3D biomaterial matrix to support long term, full thickness, immuno-competent human skin equivalents with nervous system components

Abstract

Current commercially available human skin equivalents (HSEs) are used for relatively short term studies (∼1 week) due in part to the time-dependent contraction of the collagen gel-based matrix and the limited cell types and skin tissue components utilized. In contrast, here we describe a new matrix consisting of a silk-collagen composite system that provides long term, stable cultivation with reduced contraction and degradation over time. This matrix supports full thickness skin equivalents which include nerves. The unique silk-collagen composite system preserves cell-binding domains of collagen while maintaining the stability and mechanics of the skin system for long-term culture with silk. The utility of this new composite protein-based biomaterial was demonstrated by bioengineering full thickness human skin systems using primary cells, including nerves and immune cells to establish an HSE with a neuro-immuno-cutaneous system. The HSEs with neurons and hypodermis, compared to in vitro skin-only HSEs controls, demonstrated higher secretion of pro-inflammatory cytokines. Proteomics analysis confirmed the presence of several proteins associated with inflammation across all sample groups, but HSEs with neurons had the highest amount of detected protein due to the complexity of the model. This improved, in vitro full thickness HSE model system utilizes cross-linked silk-collagen as the biomaterial and allows reduced reliance on animal models and provides a new in vitro tissue system for the assessment of chronic responses related to skin diseases and drug discovery.

Construction of a dermis–fat composite in vivo: Optimizing heterogeneous acellular dermal matrix with in vitro pretreatment

“…Previous studies have mainly used single hydrogel‐type materials as scaffolds such as a collagen–elastin matrix (Keck et al, ), fibrin hydrogel (Kober, Gugerell, Schmid, Kamolz, & Keck, ), human plasma hydrogel (Monfort, Soriano‐Navarro, García‐Verdugo, & Izeta, ), and collagen type I hydrogel (Birgit et al, ) to construct a dermis–fat composite. ADSCs cell sheets have been used to construct a three‐layer skin substitute containing subcutaneous fat in vitro without application of scaffold materials (Vidal et al, ); in another study, the dermis and hypodermis were constructed using a silk–collagen hydrogel and silk sponge respectively, and the two layers were then overlapped to form a full‐thickness skin equivalent (Bellas, Seiberg, Garlick, & Kaplan, ). Although these hydrogel‐based products are useful skin substitutes for skin biology research or cosmetic and pharmaceutical testing, their mechanical properties should be improved for in vivo applications.…”

Patient‐derived in vitro skin models for investigation of small fiber pathology

“…One group developed a hybrid model of human skin with axonal growth of rat DRG neurites into the dermis . In another study, the authors developed a skin model using neonatal foreskin fibroblasts and keratinocytes, coated with human‐induced neural stem cells . Recently, a fully human‐derived and innervated skin model using fibroblasts and keratinocytes from breast reductive surgeries was developed .…”

“…Recently, a fully human‐derived and innervated skin model using fibroblasts and keratinocytes from breast reductive surgeries was developed . The major drawbacks for the methodological transfer into clinical studies are the need of large skin samples, juvenile and highly proliferative cells as prerequisites, or hybrid models obtained from different species, which are either not practicable in clinical routine or does not reflect real‐life conditions. Moreover, these tools do not model disease conditions since skin cells and neurons derived from healthy donors were included.…”

The importance of the neuro‐immuno‐cutaneous system on human skin equivalent design

“…However, only one model system has addressed all components simultaneously. 16,22 However, the human-induced neural stem cells (hiNSCs) employed by these studies have additional considerations. First, although these are primary human-derived cells and they express several relevant neuronal markers, 23 an ideal innervation model would utilize human dorsal root ganglia, which are not readily accessible for in vitro research, thus presenting a challenge.…”