title: The effect of ascorbic acid and fluid flow stimulation on the mechanical properties of a tissue engineered pelvic floor repair material.
abstract: Synthetic non-degradable meshes used in pelvic floor surgery can cause serious complications such as tissue erosion. A repair material composed of an autologous oral fibroblast seeded degradable polylactic acid scaffold may be a viable alternative. The aims of this study were to investigate the effects of media supplementation with additives (ascorbic acid-2-phosphate, glycolic acid and 17-β-oestradiol) on the mechanical properties of these scaffolds. Oral fibroblasts were isolated from buccal mucosa. The effects of the three additives were initially compared in two-dimensional culture to select the most promising collagen stimulating additive. Sterile electrospun scaffolds were seeded with 500,000 oral fibroblasts and fixed in 6-well plates and subjected to ascorbic acid-2-phosphate (the best performing additive) and/or mechanical stimulation. Mechanical stimulation by fluid shear stress was induced by rocking scaffolds on a platform shaker for 1 h/day for 10 of 14 days of culture. In two-dimensional culture, ascorbic acid-2-phosphate (concentrations from 0.02 mM to 0.04 M) and glycolic acid (10 µM) led to significantly greater total collagen production, but ascorbic acid-2-phosphate at 0.03 mM produced the greatest stimulation (of the order of >100%). In three-dimensional culture, mechanical stimulation alone gave non-significant increases in stiffness and strength. Ascorbic acid-2-phosphate (0.03 mM) significantly increased collagen production in the order 280% in both static and mechanically stimulated scaffolds (p < 0.0001). There was no additional effect of mechanical stimulation. Dense collagen I fibres were observed with ascorbic acid-2-phosphate supplementation. Uniaxial tensiometry showed that strength (p < 0.01) and stiffness (p <0.05) both improved significantly. A combination of ascorbic acid-2-phosphate and mechanical stimulation led to further non-signficant increases in strength and stiffness. In conclusion, a pelvic floor repair material with improved mechanical properties can be developed by supplementing culture media with ascorbic acid-2-phosphate to increase collagen I production. Future studies will assess the change in mechanical properties after implantation in an animal model.
authors.affiliation: Kroto Research Institute, The University of Sheffield, Sheffield, UK Department of Urology, Royal Hallamshire Hospital, Sheffield, UK nadir.osman@sheffield.ac.uk.
authors.forename: Nadir I
authors.initials: NI
authors.lastname: Osman
authors.affiliation: Kroto Research Institute, The University of Sheffield, Sheffield, UK.
authors.forename: Sabiniano
authors.initials: S
authors.lastname: Roman
authors.affiliation: Kroto Research Institute, The University of Sheffield, Sheffield, UK.
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Bullock
authors.affiliation: Department of Urology, Royal Hallamshire Hospital, Sheffield, UK.
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Chapple
authors.affiliation: Kroto Research Institute, The University of Sheffield, Sheffield, UK.
authors.forename: Sheila
authors.initials: S
authors.lastname: MacNeil
mesh_terms: D001205:Ascorbic Acid / Q000494:pharmacology*; D001672:Biocompatible Materials / Q000737:chemistry*; D001696:Biomechanical Phenomena / Q000187:drug effects / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D024042:Collagen Type I / Q000032:analysis / Q000378:metabolism; D055119:Elastic Modulus / Q000187:drug effects; D004958:Estradiol; D005347:Fibroblasts / Q000187:drug effects; D006016:Glycolates; D006801:Humans; D008422:Materials Testing; D013526:Surgical Mesh; D023822:Tissue Engineering / Q000379:methods; D054457:Tissue Scaffolds
chemical_list: D001672:Biocompatible Materials; D024042:Collagen Type I; D006016:Glycolates; C031149:glycolic acid; D004958:Estradiol; D001205:Ascorbic Acid
keywords: Artificial tissue (biomechanics); biodegradable materials; biomechanical; culture techniques; scaffold development (tissue engineering); testing/analysis; tissue