Translation of complex 3D microarchitecture of native Extracellular matrix into our in vitro 3D model using state-of-the-art Direct Write Assembly technique allows RegenArtis to achieve first-mover advantage in India for developing 3D Bioprinted skin disease models. Our team tactfully utilizes the bio-mimetic properties of our biomaterial in combination with different cell types (including the patient’s own cells) and a wide range of biochemical stimulators to closely recapitulate the anatomy and pathophysiology of the diseased tissue niche.
Optimization of rheological properties of the biomaterial to ensure smooth extrusion plays an integral role in bioink development. Silk fibroin is an FDA-approved protein-based biomaterial purposed for tissue engineering applications. However, the rheological characteristics of standalone silk fibroin render it unsuitable for direct writing applications due to lack of shear-thinning behavior, a pre-requisite for smooth extrusion. The addition of gelatin to silk-fibroin imparts a shear-thinning nature to the polymer blend and enhances cellular attachment along with quicker gelation kinetics.
Proprietary Silk fibroin-Gelatin bioink
The three-dimensional computer-aided design (CAD) model was generated using Robocad V 4.0 software to simulate the required topographical features and structural dimension. The optimized bioink consisting of cells and biochemical mediators is loaded onto the syringe at a temperature around 22 ± 20C and printed at a speed of 1mm/s and 25 ± 2 psi pressure. 3D Bioprinting technology provides the necessary structural, mechanical and biochemical cues through precise deposition of cells, and patterning the surrounding matrix enables the homogenous distribution of cells throughout the tissue construct.
Homogeneous cell distribution in 3D Bioprinted construct
Live/dead assay of the 3-dimensional tissue construct using ethidium bromide, and calcein demonstrated high cell viability and homogenous cellular distribution in the construct during the entire period of cell culture. While the fibroblasts dwelled in the deeper regions of the construct, the keratinocytes showed efficient migration towards the pores until day 14 and wholly covered them by day 21. Furthermore, morphological analysis by SEM revealed extended morphology in cells constituting the scar model with multiple nuclei and fibrotic matrix deposition in differentiated myofibroblasts, a salient characteristic of scar tissue cellular architecture. Immunofluorescence imaging was utilized to evaluate the expression of tissue-specific biomarkers directly from the construct itself. For example, the expressions of COL1 and FN1 (ECM-specific proteins) and myofibroblast differentiation biomarker Alpha-SMA exhibited high-intensity fluorescence in the 3-dimensional tissue construct under the influence of a cocktail of cytokines elucidating their role in the fabrication of 3D in-vitro skin scar model.
Immunofluorescence staining of fibroblast cells
Direct 3D bioprinted full thickness skin constructs recapitulate regulatory signaling pathways and physiology of human skin, P Admane, AC Gupta, P Jois, S Roy, CC Lakshmanan, B Bandyopadhyay, S Ghosh, Bioprinting, 15, 2019, e00051
Regulation of fibrotic changes by the synergistic effects of cytokines, dimensionality and matrix: towards the development of an in vitro human dermal hypertrophic scar model, S Chawla, S Ghosh, Acta Biomaterialia, 2018, 69, 131-145
Establishment of in vitro model for corneal scar pathophysiology, S Chawla, S Ghosh, Journal of Cellular Physiology, 2018, 233(5), 3817-3830
- 3D bioprinted scar tissue model:
– World Patent WO 2019/106695, published on 6 June 2019;
– Indian Patent Application Number 201711043083;
– European Patent application number 18882941.0, filed on 26 June 2020;
– US application number 16/768,819 filed on 1 June 2020 (priority date 30 Nov 2017)