In vitro comparison of human plasma-based and self-assembled tissue-engineered skin substitutes: two different manufacturing processes for the treatment of deep and difficult to heal injuries

Abstract

Background: The aim of this in vitro study was to compare side-by-side two models of human bilayered tissue-engineered skin substitutes (hbTESSs) designed for the treatment of severely burned patients. These are the scaffold-free self-assembled skin substitute (SASS) and the human plasma-based skin substitute (HPSS). Methods: Fibroblasts and keratinocytes from three humans were extracted from skin biopsies (N = 3) and cells from the same donor were used to produce both hbTESS models. For SASS manufacture, keratinocytes were seeded over three self-assembled dermal sheets comprising fibroblasts and the extracellular matrix they produced (n = 12), while for HPSS production, keratinocytes were cultured over hydrogels composed of fibroblasts embedded in either plasma as unique biomaterial (Fibrin), plasma combined with hyaluronic acid (Fibrin-HA) or plasma combined with collagen (Fibrin-Col) (n/biomaterial = 9). The production time was 46–55 days for SASSs and 32–39 days for HPSSs. Substitutes were characterized by histology, mechanical testing, PrestoBlue™-assay, immunofluorescence (Ki67, Keratin (K) 10, K15, K19, Loricrin, type IV collagen) and Western blot (type I and IV collagens). Results: The SASSs were more resistant to tensile forces (p-value < 0.01) but less elastic (p- value < 0.001) compared to HPSSs. A higher number of proliferative Ki67+ cells were found in SASSs although their metabolic activity was lower. After epidermal differentiation, no significant difference was observed in the expression of K10, K15, K19 and Loricrin. Overall, the production of type I and type IV collagens and the adhesive strength of the dermal-epidermal junction was higher in SASSs. Conclusions: This study demonstrates, for the first time, that both hbTESS models present similar in vitro biological characteristics. However, mechanical properties differ and future in vivo experiments will aim to compare their wound healing potential.

Keywords

Biomaterial / Burn / Fibrin / Human plasma / Self-assembly / Skin substitute / Tissue engineering

References

[1] Germain L, Larouche D, Nedelec B, Perreault I, Duranceau L, Bortoluzzi P, et al. Autologous bilayered self-assembled skin substitutes (SASSs) as permanent grafts: a case series of 14 severely burned patients indicating clinical effectiveness. Eur Cell Mater. 2018; 36: 128-41.

[2] Naves LB, Dhand C, Almeida L, Rajamani L, Ramakrishna S. In vitro skin models and tissue engineering protocols for skin graft applications. Essays Biochem. 2016; 60: 357-69.

[3] Sierra-Sánchez Á, Kim KH, Blasco-Morente G, Arias-Santiago S. Cellular human tissue-engineered skin substitutes investigated for deep and difficult to heal injuries. NPJ Regen Med. 2021; 6: 35.

[4] O’Connor NE, Mulliken JB, Banks-Schlegel S, Kehinde O, Green H. Grafting of burns with cultured epithelium prepared from autologous epidermal cells. Lancet. 1981; 317: 75-8.

[5] Donati L, Magliacani G, Bormioli M, Signorini M, Baruffaldi Preis FW. Clinical experiences with keratinocyte grafts. Burns. 1992; 18: S19-26.

[6] Gardien KLM, Marck RE, Bloemen MCT, Waaijman T, Gibbs S, Ulrich MMW, et al. Outcome of burns treated with autologous cultured proliferating epidermal cells: a prospective randomized multicenter intrapatient comparative trial. Cell Transplant. 2016; 25: 437-48.

[7] Kashiwa N, Ito O, Ueda T, Kubo K, Matsui H, Kuroyanagi Y. Treatment of full-thickness skin defect with concomitant grafting of 6-fold extended mesh auto-skin and allogeneic cultured dermal substitute. Artif Organs. 2004; 28: 444-50.

[8] Morimoto N, Ito T, Takemoto S, Katakami M, Kanda N, Tada H, et al. An exploratory clinical study on the safety and efficacy of an autologous fibroblast-seeded artificial skin cultured with animal product-free medium in patients with diabetic foot ulcers. Int Wound J. 2014; 11: 183-9.

[9] Momeni M, Fallah N, Bajouri A, Bagheri T, Orouji Z, Pahlevanpour P, et al. A randomized, double-blind, phase I clinical trial of fetal cell-based skin substitutes on healing of donor sites in burn patients. Burns. 2019; 45: 914-22.

[10] Hansbrough JF, Boyce ST, Cooper ML, Foreman TJ. Burn wound closure with cultured autologous keratinocytes and fibroblasts attached to a collagen-glycosaminoglycan substrate. JAMA. 1989; 262: 2125-30.

[11] Llames S, García E, García V, del Río M, Larcher F, Jorcano JL, et al. Clinical results of an autologous engineered skin. Cell Tissue Bank. 2006; 7: 47-53.

[12] Gómez C, Galán JM, Torrero V, Ferreiro I, Pérez D, Palao R, et al. Use of an autologous bioengineered composite skin in extensive burns: clinical and functional outcomes. A multicentric study. Burns. 2011; 37: 580-9.

[13] Boyce ST, Simpson PS, Rieman MT, Warner PM, Yakuboff KP, Bailey JK, et al. Randomized, paired-site comparison of autologous engineered skin substitutes and split-thickness skin graft for closure of extensive, full-thickness burns. Journal of Burn Care and Research Lippincott Williams and Wilkins. 2017; 38: 61-70.

[14] Fernández-González A, Lizana-Moreno A, Fernández N, Guerrero-Calvo J, Ruiz-Garcia A, Espinosa O, et al. Clinical, histological and homeostasis evaluation of an autologous tissue bio-engineered skin substitute in a patient with 70% of total body surface area (TBSA) burn. Cytotherapy. 2017; 19: S233. http://www.isct-cytotherapy.org/article/S1465324917303973/fulltext.

[15] Meuli M, Hartmann-Fritsch F, Hüging M, Marino D, Saglini M, Hynes S, et al. A cultured autologous Dermo-epidermal skin substitute for full-thickness skin defects: a phase I, open, prospective clinical trial in children. Plast Reconstr Surg. 2019; 144: 188-98.

[16] Egea-Guerrero JJ, Carmona G, Correa E, Mata R, Arias-Santiago S, Alaminos M, et al. Transplant of tissue-engineered artificial autologous human skin in Andalusia: an example of coordination and institutional collaboration. Transplant Proc. 2019; 51: 3047-50.

[17] Cortez Ghio S, Larouche D, Doucet EJ, Germain L. The role of cultured autologous bilayered skin substitutes as epithelial stem cell niches after grafting: a systematic review of clinical studies. Burns Open. 2021; 5: 56-66.

[18] Alexaline MM, Magne B, Zuleta Rodríguez A, Nivet M, Bacqueville D, Lataillade JJ, et al. Influence of fibrin matrices and their released factors on epidermal substitute phenotype and engraftment. J Tissue Eng Regen Med. 2019; 13: 1362-74.

[19] Sierra-Sánchez Á, Fernández-González A, Lizana-Moreno A, Espinosa-Ibáñez O, Martinez-Lopez A, Guerrero-Calvo J, et al. Hyaluronic acid biomaterial for human tissue-engineered skin substitutes: preclinical comparative in vivo study of wound healing. J Eur Acad Dermatol Venereol. 2020; 34: 2414-27.

[20] Michel M, L’Heureux N, Pouliot R, Xu W, Auger FA, Germain L. Characterization of a new tissue-engineered human skin equivalent with hair. In Vitro Cell Dev Biol Anim. 1999; 35: 318-26.

[21] Larouche D, Paquet C, Fradette J, Carrier P, Auger FA, Germain L. Regeneration of skin and cornea by tissue engineering. Methods Mol Biol. 2009; 482: 233-56.

[22] Larouche D, Cantin-Warren L, Desgagné M, Guignard R, Martel I, Ayoub A, et al. Improved Methods to produce tissue-engineered skin substitutes suitable for the permanent closure of full-thickness skin injuries. Biores Open Access. 2016; 5: 320.

[23] Kelly C, Wallace D, Moulin V, Germain L, Zuccaro J, Galdyn I, et al. Surviving an extensive burn injury using advanced skin replacement technologies. J Burn Care Res. 2021; 42: 1288-91.

[24] Efanov JI, Tchiloemba B, Duong A, Bélisle A, Izadpanah A, Coeugniet E, et al. Use of bilaminar grafts as life-saving interventions for severe burns: a single-center experience. Burns. 2018; 44: 1336-45.

[25] Bisson F, Rochefort É, Lavoie A, Larouche D, Zaniolo K, Simard-Bisson C, et al. Irradiated human dermal fibroblasts are as efficient as mouse fibroblasts as a feeder layer to improve human epidermal cell culture lifespan. Int J Mol Sci. 2013; 14: 4684-704.

[26] Lavoie A, Fugère C, Beauparlant A, Goyer B, Larouche D, Paquet C, et al. Human epithelial stem cells persist within tissue-engineered skin produced by the self-assembly approach. Tissue Eng Part A. 2013; 19: 1023-38.

[27] Gauvin R, Larouche D, Marcoux H, Guignard R, Auger FA, Germain L. Minimal contraction for tissue-engineered skin substitutes when matured at the air-liquid interface. J Tissue Eng Regen Med. 2013; 7: 452-60.

[28] Scionti G, Moral M, Toledano M, Osorio R, Durán JDG, Alaminos M, et al. Effect of the hydration on the biomechanical properties in a fibrin-agarose tissue-like model. J Biomed Mater Res A. 2014; 102: 2573-82.

[29] Larose AE, Dakiw-Piaceski A, Barbier MA, Larouche D, Gauvin R, Caruso M, et al. Peel test to assess the adhesion strength of the dermal-epidermal junction in tissue-engineered skin. Tissue Eng Part C Methods. 2020; 26: 180-9.

[30] Cantin-Warren L, Guignard R, Ghio SC, Larouche D, Auger FA, Germain L. Specialized living wound dressing based on the self-assembly approach of tissue engineering. J Funct Biomater. 2018; 9: 53.

[31] Mountford EM, Blight A, Cheshire IM. Implications for would healing of patient age and time elapsed since burn injury. J Wound Care. 1995; 4: 32-5.

[32] Finnerty CC, Jeschke MG, Qian WJ, Kaushal A, Xiao W, Liu T, et al. Determination of burn patient outcome by large-scale quantitative discovery proteomics. Crit Care Med. 2013; 41: 1421-34.

[33] Pavoni V, Gianesello L, Paparella L, Buoninsegni LT, Barboni E. Outcome predictors and quality of life of severe burn patients admitted to intensive care unit. Scand J Trauma Resusc Emerg Med. 2010; 18: 24. https://doi.org/10.1186/1757-7241-18-24.

[34] Beaudoin Cloutier C, Guignard R, Bernard G, Gauvin R, Larouche D, Lavoie A, et al. Production of a Bilayered self-assembled skin substitute using a tissue-engineered acellular dermal matrix. Tissue Eng Part C Methods. 2015; 21: 1297-305.

[35] Velez AMA, Howard MS. Collagen IV in normal skin and in pathological processes. N Am J Med Sci. 2012; 4: 1-8.

[36] Caliari SR, Burdick JA. A practical guide to hydrogels for cell culture. Nat Methods. 2016; 13: 405-14.

[37] Tang L, Sierra JO, Kelly R, Kirsner RS, Li J. Wool-derived keratin stimulates human keratinocyte migration and types IV and VII collagen expression. Exp Dermatol. 2012; 21: 458-60.

[38] Li Y, Won KJ, Kim DY, Bin KH, Kang HM, Lee SY, et al. Positive promoting effects of smilax China flower absolute on the wound healing/skin barrier repair-related responses of HaCaT human skin keratinocytes. Chem Biodivers. 2021; 18: e2001051.

[39] Llames SG, Del Rio M, Larcher F, García E, Escamez MJ, Jorcano JL, et al. Human plasma as a dermal scaffold for the generation of a completely autologous bioengineered skin. Transplantation. 2004; 77: 350-5.

[40] Carriel V, Garzón I, Jiménez JM, Oliveira ACX, Arias-Santiago S, Campos A, et al. Epithelial and stromal developmental patterns in a novel substitute of the human skin generated with fibrin-agarose biomaterials. Cells Tissues Organs. 2012; 196: 1-12.

[41] Liu T, Qiu C, Lu H, Li H, Zhu S, Ma L. A novel recombinant human collagen hydrogel as minced split-thickness skin graft overlay to promote full-thickness skin defect reconstruction. Burns. 2023; 49: 169-81.

[42] Braziulis E, Diezi M, Biedermann T, Pontiggia L, Schmucki M, Hartmann-Fritsch F, et al. Modified plastic compression of collagen hydrogels provides an ideal matrix for clinically applicable skin substitutes. Tissue Eng Part C Methods. 2012; 18: 464-74.

[43] Alexaline MM, Trouillas M, Nivet M, Bourreau E, Leclerc T, Duhamel P, et al. Influence of fibrin matrices and their released factors on epidermal substitute phenotype and engraftment. J Tissue Eng Regen Med. 2019; 13: 1362-74.