Fabrication of collagen scaffolds using collagen isolated from Aplysia californica

Abstract

Event Abstract Back to Event Fabrication of collagen scaffolds using collagen isolated from Aplysia californica Victoria A. Webster1, Katherine J. Chapin1, Ozan Akkus1, 2, 3, Hillel J. Chiel2, 4, 5 and Roger D. Quinn1 1 Case Western Reserve University, Mechanical and Aerospace Engineering, United States 2 Case Western Reserve University, Biomedical Engineering, United States 3 Case Western Reserve University, Orthopaedics, United States 4 Case Western Reserve University, Biology, United States 5 Case Western Reserve University, Neurosciences, United States Introduction: Electrocompacted collagen has extensive versatility as a cell culture scaffold. Using electrocompaction techniques, aligned fibers can be produced which natively promote attachment and alignment of cells[1]. Such aligned collagen scaffolds have previously been used to fabricate organic, locomoting devices powered by cardiomyocytes[2]. However, previously developed biohybrid devices have relied on mammalian or avian cells for actuation. These cells require a narrow range of physiological conditions to function, greatly reducing their utility in non-physiological environments (i.e. environmental testing). In comparison, Aplysia californica functions across a wider range of conditions[3], potentially providing a source of more robust actuating cells. To utilize such cells, an appropriate scaffold is needed. Collagen has been isolated from the skin of Aplysia californica, and can be fabricated into ELAC scaffolds for use in biohybrid devices. Methods: Skin was harvested from Aplysia californica (Marinus Scientific) and rinsed with deionized water. Pepsin solubilized collagen (PSC) was extracted using a procedure adapted from Mizuta et al[4]. The PSC was then collected via salting out and dialyzed against several changes of deionized water over 18 hrs. Electrocompaction of the dialyzed collagen was performed at 15 V for 30 seconds, using a dual wire electrode set-up[2]. Additionally, collagen was gelled at 25o C (Fig. 1). Figure 1. Collagen is isolated from the skin of the Aplysia and isolated, yielding a viscous solution which can be compacted into ELAC threads (top-right), or gelled at 25o C (bottom-right). Both electrochemically aligned collagen (ELAC) threads and collagen gels were then cross-linked using a combination of 1-ethyl-3-(3-dimethylaminopropyl carboiimide) and N-hydroxy succinimide (EDC-NHS, Sigma) [5]. The cross-linked threads were tested in tension until failure. Commercially derived collagen (Collagen Solutions, CA) was processed in the same way and used as a benchmark of performance. Results: Using the techniques described, collagen has been isolated from the skin of Aplysia californica. Isolation resulted in a yield of 1.8% of the initial wet skin weight. The isolation process resulted in a viscous collagen solution at a concentration of 5 mg/ml which was capable of compaction resulting in aligned threads (Fig. 2). Compacted threads had a load to failure of 0.096 ± 0.034 N in tension, undergoing a strain of 19.1 ± 7.13. Commercially derived collagen threads had a higher load to failure of 0.94 ± 0.34 N. Gelled collagen resulted in robust hydrogels. Figure 2. Polarized light microscopy of electrocompacted Aplysia collagen threads shows that the collagen is aligned along the axis of the thread as indicated by the blue coloration and double headed arrow. Discussion: Isolating collagen from the skin of Aplysia californica resulted in a higher yield than reported for material isolation from the mantle of the oyster Crassostrea gigas[4]. While the strength of the Aplysia collagen threads was lower than that of the telo-collagen threads, the Aplysia collagen was isolated using pepsin digestion rather than acid extraction, which may cause more damage to the collagen, resulting in lower strength threads. However, the Aplysia collagen was still capable of compaction into robust ELAC threads. Therefore, it may be applicable as a scaffold for Aplysia muscle powered biohybrid devices. Victoria Webster and Katherine Chapin contributed equally to this work. This material is based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. DGE-0951783. This study was also funded in part by grants from the National Science Foundation (Grant Number DMR-1306665) and National Institute of Health (Grant Number R01 AR063701). Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation.