Skin Equivalent Tissue-Engineered Construct: Co-Cultured Fibroblasts/ Keratinocytes on 3D Matrices of Sericin Hope Cocoons

Abstract

The development of effective and alternative tissue-engineered skin replacements to autografts, allografts and xenografts has became a clinical requirement due to the problems related to source of donor tissue and the perceived risk of disease transmission. In the present study 3D tissue engineered construct of sericin is developed using co-culture of keratinocytes on the upper surface of the fabricated matrices and with fibroblasts on lower surface. Sericin is obtained from “Sericin Hope” silkworm of Bombyx mori mutant and is extracted from cocoons by autoclave. Porous sericin matrices are prepared by freeze dried method using genipin as crosslinker. The matrices are characterized biochemically and biophysically. The cell proliferation and viability of co-cultured fibroblasts and keratinocytes on matrices for at least 28 days are observed by live/dead assay, Alamar blue assay, and by dual fluorescent staining. The growth of the fibroblasts and keratinocytes in co-culture is correlated with the expression level of TGF-β, b-FGF and IL-8 in the cultured supernatants by enzyme-linked immunosorbent assay. The histological analysis further demonstrates a multi-layered stratified epidermal layer of uninhibited keratinocytes in co-cultured constructs. Presence of involucrin, collagen IV and the fibroblast surface protein in immuno-histochemical stained sections of co-cultured matrices indicates the significance of paracrine signaling between keratinocytes and fibroblasts in the expression of extracellular matrix protein for dermal repair. No significant amount of pro inflammatory cytokines (TNF-α, IL-1β and nitric oxide) production are evidenced when macrophages grown on the sericin matrices. The results all together depict the potentiality of sericin 3D matrices as skin equivalent tissue engineered construct in wound repair.

Silkworm Sericin: Properties and Biomedical Applications

“…Nayak et al [164] constructed 3D porous sericin matrices using genipin as crosslinked and used chitosan matrices as control, with the objective of developing an effective tissue-engineered skin replacement. Histological analysis indicates a multilayered stratified epidermal layer of keratinocytes, demonstrating that sericin matrices form epidermal and dermal components in vitro .…”

Non‐animal models of wound healing in cutaneous repair: In silico, in vitro, ex vivo, and in vivo models of wounds and scars in human skin

“…To overcome the limitations of single cell models, several three‐dimensional in vitro models have been utilised . Fibroblasts are seeded in a type I collagen solution which is solidified to gel, resulting in cells being embedded in a three‐dimensional collagen lattice that is then transferred to a culture dish and covered with medium . This model enables the evaluation of cell contraction and matrix compaction.…”

Are melanocortin peptides future therapeutics for cutaneous wound healing?

“…Three‐dimensional in vitro models, for example skin equivalents, may further be used to corroborate such findings employing collagen matrices and more than one cutaneous cell type . Indeed, mouse models into which excisional wounds are inflicted would be the next step to validate promising in vitro data.…”