Introduction:: The corneal stroma consists of orthogonal stacks of collagen lamellae consisting of aligned collagen fibrils. Corneal keratocytes reside in this stroma and bind to the collagen fibrils via integrin containing Focal Adhesions (FA). In the event of an injury, corneal fibroblasts migrate in long chains to repopulate the wound aligning their cell bodies with the underlying fibrils. The aligned collagen fibrils provide topographical cues for corneal keratocyte alignment and migration. Previous studies have shown that the ECM topography can dictate FA maturation, size and alignment. However, the geometric effects of aligned fibril topography on FA size and area in corneal fibroblasts remains to be investigated. FAs are also dynamically involved in migration, forming associations with the Extracellular Matrix.
In this study we used micropatterns of aligned collagen fibrils and collagen monomers (100um and 750um) and determined if the micropattern geometry and topography caused changes in FA size and number.
Materials and Methods:: Polydimethylsiloxane (PDMS) Sylgard 184 Elastomer was spin coated on glass coverslips, and cured for 2 hours in a PDMS oven. Aligned collagen fibrils were patterned on the PDMS using microchannels of widths 100 and 750 µm. To pattern aligned collagen fibrils, collagen solution was perfused into the microchannels as described previously.1 Human Corneal fibroblast cell line (HTKs)2 was plated on these substrates in basal media for 24 hours. Following 24 hours of culture, the cells were fixed and permeabilized using standard immunofluorescence procedures. In some substrates HTKs were seeded in media containing 10% FBS. Samples were then blocked with 2% Bovine Serum Albumin overnight at 4°C and then fluorescently labeled for Vinculin, Actin and the Nucleus. Fluorescent images were acquired and FA Size and Area for each of the samples was computed using ImageJ software.
Results, Conclusions, and Discussions:: Results and Discussion: Our results indicate that FA size and number was modulated by the micropattern size and the underlying ECM. While the FA localized to the cell tips when cultured on aligned fibrils (100 um and 750um), significant differences were obtained when HTKs were cultured on 750 um wide monomer micropatterns or unpatterned substrates, with higher FA number and size.
Conclusions: These results suggest that the geometry and underlying ECM modulates FA size and area in corneal fibroblast. In future experiments we will investigate how geometry affects migration speed in corneal fibroblasts in response to a freeze injury and the changes in distribution of contractile filaments through phospho-myosin light chain (pMLC) staining.
Acknowledgements (Optional): : Acknowledgments: This work was supported by the NIH grant R01 EY030190 as well as a Predoctoral Fellowship Award from International Foundation for Ethical Research awarded to Divya Subramanian.
References (Optional): : 1) Lam KH, et al. “A High-Throughput Microfluidic Method for Fabricating Aligned Collagen Fibrils to Study Keratocyte Behavior”, Biomedical Microdevices. 21:99, 2019.
2) Jester, James V., et al. "Myofibroblast differentiation of normal human keratocytes and hTERT, extended-life human corneal fibroblasts." Investigative ophthalmology & visual science 44.5 (2003): 1850-1858.
3) Vinje, Jakob B., et al. "Analysis of actin and focal adhesion organisation in U2OS cells on polymer nanostructures." Nanoscale Research Letters 16 (2021): 1-14.
4) Teixeira, Ana I., Paul F. Nealey, and Christopher J. Murphy. "Responses of human keratocytes to micro‐and nanostructured substrates." Journal of Biomedical Materials Research Part A: An Official Journal of The Society for Biomaterials, The Japanese Society for Biomaterials, and The Australian Society for Biomaterials and the Korean Society for Biomaterials 71.3 (2004): 369-376..
