Skin Tissue Regeneration: An Investigation of Melt-electrowriting with a Novel Biomaterial from Polycaprolactone and Milk Proteins

In 2017, only 28% of patients awaiting tissue transplants received treatment that same year. Coupled with a growing ageing population, demand for tissue replacements is increasing rapidly. Furthermore, the treatment of dermal skin tissue wounds proves difficult due to the low regeneration capability of the innate tissue, and the physical drawbacks of current substitutes. Polycaprolactone (PCL) is a bioresorbable and biocompatible polymer that has Food and Drug Administration approval for implantation into the human body. PCL is mechanically stable, flexible, and shows superior melt processing properties. However, PCL lacks biological functionality. Therefore, the current study sought to add bioactive milk proteins, lactoferrin (LF) and whey protein (WP), to PCL at concentrations of 0.05%, 0.1%, 0.25%, and a combination (COMB) of both LF and WP at concentrations of 0.25%. The biomaterial was used to create novel bioactive scaffolds that would encourage tissue regeneration and have the structural characteristics needed to regenerate thick dermal skin tissue. The biomaterial was processed into tissue regenerative scaffolds using a GeSiM Bioplotter equipped with an emerging 3D-bioprinting technique – melt-electrowriting (MEW). The scaffolds were characterised, and their biological activity assessed in an in vitro model of skin tissue (using HaCaTs and Nhdf cells). Physical characterisation showed that reproducible, layered microarchitecture scaffolds (~56% porosity) could be created from the biomaterial using MEW. The biodegradability and swelling of the scaffolds was low (≤3.3% mass loss over 21 days and 24 hour swelling ratio ≤6.4%), with COMB scaffolds showing significantly higher results than PCL alone (p≤0.05). Protein release of LF and WP from scaffolds was rapid, with an initial release of >40% after 1 hour. Biological analysis showed that neat LF did not improve proliferation or migration in 2D cell culture, whereas neat WP increased cell proliferation compared to PCL in both cell lines, but only increased migration in Nhdf cells at 0.25% (p≤0.05). COMB treatment increased migration of HaCaTs, Nhdfs, and a co-culture of both cell lines. Contrastingly, cells seeded onto scaffolds (3D cell culture) showed improved biological activity in response to scaffolds containing LF (including COMB scaffolds). Cell growth, spreading, and infiltration into the scaffolds significantly increased for LF containing scaffolds compared to PCL alone and WP scaffolds (p≤0.05). Fluorescence microscopy showed a high ratio of live/dead cells attached to scaffolds. COMB scaffolds indicated a potential synergistic mechanism, showing significantly improved cell migration and growth compared to PCL alone. These findings demonstrated that the addition of LF and WP increased the biological activity of 3D PCL scaffolds, possibly through immunomodulatory mechanisms (such as a decrease of harmful inflammation and/or production of oxidative species). These scaffolds showed promising tissue regenerative capacity and could act as suitable tissue regenerative constructs for hard-to-treat thick dermal tissue wounds. Moreover, the outcomes from this study not only illustrate the currently accepted practises for bioengineering research, as well as a validation of the GeSiM Bioplotter 3.1 for fabrication of 3D tissue regenerative constructs, but also contribute to the understanding of milk derived biomaterial-cell/tissue interaction, bioactive efficacy of WP or LF, and tissue regenerative medicine