(F-211) The Role of Substrate Stiffness on Diabetic Wound Healing

Introduction::
In people living with both type I and type II diabetes, impaired wound healing is a major concern as the formation of ulcerated wounds can drastically reduce both the effectiveness of the healing process and the quality of life of the patient. The healing of dermal wounds particularly, involves a patients fibroblasts building up a strong extracellular matrix of mostly collagen I and collagen III fibers, which the cells of diabetic patients struggle to build. Extracellular matrix stiffness, and growth substrate stiffness in general, have already been shown to have a significant effect on the growth and development of already existent cells, and in diabetic dermal fibroblasts, morphological and physiological characteristics associated with the healing process appear to be altered from their healthy state. We hypothesize altered substrate stiffness regulate diabetic fibroblast function and can be used to develop therapeutic strategies with targeted stiffness.




Materials and Methods::
Preparing Polyacrylamide Gels Three different stiffnesses of polyacrylamide gels were created 2 kPa, 22 kPa, and one set was left as just glass coverslips on 12 mm coverslips prepared in 24 well plates[1]. The gels were created with varying amounts of 2% Bis-acrylamides, 30% acrylamide, 250 mM HEPES pH 8, H2O, TEMED, and 10% APS for creating different stiffnesses. The gels were made to have a 125 mm in thickness. Measuring the Metabolic Activity of hDF and dhDF The metabolic activity of both normal primary human dermal fibroblasts (hDF) and primary diabetic human dermal fibroblasts (dhDF) were measured using a Presto Blue Assay. The cells were seeded at varying cell seeding densities on PA gels and glass accordingly (1000, 5000 and 10,000 cells/1.13 cm²). Quantitative Immunofluorescence Analysis of Cellular Mechanotransduction The data was obtained through using three different antibodies during immunostaining. Over three different experiments, the dhDF and hDF were stained with Ki67 primary antibodies, YAP primary antibodies, and a-SMA primary antibodies. The cells were seeded at different densities and allowed to adhere for 48 hours before the immunostaining experiment following our earlier protocol including secondary antibody controls[2]. Based on the observed differences in the markers we plan to further investigate the tension generated by hDF and dhDFs to the underlying surface using traction force microscopy[3].




Results, Conclusions, and Discussions::





Results and Discussion: Presto Blue analysis of fibroblasts with varying seeding density show significant differences in cell metabolic activity between diabetic and normal fibroblasts (Figure 1). The ongoing experiments are focusing on understanding the role of stiffness in regulating the differences in metabolic activity. The results of this experiment will shed light into how substrate stiffness will cause the dhDF to behave more like the hDF in both metabolically and morphologically.








Conclusions and Acknowledgements:

The goal of this study was to find a relationship between substrate stiffness and the characteristics of these

of these diabetic cells, and to form a preliminary link between these disease state characteristics and the stiffness of the cells’ growth substrate, hopefully clearing the way for further studies to investigate this relationship and potentially find a way to utilize it in finding a way to facilitate and speed the healing process for diabetic wounds.












Acknowledgements (Optional): :

References (Optional): :
1. Olsen, A.L., et al., Hepatic stellate cells require a stiff environment for myofibroblastic differentiation. American Journal of Physiology-Gastrointestinal and Liver Physiology, 2011. 301(1): p. G110-G118.




2. Ozdemir, T., et al., Substrate curvature sensing through Myosin IIa upregulates early osteogenesis. Integrative Biology, 2013. 5(11): p. 1407-1416.




3. Driscoll, T.P., et al., Cytoskeletal to nuclear strain transfer regulates YAP signaling in mesenchymal stem cells.Biophysical journal, 2015. 108(12): p. 2783-2793.