Tailored nanofibers with calreticulin for tissue regeneration of diabetic wounds
Major missions of the DIACOMP Consortium are targeted delivery of therapeutics addressing complications of diabetes and devising strategies for repair and regeneration of wound tissue. A high impact complication of diabetes mellitus (US: 30.3 million cases) is chronic non-healing diabetic foot ulcers (DFUs), which occur at a rate of 25%, can lead to amputation and death, and impose a healthcare burden of over $20 billion. DFUs remain a serious unmet medical need due to the lack of effective therapies. This proposal will test the feasibility of engineering a novel scaffold for healing chronic DFUs by synergizing the advantages of electro-spun nanofibers (NF) and calreticulin (CRT), a protein that enhances the rate and quality of healing in porcine and diabetic mouse wound models. Moreover, CRT is the first potential biotherapeutic that heals full-thickness wounds by a tissue regenerative process replete with epidermal appendage neogenesis and lack of scarring. Lack of cell recruitment, cell proliferation, and a paucity of granulation tissue are major defects that cause the chronicity of DFUs. Mechanism of action studies in vitro using human keratinocytes, fibroblasts, and macrophages show that CRT corrects these serious defects that prevent healing of DFUs. Electrospun poly(?-caprolactone) (PCL)/collagen I (Coll) NFs are engineered to mimic native extracellular matrix (ECM) to stimulate cellular functions by modulating composition, alignment, diameter, and pore size. The broad long-term objective of this proposal is to create a tailored pro-healing microenvironment with CRT-laden hybrid fibrous matrices, which afford an optimal ECM-like environment to sustain and amplify tissue regeneration as an innovative therapeutic approach to solving the problem of recalcitrant healing of chronic DFUs. Specific Aim 1 will determine whether CRT spun into NF hybrid scaffolds or coated onto NFs exhibits optimal release kinetics of CRT, and in conjunction with ECM-like matrices, supports wound healing-related cellular responses of human keratinocytes, macrophages, and fibroblasts (e.g., migration, proliferation, ECM and integrin induction, and macrophage activation) and is suitable as a novel wound care agent (e.g,, tensile strength). Human diabetic fibroblasts will be analyzed and compared with normal fibroblast phenotypic behavior. Using a diabetic mouse full-thickness wound model for proof of principle studies, Specific Aim 2 will test the efficacy of optimized CRT-PCL/Coll NFs to engineer an ideal wound scaffold that recruits and harbors cells for induction of tissue regeneration. These pilot studies will support a full proposal for the development of CRT-NFs to advance to the clinic as a successful treatment for patients suffering from DFUs.