Cross-linked Collagen Hydrogel Matrix Resisting Contraction To Facilitate Full-Thickness Skin Equivalents

Abstract

Full-thickness skin equivalents are gathering increased interest as skin grafts for the treatment of large skin defects or chronic wounds or as nonanimal test platforms. However, their fibroblast-mediated contraction and poor mechanical stability lead to disadvantages toward their reproducibility and applicability in vitro and in vivo. To overcome these pitfalls, we aimed to chemically cross-link the dermal layer of a full-thickness skin model composed of a collagen type I hydrogel. Using a noncytotoxic four-arm succinimidyl glutarate polyethylene glycol (PEG-SG), cross-linking could be achieved in cell seeded collagen hydrogels. A concentration of 0.5 mg of PEG-SG/mg of collagen led to a viability comparable to non-cross-linked collagen hydrogels and no increased release of intracellular lactate dehydrogenase. Cross-linked collagen hydrogels were more mechanically stable and less prone to enzymatic degradation via collagenase when compared with non-cross-linked collagen hydrogels. Remarkably, during 21 days, cross-linked collagen hydrogels maintain their initial surface area, whereas standard dermal models contracted up to 50%. Finally, full-thickness skin equivalents were generated by seeding human epidermal keratinocytes on the surface of the equivalents and culturing these equivalents at an air-liquid interface. Immunohistochemical stainings of the cross-linked model revealed well-defined epidermal layers including an intact stratum corneum and a dermal part with homogeneously distributed human dermal fibroblasts. These results indicate that cross-linking of collagen with PEG-SG reduces contraction of collagen hydrogels and thus increases the applicability of these models as an additional tool for efficacy and safety assessment or a new generation of skin grafts.

Bio‐Orthogonally Crosslinked, In Situ Forming Corneal Stromal Tissue Substitute

“…From the release of EGF from the matrices by enzymatic degradation, the physical gels released most of their EGF cargo within 1 hr, and SPAAC-crosslinked gels were still slower to release EGF, although no difference was seen between the two concentrations (Figure S1B). Chemical crosslinking is known to increase resistance to enzymatic degradation by collagenase, [15] and thus our results indicate that the SPAAC reaction effectively crosslinks collagen.…”

“…[15] The transparency of SPAAC gel was increased compared to the non-crosslinked collagen by reducing the fibrillar structure formation through chemical crosslinking, which has been posited in other work as the reason that crosslinked collagen matrix is more transparent than non-crosslinked collagen gels. [15] In addition, the groups conjugated to collagen could alter collagen’s physical properties, which was seen when the azide-conjugated collagen did not form a gel at room temperature that normally forms with the same concentration of unconjugated collagen upon neutralization.…”

Construction of low contracted 3D skin equivalents by genipin cross‐linking

“…For example, the Graftskin decreased its thickness from 2.3 mm to 0.5‐1 mm, indicating the 57%‐78% of gel contraction during its construction . On the other hand, introducing large amount of synthetic polymers such as PEG into collagen, though completely inhibited the contraction of skin equivalents, possibly impaired the wound healing as skin grafts, because the nonprotein components would hamper the development of new dermal layers . Nevertheless, the genipin cross‐linked skin equivalents still showed the limitation as skin grafts due to visible light blue tint by genipin treatment (Figure B).…”

“…Although the concentrated collagen, plastic compressed collagen and de‐epidermized dermis have been used to reduce the contraction, the severe contracts still existed by up to 60% . Introducing other polymers (eg, PEG) into skin equivalents, though effectively reduced the contraction, might delay the wound healing via slowing down the collagen remodelling and replacement . Relatively, the chemical cross‐linking of collagen scaffolds by small molecules might be an effective strategy in reducing the contraction without introducing noncollagen components into skin equivalents, but the residual of cross‐linkers (eg, glutaraldehyde) is possible to elicit potential cytotoxicity…”

Three‐Dimensional (3D) cell culture monitoring: Opportunities and challenges for impedance spectroscopy

“…Nevertheless, compared to EIS, additional information can be gathered using EIT including the position and potentially the size of spheroids and tissues in culture systems. Given the issue of contraction in some collagen‐based 3D models, as well as the presence of contraction in wound healing models (Lotz et al, 2017), a potential application for EIT could be to monitor contraction over time or after wounding in these models. Others have designed similar set ups to those demonstrated in Figure 7, with the eventual aim of monitoring cell cultures, but have not yet tested it using cells (Xu et al, 2018).…”