Curtil A, Pegg D E, Wilson A
Medical Cryobiology Unit, University of York, United Kingdom.
Cryobiology. 1997 Feb;34(1):13-22. doi: 10.1006/cryo.1996.1982.
When freeze-dried cardiac valves have been implanted they remained acellular. This study is the initial step in the development of a method designed to repopulate the substance of the freeze-dried valve with fibroblasts and the lumenal surface with endothelial cells. In this scheme, the freeze-drying process performs three functions; it provides a porous matrix, it kills the donor cells, and it preserves the collagen structure and hence the mechanical strength of the valve. This paper describes the production of appropriate porosity in freeze-dried porcine pulmonary valve leaflets. We found that Tg' for this material is -83 degrees C, which made it impracticable to freeze-dry exclusively from the glassy state. Uncontrolled freeze-drying produced a variable structure with most of the pores considerably smaller than the desired size and a dense layer, apparently devoid of perforations, on the surface. Compacted layers also occurred within the substance of the leaflets. These appearances suggested that extensive collapse had occurred during the drying process. Variation of the cooling rate, the primary drying temperature, and the warming rate during secondary drying enabled us to identify the following conditions that provided satisfactory internal porosity: cooling at 5 degrees C/min, vacuum drying for 6 h at -20 degrees C, and secondary drying for 10 h during rewarming at 0.06-0.08 degrees C/min. The internal cavities measured 100-350 microns2 by ca. 400 microns2, which is adequate to provide access for the fibroblasts (cross-sectional area ca. 150-200 microns2 when rounded but fusiform when attached. However, the internal porous structure rarely communicated with the surface and mechanical perforation was required to provide continuity between the surface and the internal sponge. The resulting method provides a basis for studies of cell colonization.
当植入冻干心脏瓣膜时，它们会保持无细胞状态。本研究是开发一种方法的第一步，该方法旨在用成纤维细胞重新填充冻干瓣膜的实质，并在内腔表面覆盖内皮细胞。在这个方案中，冻干过程有三个作用：它提供了一个多孔基质，杀死了供体细胞，并保留了胶原蛋白结构，从而保持了瓣膜的机械强度。本文描述了在冻干猪肺动脉瓣叶中产生合适孔隙率的方法。我们发现这种材料的玻璃化转变温度（Tg'）为-83℃，这使得仅从玻璃态进行冻干变得不切实际。不受控制的冻干产生了一种可变结构，大部分孔隙比所需尺寸小得多，并且在表面有一层明显没有穿孔的致密层。在瓣叶实质内也出现了压实层。这些现象表明在干燥过程中发生了广泛的塌陷。通过改变冷却速率、一次干燥温度和二次干燥期间的升温速率，我们确定了以下能提供令人满意内部孔隙率的条件：以5℃/分钟的速度冷却，在-20℃下真空干燥6小时，以及在以0.06 - 0.08℃/分钟的速度升温期间进行10小时的二次干燥。内部腔室的尺寸约为100 - 350微米²乘以约400微米²，这足以让成纤维细胞进入（成纤维细胞呈圆形时横截面积约为150 - 200微米²，但附着时呈梭形）。然而，内部多孔结构很少与表面连通，需要进行机械穿孔以在表面和内部海绵之间提供连续性。所得方法为细胞定植研究提供了基础。
