Sustained release of stromal cell derived factor-1 from an antioxidant thermoresponsive hydrogel enhances dermal wound healing in diabetes.

Diabetic foot ulcers (DFUs) are a severe complication of diabetes mellitus. Altered cell migration due to microcirculatory deficiencies as well as excessive and prolonged reactive oxygen species production are implicated in the delayed healing of DFUs. The goal of this research was to assess whether sustained release of SDF-1, a chemokine that promotes endothelial progenitor cell homing and angiogenesis, from a citrate-based antioxidant thermoresponsive polymer would significantly improve impaired dermal wound healing in diabetes. Poly (polyethylene glycol citrate-co-N-isopropylacrylamide) (PPCN) was synthesized via sequential polycondensation and free radical polymerization reactions. SDF-1 was entrapped via gelation of the PPCN+SDF-1 solution above its lower critical solution temperature (LCST) and its release and bioactivity was measured. The effect of sustained release of SDF-1 from PPCN (PPCN+SDF-1) versus a bolus application of SDF-1 in phosphate buffered saline (PBS) on wound healing was evaluated in a diabetic murine splinted excisional dermal wound model using gross observation, histology, immunohistochemistry, and optical coherence tomography microangiography. Increasing PPCN concentration decreased SDF-1 release rate. The time to 50% wound closure was 11days, 16days, 14days, and 17days for wounds treated with PPCN+SDF-1, SDF-1 only, PPCN only, and PBS, respectively. Wounds treated with PPCN+SDF-1 had the shortest time for complete healing (24days) and exhibited accelerated granulation tissue production, epithelial maturation, and the highest density of perfused blood vessels. In conclusion, sustained release of SDF-1 from PPCN is a promising and easy to use therapeutic strategy to improve the treatment of chronic non-healing DFUs.

A thermoresponsive biodegradable polymer with intrinsic antioxidant properties.

Oxidative stress in tissue can contribute to chronic inflammation that impairs wound healing and the efficacy of cell-based therapies and medical devices. We describe the synthesis and characterization of a biodegradable, thermoresponsive gel with intrinsic antioxidant properties suitable for the delivery of therapeutics. Citric acid, poly(ethylene glycol) (PEG), and poly-N-isopropylacrylamide (PNIPAAm) were copolymerized by sequential polycondensation and radical polymerization to produce poly(polyethylene glycol citrate-co-N-isopropylacrylamide) (PPCN). PPCN was chemically characterized, and the thermoresponsive behavior, antioxidant properties, morphology, potential for protein and cell delivery, and tissue compatibility in vivo were evaluated. The PPCN gel has a lower critical solution temperature (LCST) of 26 °C and exhibits intrinsic antioxidant properties based on its ability to scavenge free radicals, chelate metal ions, and inhibit lipid peroxidation. PPCN displays a hierarchical architecture of micropores and nanofibers, and contrary to typical thermoresponsive polymers, such as PNIPAAm, PPCN gel maintains its volume upon formation. PPCN efficiently entrapped and slowly released the chemokine SDF-1α and supported the viability and proliferation of vascular cells. Subcutaneous injections in rats showed that PPCN gels are resorbed over time and new connective tissue formation takes place without signs of significant inflammation. Ultimately, this intrinsically antioxidant, biodegradable, thermoresponsive gel could potentially be used as an injectable biomaterial for applications where oxidative stress in tissue is a concern.

Advanced nanocomposites for bone regeneration.

The field of orthopedic tissue engineering is quickly expanding with the development of novel materials and strategies designed for rapid bone regeneration. While autologous bone grafts continue to be the standard of care, drawbacks include donor-site morbidity and short tissue supplies. Herein we report a novel nanocomposite sponge composed of poly(1,8-octanediol-co-citrate) (POC) and the bioactive ceramic ß-tricalcium phosphate (TCP). We show that these nanocomposite sponges can be used as a depot for bone-producing (a.k.a. osteogenic) growth factors. In vitro bioactivity is demonstrated by significant upregulation of osteogenic genes, osteopontin (∼3 fold increase), osteocalcin (∼22 fold increase), alkaline phosphatase (∼10 fold increase), and transcription factor, RUNX2 (∼5 fold increase) over basal expression levels in mesenchymal stem cells. In vivo osteogenicity and biocompatibility is demonstrated in a standard subcutaneous implant model in rat. Results show that the nanocomposite sponge supports complete cell infiltration, minimal adverse foreign body response, positive cellular proliferation, and cellular expression of osteogenic markers in subcutaneous tissue. The results shown herein are encouraging and support the use of this sponge for future bone tissue engineering efforts.

The blood and vascular cell compatibility of heparin-modified ePTFE vascular grafts.

Prosthetic vascular grafts do not mimic the antithrombogenic properties of native blood vessels and therefore have higher rates of complications that involve thrombosis and restenosis. We developed an approach for grafting bioactive heparin, a potent anticoagulant glycosaminoglycan, to the lumen of ePTFE vascular grafts to improve their interactions with blood and vascular cells. Heparin was bound to aminated poly(1,8-octanediol-co-citrate) (POC) via its carboxyl functional groups onto POC-modified ePTFE grafts. The bioactivity and stability of the POC-immobilized heparin (POC-Heparin) were characterized via platelet adhesion and clotting assays. The effects of POC-Heparin on the adhesion, viability and phenotype of primary endothelial cells (EC), blood outgrowth endothelial cells (BOECs) obtained from endothelial progenitor cells (EPCs) isolated from human peripheral blood, and smooth muscle cells were also investigated. POC-Heparin grafts maintained bioactivity under physiologically relevant conditions in vitro for at least one month. Specifically, POC-Heparin-coated ePTFE grafts significantly reduced platelet adhesion and inhibited whole blood clotting kinetics. POC-Heparin supported EC and BOEC adhesion, viability, proliferation, NO production, and expression of endothelial cell-specific markers von Willebrand factor (vWF) and vascular endothelial-cadherin (VE-cadherin). Smooth muscle cells cultured on POC-Heparin showed increased expression of α-actin and decreased cell proliferation. This approach can be easily adapted to modify other blood contacting devices such as stents where antithrombogenicity and improved endothelialization are desirable properties.

Sustained transgene expression via citric acid-based polyester elastomers.

Polymeric scaffolds are an important tool in tissue engineering and gene delivery using porous scaffolds can be a viable approach to control tissue response. Herein we describe the use of a biodegradable polyester elastomer, poly(1,8-octanediol-co-citrate) (POC), as a substrate for plasmid immobilization and cellular transfection of colonizing cells. Plasmid (pDNA), either complexed with poly(ethyleneimine) (PEI) forming polyplexes or in its native state, was surface-immobilized onto POC scaffolds via adsorption. Polyplex-containing scaffolds showed higher loading and slower initial rates of release than naked pDNA-containing scaffolds. Seeding of HEK293 cells and porcine aortic smooth muscle cells (PASMC) onto polyplex loaded-scaffolds demonstrated cell proliferation and transfection in vitro up to 12 days, significantly longer relative to bolus transfection. In vivo, transfection was evaluated using the mouse intraperitoneal (IP) fat model. In contrast to the in vitro study, successful long-term transgene delivery was only achieved with the naked pDNA-containing scaffolds. In particular, naked pDNA-containing scaffolds promoted high levels of both luciferase and green fluorescent protein (GFP) expression in vivo for 2 weeks. The results demonstrate that POC scaffolds are a suitable material for substrate-mediated gene delivery. POC scaffolds can potentially support long-term biological cues to mediate tissue formation through non-viral gene delivery.

Hemocompatibility evaluation of poly(diol citrate) in vitro for vascular tissue engineering.

One of the ongoing challenges in tissue engineering is the synthesis of a hemocompatible vascular graft. Specifically, the material used in the construct should have antithrombogenic properties and support the growth of vascular cells. Our laboratory has designed a novel biodegradable, elastomeric copolymer, poly(1,8-octanediol citrate) (POC), with mechanical and degradation properties suitable for vascular tissue engineering. The hemocompatibility of POC in vitro and its ability to support the attachment and differentiation of human aortic endothelial cell (HAEC) was assessed. The thrombogenicity and inflammatory potential of POC were assessed relative to poly(l-lactide-co-glycolide) and expanded poly(tetrafluoroethylene), as they have been used in FDA-approved devices for blood contact. Specifically, platelet aggregation and activation, protein adsorption, plasma clotting, and hemolysis were investigated. To assess the inflammatory potential of POC, the release of IL-1beta and TNF-alpha from THP-1 cells was measured. The cell compatibility of POC was assessed by confirming HAEC differentiation and attachment under flow conditions. POC exhibited decreased platelet adhesion and clotting relative to control materials. Hemolysis was negligible and protein adsorption was comparable to reference materials. IL-1beta and TNF-alpha release from THP-1 cells was comparable among all materials tested, suggesting minimal inflammatory potential. POC supported HAEC differentiation and attachment without any premodification of the surface. The results described herein are encouraging and suggest that POC is hemocompatible and an adequate candidate biomaterial for in vivo vascular tissue engineering.