Development of 3D microvascularized gelatin hydrogel for skin tissue engineering

Abstract

Event Abstract Back to Event Development of 3D microvascularized gelatin hydrogel for skin tissue engineering Jung Bok Lee1, 2, Shannon Faley2, Hak-Joon Sung1 and Leon M. Bellan1, 2 1 Vanderbilt University, Biomedicl engineering, United States 2 Vanderbilt University, Mechanical Engineering, United States Introduction: Numerous strategies have been approached to regenerate skin defects resulting from burns, trauma, or non-self-healing chronic wounds.[1][2] Among the various types of scaffolds employed, hydrogels, consisting of natural or synthetic polymers with high water content, have been widely used as engineered tissue constructs for skin regeneration due to their tunable properties to mimic extracellular matrix (ECM).[3][4][5] Despite of continuous progress, a significant challenge still remains in using hydrogels to produce tissue constructs due to inefficient delivery of nutrients and oxygen to cells embedded deep within the hydrogels. The aim of the current study is to investigate whether perfusion through microfluidic hydrogels improves cell survival in co-culture of human umbilical vein endothelial cells (HUVECs) and human neonatal dermal fibroblasts (HNDF), an in vitro model of skin tissue. We fabricated 3D microvascular networks in gelatin hydrogels using thermo-responsive sacrificial poly(N-isopropylacrylamide) (PNIPAM) microfibers. These microchannels allow constant perfusion of culture media throughout the hydrogel, and significantly improve the viability and proliferation of embedded HUVECs and HNDF. These perfused co-culture models will prove useful in highlighting key factors associated with the development of 3D vascularized, tissue engineered construct for skin regeneration. Materials and Methods: To make microchannels in gelatin hydrogels, PNIPAM fibers were obtained by high-speed spinning of 50% w/v PNIPAM solution in methanol. To fabricate cell-laden gels, 10 % w/v gelatin in PBS was prepared and mixed with red fluorescent protein-expressing HNDF (RFP-HNDF) (5x106 cells/ml) and 2.5 w/v% microbial transglutaminase (mTG), a crosslinking mediator. Gelatin solution containing RFP-HNDF and mTG was cast into PDMS molds with spun PNIPAM fibers and gelled with crosslinking at 37 ºC. PNIPAM fibers were then removed by continuously shaking and perfusing DMEM into the hydrogel at room temperature, thereby introducing microchannels. Next, a high concentration of HUVECs constitutively expressing green fluorescent protein (GFP-HUVEC) were injected into the microchannels in suspension (seeding density: 5 million cells in 100 μl) to seed the inner channel surface. The gelatin hydrogel was then connected to a peristaltic pump to apply gentle media flow. Results and Discussion: The microchannels were interconnected with formation of a network, as visualized by perfusion of Fluospheres into macrochannels in the hydrogel (Fig 1A). The media perfusion allowed HNDF to spread, elongate and form cell networks after 7 days of culture (Fig. 1C) whereas the lack of media perfusion inhibited this HNDF behavior (Fig.1B). The media perfusion enhanced exchange of necessary soluble compounds, as seen from cell viability data. Conclusion: This study demonstrated the feasibility of making PNIPAM microfibers and using these as sacrificial templates to obtain interconnected microchannels in gelatin scaffolds. Cell culture studies showed improved cell viability and stretched cell morphology with cell network formation in this perfused system. The results suggest this perfusable gelatin hydrogel system supports co-cultured HUVECs and HNDF cells, indicating significant potential to apply for engineering soft tissues including skin tissues. Figure 1. A confocal microscope image of microchannels in the gelatin hydrogel (3D image) (A) and confocal microscope images of HUVECs (green) and HNDF (red) in the gelatin hydrogel without perfusion (B) and with perfusion (C) after 7 days culture. NIH R00EB013630 (to L.M.B); AHA Grant-in-Aid 15GRNT25710148 (to L.M.B. and H-J. S.); NSF 1506717 (to H-J. S. and L.M.B.); NIH EB019509 (to H-J.S)