Bioengineering a Human Plasma-Based Epidermal Substitute With Efficient Grafting Capacity and High Content in Clonogenic Cells

Abstract

Cultured epithelial autografts (CEAs) produced from a small, healthy skin biopsy represent a lifesaving surgical technique in cases of full-thickness skin burn covering >50% of total body surface area. CEAs also present numerous drawbacks, among them the use of animal proteins and cells, the high fragility of keratinocyte sheets, and the immaturity of the dermal-epidermal junction, leading to heavy cosmetic and functional sequelae. To overcome these weaknesses, we developed a human plasma-based epidermal substitute (hPBES) for epidermal coverage in cases of massive burn, as an alternative to traditional CEA, and set up critical quality controls for preclinical and clinical studies. In this study, phenotypical analyses in conjunction with functional assays (clonal analysis, long-term culture, or in vivo graft) showed that our new substitute fulfills the biological requirements for epidermal regeneration. hPBES keratinocytes showed high potential for cell proliferation and subsequent differentiation similar to healthy skin compared with a well-known reference material, as ascertained by a combination of quality controls. This work highlights the importance of integrating relevant multiparameter quality controls into the bioengineering of new skin substitutes before they reach clinical development. STEM CELLS TRANSLATIONAL MEDICINE 2015;4:643-654 SIGNIFICANCEThis work involves the development of a new bioengineered epidermal substitute with pertinent functional quality controls. The novelty of this work is based on this quality approach.

Influence of fibrin matrices and their released factors on epidermal substitute phenotype and engraftment

“…Human keratinocytes and human dermal fibroblasts were isolated from skin obtained from female patients undergoing breast reduction surgeries after informed consent. As described previously (Alexaline et al, 2015), skin pieces were incubated overnight at 4°C in 1.8 UI/ ml Dispase II (Roche) and 0.0625% trypsin (Biochrom). Epidermis pieces were separated from dermis using forceps, and further digested at 37°C in 0.05% trypsin/EDTA (Gibco) for 30 min.…”

“…Cryopreserved human dermal fibroblasts were thawed, received a dose of 60 Grays of γ rays to prevent further proliferation, and were seeded at 20,000 cells/cm 2 . Four to 12 hr later after this step, primary keratinocytes were thawed and plated at a density of 2,400 cells/cm 2 on growth-arrested fibroblasts in a medium previously described (Alexaline et al, 2015). Before reaching 70% of keratinocyte confluence, remaining fibroblasts were removed from the flasks by flushing, and keratinocytes were detached from plastic with 0.017% and 0.05% trypsin/EDTA, respectively.…”

“…Percentage of keratinocyte expressing Kera-tin10 was quantified as the ratio between positive staining and total epidermal surface and the result was normalized by the average number of cell layers. For in vivo experiment, as previously described (Alexaline et al, 2015), human reepithelialization percentage was calculated as the ratio between the length of the Integrin β1 positive stained region (human specific) and the total length of the wound (zone deprived of epidermal appendages). For each epidermal substitute, animals were assigned to a group corresponding to 0% to 10%, 10% to 50%, or 50% to 100% of human reepithelialization.…”

“…But one way to combine both approaches is the use of clotted human plasma that results in the formation of a plasma-based fibrin matrix (hPBM): a fibrin scaffold containing all plasma factors without platelets. hPBM have been more recently used as skin scaffolds in vitro, in vivo (Alexaline et al, 2015;Llames et al, 2004;Monfort, Soriano-Navarro, Garcia-Verdugo, & Izeta, 2013) and on patients (Gomez et al, 2011;Llames et al, 2006;Mohamed Haflah et al, 2018) showing interesting properties in skin bioengineering. Indeed, we have previously shown that human plasma-based epidermal substitutes (hPBES) have a better regenerative potential than conventional CEAs, with less clonal conversion (Alexaline et al, 2015).…”

“…Moreover, both FPF and hPBM are biodegradable materials that are removed physiologically after grafting by the fibrinolytic system (Janmey, Winer, & Weisel, 2009). Additionally, the detachment procedure of these two matrices (Alexaline et al, 2015;Meana et al, 1998) do not necessitate enzymatic treatment (Osada, Sekine, Soejima, Sakurai, & Shimizu, 2016), thus allowing the preservation of DEJ proteins. At last, rete ridges-like structures were observed 1 month after grafting epidermal substitutes grown on FPF but not CEA (Ronfard et al, 2000).…”