Microfluidic bioprinter for in situ patterning of engineered skin grafts

Cheng, Richard 1; Eylert, Gertraud 3; He, Sijin 2; Gariepy, Jean-Michel 2; Jeschke, Marc 3; Guenther, Axel 1, 2

1. Institute of Biomaterials & Biomedical Engineering, University of Toronto; 
2. Department of Mechanical & Industrial Engineering, University of Toronto; 
3. Ross Tilley Burn Centre, Sunnybrook Research Institute

The current standard of care for patients with severe burns lacking the dermal and epidermal layer is the application of acellular extracellular matrix (ECM) materials which provide a temporary barrier against infection, but drawbacks include the high cost of treatment and the need for multiple surgeries [1]. We report the development of a handheld bioprinter to organize fibrin and hyaluronic acid based soft biomaterials through a microfluidic device to pattern mesenchymal stem cells (MSCs) from human burn patients directly on the wound site. In contrast to alternative methods of cell transplantation like spraying keratinocytes [2] or injecting cell-laden microparticles [3], our approach of delivering cells within a patterned biomaterial enables reconstitution of the dermal-epidermal interfacial architecture of intact skin and utilizing ECM proteins critical during the wound healing process.

The handheld bioprinter contains on-board syringes driven by a stepper motor that controllably delivers cell-containing biomaterials through a 3D-printed microfluidic device to pattern sixteen 550µm wide, 150µm thick stripes in parallel to form a single layered sheet. Upon exiting the microfluidic device, the fibrinogen stripe gels within 25 seconds of contact with the thrombin crosslinker delivered simultaneously in laterally flanking channels. Optimization of the material concentration allows consistent patterning by limiting excessive diffusion of non-gelled biomaterials even on inclined surfaces, as demonstrated by a 33% increase in stripe width consistency at an angular deposition of 28 degrees compared to 4-times less material concentration. A 3.4 N-cm torsion spring and a two-degree of freedom printhead with 45/10 degrees of flexibility in the X/Y axis of rotation have also been incorporated to improve uniform deposition on non-homogenous surfaces and to mitigate the effects of hand-induced variations during the printing process. In vitro experiments where 2x10^6 cells/mL MSCs were patterned in tissue culture conditions resulted in 40% increased area coverage by three days, suggesting active cell migration and proliferation. In vivo experiments where cell-containing stripes were deposited on a porcine burn wound model led to complete granulation tissue formation after three weeks of treatment, supporting in situ patterning of cell-containing engineered skin grafts using the handheld bioprinter as a potential treatment for full-thickness burns.

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3. Griffin D, Weaver W, Scumpia P, Di Carlo D, Segura T. Accelerated wound healing by injectable microporous gel scaffolds assembled from annealed building blocks. Nature Materials 14(7):737-44, 2015