Introduction:: Small-diameter synthetic vascular grafts (SDSVGs) are in critical need due to the limited availability of viable autologous grafts in some patient populations. However, SDSVGs suffer from low patency rates, limiting their utility in the clinic. Thrombosis and neointimal hyperplasia, cause the majority of SDVG failures. Both biochemical processes interact profoundly with endothelial cell signaling, which can determine whether the resultant graft-tissue-blood interaction is salubrious or pathologic. Introducing a layer of endothelial cells on the graft luminal surface alters the surface’s interaction with blood and should result in higher patency rates for SDVGs. Therefore, measuring endothelialization, which includes the attachment, migration, proliferation, and extracellular matrix (ECM) deposition of endothelial cells, is a critical step for evaluating potential biomaterials for use as SDSVGs. Currently, there is no standard testing procedure for measuring endothelialization. Here, we propose an endothelialization measurement workflow for biomaterials that is automated and scalable. We utilize the workflow to measure the endothelialization of polyvinyl alcohol (PVA) exposed to different reactive ion plasma (RIP) treatments. This workflow offers an automated, standardized, unbiased, and efficient process for measuring and comparing endothelialization of current and potential SDSVG materials.
Materials and Methods:: PVA samples were prepared and RIP treated with one of 3 carrier gases (oxygen, nitrogen, or argon) at one of 2 radiofrequency powers (50 W or 100 W) for 1 minute under 100 mTorr of pressure. A culture insert (ibidi USA, Fitchburg, WI) was placed on each sample. The samples were placed into six-well plates, and human umbilical vein endothelial cells (HUVECs) were seeded onto the PVA samples at a seeding density of ~208,000 cells/cm2 (~2,000,000 cells). The Etaluma LS720 (Etaluma, Carlsbad, CA) microscope with a 20x objective was used to capture brightfield and fluorescence images. The LS720’s automated stage reliably returns to pre-programmed positions, allowing multiple locations on the same sample to be imaged repeatedly. HUVECs were imaged 2 hours after seeding, washed 4 times using phosphate-buffered saline (PBS) 24 hours after seeding, imaged after the wash, and imaged 48 hours after seeding. Next, removal of the culture insert creates a cell free area, which was imaged every 10 minutes for 48 hours. Bromodeoxyuridine (brdU) incorporation was then performed (Cell Signaling Technology, Danvers, MA) with a 24-hour incorporation time. A plate reader was used to obtain absorbance values. The surfaces were decellularized and immunofluorescence staining was performed for 1 of 3 ECM components (collagen I, laminin, or fibronectin). The deposited ECM components were then quantified using fluorescence microscopy. ImageJ was used for the postprocessing of images and was automated to provide live/dead cell counts, wound area quantification, healing rate, and fluorescence intensity quantification.
Results, Conclusions, and Discussions:: Results and Discussion: The overall automated workflow is shown in Figure 1. biomaterial characterization, in this case optical profilometer data, shown in Phase 1 indicates that the RIP treatment with Argon at 100W, Nitrogen at 100W, and Oxygen at 100W resulted in a change in surface texture. Phase 2 outputs show cells attaching to the surface, reducing in quantity after the wash, and increasing in quantity during the 24 hours after the wash. Bar graphs indicate the number of live cells present for each image shown. Although cells were manually counted, an automated cell counter script is being written. Further validation of the workflow including assays measuring the soluble factors associated with proliferation are currently underway.
Conclusion: We have demonstrated that that workflow can be used to automatically image cells. The resulting workflow provides an automated, repeatable, and scalable process for measuring endothelialization of vascular biomaterials.
