Consolo F, Brizzola S, Tremolada G, Grieco V, Riva F, Acocella F, Fiore G B, Soncini M
Dipartimento di Elettronica, Informazione e Bioingegneria, Politecnico di Milano, Italy.
Department of Health, Animal Science and Food Safety, Surgery Unit, Faculty of Veterinary Medicine, Università delgi Studi di Milano, Italy.
J Tissue Eng Regen Med. 2016 Feb;10(2):E101-12. doi: 10.1002/term.1767. Epub 2013 Jun 4.
A combined physical-chemical protocol for whole full-thickness bladder decellularization is proposed, based on organ cyclic distention through repeated infusion/withdrawal of the decellularization agents through the urethra. The dynamic decellularization was intended to enhance cell removal efficiency, facilitating the delivery of detergents within the inner layers of the tissue and the removal of cell debris. The use of mild chemical detergents (hypotonic solution and non-ionic detergent) was employed to limit adverse effects upon matrix 3D ultrastructure. Inspection of the presence of residual DNA and RNA was carried out on decellularized matrices to verify effective cell removal. Histological investigation was focused on assessing the retention of adequate structural and functional components that regulate the biomechanical behaviour of the acellular tissue. Biomechanical properties were evaluated through uniaxial tensile loading tests of tissue strips and through ex vivo filling cystometry to evaluate the whole-organ mechanical response to a physiological-like loading state. According to our results, a dynamic decellularization protocol of 17 h duration with a 5 ml/min detergent infusion flow rate revealed higher DNA removal efficiency than standard static decellularization, resulting in residual DNA content < 50 ng/mg dry tissue weight. Furthermore, the collagen network and elastic fibres distribution were preserved in the acellular ECM, which exhibited suitable biomechanical properties in the perspective of its future use as an implant for bladder augmentation.
本文提出了一种用于全层膀胱脱细胞的物理化学联合方案，该方案基于通过尿道反复注入/抽出脱细胞剂对器官进行循环扩张。动态脱细胞旨在提高细胞去除效率，促进洗涤剂在组织内层的递送以及细胞碎片的清除。使用温和的化学洗涤剂（低渗溶液和非离子洗涤剂）以限制对基质三维超微结构的不利影响。对脱细胞基质进行残留DNA和RNA检测，以验证细胞是否有效去除。组织学研究重点评估调节无细胞组织生物力学行为的适当结构和功能成分的保留情况。通过组织条带的单轴拉伸加载试验和离体充盈膀胱测压法评估生物力学性能，以评估全器官对类似生理加载状态的机械反应。根据我们的结果，持续17小时、洗涤剂注入流速为5毫升/分钟的动态脱细胞方案显示出比标准静态脱细胞更高的DNA去除效率，导致残留DNA含量低于50纳克/毫克干组织重量。此外，无细胞细胞外基质中的胶原网络和弹性纤维分布得以保留，从其未来用作膀胱扩大植入物的角度来看，该基质具有合适的生物力学性能。
