Highly Elastic Biomaterials for the Lower Urinary Tract: Materials Testing and In vitro Analysis
Renea Sturm, M.D.1, Yadi Huo, B.S.2, Felix Yiu, B.S.1, Yimin Gu, B.S.2, Ronak Afshari, Ph.D.2, Mahsa Ghovvati, Ph.D.2, Nasim Annabi, Ph.D.2.
1Department of Urology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA, 2Department of Chemical Engineering, University of California Los Angeles, Los Angeles, CA, USA.
BACKGROUND: Commonly applied biologic scaffolds have significantly higher stiffness than native urethral tissue. An example is one-ply small intestinal submucosa (SurgisisTM) which has a mean tensile strength of 5MPa, approximately 10-fold higher than healthy urethral tissue (Huo/Sturm). The mechanical niche is an important parameter controlling cell fate and extracellular matrix (ECM) deposition. Additionally, tissue mismatch of vastly different substrates can minimize early functionality and increase regional wound tension when applied in reconstructive procedures. The aim of this study was to engineer and complete an in vitro analysis of a novel biomimetic and biocompatible scaffold that mimics the mechanical properties of lower urinary tract tissue.
Methods: Hybrid nanofibrous scaffolds were synthesized by electrospinning varied concentrations of two naturally derived polymers in random orientation onto a mat: elastin-like polypeptide (ELP) and gelatin methacryloyl (GelMA) to achieve target mechanical properties for 1) soft (tensile modulus, TM: 50-250kPa) and 2) soft and elastic (300-500% extensibility) scaffolds, respectively. Scaffolds were characterized using NMR spectroscopy, degradation, swelling, tensile and suture testing, light and scanning electron microscopy. Human bladder-derived urothelial and smooth muscle cells, and neonatal foreskin fibroblasts (ATCC) were seeded in mono cell culture conditions using cell-specific media directly onto 1cm2 scaffolds in 24-well plates at a 1x104 cells/mL concentration. A systematic evaluation of cell viability (Presto blue), adhesion/spreading (Actin/dapi), cytotoxicity and proliferation (Live/dead, counts) using light/fluorescent and confocal microscopy occurred between day 1 and 7 endpoints. T-tests were used for pairwise comparisons; one-way ANOVA to evaluate between-group differences.
Results: The material composition and fabrication process were optimized to achieve target mechanical properties for each cohort (Fig A-E). Specifically, percent extensibility significantly increased with the substitution of 3 to 7% ELP for GelMA weight/volume (w/v) versus 10% GelMA alone (A). Furthermore, 5% GelMA/5% ELP w/v achieved TM and ultimate tensile strength in static (B-C) and cyclic (D-E) testing that did not significantly differ from anterior urethral or bladder tissue in the New Zealand White rabbit and was consistent with soft and elastic target parameters. Scaffolds were suturable (F), with improved performance in suture tensile testing with the addition of ELP. Excellent cell viability was observed from day 1 to 7 across cell lines and scaffolds (H-J). Proliferation and spreading of SMCs (G) and fibroblasts was robust, while urothelium demonstrated less scaffold adherence and proliferation as compared to the other two cell lines.
Conclusion: Highly elastic engineered GelMA/ELP hybrid scaffolds are suitable substrates for bladder smooth muscle and fibroblast cell line proliferation. Future scaffold modifications are ongoing to optimize parameters for urothelial proliferation, and to evaluate the effects of mechanical properties on in vitro ECM production and mechanotransduction of lower urinary tract cell lines. In the future, these scaffolds may provide an alternative strategy for suturable, elastic lower urinary tract replacement tissue that can restore early functionality in hypospadias and complex stricture repairs.
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