Skip to main content
2024 BMES Annual Meeting Main banner
Icon Legend
This session is not in your schedule.
This session is in your schedule. Click again to remove it.
Back

Biomechanics

Biomechanics - Poster Session E

Poster Q3 - The combined effect of Diabetic Plantar skin stiffness and hyperglycemia on wound closure

Saturday, October 26, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Diabetic foot ulcers (DFU) occur due to decreased fibroblast migration and the shift away from the homeostatic balance between matrix metalloproteinase (MMP) mediated extracellular matrix (ECM) degradation and assembly [1]. Hyperglycemia, defined as blood glucose levels higher than 11.1 mM in diabetic patients, induces osmotic stress that causes cell damage, it hinders ulcer healing through impaired cell migration, dysregulated actin assembly and polymerization [2] and decreased matrix metalloproteinase expression [3]. Moreover, diabetic plantar skin demonstrates higher tissue stiffness than healthy plantar skin [4–7]. Increased tissue stiffness in healthy conditions enhances cell migration by increasing cell adhesion and actin contraction, driving wound closure [8]. However, DFU, despite having an increase in plantar tissue stiffness, demonstrate a lack of wound closure due to decreased migration and dysregulated extracellular matrix structure function [1]. It remains unclear how hyperglycemia alters mechanosenstive cellular migration in the context of DFU.
Therefore, we seek to investigate wound migration dynamics in the context of (i) mechanical substrate stiffnesses mimicking normal and diabetic plantar skin stiffnesses and (ii) glucose levels mimicking diabetic and healthy patients.

Materials and

Methods:  Target plantar skin stiffnesses were simulated using polydimethylsiloxane (PDMS) substrates: normal, 55 kPa, and diabetic, 90 kPa. Two glucose levels were simulated by adjusting glucose concentrations in DMEM media: healthy, 5.5 mM, and diabetic, 11.1 mM, Human dermal fibroblasts were seeded on plasma fibronectin-coated PDMS with 1 mm cylindrical PDMS masks and cultured for 48 hours before simulating wounding via mask removal (Fig. 1). Plates were imaged with a Keyence BZ-X800 microscope, 10x objective for 24 hours at 10 min intervals, cell actin labeled with Sir-ACTIN, and nuclei labeled with SPY595-DNA. Time-lapse videos were analyzed with ImageJ to measure wound closure according to the following protocol [9], ImageJ Trackmate was used to evaluate cell migration, and the resulting data was used to calculate cell velocity and directionality using Chemotaxis and Migration Tool 2.0 (ibidi, http://www.ibidi.de/applications/ap_chemo.html), GraphPad-Prism 9 calculated one-way ANOVAs and regression best-fit analysis, and MATLAB graphed the directionality tracks.

Results, Conclusions, and Discussions: Gap closure analysis of the simulated DFU model (Fig 2A,C) demonstrates fastest wound closure in the diabetic tissue stiffness (90kPa) condition with slope = -3.44 (R2 = 0.98 ) on 5.5 mM normal glucose and -2.62 (R2 = 0.88) on 11.1 mM higher glucose, while the slowest condition to close was the 50 kPa condition slope = -2.19 (R2 = 0.89) ) on 5.5 mM normal glucose and -2.27 (R2 = 0.99) on 11.1 mM higher glucose (Fig 2B). From a mechanical substrate stiffness standpoint, these results coincide with literature [10,11]; however, DFU lack this rapid wound closure. This indicates that mechanical substrate stiffness alone does not recover lack of cellular function induced by hyperglycemia-induced responses, or that hyperglycemia interferes with cell mechanosensing.
Individual cell migration tracks demonstrated cells in all conditions similarly migrated towards the center of the wound (Fig 3A,B). Cell directionality (Euclidean Distance/Accumulated Distance) lowered significantly in the hyperglycemic (55kPa) condition compared to its normal (55kPa) control. Interestingly, the diabetic increased tissue stiffness 90kPa, on diabetic hyperglycemia condition recovered the directionality observed in the healthy phenotype 55kPa and normal glucose. Further, cells migrating on softer 55 kPa substrates under hyperglycemic conditions migrated slower than that of cells migrating under normal glucose conditions, with similar results on the stiffer 90 kPa substrate (Fig 3D). Additionally, regardless of substrate stiffness differences, cells cultured under the same glucose conditions migrated at similar speeds.
This data shows how the role of stiffness on wound closure differentially affects cell migration under the influence of hyperglycemia. Higher substrate stiffness increased wound closure rate, did not affect cell velocity, and neutralized the effect of directionality under hyperglycemia. This indicates a possible overlap between the mechanoresponse of cells to the stiffness and hyperglycemia in diabetes, but further investigation is required to detect the molecular mechanisms affected.. One hypothesized pathway that may be affected includes integrin binding to connect the extracellular matrix to the cell cytoskeleton through integrin binding, which in turn will activate YAP/TAZ downstream cellular mechanosignaling to regulate gene expression controlling migration and wound closure[8].

Acknowledgements (Optional):  

References:
[1] Kanta, J., Int Wound J 2023:20:1750–1761.
[2] Lamers, M. L., PLoS One 2011:6:22865.
[3] Pahwa, H., Mol Cell Biochem 2020:469:109–118.
[4] Chao, C. Y. L., Ultrasound Med Biol 2011:37:1029–1038.
[5] Cheing, G. L. Y., Clinical Biomechanics 2013:28:88–92.
[6] Sun, J. H., Arch Phys Med Rehabil 2011:92:1484–1489.
[7] Zheng, Y. P., Ultrasound Med Biol 2000:26:451–456.
[8] Yi, B., Bioact Mater 2021:15:82–102.
[9] De Ieso, M. L., Biosci Rep 2018:38:20180698.