Effective use of mesenchymal stem cells in human skin substitutes generated by tissue engineering

Abstract

Mesenchymal stem cells (MSCs) can differentiate toward epithelial cells and may be used as an alternative source for generation of heterotypical artificial human skin substitutes, thus, enhancing their development and translation potential to the clinic. The present study aimed at comparing four types of heterotypical human bioengineered skin generated using MSCs as an alternative epithelial cell source. Adipose-tissue-derived stem cells (ADSCs), dental pulp stem cells (DPSCs), Wharton's jelly stem cells (WJSCs) and bone marrow stem cells (BMSCs) were used for epidermal regeneration on top of dermal skin substitutes. Heterotypic human skin substitutes were evaluated before and after implantation in immune-deficient athymic mice for 30 d. Histological and genetic studies were performed to evaluate extracellular matrix synthesis, epidermal differentiation and human leukocyte antigen (HLA) molecule expression. The four cell types differentiated into keratinocytes, as shown by the expression of cytokeratin 10 and filaggrin 30 d post-grafting; also, they induced dermal fibroblasts responsible for the synthesis of extracellular fibrillar and non-fibrillar components, in a similar way among each other. WJSCs and BMSCs showed higher expression of cytokeratin 10 and filaggrin, suggesting these cells were more prone to epidermal regeneration. The absence of HLA molecules, even when the epithelial layer was differentiated, supports the future clinical use of these substitutes-especially ADSCs, DPSCs and WJSCs-with low rejection risk. MSCs allowed the generation of bioengineered human skin substitutes with potential clinical usefulness. According to their epidermal differentiation potential and lack of HLA antigens, WJSCs should preferentially be used.

Evaluation of the optical and biomechanical properties of bioengineered human skin generated with fibrin-agarose biomaterials

“…Once primary cell cultures of human skin keratinocytes and fibroblasts were obtained, bioengineered tissues were generated using fibrin-agarose biomaterials following previously published methods. 10,[23][24][25][26][27] Briefly, human plasma was used as a source of fibrin and 0.1% agarose, tranexamic acid, and calcium chloride were added and immediately aliquoted on culture plates. Four types of samples were used in this study: (1) acellular fibrin-agarose substitutes (AS), in which fibrin-agarose biomaterials were generated without cells; (2) dermal skin (DS) substitutes consisting of 800,000 human skin fibroblasts immersed within fibrin-agarose biomaterials;…”

Generation of a novel human dermal substitute functionalized with antibiotic-loaded nanostructured lipid carriers (NLCs) with antimicrobial properties for tissue engineering

“…Generation of non-functionalized human dermal skin substitutes (SS) by tissue engineering Primary cell cultures of dermal broblast cells were obtained from skin biopsies taken from healthy donors. Biopsies were carefully rinsed in phosphate-buffered saline and enzymatically digested in a 2 mg∕ml Clostridium histolyticum collagenase I (Gibco -Thermo Fisher Scienti c, Waltham, MA) solution at 37 °C for 6 h to obtain primary cell cultures of skin broblasts following previously described protocols (14,15). Isolated human dermal broblasts were then cultured in Dulbecco's modi ed Eagle's medium -DMEM-(Merck Life Science, St. Louis, MO) supplemented with 10% fetal bovine serum (Merck Life Science) and 1% antibiotics/antimycotics (100 U/mL penicillin G, 100 mg/ mL streptomycin and 0.25 mg/mL amphotericin B; Merck Life Science) under standard cell culture conditions.…”

“…Bioengineered human dermal skin substitutes (SS) were generated using brin-agarose biomaterials as previously described (7,9,11,14,15,28). In brief, the following components were mixed per each ml of dermal skin substitute: 760 µl of human plasma -as a brin source-, 75 µl of DMEM containing 140,000 cultured human broblasts, 15 µl of tranexamic acid -as anti brinolytic agent-(Amcha brin, Fides-Ecofarma, Valencia, Spain), 50 µl of a 2% solution of type VII agarose (Merck Life Science) melted in PBS, and 100 µl of 1% CaCl 2 solution (Merck Life Science) -to promote brin polymerization-.…”

“…Among the different biomaterials that showed preclinical and clinical usefulness for the generation of human tissue substitutes, brin-agarose hydrogels show excellent biocompatibility and biomechanical properties and its porous brillar pattern allows diffusion and interchange of oxygen and nutrients (2,3,5,6). Fibrin-agarose was previously used to generate bioarti cial substitutes of the human cornea (7), sclera (8), oral mucosa (9), palate (10), nerve (11,12), cartilage (13) and skin (14,15). In fact, a bioarti cial model of tissue-engineered human skin (UGRSKIN), consisting of a dermal skin substitute based on brin-agarose biomaterials and dermal broblasts and an overlying epithelial layer, has been successfully translated to the clinical setting (16).…”

Tissue engineering, university and health system. History of an experience

“…Fruto de esta actividad investigadora ha sido la generación de distintos tejidos artificiales como córnea (14) , piel (15,16), mucosa oral (17), nervio periférico (18), paladar (19), cartílago (20), etc. utilizando distintos tipos celulares, biomateriales , procesos de biofabricación (21, 22, 23) y protocolos de control de calidad de los que se ha dado cuenta en publicaciones científicas (24,25,26,27,28,29,30).…”