Wound Healing on Athymic Mice With Engineered Skin Substitutes Fabricated with Keratinocytes Harvested from an Automated Bioreactor

Abstract

The Kerator is a computer controlled bioreactor for the automated culture and harvest of keratinocytes that can reduce labor and materials involved in the fabrication of engineered skin substitutes (ESS). Previous studies have shown that Kerator is comparable to tissue culture flasks by keratinocyte confluence during culture, clonogenic potential of harvested keratinocytes and microanatomy, cell viability and surface hydration of ESS fabricated with the harvested keratinocytes. In this study, the Kerator and tissue culture flasks were further compared by keratinocyte proliferation in vitro and wound healing after transplantation of ESS to athymic mice. The number of BrdU positive keratinocytes in ESS fabricated with keratinocytes harvested from Kerator after 2 weeks of in vitro maturation was 34 ± 3 per high power field (hpf) (mean ± SEM), which was not significantly different from that fabricated with keratinocytes harvested from flasks (34 ± 1.5 per hpf). Percentage original wound area 6 weeks after surgery of ESS fabricated with keratinocytes from the Kerator was 36 ± 3.3%, which was not significantly different from that of ESS fabricated with keratinocytes from flasks (30 ± 4.3%). In both cases, 78% (7 of 9) mice transplanted were positive for engraftment of human keratinocytes by direct immunofluoresence for HLA-ABC antigens. These results further confirm that the ESS fabricated with keratinocytes harvested from Kerator and flasks are equivalent in vitro and in vivo. Therefore, use of Kerator for large scale production of ESS can lead to increased availability at reduced cost while maintaining ESS quality for grafting.

In Vivo Assessment of Printed Microvasculature in a Bilayer Skin Graft to Treat Full-Thickness Wounds

“…27 In studies that used human fibroblast and human keratinocytes in a collagenglycosaminoglycan sponge, the contraction was *64% after 6 weeks postgraft. 28 In the control group the percentage of the wound contraction slightly increased over time, as did those in the commercial skin graft. Again, this is expected and was found in previous research, which also documented a peak of wound contraction between the second and third week 28,29 and is attributed to the scar formation, which 30 we documented by histology.…”

“…28 In the control group the percentage of the wound contraction slightly increased over time, as did those in the commercial skin graft. Again, this is expected and was found in previous research, which also documented a peak of wound contraction between the second and third week 28,29 and is attributed to the scar formation, which 30 we documented by histology. 29 The wound contractions reported in this study are consistent with the previous studies done by Kalyanaraman & Boyce, who fabricated an ESS using a Kerator bioreactor.…”

“…29 The wound contractions reported in this study are consistent with the previous studies done by Kalyanaraman & Boyce, who fabricated an ESS using a Kerator bioreactor. 28 In those studies, a collagenglycosaminoglycan matrix was employed with seeded human fibroblast and human keratinocytes. Other studies performed with athymic mouse model revealed that when keratinocytes and fibroblast cells are seeded onto an acellular collagenglycosaminoglycan matrix augmented with vitamin C is implanted on a full-thickness wound, the wound contraction is *70% at week 6.…”

“…33 Other studies have shown that when a collagen matrix with keratinocytes and fibroblasts is implanted in a full-thickness wound, the epidermal and dermal layers are present in the neoskin. 28,29,34 There is clearly room for improvement in existing skin grafts, and adding endothelial cells to a network is a step in this direction. There is some evidence that the addition of endothelial vessel networks will achieve viable integration.…”

Epidermal cells delivered for cutaneous wound healing

“…Nevertheless, these processes are very intensive in terms of the labor and materials used, which limits the amount of engineered skin substitutes that can be fabricated at a given time, and also impacts the cost of the final product (96).…”