Developing biodegradable scaffolds for tissue engineering of the urethra

Abstract

Objective: To develop a synthetic biodegradable alternative to using human allodermis for the production of tissue-engineered buccal mucosa for substitution urethroplasty, looking specifically at issues of sterilization and cell-seeding protocols and, comparing the results to native buccal mucosa.

Material and methods: Three methods of sterilization, peracetic acid (PAA), γ-irradiation and ethanol, were evaluated for their effects on a biodegradable electrospun scaffold of polylactide-co-glycolide (PLGA, 85:15), to identify a sterilization method with minimal adverse effects on the scaffolds. Two protocols for seeding oral cells on the scaffold were compared, co-culture of fibroblasts and keratinocytes on the scaffolds for 14 days, and seeding fibroblasts for 5 days then adding keratinocytes for a further 10 days. Cell viability and proliferation on the scaffolds, scaffold contraction and mechanical properties of the scaffolds with and without cells were examined.

Results: γ-irradiation and PAA sterilized scaffolds remained sterile for >3 months when incubated in antibiotic-free culture medium, while ethanol sterilized and unsterilized samples became infected within 2-14 days. All scaffolds showed extensive contraction (up to 50% over 14 days) irrespective of the method of sterilization or the presence of cells. All methods of sterilization, particularly ethanol, reduced the tensile strength of the scaffolds. The addition of cells tended to further reduce mechanical properties but increased elasticity. The cell-seeding protocol of adding fibroblasts for 5 days followed by keratinocytes for 10 days was the most promising, achieving a mean (sem) ultimate tensile stress of 1.20 (0.24) × 10⁵ N/m² compared to 3.77 (1.05) × 10⁵ N/m² for native buccal mucosa, and a Young's modulus of 2.40 (0.25) MPa, compared to 0.73 (0.09) MPa for the native buccal mucosa.

Conclusion: This study adds to our understanding of how sterilization and cell seeding affect the physical properties of scaffolds. Both PAA and γ-irradiation appear to be suitable methods for sterilizing PLGA scaffolds, although both reduce the tensile properties of the scaffolds. Cells grow well on the sterilized scaffolds, and with our current protocol produce constructs which have ≈ 30% of the mechanical strength and elasticity of the native buccal mucosa. We conclude that sterilized PLGA 85:15 is a promising material for producing tissue-engineered buccal mucosa.