IN VITRO COMPARISON OF SELF-ASSEMBLED AND PLASMA-BASED TISSUE ENGINEERED SKIN SUBSTITUTES: TWO DIFFERENT MANUFACTURING PROCESSES FOR THE TREATMENT OF SEVERE BURN PATIENTS

Speaker

Sierra-Sánchez, Álvaro (Unidad de Producción Celular e Ingeniería Tisular (UPCIT). Virgen de las Nieves University Hospital, Andalusian Network of Design and Translation of Advanced Therapies )

Description

"Introduction
The use of human bilayer tissue-engineered skin substitutes (hbTESSs) for the treatment of dermatological pathologies is a promising therapy, especially for severe burn patients where there is a lack of donor tissue and wound healing process is disrupted, increasing risk of infection and mortality. In search of personalized medicine, several hbTESSs are under research; their comparison would help understand which technique is more appropriate according to the patient’s pathology and condition, however, even at in vitro level, such comparison is complicated because of the high costs or specific requirements. Among hbTESSs, two models have been already applied on more than ten patients as part of respective clinical trials1,2. On the one hand, the Self-Assembly (SA) approach (LOEX-Canada) uses appropriate culture and mechanical conditions to induce fibroblasts to secrete significant amount of extracellular matrix (ECM) as during organogenesis3. On the other hand, human plasma fibrin-based strategy (UPCIT-Spain) generates a dermal layer of fibroblasts embedded into a hydrogel composed, mainly, of human plasma (clotting factor: fibrin) which can be mixed with biomaterials such as hyaluronic acid or collagen4. The aim of this study was to compare these two hbTESSs models.

Methodology
Three human skin samples were collected, and fibroblasts and keratinocytes were extracted for manufacturing both hbTESS models (N=3). Skin substitutes produced by SA approach were composed of three dermal (fibroblasts+ECM) and one epidermal (keratinocytes) layers. Using human plasma fibrin-based strategy, fibroblasts were embedded into three different dermal matrices (fibrin only, fibrin-hyaluronic acid (HA) or fibrin-collagen (COL)) and epithelialized with a layer of cultured keratinocytes on top. Mechanical properties were analyzed using a tensile testing machine. Immunofluorescence (Ki67, Keratin (K) 19, Collagen-IV, K10, Loricrin), western blot (Collagen-I and -IV) and PrestoBlueTM assay (cell metabolic activity indicator) were performed to compare the results. The same culture media were used for both protocols, but initial number of cells and time of culture followed the original clinical guidelines of each process.

Results
SA approach generates skin substitutes more resistant to tensile forces and with higher adhesion at the dermo-epidermal junction (2 times higher), however plasma-based hbTESSs are thicker, more elastic, and their production is less time-consuming (18 vs. 32 days). Higher number of cells and proliferative cells (Ki67+) is found in SA substitutes although their metabolic activity is lower. After epidermal differentiation, no significant differences were observed between both models, for the number of epidermal stem cells (K19+), and the K10 and Loricrin expression. Overall, production of collagen (I and IV) is higher in SA substitutes, but Collagen-IV is more specifically located at the basement membrane for plasma-based hbTESSs. Finally, properties of plasma-based subtypes are quite similar and only in some specific studies, significant differences are observed (higher amount of Collagen-I in fibrin-COL substitutes -p<0.01-).

Conclusions
Our study characterizes two hbTESS models, demonstrating that manufacturing time as well as mechanical and some biological properties are different, however previous clinical studies have already shown their safety. Future in vivo experiments should compare their wound healing potential and long-term persistence after grafting to complete their characterization."

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