(27bv) Investigating the Effects of Surface Stiffness on Human Mesenchymal Stem Cell Immunomodulatory Potential

Introduction: Chronic, non-healing wounds are a growing financial and medical burden (1). Current treatment methods often fall short of desired outcomes, such as the autologous split-thickness skin graft, which has unfavorable scar and graft contracture (2). Human mesenchymal stem cells (hMSCs) implanted at the wound site are currently being explored as a treatment for chronic wounds due to their immune cell regulatory function (3). However, hMSCs have low viability, poor engraftment, and a risk of eliciting an immune response after injection at the site of a wound (4). In addition, they often have heterogeneous behaviors due to variability in donor, age, and tissue source. The physical properties of the cell culture microenvironment, such as the stiffness of the underlying extracellular matrix, has been shown to direct the maturation of hMSCs (5). While the effects of substrate stiffness on hMSC behavior such as differentiation have been investigated (6), the effects of physical properties on hMSC immunomodulation have yet to be elucidated. Thus, to address the limitations of hMSC therapies for chronic wounds, we will investigate culture of hMSCs on polyacrylamide (PAAm) gels, which have easily tunable mechanical properties and are commonly used as a cell culture substrate, with differing stiffness properties. We will assess hMSC viability, morphology, and immunomodulatory cytokine secretion and expression on these gels as a function of stiffness, ultimately leading to an increased understanding of the foundational relationship between the physical cues of the cellular microenvironment and hMSC immunomodulation.

Materials and Methods: PAAm gels were fabricated following the protocol by Engler et al. (7). Briefly, solutions of varying concentrations of acrylamide (4-8% v/v) and bis-acrylamide (0.06-0.264% v/v) in 1× phosphate buffered saline (PBS) were polymerized with ammonium persulfate (APS) and N, N, N’, N’-tetramethylethylenediamine (TEMED) in between an amino-salinized coverslip and chloro-salinized coverglass in order to achieve thin gels (thickness = 102 µm) with three different expected stiffness values, 1.16 ± 0.54 kPa (soft gels), 4.47 ± 1.19 kPa (medium gels), and 19.66 ±1.19 kPa (stiff gels). The PAAm gels were then tested using atomic force microscopy (AFM) to measure their stiffness (Young’s Modulus, E). Briefly, an AFM probe with a spring constant of 0.01 N/m was used to measure stiffness over a 1 by 1 µm section of each PAAm gel, in a 4 by 4 grid, and an average stiffness was recorded for each gel. To prepare for cell culture, sulfo-SANPAH was covalently bound to the PAAm gel surface and subsequently covalently bound to fibronectin. Passage 5 bone marrow-derived hMSCs were seeded on PAAm gels at 5,000 cells/cm² after the protein coating and evaluated for viability using a live/dead assay and morphology using fluorescent F-actin and nucleus staining.

Results: PAAm gels with three different stiffnesses, soft, medium, and stiff were fabricated, protein coated, and seeded with hMSCs as described above. The Young’s modulus was measured to be 1.99 kPa for the soft gel, 4.50 kPa for the medium gel, and 15.14 kPa for the stiff gel (n=1 each), which are within expected ranges for these acrylamide/bis-acrylamide ratios (reported in the literature as 1.16 ± 0.54 kPa, 4.47 ± 1.19 kPa, and 19.66 ± 1.19 kPa, respectively). Live/dead staining confirmed that the fibronectin coated PAAm gels are cytocompatible for all stiffness values, with medium stiffness gels having the lowest number of dead (red) cells (Figure 1A). In addition, we demonstrate that morphology of the cells, which is known to be correlated to cell function, was impacted by gel stiffness (Figure 1B). Phase contrast microscopy and F-actin and nuclei staining show that hMSCs cultured on stiff gels exhibited a spindle-like morphology, hMSCs on medium stiffness gels exhibited the most spreading, and hMSCs on soft gels were more rounded.

Figure 1. Viability and morphology of hMSCs cultured on soft, medium, and stiff PAAm gels. A) Live/dead assay for hMSCs cultures on a fibronectin coated PAAm gel for a soft (left), medium (middle), and stiff (right) PAAm gel with phase contrast images on the top and live/dead staining on the bottom. Live cells=green, dead cells=red. Scale bar=200 µm. B) Morphology of hMSCs cultured on fibronectin coated PAAm gels for soft (left), medium (middle), and stiff (right) PAAm gels depicted using fluorescence staining of F-actin (green) and nuclei (blue) on the bottom. Scale bar=200 µm.

Conclusions: In conclusion, we demonstrate that fibronectin coated PAAm gels are a suitable culture substrate for hMSCs. Preliminary data indicates that there are morphological differences as a function of gel stiffness, and future work will focus on evaluating the immunomodulatory potential of hMSCs on these gels.

References:

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