system. We investigated in this study the potential of polyphosphoester microspheres as carriers for the sustained release of nerve growth factor (NGF), a water-soluble neurotrophic protein. Two polyphosphoesters (PPEs), P(BHET-EOP/TC) and P(DAPG-EOP), as well as poly(lactide/glycolic acid) (PLGA), were used to fabricate microspheres by a W/O/W emulsion and solvent evaporation/extraction method. With bovine serum albumin as a model protein to optimize processing parameters. P(DAPG-EOP) microspheres exhibited a lower burst effect but similar protein entrapment levels and efficiencies when compared with those made of PLGA. Bioactive NGF could be released for at least 10 weeks from the P(DAPG-EOP) microspheres, as confirmed by a neurite outgrowth assay of the PC12 cells. These NGF containing microspheres were incorporated into the nerve guide conduits that were implanted to bridge a 10 mm gap in a rat sciatic nerve model. Two weeks after implantation, immunostaining with an antibody against the neurofilament protein confirmed the presence of axons at the distal end of regenerated cables within the NGF microsphere-loaded conduits. These results demonstrated the feasibility of using biodegradable PPEs for microencapsulation of NGF and provided a basis for future therapeutic application of the microspheres.     

The pro-fibrogenic effect of nerve growth factor on conjunctival fibroblasts is mediated by transforming growth factor-β

BACKGROUND: Nerve growth factor (NGF) and nerve growth factor receptor (NGFR) expressions have been found to be increased in sub-conjunctival scarring. OBJECTIVE: The aim of this study was to investigate the in vitro effects of NGF on some pro-fibrogenic properties of human conjunctival fibroblasts. METHODS: Expression of NGF, trkA(NGFR) and p75NTR on human fibroblasts grown from conjunctival biopsies and incubated for 2 or 6 days with NGF were evaluated by immunofluorescence, RT-PCR, flow cytometry and ELISA. The fibrogenic effect of NGF on conjunctival fibroblasts was investigated by evaluating their migration (wound model), proliferation ([3H]-thymidine incorporation), collagen production (3H]-proline incorporation), expression of alpha-smooth muscle actin (alpha-SMA) (cell surface ELISA) and contraction of 3D collagen gels. RESULTS: NGF induced the expression of p75NTR in the fibroblasts that constitutively expressed only trkA(NGF) and increased the migration of wounded fibroblasts, but not their proliferation and collagen production. NGF induced the conversion of fibroblasts into myofibroblasts expressing alpha-SMA, and enhanced their contraction of a collagen matrix. Interestingly, chronic NGF treatment induced transforming growth factor-beta1 (TGF-beta1) production by fibroblasts, and following specific TGF-beta neutralization, all the NGF-induced effects were completely abrogated. CONCLUSION: Our findings indicate that NGF, via TGF-beta induction, is likely to be involved in the healing or fibrotic processes occurring in conjunctiva during some pathological conditions.     

Magnetic alginate microspheres: system for the position controlled delivery of nerve growth factor

The use of polymeric carriers containing dispersed magnetic nanocrystalline particles for targeted delivery of drugs in clinical practice has attracted the interest of the scientific community. In this paper a system comprised of alginate microparticles with a core of magnetite and carrying nerve growth factor (NGF) is described. The magnetic properties of these microspheres, typical of superparamagnetic materials, allow precise and controlled delivery to the intended tissue environment. Experiments carried out on PC12 cells with magnetic alginate microspheres loaded with NGF have confirmed the induction of cell differentiation which is strongly dependent on the distance from the microsphere cluster. In addition, finite element modelling (FEM) of the release profile from the microspheres in culture, indicated the possibility of creating defined and predictable NGF gradients from the loaded microspheres. These observations on the carriage and release of growth factors by the proposed microparticles open new therapeutic options for both neuronal regeneration and of the development of effective neuronal interfaces.     

Affinity-based release of glial-derived neurotrophic factor from fibrin matrices enhances sciatic nerve regeneration

Glial-derived neurotrophic factor (GDNF) promotes both sensory and motor neuron survival. The delivery of GDNF to the peripheral nervous system has been shown to enhance regeneration following injury. In this study, we evaluated the effect of affinity-based delivery of GDNF from a fibrin matrix in a nerve guidance conduit on nerve regeneration in a 13 mm rat sciatic nerve defect. Seven experimental groups were evaluated which received GDNF or nerve growth factor (NGF) with the delivery system within the conduit, control groups excluding one or more components of the delivery system, and nerve isografts. Nerves were harvested 6 weeks after treatment for analysis by histomorphometry and electron microscopy. The use of the delivery system (DS) with either GDNF or NGF resulted in a higher frequency of nerve regeneration vs. control groups, as evidenced by a neural structure spanning the 13 mm gap. The GDNF DS and NGF DS groups were also similar to the nerve isograft group in measures of nerve fiber density, percent neural tissue and myelinated area measurements, but not in terms of total fiber counts. In addition, both groups contained a significantly greater percentage of larger diameter fibers, with GDNF DS having the largest in comparison to all groups, suggesting more mature neural content. The delivery of GDNF via the affinity-based delivery system can enhance peripheral nerve regeneration through a silicone conduit across a critical nerve gap and offers insight into potential future alternatives to the treatment of peripheral nerve injuries.