3D Porous Polymer Scaffold-Conjugated KGF-Mimetic Peptide Promotes Functional Skin Regeneration in Chronic Diabetic Wounds.

The re-epithelialization process gets severely dysregulated in chronic nonhealing diabetic foot ulcers/wounds. Keratinocyte growth factor (KGF or FGF-7) is the major modulator of the re-epithelialization process, which regulates the physiological phenotypes of cutaneous keratinocytes. The existing therapeutic strategies of growth factor administration have several limitations. To overcome these, we have designed a KGF-mimetic peptide (KGFp, 13mer) based on the receptor interaction sites in murine KGF. KGFp enhanced migration and transdifferentiation of mouse bone marrow-derived MSCs toward keratinocyte-like cells (KLCs). A significant increase in the expression of skin-specific markers Bnc1 (28.5-fold), Ck5 (14.6-fold), Ck14 (26.1-fold), Ck10 (187.7-fold), and epithelial markers EpCam (23.3-fold) and Cdh1 (64.2-fold) was associated with the activation of ERK1/2 and STAT3 molecular signaling in the KLCs. Further, to enhance the stability of KGFp in the wound microenvironment, it was conjugated to biocompatible 3D porous polymer scaffolds without compromising its active binding sites followed by chemical characterization using Fourier transform infrared spectroscopy, field-emission scanning electron microscopy, dynamic mechanical analysis, and thermogravimetry. In vitro evaluation of the KGFp-conjugated 3D polymer scaffolds revealed its potential for transdifferentiation of MSCs into KLCs. Transplantation of allogeneic MSCGFP using KGFp-conjugated 3D polymer scaffolds in chronic nonhealing type 2 diabetic wounds (db/db transgenic, 50-52 weeks old male mice) significantly enhanced re-epithelialization-mediated wound closure rate (79.3%) as compared to the control groups (Untransplanted -22.4%, MSCGFP-3D polymer scaffold -38.5%). Thus, KGFp-conjugated 3D porous polymer scaffolds drive the fate of the MSCs toward keratinocytes that may serve as potential stem cell delivery platform technology for tissue engineering and transplantation.

Fibrin Nanoparticles Coupled with Keratinocyte Growth Factor Enhance the Dermal Wound-Healing Rate.

Expediting the wound-healing process is critical for patients chronically ill from nonhealing wounds and diseases such as hemophilia or diabetes or who have suffered trauma including easily infected open wounds. FDA-approved external tissue sealants include the topical application of fibrin gels, which can be 500 times denser than natural fibrin clots. With lower clot porosity and higher polymerization rates than physiologically formed fibrin clots, the commercial gels quickly stop blood loss but impede the later clot degradation kinetics and thus retard tissue-healing rates. The fibrin nanoparticles (FBNs) described here are constructed from physiologically relevant fibrin concentrations that support new tissue and dermal wound scaffold formation when coupled with growth factors. The FBNs, synthesized in a microfluidic droplet generator, support cell adhesion and traction generation, and when coupled to keratinocyte growth factor (KGF), support cell migration and in vivo wound healing. The FBN-KGF particles enhance cell migration in vitro greater than FBN alone or free KGF and also improve healing outcomes in a murine full thickness injury model compared to saline, bulk fibrin sealant, free KGF, or bulk fibrin mixed with KGF treatments. Furthermore, FBN can be potentially administered with other tissue-healing factors and inflammatory mediators to improve wound-healing outcomes.

Dual growth factor-induced angiogenesis in vivo using hyaluronan hydrogel implants.

Crosslinked hyaluronan (HA) hydrogels preloaded with two cytokine growth factors, vascular endothelial growth factor (VEGF) and keratinocyte growth factor (KGF), were employed to elicit new microvessel growth in vivo. As a major glycosaminoglycan (GAG) component of extracellular matrix (ECM), HA is an excellent biopolymeric building block for new biomimetic, biocompatible therapeutic materials. HA hydrogel film samples were surgically implanted in the ear pinnae of mice, and the ears were harvested at 7 or 14 days post-implantation. Histologic analysis showed that each of the groups receiving an implant demonstrated significantly more microvessel density than control ears undergoing surgery but receiving no implant (p<0.001). Treatment groups receiving either co-delivery of both KGF and VEGF, an HA hydrogel lacking a growth factor or HA hydrogels containing a single cytokine were statistically unchanged with time, whereas treatment with KGF alone produced continuing increases in vascularization from day 7 to day 14. Strikingly, presentation of both VEGF and KGF in crosslinked HA generated intact microvessel beds with well-defined borders. In addition, an additive response to co-delivery of both cytokines in the HA hydrogel was observed. The HA hydrogels containing KGF+VEGF produced the greatest angiogenic response of any treatment group tested (NI=5.4 at day 14, where NI is a neovascularization index). This was 33% greater vessel density than in the next largest treatment group, that received HA+KGF (NI=4.0, p<0.002). New therapeutic approaches for numerous pathologies could be notably enhanced by the localized, sustained angiogenic response produced by release of both VEGF and KGF from crosslinked HA films.

A novel solid-phase site-specific PEGylation enhances the in vitro and in vivo biostabilty of recombinant human keratinocyte growth factor 1.

Keratinocyte growth factor 1 (KGF-1) has proven useful in the treatment of pathologies associated with dermal adnexae, liver, lung, and the gastrointestinal tract diseases. However, poor stability and short plasma half-life of the protein have restricted its therapeutic applications. While it is possible to improve the stability and extend the circulating half-life of recombinant human KGF-1 (rhKGF-1) using solution-phase PEGylation, such preparations have heterogeneous structures and often low specific activities due to multiple and/or uncontrolled PEGylation. In the present study, a novel solid-phase PEGylation strategy was employed to produce homogenous mono-PEGylated rhKGF-1. RhKGF-1 protein was immobilized on a Heparin-Sepharose column and then a site-selective PEGylation reaction was carried out by a reductive alkylation at the N-terminal amino acid of the protein. The mono-PEGylated rhKGF-1, which accounted for over 40% of the total rhKGF-1 used in the PEGylation reaction, was purified to homogeneity by SP Sepharose ion-exchange chromatography. Our biophysical and biochemical studies demonstrated that the solid-phase PEGylation significantly enhanced the in vitro and in vivo biostability without affecting the over all structure of the protein. Furthermore, pharmacokinetic analysis showed that modified rhKGF-1 had considerably longer plasma half-life than its intact counterpart. Our cell-based analysis showed that, similar to rhKGF-1, PEGylated rhKGF-1 induced proliferation in NIH 3T3 cells through the activation of MAPK/Erk pathway. Notably, PEGylated rhKGF-1 exhibited a greater hepatoprotection against CCl(4)-induced injury in rats compared to rhKGF-1.

Determining the protein drug release characteristics and cell adhesion to a PLLA or PLGA biodegradable polymer membrane.

Biodegradable polymers are of interest for developing controlled protein drug delivery platforms. In this study, two poly (alpha-hydroxy) esters were formulated with Aerosol-OT, a surfactant stabilizer, to encapsulate the protein keratinocyte growth factor (KGF) for controlled release KGF is involved in a number of crucial biologic processes, most notably epithelial growth and repair. The concentration of KGF that caused a biological response in vitro was determined (optimally 10 ng/mL) and compared with the release of KGF from the two biodegradable polymer membrane formulations. Each polymer formulation released biologically relevant levels, 10 ng/mL of active KGF, although with different times release kinetics. The membrane composed of PLGA/AOT/KGF exhibited a faster release rate of KGF into solution after 120 h of degradation time than the release rate of the PLLA/AOT/KGF matrices. Cell seeding assays showed that both polymer matrices, when formulated with AOT, sustained cell growth. Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS) was used to characterize the distribution of AOT and KGF through the polymer membrane. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.