Chen Ming, Michaud Heather, Bhowmick Sankha
Biomedical Engineering and Biotechnology Program, University of Massachusetts Dartmouth, North Dartmouth, MA 02747, USA.
J Biomech Eng. 2009 Jul;131(7):074521. doi: 10.1115/1.3173283.
A major challenge encountered in using electrospun scaffolds for tissue engineering is the non-uniform cellular distribution in the scaffold with increasing depth under normal passive seeding conditions. Because of the small surface pores, typically few microns in diameter, cells tend to congregate and proliferate on the surface much faster compared to penetrating the scaffold interior. In order to overcome this problem, we used a vacuum seeding technique on polycaprolactone electrospun scaffolds while using NIH 3T3 fibroblasts as the model cell system. This serves as a precursor to the bilayer skin model where the fibroblasts would be residing at an intermediate layer and the keratinocytes would be on the top. Vacuum seeding was used in this study to enhance fibroblasts seeding and proliferation at different depths. Our results show that the kinetics of cell attachment and proliferation were a function of varying vacuum pressure as well as fiber diameter. Cell attachment reached a maxima somewhere between 2-8 in. Hg vacuum pressure and fell for lower vacuum pressures presumably because of cell loss through the filtration process. Cell proliferation and collagen secretion over five days indicated that vacuum pressure did not affect cellular function adversely. We also compared the combined impact of scaffold architecture (400 nm versus 1100 nm average diameter fiber scaffolds) and vacuum pressure. At a given pressure, more cells were retained in the 400 nm scaffolds compared to 1100 nm scaffolds. In addition, the cell intensity profile shows cell intensity peak shift from the top to the inner layers of the scaffold by lowering the vacuum pressure from 0 in. Hg to 20 in. Hg. For a given vacuum pressure the cells were seeded deeper within the 1100 nm scaffold. The results indicate that cells can be seeded in electrospun scaffolds at various depths in a controlled manner using a simple vacuum seeding technique. The depth of seeding is a function of pressure and scaffold fiber diameter.
在组织工程中使用电纺支架时遇到的一个主要挑战是，在正常被动接种条件下，随着支架深度增加，细胞在支架内分布不均匀。由于表面孔隙小，直径通常只有几微米，与穿透支架内部相比，细胞往往更快地聚集并在表面增殖。为了克服这个问题，我们在聚己内酯电纺支架上使用了真空接种技术，同时使用NIH 3T3成纤维细胞作为模型细胞系统。这是双层皮肤模型的前身，其中成纤维细胞将位于中间层，角质形成细胞将位于顶部。本研究中使用真空接种来增强成纤维细胞在不同深度的接种和增殖。我们的结果表明，细胞附着和增殖的动力学是真空压力和纤维直径变化的函数。细胞附着在2 - 8英寸汞柱的真空压力之间达到最大值，而在较低真空压力下下降，可能是因为在过滤过程中细胞损失。五天内的细胞增殖和胶原蛋白分泌表明，真空压力不会对细胞功能产生不利影响。我们还比较了支架结构（平均直径400纳米与1100纳米的纤维支架）和真空压力的综合影响。在给定压力下，与1100纳米的支架相比，400纳米的支架中保留了更多细胞。此外，细胞强度分布图显示，通过将真空压力从0英寸汞柱降至20英寸汞柱，细胞强度峰值从支架顶部转移到内层。对于给定的真空压力，细胞在1100纳米的支架中接种得更深。结果表明，使用简单的真空接种技术，可以以可控方式将细胞接种到电纺支架的不同深度。接种深度是压力和支架纤维直径的函数。
