Carlos Luis Arellano

(1) Background: Meshed split skin grafting (SSG) still remains the gold standard of care for deep wound treatment. The aim of this study was to evaluate an autologous bioengineered dermal–epidermal composite skin for treating full-thickness wounds in a porcine model using a clinically relevant two-stage grafting approach. (2) Methods: Multiple full-thickness wounds were created on the dorsal sites of animals. Wounds were initially grafted with a polyurethane dermal template, followed three weeks later by either Platelet hydroGel–Pig Skin Equivalent (PG-PSE) or meshed SSG control. Human platelet lysate was also assessed as a substitute for foetal calf serum during skin maturation. (3) Results: By two weeks post-grafting, epidermisation of PG-PSE-treated wounds were comparable to SSG-treated wounds. EdU labelling confirmed early engraftment and contribution of graft-derived cells to both neo-epidermis and neo-dermis. Cytokeratin 5+ immunofluorescence staining, showed a hyperproliferative neo-epidermis, enriched in progenitor keratinocytes, typical of early-stage skin grafts in pigs. Neo-dermis was marked with abundance of vimentin⁺ fibroblasts and rete ridges indicating a mature dermal–epidermal junction. Robust neovascularisation was observed in both superficial and deep neo-dermis, with platelet lysate-matured grafts showing greater microvessel formation in superficial neo-dermis in comparison with native skin. Picrosirius red staining of the superficial neo-dermis revealed a lower abundance of thick, red collagen fibres in PG-PSE grafts compared to SSGs, consistent with ongoing collagen deposition and tissue integration in PG-PSE. However, the ratio of thin green collagen fibres to thick red collagen fibres in the deep neo-dermis was largely comparable between group conditions, suggesting that bioengineered grafting does not alter dermal template maturation. (4) Conclusion: Overall, autologous dermal–epidermal bioengineered skin effectively vascularises and repairs wounds over a dermal template. Platelet lysate is a promising alternative to foetal calf serum, although PG-PSE may require longer time for complete dermal maturation and warrants further long-term evaluation.

(1) Background: Meshed split skin grafting (SSG) still remains the gold-standard of care for deep wound treatment. The aim of this study was to evaluate an autologous bioengineered dermal–epidermal composite skin for treating full-thickness wounds in a porcine model using a clinically relevant two-stage grafting approach. (2) Methods: Multiple full thickness wounds were created on the dorsal sites of animals. Wounds were initially grafted with a polyurethane dermal template, followed three weeks later by either Platelet hydroGel–Pig Skin Equivalent (PG-PSE) or meshed SSG control. Human platelet lysate was also assessed as a substitute for foetal calf serum during skin maturation. (3) Results: By two weeks post grafting, epidermisation of PG-PSE–treated wounds was comparable to SSG-treated wounds. EdU labelling confirmed early engraftment and contribution of graft-derived cells to both neo-epidermis and neo-dermis. Immunofluorescence staining for vimentin+ fibroblasts and cytokeratin 5+ keratinocytes demonstrated formation of a neo-dermis and a hyperproliferative neo-epidermis enriched in progenitor keratinocytes, with rete ridge architecture indicative of dermal–epidermal maturation. Robust neovascularisation was observed in both papillary and reticular neo-dermis, with platelet lysate–matured grafts showing greater microvessel formation than foetal calf serum–matured grafts. Collagen deposition in PG-PSE grafts, particularly collagen I in the papillary neo-dermis, remained lower than in SSG-treated wounds, consistent with ongoing integration. However, collagen type I/type III composition in the reticular neo-dermis was largely comparable between PG-PSE and SSG, suggesting that bioengineered grafting does not alter dermal template maturation. (4) Conclusion: Overall, autologous dermal–epidermal bioengineered skin effectively vascularises and repairs wounds over a dermal template. Platelet lysate is a promising alternative to foetal calf serum, although PG-PSE may require longer for complete dermal maturation and warrants further long-term evaluation.

Despite advancement in skin engineering, native skin grafting remains the gold standard in clinical settings. We have previously demonstrated that a platelet-derived hydrogel (PG) can act as a scaffold to engineer a semi-mature bilaminar human skin equivalent (PG-HSE). In this study, PG-HSE was grafted on full thickness wounds in athymic mice. PG-HSE was compared with native skin autografts and a clinically proven bilaminar skin graft that utilises a single layer NovoSorb^® polyurethane biodegradable temporising matrix (plus plasma) as the scaffold (BTM-HSE). The graft analysis revealed PG-HSE-grafted wounds were fully epidermised in two weeks and the level of inflammatory markers, CXCl1, CXCl2, IL1β, and IL-6 transcripts, in grafts were at similar levels to their levels in autografts. This coincided with higher expression of COL1A2, COL3A1, and COL5A1 transcripts in PG-HSE grafts, compared to autografts and BTM-HSE grafts. Moreover, a higher deposition of both Col I and Col III was detected in the PG-HSE graft wound bed, when compared to the BTM-HSE graft wound bed. Conversely, BTM-HSE grafts showed a higher level of integrins, ITGA2, ITGA3, ITGA5, ITGA6, ITGAV, and ITGB1, at the RNA level, suggesting a stronger cell–scaffold interaction. In summary, we have shown although both PG and single layer BTM foam (plus plasma) are effective scaffolds for skin engineering, some key aspects of wound repair, including a reduction in inflammation and an increase in collagen deposition, are achieved with the platelet-derived hydrogel. The long-term effect of these scaffolds on wound scarring remains to be investigated.

Autologous skin grafting has been commonly used in clinics for decades to close large wounds, yet the cellular and molecular interactions between the wound bed and the graft that mediates the wound repair are not fully understood. The aim of this study was to better understand the molecular changes in the wound triggered by autologous and synthetic grafting. Defining the wound changes at the molecular level during grafting sets the basis to test other engineered skin grafts by design. In this study, a full-thickness skin graft (SKH-1 hairless) mouse model was established. An autologous full-thickness skin graft (FTSG) or an acellular fully synthetic Biodegradable Temporising Matrix (BTM) was grafted. The wound bed/grafts were analysed at histological, RNA, and protein levels during the inflammation (day 1), proliferation (day 5), and remodelling (day 21) phases of wound repair. The results showed that in this mouse model, similar to others, inflammatory marker levels, including Il-6, Cxcl-1, and Cxcl-5/6, were raised within a day post-wounding. Autologous grafting reduced the expression of these inflammatory markers. This was different from the wounds grafted with synthetic dermal grafts, in which Cxcl-1 and Cxcl-5/6 remained significantly high up to 21 days post-grafting. Autologous skin grafting reduced wound contraction compared to wounds that were left to spontaneously repair. Synthetic grafts contracted significantly more than FTSG by day 21. The observed wound contraction in synthetic grafts was most likely mediated at least partly by myofibroblasts. It is possible that high TGF-β1 levels in days 1–21 were the driving force behind myofibroblast abundance in synthetic grafts, although no evidence of TGF-β1-mediated Connective Tissue Growth Factor (CTGF) upregulation was observed.

An efficient and safe method to deliver active proteins into the cytosol of targeted cells is highly desirable to advance protein‐based therapeutics. A novel protein delivery platform has been created by combinatorial design of cationic lipid‐like materials (termed “lipidoids”), coupled with a reversible chemical protein engineering approach. Using ribonuclease A (RNase A) and saporin as two representative cytotoxic proteins, the combinatorial lipidoids efficiently deliver proteins into cancer cells and inhibit cell proliferation. A study of the structure–function relationship reveals that the electrostatic and hydrophobic interactions between the lipidoids and the protein play a vital role in the formation of protein–lipidoid nanocomplexes and intracellular delivery. A representative lipidoid (EC16‐1) protein nanoparticle formulation inhibits cell proliferation in vitro and suppresses tumor growth in a murine breast cancer model.