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      3D bioprinting of functional human skin: production andin vivoanalysis

      , , , ,
      Biofabrication
      IOP Publishing

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          Abstract

          Significant progress has been made over the past 25 years in the development of in vitro-engineered substitutes that mimic human skin, either to be used as grafts for the replacement of lost skin, or for the establishment of in vitro human skin models. In this sense, laboratory-grown skin substitutes containing dermal and epidermal components offer a promising approach to skin engineering. In particular, a human plasma-based bilayered skin generated by our group, has been applied successfully to treat burns as well as traumatic and surgical wounds in a large number of patients in Spain. There are some aspects requiring improvements in the production process of this skin; for example, the relatively long time (three weeks) needed to produce the surface required to cover an extensive burn or a large wound, and the necessity to automatize and standardize a process currently performed manually. 3D bioprinting has emerged as a flexible tool in regenerative medicine and it provides a platform to address these challenges. In the present study, we have used this technique to print a human bilayered skin using bioinks containing human plasma as well as primary human fibroblasts and keratinocytes that were obtained from skin biopsies. We were able to generate 100 cm2, a standard P100 tissue culture plate, of printed skin in less than 35 min (including the 30 min required for fibrin gelation). We have analysed the structure and function of the printed skin using histological and immunohistochemical methods, both in 3D in vitro cultures and after long-term transplantation to immunodeficient mice. In both cases, the generated skin was very similar to human skin and, furthermore, it was indistinguishable from bilayered dermo-epidermal equivalents, handmade in our laboratories. These results demonstrate that 3D bioprinting is a suitable technology to generate bioengineered skin for therapeutical and industrial applications in an automatized manner.

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          Most cited references44

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          Printing and prototyping of tissues and scaffolds.

          New manufacturing technologies under the banner of rapid prototyping enable the fabrication of structures close in architecture to biological tissue. In their simplest form, these technologies allow the manufacture of scaffolds upon which cells can grow for later implantation into the body. A more exciting prospect is the printing and patterning in three dimensions of all the components that make up a tissue (cells and matrix materials) to generate structures analogous to tissues; this has been termed bioprinting. Such techniques have opened new areas of research in tissue engineering and regenerative medicine.
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            Three clonal types of keratinocyte with different capacities for multiplication.

            Colony-forming human epidermal cells are heterogeneous in their capacity for sustained growth. Once a clone has been derived from a single cell, its growth potential can be estimated from the colony types resulting from a single plating, and the clone can be assigned to one of three classes. The holoclone has the greatest reproductive capacity: under standard conditions, fewer than 5% of the colonies formed by the cells of a holoclone abort and terminally differentiate. The paraclone contains exclusively cells with a short replicative lifespan (not more than 15 cell generations), after which they uniformly abort and terminally differentiate. The third type of clone, the meroclone, contains a mixture of cells of different growth potential and is a transitional stage between the holoclone and the paraclone. The incidence of the different clonal types is affected by aging, since cells originating from the epidermis of older donors give rise to a lower proportion of holoclones and a higher proportion of paraclones.
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              Progress and opportunities for tissue-engineered skin.

              Tissue-engineered skin is now a reality. For patients with extensive full-thickness burns, laboratory expansion of skin cells to achieve barrier function can make the difference between life and death, and it was this acute need that drove the initiation of tissue engineering in the 1980s. A much larger group of patients have ulcers resistant to conventional healing, and treatments using cultured skin cells have been devised to restart the wound-healing process. In the laboratory, the use of tissue-engineered skin provides insight into the behaviour of skin cells in healthy skin and in diseases such as vitiligo, melanoma, psoriasis and blistering disorders.
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                Author and article information

                Journal
                Biofabrication
                Biofabrication
                IOP Publishing
                1758-5090
                March 01 2017
                December 05 2016
                : 9
                : 1
                : 015006
                Article
                10.1088/1758-5090/9/1/015006
                27917823
                d57f8301-b399-4798-ad1d-8c16b1e3d6d4
                © 2016

                http://iopscience.iop.org/info/page/text-and-data-mining

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