Department of Urology, Menoufia Teaching Hospital, Menoufia, Egypt.
BJU Int. 2011 Jan;107(2):296-302. doi: 10.1111/j.1464-410X.2010.09310.x.
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.
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.
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.
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.
开发一种合成可生物降解的替代物，以替代使用人类异体真皮来生产组织工程颊黏膜，用于替代尿道成形术，特别关注灭菌和细胞接种方案的问题，并将结果与天然颊黏膜进行比较。
评估了三种灭菌方法，过氧乙酸（PAA）、γ-辐照和乙醇，以确定对聚乳酸-共-乙醇酸（PLGA，85:15）的可生物降解电纺支架具有最小不利影响的灭菌方法。比较了两种在支架上接种口腔细胞的方案，即成纤维细胞和角质形成细胞在支架上共培养 14 天，以及先接种成纤维细胞 5 天，然后再添加角质形成细胞 10 天。检查了细胞在支架上的活力和增殖、支架收缩和有/无细胞时支架的机械性能。
γ-辐照和 PAA 灭菌的支架在无抗生素的培养基中孵育时可保持无菌>3 个月，而乙醇灭菌和未灭菌的样品在 2-14 天内被感染。所有支架均表现出广泛的收缩（14 天内高达 50%），无论灭菌方法或细胞存在与否。所有的灭菌方法，特别是乙醇，都降低了支架的拉伸强度。添加细胞往往会进一步降低机械性能，但会增加弹性。先接种成纤维细胞 5 天，然后再接种角质形成细胞 10 天的细胞接种方案最有希望，达到 1.20（0.24）×10⁵ N/m²的平均（标准差）最大拉伸应力，而天然颊黏膜为 3.77（1.05）×10⁵ N/m²，杨氏模量为 2.40（0.25）MPa，而天然颊黏膜为 0.73（0.09）MPa。
本研究增加了我们对灭菌和细胞接种如何影响支架物理性能的理解。过氧乙酸（PAA）和γ-辐照似乎都是 PLGA 支架的合适灭菌方法，尽管两者都降低了支架的拉伸性能。细胞在经过消毒的支架上生长良好，并且根据我们目前的方案，所产生的构建体具有约 30%的天然颊黏膜的机械强度和弹性。我们得出结论，消毒的聚乳酸-共-乙醇酸 85:15 是一种有前途的生产组织工程颊黏膜的材料。
