Quick Layer-by-Layer Assembly of Aligned Multilayers of Vascular Smooth Muscle Cells in Deep Microchannels
Bypass grafting is a common procedure used in cases of arterial disease. However, the availability of suitable native arteries/veins is limited, particularly when the patient has undergone the procedure before. The use of synthetic prostheses has not been met with much success for small diameter grafts, and so it is desirable for tissue engineering to produce blood vessels. The major challenge in this case is to reproduce the heterogeneous but well-organized 3D microstructure in human tissue. Native blood vessels usually consist of many layers of vascular smooth muscle cells (SMCs) that are aligned circumferentially. Reproducing this in vivo is a major challenge of tissue engineering.
Physical restrictions on SMCs during their culture has been shown to achieve aligned cellular microstructure. Thus, Feng et al. attempted to use microchanneled scaffolds, essentially wells with thin wall widths in contrast to a fairly wide and deep well on which to culture SMCs on. First, it was shown that monolayers of SMCs grew well in the microchannels and aligned uniformly when the layer was confluent. This required manipulating the channel widths to one where the cells grew well in. After this, a layer-by-layer (LBL) process was attempted. In the past, SMCs has been shown to grow when encapsulated in a collagen type I hydrogel, but these SMCs had a much lower proliferation and alpha-actin expression. Thus, the idea here was to use the hydrogel in thin layers to separate confluent layers of SMCs. When the first layer of SMCs were confluent, a layer of hydrogel was applied, upon which more SMCs were seeded. In this paper, four layers were attempted, which survived even after six days. In an extension to this, the hydrogel step was skipped, and five layers of SMCs survived for also the required six days.
Different initial cell seeding densities of SMCs for each layer was also explored. When the cell seeding density reached 200000 cells/cm2, the time until the layer reached confluence was about half a day. This allows a thick layer of SMCs to be produced quickly, and the implication for clinical applications is apparent. This paper showed that a simple technique, such as the LBL, may go a long way toward creating blood vessel grafts. The authors showed that cells between the channels were connected by some cells that would grow over the walls, ensuring that cell-cell interaction was still present. The authors also proposed further work on creating the scaffold with different degradable materials. When the perfect material is achieved, hopefully timed to the growth of the SMCs, a good multilayer of SMCs will be easily producible, all perfectly aligned. This paper was also very interesting, in that many techniques used were similar to those done in the 115 labs. Simple live/dead assays with Trypan Blue was used, as well as staining of F-actin and alpha-actin to determine alignment of the expressed proteins.
15 comments:
I'm confused about Terry's comment. Isn't the point of the whole thing to create vascularization? Aren't there already empty spaces in the layers?
Did they see how thick or thin the layers could be in order to optimize cell proliferation, viability, etc? And how did they get the interfaces of each layer to stick together? I don't know much about the hydrogels, but since there's usually a force in blood vessels, could it cause the gels to slide with each other? Or would the cells from the layers eventually grow on top of another?
The paper proposes to create blood vessels of small diameter, and so only several layers of cells were used. Vascularization is probably not needed in such a case. However, to use this technique to specifically create empty spaces in a tissue is certainly a possibility, but the Layer-by-layer technique described in the paper would take an awfully long time to create a significantly sized tissue.
The walls of the micropattern are to help align the cells. Eventually, the walls can be degraded, and the cells should proliferate into the gap, creating a large aligned blood vessel.
The layers in the paper are all monolayers of cells. The optimal well width have already been determined in another paper this group cited. The gels I believe are very thin so as to allow another layer of cells to grow on them. The idea is that the cells in each monolayer is held together via the ECM the cells normally develop and each layer is held together by the hydrogel. This Layer-by-layer method is superior to growing cells in a 3D gel since the cell growth in those are shown to be hindered.
Here is the link to the article on pubmed.
Was alpha-actin or other gene expression affected when using the LBL technique to grow smooth muscle cells or was this problem completely resolved by their method?
Does this paper mention anything about dealing with in vivo reactions with the scaffold?
such as thrombosis or plasma protein deposition on SMCs layers?
Were any tests done to see if this system would be potentially able to withstand fluid mechanics/forces?
In this situation, why would a product created by cells be superior to something that's made out of more resilient synthetic materials?
Do you think the KISS (keep it simple stupid) principle could have applied here in that a synthetic approach could have been easier?
In my opinion, I am forseeing numerous headaches not only in growing the smooth muscle cells but also characterizing them under different stresses and conditions. Of course, in the interest of progress it would be much cooler to have a product made of only cells...but would that product function better?
This paper explored creating the 3D structure of SMCs. Although alpha-actin expression might have changed, it should not be an issue as the final aim is to create a structure similar to that of blood vessels.
The experiments did not proceed to in vivo testing, as it was only an exploration of the LBL method.
No studies were done on the mechanical properties of the final product. However, as the final aim is to create an exact copy of a capillary, that should not be much of an issue, as in the final product, there is only cells and no scaffold.
In the beginning of the paper, the authors have already discussed other current methods, including synthetic approaches, which were met with limited success. That is part of the impetus for pursuing this project.
After LBL was imposed, were proteins and such able to diffuse through separate layers? In other words, were cells from different layers possible be able to "communicate" with each other?
This paper did not explore those areas.
Did they speculate how this layer-by-layer procedure might work on different cells such as endothelial cells? From what I know, standard materials to engineer blood vessels are not used for small diameter vessels because protein and platelet deposition will end up clogging the vessel. I specifically ask about endothelial cells because the innermost layer of endothelial cells in a blood vessel has been characterized in preventing this deposition from happening. So obviously to make a final small diameter blood vessel product, a similar layer of endothelial cells is important.
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