Shiva Akbarzadeh

(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.

Healing after split‐skin grafting remains difficult to anticipate in small‐area burns, particularly when wounds show no clinically diagnosed infection. We tested whether the burn wound microbiome before grafting captures early wound states linked to subsequent healing. Twenty‐six adults with mid‐dermal to full‐thickness burns involving < 10% total body surface area underwent longitudinal sampling at surgery, graft check and clinic review. At surgery, swabs were collected from the burn wound before grafting, the split‐skin donor site and anatomically matched uninjured control skin. Across 170 infection‐free samples, microbiome profiling showed that pre‐grafting burn wounds already had reduced microbial diversity and distinct community structure compared with matched control skin. This baseline dysbiosis included depletion of commensal‐associated anaerobes, including Finegoldia and Anaerococcus and enrichment of opportunistic burn‐associated taxa, including Pseudomonas and Acinetobacter . Donor sites acquired a related wound‐like microbiome and remodelled in parallel with burn wounds during healing, but post‐grafting burn microbiota were not preferentially similar to matched donor sites, arguing against a donor‐to‐burn microbial transfer. The clearest clinical signal was present before grafting: lower burn wound diversity at surgery was associated with delayed healing, and baseline community structure differed in wounds that later required regrafting. These findings suggest that the pre‐grafting burn wound microbiome may provide an early, infection‐independent marker of healing trajectory and should be tested as a predictive biomarker in larger prospective cohorts.

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.

Safety concerns associated with foetal bovine serum (FBS) have restricted its translation into clinics. We hypothesised that platelet lysate (PL) can be utilised as a safe alternative to produce serum-free 3D-engineered skin. PL supported a short-term expansion of fibroblasts, with negligible replication-induced senescence and directed epidermal stratification. PL-expanded fibroblasts were phenotypically separated into three subpopulations of CD90⁺FAP⁺, CD90⁺FAP⁻ and CD90⁻FAP⁺, based on CD90 (reticular marker) and FAP (papillary marker) expression profile. PL drove the expansion of the intermediate CD90⁺ FAP⁺ subpopulation in expense of reticular CD90⁺FAP⁻, which may be less fibrotic once grafted. The 3D-engineered skin cultured in PL was analysed by immunofluorescence using specific markers. Detection of ColIV and LMN-511 confirmed basement membrane. K10 confirmed near native differentiation pattern of neo-epidermis. CD29- and K5-positive interfollicular stem cells were also sustained. Transmission and scanning electron microscopies detailed the ultrastructure of the neo-dermis and neo-epidermis. To elucidate the underlying mechanism of the effect of PL on skin maturation, growth factor contents in PL were measured, and TGF-β1 was identified as one of the most abundant. TGF-β1 neutralising antibody reduced the number of Ki67-positive proliferative cells, suggesting TGF-β1 plays a role in skin maturation. Moreover, the 3D-engineered skin was exposed to lucifer yellow on days 1, 3 and 5. Penetration of lucifer yellow into the skin was used as a semi-quantitative measure of improved barrier function over time. Our findings support the concept of PL as a safe and effective serum alternative for bioengineering skin for cell therapies.

Background: Platelet-rich plasma (PRP) and its derivatives are an emerging biotechnology whereby concentrated platelets provide damaged tissue with growth factors, cytokines, and other mediators to improve healing outcomes. Although there is strong evidence in the benefits of autologous PRP for both acute and chronic wounds, allogeneic PRP has been studied far less in comparison. Summary: In this mini-review, we discuss critical steps of allogenic PRP (and its derivatives) preparation. We performed a non-systematic review of the literature to identify animal and human subject studies testing allogenic PRP for wound treatment. We searched OVID Medline and PubMed for articles using the keywords “wound, ulcer, lesion, skin, and cutaneous” and “PRP, or platelet-rich plasma, or platelet-rich fibrin, or PRF, or platelet releasate” and “homologous, allogeneic or allogenic,” which were limited to non-review articles and English language. Two studies in animal models and 8 studies in patients were reviewed. There were inconsistencies in preparation methods, treatment regimens, and some lacked a control group in their studies. Despite these variations, none of the studies identified any major side effects or adverse events. The treatment resulted in a reduced time to heal and/or reduced wound size in most cases. Key Messages: In situations where autologous PRP is not available or suitable, allogeneic PRP appears to provide a safe alternative. Its efficacy, however, requires larger-scale studies with appropriate controls. Standardization in PRP preparation and treatment regime are also needed to be able to interpret allogenic PRP efficacy.

Platelets are a reservoir of growth factors, cytokines and chemokines involved in spontaneous wound repair. In this study, a novel platelet-rich and fibrin-rich hydrogel was generated from expired platelet components that would otherwise have been transfused. The material contained physiological concentrations of transforming growth factor β1 (TGF-β1, platelet-derived growth factor AB (PDGF-AB), PDGF-BB, insulin-like growth factor-1 (IGF-1), fibroblast growth factor 2 (FGF-2), and epidermal growth factor (EGF). The effect of the hydrogel on wound repair was investigated in SKH-1 mice. Full thickness dorsal wounds were created on the mice and treated with the hydrogel at various concentrations. Immunohistochemical staining with CD31 (endothelial cell marker) revealed that wounds treated with the hydrogel showed significantly enhanced vascularisation in the wound bed. Moreover, high levels of interleukin-6 (IL-6) and KC (IL-8 homologue) in treated wounds were sustained over a longer period of time, compared to untreated wounds. We postulate that sustained IL-6 is a driver, at least partly, of enhanced vascularisation in full thickness wounds treated with the hydrogel. Future work is needed to explore whether this novel hydrogel can be utilised as a treatment option when vascularisation is a critical limitation.