Fibrin Based Biomaterial Organization Using Thermoplastic Microfluidic Device

Cheng, Richard 1 ;  Hakimi, Navid 2 ; Guenther, Axel 1, 2

1. Institute of Biomaterials & Biomedical Engineering, University of Toronto; 2. Department of Mechanical & Industrial Engineering, University of Toronto

Burn injuries affect more than 1.3 million people in Canada, leading to an economic burden of $366 million annually. The current gold standard in surgical practice is the use of split-surface autographs, allographs, or skin substitutes such as Integra, but limitations range from the lack of tissue organization, the potential for immunological rejection, and the need for high quantities of donated tissue. 3D printers that utilize patient cells to print biomaterials mimicking the architecture of human skin are emerging as potential solutions for burn victims, but major drawbacks of commercially-available bioprinters include low throughput and the inability to create complex three-dimensional structures from soft biomaterials. 

At the Guenther laboratory, we have made progress in addressing these issues by constructing a microfluidic-chip containing handheld bioprinter which enables the patterning of cell-embedded, multilayered hydrogels in a single continuous process directly on the wound site. Design aspects of the microfluidic device include the incorporation of on-chip Strook mixers to control onset of fibrin gelation, symmetrical branching architecture optimized to minimize sheet inconsistency, and multilayered bonding to enable production of a bilayered sheet mimicking the native skin organization. The use of thermoplastics as the material to fabricate these microfluidic devices were selected to enable manufacturing techniques such as hot embossing, such that these bioprinting cartridges can be disposed and replaced after every use. We have developed a deep reactive ion etching protocol to fabricate silicon wafers with positive sidewall profiles for demoulding of thermoplastic materials following the embossing procedure. After device bonding using thermal and plasma treatment, the devices were tested for burst pressure and capacity to extrude fibrin-based biomaterials, with comparable results with traditional PDMS and 3D-printed devices. 

We hypothesize that that the in-situ deposition of bilayered biomaterials containing dermal fibroblasts and keratinocytes organized into striped patterns will conserve the starting quantity of cells while maximizing cell proliferation for efficient wound coverage. To more accurately model the dermis and epidermis architecture of the mammalian skin, dermal fibroblasts and keratinocytes were embedded in fibrin hydrogels and co-extruded in separate layers using a microfluidic device. To maximize conservation of patient-derived cells, the keratinocytes were prepared in distinct concentrations to facilitate optimal cell clustering and proliferation as assayed using K14 and Ki67 immunostaining. Cells were patterned in stripes with optimized gap distances to generate maximum wound area coverage within a three-day recovery time frame. Taken together, we have demonstrated the capacity of thermoplastic microfluidic devices to organize fibrin-based biomaterials to conserve keratinocytes for skin tissue engineering.