3D Bioprinted Hypertrophic Skin Scar Model

The three-dimensional in-vitro skin scar model developed by us successfully recapitulated the complex microarchitecture and pathophysiology of native scarred skin. The precise patterning of ECM using 3D Bioprinting technology, the cocktail of cytokines, and homogenous cell distribution allowed differentiation of dermal fibroblast to contractile myofibroblast. This demonstrated the hallmark features of scar tissue like an excessive contraction, synthesis of fibrotic extracellular matrix, and reduced MMP synthesis.

Actin staining demonstrating myofibroblast contraction

Characterization:

MTT assay: The basic principle of this assay lies in the reduction of tetrazolium dye to a purple-colored compound by NADPH-dependent cellular oxidoreductase enzyme produced during cellular metabolism. The 3D hypertrophic scar tissue exhibited enhanced metabolic activity, corroborating an essential parameter of enhanced cellular viability in the provided 3D microenvironment.

Hydroxyproline assay (Total Collagen content): The measured quantity of collagen in the fabricated tissue construct showed an increasing tendency, a desirable characteristic of scar tissue.

Cellular morphology: Demonstrated differentiation of dermal fibroblast to myofibroblasts indicated by spread morphology akin to native scar skin.

Gene expression: RT-PCR analysis revealed enhanced expression of ECM-specific genes (COL1, FN1), myofibroblast-specific genes (HDAC, TNC, SPARC), and a reduced expression pattern for matrix remodeling enzymes (MMP1, MMP13). The accelerated expression of SMAD4 and Beta-catenin genes validates the involvement of TGF-beta and Wnt signaling pathways in hypertrophic scar formation.

Protein expression: Immunohistochemical analysis reported increased expression of COL1, SPARC protein and alpha-SMA fiber protein.

3D contraction: The fibrotic scar tissue developed exhibited upregulated contractile property independent of the collagen presence.