Design, development, analysis and comparison of human tissue-engineered skin substitute models

Tissue engineering is a multidisciplinary field which involves several areas such as cell biology, material science, engineering, or medicine. It appears as a necessity to solve the lack of organ donors or another efficient substitute for the tissue required. In the case of skin, tissue-engineered skin substitutes (TESSs) have been developed since more than forty years ago, however, due to the advances in technology, they have emerged as a promising therapeutic strategy in the last fifteen years. The main purpose in dermatology of these advanced therapies is to resemble as much as possible the native human skin and be an alternative to the gold standard treatment with autografts. In the last years, many different TESSs have been developed, based on different characteristics such as cellular composition or biomaterials used as scaffold. Among the first, keratinocytes and fibroblasts have been the main cell types used, allowing the manufacture of cultured epithelial or dermal substitutes (monolayer), respectively, and composite skin substitutes (bilayer) where epidermis and dermis are resembled. However, trying to develop more complex skin substitutes, in the last years, more cell types have been incorporated and studied, such as melanocytes, adipocytes or mesenchymal stem cells. This fact has allowed to produce trilayer substitutes that are under research trying to improve the clinical TESSs already used. Regarding the biomaterial composition, collagen, hyaluronic acid or human plasma/fibrin are the most studied, alone or combined with others, however, in some cases, no biomaterial-based TESSs have been also developed. These can be constituted of an acellular dermal matrix where fibroblasts and keratinocytes are cultured or, even, the cultured fibroblasts, under specific conditions, are able to produce and secrete their own extracellular matrix that serves as scaffold. In this context, the Unidad de Producción Celular e Ingeniería Tisular (UPCIT), the laboratory where this Doctoral Thesis has been developed, is able to produce a clinical human plasma-based skin substitute (HPSS) constituted of human plasma and agarose as secondary biomaterial and human primary keratinocytes and fibroblasts as cellular composition. However, agarose it is not naturally found in skin or human body and moreover, it is difficult to handle under Good Manufacturing Practices (GMP) conditions (all advanced therapy medicinal products must comply with this regulation to be used in patients). Moreover, the HPSS manufactured in the UPCIT it is a bilayer substitute constituted of keratinocytes and fibroblasts, however, the development of more complex or different HPSSs is an interesting approach for the treatment of different types of skin injuries or wounds. Therefore, the objectives of this Doctoral Thesis are, I) to evaluate the skin cell isolation protocol used for the extraction of the human primary keratinocytes and fibroblasts used during the UPCIT’s manufacturing process, II) to analyze several biological properties and culture methodologies of different versions of the HPSS model manufactured at UPCIT, combining, individually, six secondary biomaterials (serine, fibronectin, collagen, laminin-1, laminin-2 and hyaluronic acid) with human plasma as scaffold (a seventh type without secondary biomaterial was also manufactured) and studying four cellular combinations [Trilayer (mesenchymal stem cells, fibroblasts and keratinocytes), Bilayer (fibroblasts and keratinocytes), Monolayer (keratinocytes) and Control (without cells) HPSSs], III) to determine the advantages and disadvantages of the HPSS model, by its comparison with another clinical TESS model where no biomaterials are used (self-assembled skin substitute – SASS) and IV) to determine the in vivo wound healing potential of a bilayer HPSS combined with hyaluronic acid as secondary biomaterial and compare the results with the gold standard treatment and secondary wound healing approaches. Firstly, to evaluate the skin cell isolation protocol, the UPCIT’s protocol, which is based on a one-step digestion strategy where the dermis and epidermis are firstly mechanically separated and subsequently digested, was compared with the two-step digestion protocol developed at LOEX laboratory (Canada) for the manufacture of the SASS clinical model. In this protocol, the dermis and epidermis are enzymatically separated by the effect of the enzyme thermolysin at the dermalepidermal junction level and then, each tissue is individually digested. The results of this study revealed that the epithelial cell viability was higher using the LOEX-Protocol compared to the UPCITProtocol (93% vs. 85%) and the number of epithelial cells extracted per cm2 of skin was also 3.4 times higher. However, when the dermal cells were isolated, no significant differences were reported. Moreover, once the keratinocytes and fibroblasts were cultured for several passages, no differences in terms of population doubling time, time of culture or percentage of expression of an epithelial stem cell marker (Keratin 19), were observed. These results proved the effectiveness of the UPCIT’s protocol for its application into a clinical environment. On the second study, several conditions of the HPSS model regarding the secondary biomaterial used, the skin cell tissue source or the cellular composition were evaluated by in vitro asssays such as cell viability, cell metabolic activity, protein secretion profile and histology. The results revealed that the in vitro properties of the HPSS model were dependent on the human plasma used more than the secondary biomaterial added and moreover, similar results were observed regardless of abdominal skin or foreskin cells were used. In addition, two culture methodologies were also compared, submerged (SUB) and air/liquid interface (ALI), demonstrating that better histological structure and higher secretion of useful wound healing proteins such as bFGF and, mainly, VEGF-A were reported when ALI was applied, although it was more time-consuming. Regarding cell composition, better results were reported when Trilayer and Bilayer HPSSs were developed, compared to Monolayer substitutes. Once the in vitro biological properties of the several variations of the HPSS model manufactured at UPCIT were determined, the purpose was to compare them and also its mechanical properties, with another clinical TESS model developed without the use of biomaterials (SASS). This is a bilayer model cultured under ALI methodology, and for this reason, bilayer HPSSs composed of human plasma and the most usually investigated secondary biomaterials (collagen and hyaluronic acid) were compared using the same skin cell populations for the manufacture of both models. A bilayer HPSS without secondary biomaterial was also included in this study. The results demonstrated that slight biological differences were observed between both models and between the HPSS subtypes, however, SASSs were more resistant to tensile forces (p-value<0.01), but HPSS manufacturing time was shorter (46- 55 days for SASSs and 32-39 days for HPSSs), something to consider when a faster treatment is required. Therefore, the previous studies demonstrated that the role of the secondary biomaterial used for the development of the HPSS model manufactured at UPCIT is not as important in vitro, however, their individual in vivo properties could determine better outcomes. To that purpose, a bilayer (because it is the type of cellular TESS most used in a clinical environment) HPSS constituted of hyaluronic acid as secondary biomaterial (for its in vivo properties, previously demonstrated in other studies) was manufactured and its wound healing potential was evaluated in a surgical excision skin wound model in mice for 8 weeks. The results reported by this HPSS were compared with the use of autografts, another bilayer HPSS constituted of agarose as secondary biomaterial (the oldest HPSS manufactured at UPCIT) and secondary, commercial or under research, wound healing approaches. Homeostasis analysis indicated similar values of transepidermal water loss and elasticity between the bilayer HPSS combined with hyaluronic acid (6.42±0.75 g/h/m2, 0.42±0.08 AU), autografts (6.91±1.28 g/h/m2, 0.40±0.08 AU) and healthy mouse skin (6.40±0.43 g/h/m2, 0.35±0.03 AU). Moreover, histological results showed that bilayer HPSSs and autografts presented better skin structuration and higher expression of keratins. On balance, the results of this Doctoral Thesis demonstrate that the design, development and manufacture of different subtypes of a HPSS model are a promising and useful strategy as advanced therapy. The possibility of using several secondary biomaterials and skin cell tissue sources without reporting significant differences in terms of their biological properties, the versatility of applying two culture methodologies depending on the needs (time vs. higher secretion of wound healing factors) and the ease of manufacturing different cellular compositions, together with the homogeneity of their in vitro results reported when compared with another clinical TESS model, determine that the HPSS model is robust and successful. This is particularly observed when hyaluronic acid was in vivo studied as secondary biomaterial, demonstrating a wound healing potential and a recovery of homeostasis parameters similar to those of autografts. Therefore, this research validates the translation of the HPSS model into a clinical environment and recommends its use as an alternative to autografts for the treatment of several skin injuries and wounds.

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