Human endothelial cell growth on mussel-inspired nanofiber scaffold for vascular tissue engineering

Authors: Sook Hee Ku and Chan Beum Park

Introduction

Small diameter vascular grafts can be improved by seeding them with endothelial cells in order to increase the patency of the graft and therefore effectively prevent thrombosis, which is a common problem with grafts in tiny vessels. The seeded endothelial cells increase vessel integrity by releasing certain factors that prevent thrombogenesis. This paper has set up experiments to determine if certain ECM-like substrates increase human umbilical vein endothelial cells' (HUVECs) adhesion and viability. The main substrates they tested were uncoated polycaprolactone (PCL) nanofibers, gelatin coated PCL nanofibers and poly(dopamine) (PDA) coated PCL nanofibers. PCL was used due to its biodegradability and good mechanical properties although it lacks cell adhesive properties on its own, while PDA was used because it mimics the adhesion mechanism of mussels through the inclusion of catecholamine moieties as adhesive agents. Gelatin coating is currently a widely used method for promoting cell adhesion so it was incorporated onto PCL fibers to compare the effectiveness of gelatin versus PDA as adhesive agents. The team then went further and looked at how changing the substrate material from PCL to a variety of commonly used substrates but still coating each with PDA affected cell viability and cytoskeleton development.

Cell viability was determined by culturing cells on the various substrates and then performing a Live/Dead cell assay as well as a MTT assay. Cell adhesion was determined through looking at cytoskleton organization in the endothelial cells by way of actin staining with rhodamine-phalloidin. Finally, to analyze functional development of the endothelial cells they looked at the expression of two markers: platelet endothelial cell adhesion molecule (PECAM-1) and von Willebrand factor (vWF).

Results

Figure 1 shows the results of how they effectively coated PCL nanofibers with both gelatin and PDA. Fig. 1A is a schematic of how the cells will spread more readily on PDA coated vs. uncoated nanofibers. Fig. 1B shos SEM images of the uncoated, gelatin coated and PDA coated (from left to right) PCL nanofibers. Fig. 1C shows Raman shift spectra of the three different substrates, with the peaks on the PDA coated PCL nanofibers (bottommost spectra) demonstrating aromatic properties within the substrate that is a consequence of the addition of aromatic PDA. Fic 1D shows how the addition of gelatin and PDA coating changes the formerly hydrophobic PCL solution into a hydrophilic solution with a water contact angle of zero. All of these figures together demonstrate that they were able to effectively coat the PCL nanofibers as they intended.

Figure 2 shows results of HUVEC viability on the three different substrates: in all three figures the leftmost data point is unmodified PCL fibers, the middle point is gelatin-coated PCL fibers and the rightmost is PDA coated PCL nanofibers. Looking at the morphology and number of the cells from a live/dead assay in Fig. 2A it can be determined that not only are there more cells as you progress from left to right but also the cells are spreading out indicating better attachment and growth, especially on the PDA coated PCL nanofibers. Fig. 2B shows simply the number of live cells per a given area on all three substrates, with the number of cells on PDA coated PCL fibers being significantly higher. Fig. 2C shows the results of an MTT assay, which is another viability measurement that looks at enzymatic activity that showed that there is an almost five-fold increase in viability for cells on the PDA coated nanofibers in comparison to the gelatin-coated and unmodified nanofibers. All three results indicate greatly increased viability of HUVECs on PDA coated PCL nanofibers.
Figure 3 shows both cytoskeleton organization on the three different substrates as well as the functional development of the HUVECs by looking at two specific commonly known markers of endothelial cells: PECAM-1 and vMF. Fig. 3A shows the increasing amount of cytoskleton development as you go from the relatively low levels of actin in the HUVECs cultured on unmodified PLG fibers to the intricate actin networks shown in cells cultured on PDA coated PLG nanofibers. Also, the assay showed a very narrow spherical morphology for the cells on unmodified fibers which represents no adhesion/no spreading of the cells while the cells on the PDA coated fibers show an attached/spread out morphology. Fig. 3B shows the presence of specific endothelial cell markers, namely PECAM-1 and vWF (shown in green fluorescence). PECAM-1 is located at cell-cell interfaces and in cell membranes, and it can be seen that it is much more prevalent in the cells on the PDA coated PLG fibers, indicating more cell-cell adhesion and normal endothelial cell functionality. vWF is produced in the cytoplasm of endothelial cells and the paper indicated that since it is more spread out within the cytoplasm of cells on PDA coated PLG fibers these cells interact better with their scaffold than do the cells on uncoated fibers. These two figures indicate that cells both attach more effectively and proliferate better on PDA coated PLG fibers than on unmodified or gelatin-coated fibers, although gelatin-coated fibers still offer a significant improvement over unmodified fibers.
Figure 4 shows the results of testing PDA and gelatin coating on different substrates other than PLG nanofibers. Fig. 4A shows actin staining of the cytoskeleton of cells on unmodified substrates (top row), gelatin coated substrates (middle row) and PDA coated substrates (bottom row). There is a noticeable difference in both the larger number of cells that show up for PDA coated substrates as well as the increased amount of cytoskeleton development in these cells. Cell viability was looked at using MTT assays and the results are shown in Fig. 4B: for all substrates other than glass there is a large increase in cell viability for cells cultured on PDA coated substrates versus those cultured on gelatin coated substrates. This final set of results shows that substrates other than PLG nanofibers can be coated with PDA and still show significant improvement in terms of HUVEC attachment and viability.

Critique
Overall this paper did a thorough job of looking at multiple factors to reach their conclusions: not only did they look at cell viability using two assays (live/dead and MTT) but they also stained for cytoskeleton development and the presence of specific markers indicating functional development of the endothelial cells. From the cumulative results of all of these assays they demonstrated that PDA coating is an effective method for increasing HUVEC viability and attachment to specific substrates, most notably to PLG nanofibers. I think they may have stretched too far by looking at multiple coated substrates however: since the majority of the paper focused on looking at PLG nanofibers as a specific scaffold for vascular grafts they should have limited their results to looking at purely the PLG nanofibers and then followed up this paper with another that looks more in depth at all of the various substrates that they kind of just threw in at the end here. Also, the results involving endothelial cell markers seemed less valid and conclusive than their other results: to me the vWF markers did not look significantly more distributed or numerous for the PDA coated PLG fibers than for the other two substrates, there were just more cells in that particular image for the PDA coated fibers.