Booth Catherine, Korossis Sotiris A, Wilcox Helen E, Watterson Kevin G, Kearney John N, Fisher John, Ingham Eileen
Division of Microbiology, University of Leeds, UK.
J Heart Valve Dis. 2002 Jul;11(4):457-62.
Several deficiencies in current heart valve prostheses make them problematic for use in younger patients. Tissue valve substitutes are non-viable with a life expectancy of only 10-15 years, while mechanical valves require long-term anti-coagulation therapy. A solution to these problems would be to develop a tissue-engineered heart valve containing autologous cells, enabling the valve to maintain its biochemical and mechanical properties, yet grow with the patient. The study aim was to optimize a protocol to produce a porcine acellular matrix scaffold for use in developing a tissue-engineered heart valve.
Fresh porcine aortic valve leaflets were treated with Triton X-100, sodium dodecyl sulfate (SDS), sodium deoxycholate, MEGA 10, TnBP, CHAPS, and Tween 20, over a range of concentrations, in the presence of protease inhibitors for up to 72 h. Histological analysis was used to detect the major structural proteins of the heart valve, collagen I, elastin and glycosaminoglycans.
After 72 h, most protocols resulted in the retention of large numbers of whole cells and cell fragments. Only SDS (0.03-1%) or sodium deoxycholate (0.5-2%) resulted in total decellularization at 24 h. Histological analysis of acellular matrices showed that the major structural proteins had been retained and appeared to be intact.
Protocols utilizing SDS or sodium deoxycholate were successful for leaflet decellularization, and histological analysis showed that the major structural components of the valve matrix had been maintained. These methods are being developed further with a view to reseeding with autologous cells to produce tissue-engineered solutions for clinical implantation.
当前心脏瓣膜假体存在的若干缺陷使其在年轻患者中的应用存在问题。组织瓣膜替代品无法存活,预期寿命仅为10至15年,而机械瓣膜则需要长期抗凝治疗。解决这些问题的一个办法是开发一种含有自体细胞的组织工程心脏瓣膜,使瓣膜能够维持其生化和机械性能,并随患者生长。本研究的目的是优化一种方案,以制备用于开发组织工程心脏瓣膜的猪脱细胞基质支架。
在蛋白酶抑制剂存在的情况下,将新鲜猪主动脉瓣叶用不同浓度的 Triton X - 100、十二烷基硫酸钠(SDS)、脱氧胆酸钠、MEGA 10、三丁基膦(TnBP)、3 - [(3 - 胆酰胺丙基)二甲氨基] - 1 - 丙磺酸(CHAPS)和吐温20处理长达72小时。采用组织学分析来检测心脏瓣膜的主要结构蛋白,即胶原蛋白I、弹性蛋白和糖胺聚糖。
72小时后,大多数方案导致大量完整细胞和细胞碎片的残留。只有SDS(0.03 - 1%)或脱氧胆酸钠(0.5 - 2%)在24小时时导致完全脱细胞。脱细胞基质的组织学分析表明,主要结构蛋白得以保留且似乎完好无损。
使用SDS或脱氧胆酸钠的方案成功实现了瓣叶脱细胞,组织学分析表明瓣膜基质的主要结构成分得以保留。这些方法正在进一步研发,以期重新接种自体细胞,生产用于临床植入的组织工程解决方案。