The major obstacle to using skin cell sheets in clinical practices is their size limit5. In real applications, the skin cell sheets need to be at least ten times larger than their current size for full wound area coverage to promote effective treatment6,7. To overcome this issue, we first considered constructing a larger cell sheet by using a larger surface area, because the area of the temperature-responsive culture surface directly affects the size of the harvested cell sheets. However, with an increased temperature-responsive culture surface size, a significantly large number of cells were required to obtain the same seeding density, which was very labor intensive and prone to technical errors. We, therefore, modified our protocol by reducing the cell density and allowing the cells to proliferate for a few days until confluence. Unfortunately, by increasing the cell culture time on the temperature-responsive polymer, the cells attached too strongly to the surface, making detaching the cells as an intact sheet challenging. During the process of cell sheet harvesting, the cell sheet was broken into smaller (Supplementary S3). Thus, we proposed the use of nylon dressing or MEEK gauzes as cell sheet supporting material during cell sheet harvesting and for cell sheet expansion. Nylon is an inexpensive material and currently used as wound dressing, while MEEK pre-folded gauze is currently used in severe burn patients, giving a high re-epithelialization rate 12,17,19. To verify their potential application with cell sheet engineering, we investigated the qualities and behavior of fibroblast cell sheet on both fabrics.
Our results showed that the cell sheets could firmly attach to the nylon dressing and MEEK gauzes with no adhesives (Figs. 3 and 4). More importantly, the fibroblast cell sheets on the MEEK gauzes did not detach from the gauze, even after being cut and stretched. The strong attachment of the cell sheets to both materials was possibly due to the adhesive protein that was harvested together with the cell sheets20. Normally, in the MEEK technique for skin grafts, the proprietary glue, Humeca® glue, is required to keep the donor skin sample in place during the cutting process and is critical to the outcome of the skin graft transfer21. Fortunately, with the adhesive protein harvested with the cell sheet, Humeca® glue was unnecessary. This would be particularly beneficial in reducing the preparation steps in the protocol and there would be no concern over the effect of the synthetic adhesive glue on the cell viability and migration.
Besides the cell attachment, both the nylon and MEEK fabrics were biocompatible with the cells and could clearly support the cell growth. The fibroblast cells on the nylon support grew well for over 7 days, and the outgrowth cells were healthy. For the MEEK samples, we only evaluated the cell viability at the expansion ratio of 1:9, as a representative for all the expansion ratio. The cell viability was found to be over 95% and the cells at the edge migrated and proliferated into healthy cells. Hence, we can assume that even though cutting the cell sheets might have caused the cell damage around the edges, it did not have any effects on the cell proliferation and migration. According to our previous study, the fibroblast cell sheets could help accelerate the wound healing process by releasing essential cytokines and growth factors that regulated the wound repair5. A higher cell number would lead to more cytokine and growth factor secretion, possibly leading to faster wound healing.
The migration patterns between the fibroblasts on the nylon dressing and MEEK gauze were quite similar, as the cells migrated from the periphery of the cell sheets and preferentially moved along the matrix fibers (Fig. 5). The aligned fibers could induce cellular elongation and the alignment of collagen secretion by fibroblast, guiding the migration of the cells to the defect area 22,23. According to the migration movie clips (Supplementary S2), individual fibroblasts initially detach themselves from the monolayer and moved away in a non-coherent pattern, but directed towards free space. The migration of fibroblasts occurring earlier on nylon dressing, as compared to MEEK gauze, was possibly the result of the lower cell density at the periphery of the cell sheets. At a lower cell density, stronger cell-substrate interactions overcome cell-cell interactions, allowing the cells to escape the monolayer24. On the other hand, in all MEEK conditions, single cells took longer to detach from the dense cells at the edge of the monolayer due to strong cell-cell interactions, leading to immediate net movement at earlier time points.
Our result showed that the migration rates of fibroblasts on the MEEK gauzes were predominantly higher than on the nylon dressing. Even though both fabrics were made of the same polymer, which is polyamide, the topography of the substrates and densities of the fibers were clearly different. These factors have been previously shown to affect the cell-substrate adhesion properties25, which directly influences the cell motility26. In addition, cutting the cell sheets into small cell sheet islands in the modified MEEK technique resembled wounding the cell monolayer in a scratch assay, which was reported to affect the cell migration by inducing changes in gene expression and signaling27. There was the evidence that cells produced chemicals or signals, such as ATP or Ca2+ after injury28,29. Increases in the level of ATP and Ca2+ have been shown to enhance and stimulate fibroblast proliferation, migration, and ECM productions which are involved in wound healing mechanisms28. Another possible explanation for a higher migration rate of fibroblast cells on the MEEK gauze is the mechanical stimulation due to the stretching of the MEEK gauze for the cell sheet expansion. Stretched fibroblasts have been reported to migrate faster and move a further distance, as compared to their non-stretched counterparts30. Stretching caused the up-regulation of matrix metalloproteinases (MMPs), which were responsible for collagen degradation, leading to lower cell-substrate interaction and resulting in the increased migration30.
When comparing between different expansion ratios in the MEEK technique, the migration rate of fibroblasts on the MEEK gauze at 1:9 expansion ratio was lower than that of the cells on the MEEK gauze at 1:6 expansion ratio, which could have resulted from a greater distance between the cell sheet islands. Generally, cells communicated with each other through the release of soluble cytokines and chemokines. These signaling molecules diffuse through the medium, bind to the cell's receptors and activate many crucial biological pathways responsible for proliferation, migration, etc31. The longer distance between each cell island on the MEEK gauze at a 1:9 expansion ratio would have led to larger diffusion length and possibly lower cytokine concentrations to induce the cell migration. Thus, the MEEK at expansion ratio of 1:6 and 1:3 would be more appropriate for wound management, as the cell sheets on MEEK gauzes at these expansion ratios could migrate faster to accelerate wound healing. However, using the expansion ratio of 1:3 may not be significantly beneficial, as it can only cover a small wound area, which commonly heals quite quickly17. Considering the cell viability and migration potential, our result suggests the use of MEEK technique at an expansion ratio of 1:6 to expand skin cell sheets to treat large chronic and burn wounds.
In conclusion, we have shown that the combination of the fibroblast cell sheet with nylon dressing or MEEK gauzes at various expansion ratios may overcome the limitation of the cell sheet applications in clinical settings, including limited treatment area and cell sheet handling. Fibroblast cells on nylon dressing and the expanded fibroblast sheets on MEEK gauzes have shown to possess high capacities of cell proliferation and migration which could be particularly beneficial in the wound care applications. This combination technique can be applied to different cell types, such as keratinocytes and endothelial cells to construct large cell sheets and apply to the wounds to speed up the re-epithelialization32 and vascularization processes33.