Biomimetic Extracellular Matrix Nanofibers Electrospun with Calreticulin Promote Synergistic Activity for Tissue Regeneration

In recognition of the potential of calreticulin (CRT) protein in enhancing the rate and quality of wound healing in excisional animal wound models, this study was to incorporate CRT via polyblend electrospinning into polycaprolactone (PCL)/type 1 collagen (Col1) nanofibers (NFs; 334 ± 75 nm diameter) as biomimetic extracellular matrices to provide a novel mode of delivery and protection of CRT with enhanced synergistic activities for tissue regeneration. Release kinetic studies using fluoresceinated CRT (CRT-FITC) polyblend NFs showed a burst release within 4 h reaching a plateau at 72 h, with further intervals of release upon incubation with fresh phosphate buffered saline for up to 8 weeks. By measuring fluorescence during the first 4 h of release, CRT-FITC-containing NFs were shown to protect CRT from proteolytic digestion (e.g., by subtilisin) compared to CRT-FITC in solution. CRT incorporated into NFs (CRT-NFs) also showed retention of biological activities and potency for stimulating proliferation and migration of human keratinocytes and fibroblasts. Fibroblasts seeded on CRT-NFs, after 2 days, showed increased amounts of fibronectin, TGF-β1, and integrin β1 in cell lysates by immunoblotting. Compared to NFs without CRT, CRT-NFs supported cell responses consistent with greater cell polarization and increased laminin-5 deposition of keratinocytes and a more motile phenotype of fibroblasts, as suggested by vinculin-capping F-actin fibers nonuniformly located throughout the cell body and the secretion of phosphorylated focal adhesion kinase-enriched migrasomes. Altogether, CRT electrospun into PCL/Col1 NFs retained its structural integrity and biological functions while having additional benefits of customizable loading, protection of CRT from proteolytic degradation, and sustained release of CRT from NFs, coupled with innate physicochemical cues of biomimetic PCL/Col1 NFs. Such synergistic activities have potential for healing recalcitrant wounds such as diabetic foot ulcers.