Medium flow rate regulates viability and barrier function of engineered skin substitutes in perfusion culture
Medium flow rate regulates viability and barrier function of engineered skin substitutes in perfusion culture
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DOI：
10.1089/tea.2007.0237
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发表时间：
2008-05-01
影响因子：
4.1
通讯作者：
Boyce, Steven T.
中科院分区：
文献类型：
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作者：
Kalyanaraman, Balaji;Supp, Dorothy M.;Boyce, Steven T.
工程组织的灌注培养可改善营养物质的传质，并为发育中的结构提供流动介导的机械刺激，从而改善其体外解剖学和生理学。在这项研究中，研究了在气液界面灌注中培养的工程皮肤替代品（ESS）对介质流速的反应。使用自体角质形成细胞、成纤维细胞和胶原糖胺聚糖 (GAG) 海绵制成的 ESS 在定制的循环生物反应器系统的气液界面上以 5、15 和 50 mL/min 的流速（每种条件 n = 8）孵育 21 天。使用组织学、3-(4,5-二甲基噻唑-2-基)-2,5-二苯基溴化四唑 (MTT) 测定、溴脱氧尿苷 (BrdU) 掺入和表面水合在体外评估 ESS。以 5 和 15 mL/min 孵育的 ESS 具有与静态条件下孵育的对照 ESS 相当的组织学组织。以 50 mL/min 孵育的 ESS 显示出杂乱无章的表皮替代物，并且在培养的后期时间点显示出真皮支架的更大降解。在培养第 14 天（平均值 +/- 标准误差平均值 1.63 +/- 0.11 对比 1.30 +/- 0.14，p < 0.05）和培养第 21 天（1.66 +/- 0.12 对比 1.11 +/- 0.15，p < 0.05），使用 MTT 测定测量的 ESS 中的细胞活力显着高于静态对照。以 15 mL/min 孵育的 ESS 活力与对照相当。在所有时间点，以 50 mL/min 孵育的 ESS 的活力均显着低于对照。 BrdU 掺入数据的结果显示，虽然 ESS 在 5 和 15 mL/min 下孵育与对照相当，但在 50 mL/min 下孵育的 ESS 在每个高倍视野中的增殖角质形成细胞比对照少（2.77 +/- 0.48 vs 28.1 +/- 0.78，p < 0.05）。以 5 mL/min 孵育的 ESS 具有与对照相当的表面水化，而在所有时间点以 15 mL/min 和 50 mL/min 孵育的 ESS 具有显着高于静态对照的表面水化。 ESS 以 5 mL/min 的流速孵育并移植到无胸腺小鼠的全层伤口上，结果显示伤口愈合与对照组相当。从这些结果可以得出结论，较低流速下的 ESS 灌注培养可增加细胞活力并维持适合移植的表皮屏障，而较高的流速会导致体外 ESS 解剖学和生理学的恶化。
Perfusion culture of engineered tissues improves mass transfer of nutrients and provides flow-mediated mechanical stimulation to the developing constructs, thereby improving their anatomy and physiology in vitro. In this study, the responses to medium flow rate of engineered skin substitutes (ESS) incubated in perfusion at the air-liquid interface were investigated. ESS fabricated with autologous keratinocytes, fibroblasts, and collagen-glycosaminoglycan (GAG) sponges were incubated for 21 days at the air-liquid interface in a custom-built recirculating bioreactor system at flow rates of 5, 15, and 50 mL/min (n = 8 per condition). ESS were evaluated in vitro using histology, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, bromodeoxyuridine (BrdU) incorporation, and surface hydration. ESS incubated at 5 and 15 mL/min had histological organization comparable with that of control ESS incubated in static conditions. ESS incubated at 50 mL/min displayed a disorganized epidermal substitute and, at later time points in culture, showed greater degradation of the dermal scaffold. Cell viability measured using MTT assay was significantly higher in ESS incubated at 5 mL/min than in static controls at day 14 (mean +/- standard error of the mean 1.63 +/- 0.11 vs 1.30 +/- 0.14, p < 0.05) and day 21 (1.66 +/- 0.12 vs 1.11 +/- 0.15, p < 0.05) of culture. Viability of ESS incubated at 15 mL/min was comparable with that of controls. ESS incubated at 50 mL/min had significantly lower viabilities than controls at all time points. Results of BrdU incorporation data showed that, although ESS incubated at 5 and 15 mL/min were comparable with controls, those incubated at 50 mL/min had fewer proliferating keratinocytes per high-power field than controls (2.77 +/- 0.48 vs 28.1 +/- 0.78, p < 0.05). ESS incubated at 5 mL/min had surface hydration comparable with that of controls, whereas those incubated at 15 mL/min and 50 mL/min had significantly higher surface hydration than static controls at all time points. ESS incubated at a 5 mL/min flow rate and transplanted onto full-thickness wounds on athymic mice demonstrated wound healing comparable with that of controls. From these results, it can be concluded that perfusion culture of ESS at lower flow rates increases cell viability and maintains an epidermal barrier suitable for grafting, whereas higher flow rates lead to deterioration of ESS anatomy and physiology in vitro.