Design and development of a collagen mimetic peptide (CMP) instructive hydrogel for wound healing

Abstract

Collagen mimetic peptides (CMPs) are synthetic peptides designed to resemble collagen in amino acid sequences, structure and function i.e., bioactivity. Hyaluronic acid (HA) also has intrinsic properties such as biocompatibility, biodegradability and hydrophilic characteristics thus, employing it as a suitable biomaterial for hydrogel formulation. This study provides a proof-of-concept for the evaluation of candidate CMP hydrogels with encoded biomimetic, instructive prompts that elicit cellular domino effects to achieve advanced wound repair. The biocompatibility and bioactivity of three CMPs namely (NL008, NL009, NL010) was established in vitro using cell viability and wound scratch assays. NL009 and NL010 were selected for further evaluations. The CMP hydrogels (NL009-HAgel and NL010-HAgel) were formulated by the addition of CMP to a solution of HA. The hydrogels were washed with phosphate buffer to remove surface bound CMPs. NL009-HAgel and NL010-HAgel were characterised for pertinent physico-chemical interactions (FTIR and XRD) and physio-mechanical properties (Texture Analyser, Rheometer and Elastosens, swelling, degradation by mass loss and water vapor transmission rate). Visualisation of the surface morphology of the lyophilised hydrogels was carried out using SEM. Release of the peptide and skin permeation studies were evaluated in vitro in SWF and over excised pig skin using Franz diffusion experiments and porcine skin tape stripping. NL009-HAgel and NL010-HAgel were subjected to biocompatibility and bioactivity tests via MTT and scratch assays, of which the candidate NL010-HAgel was selected as the candidate formulation for in vivo studies. Wound closure was evaluated on rat models for the candidate formulation against a market comparator. To evaluate how treatment with NL010-HAgels influenced cutaneous healing at a cellular level, histological analysis was conducted on the excised tissue. Masson’s trichome staining of wound tissue sections was used to comparatively examine collagen deposition. The pristine CMPs enhanced cell viability and migration in HaCaT keratinocytes and 3T3 fibroblasts cell models in vitro. Formulations of varying w/t % were prepared. The hydrogel showed an optimal microporous ( G” 120 Pa). The NL010-HAgel showed high swelling capacities (~390%) and water vapor transmission rates (165 g/m2 ). In Vitro bioactive release profile showed an initial burst CMP release (83%), thereafter steady state was achieved over a period of 7 days. Across an ex vivo porcine skin membrane, the CMP hydrogel showed good permeation (228 µg/cm-2 /min-1 ) and was retained in the epidermis and superficial dermis. Local CMP delivery was further validated by porcine skin tape stripping, wherein CMP content was retained locally within the epidermis and dermis. The CMP hydrogels showed enhanced cell viability and migration (99.9% closure within 24 hours) when evaluated in vitro on cell-based models. Owing to their promising characterisations in vitro, a candidate CMP formulation was assessed against a market comparator (Corning® Puramatrix™) in vivo. Herein, the formulation proved to enhance wound closure within the first few days of healing (30% and 75% closure in 3 and 7 days, respectively). While the market comparator (93.53%) exhibited better wound closure rates over the CMP hydrogel (93.25%) at the close of the study, the finding was not statistically significant (p>0.05). It was inferred that the CMP hydrogel was responsible for improved epidermal regeneration, granulation tissue deposition and quantity and quality of collagen deposition in the wound beds. The findings in this study promote the candidate CMP cell-instructive hydrogel as a potential system for enhanced wound healing