Adipose stem cells exhibit mechanical memory and reduce fibrotic contracture in a rat elbow injury model

Abstract

Fibrosis is driven by a misdirected cell response causing the overproduction of extracellular matrix and tissue dysfunction. Numerous pharmacological strategies have attempted to prevent fibrosis but have attained limited efficacy with some detrimental side effects. While stem cell treatments have provided more encouraging results, they have exhibited high variability and have not always improved tissue function. To enhance stem cell efficacy, we evaluated whether mechanical memory could direct cell response. We hypothesized that mechanically pre-conditioning on a soft matrix (soft priming) will delay adipose-derived stem cell (ASC) transition to a pro-fibrotic phenotype, expanding their regenerative potential, and improving healing in a complex tissue environment. Primary ASCs isolated from rat and human subcutaneous fat exhibited mechanical memory, demonstrated by a delayed cell response to stiffness following two weeks of soft priming including decreased cell area, actin coherency, and extracellular matrix production compared to cells on stiff substrates. Soft primed ASCs injected into our rat model of post-traumatic elbow contracture decreased histological evidence of anterior capsule fibrosis and increased elbow range-of-motion when evaluated by joint mechanics. These findings suggest that exploiting mechanical memory by strategically controlling the culture environment during cell expansion may improve the efficacy of stem cell-based therapies targeting fibrosis.

Keywords: anterior capsule; mechanical priming; post‐traumatic joint contracture; pre‐conditioning; range‐of‐motion.

Figure 1.

Schematic of the in vitro experimental method timeline. Adipose-derived stem cells (ASCs) were isolated from rat subcutaneous adipose tissue expanded for four passages and frozen down for storage. ASCs were cultured on either 1 or 120 kPa polyacrylamide gels functionalized with 10 μg/mL fibronectin. (· · · = enzymatically transferred to a new gel using 0.25% trypsin-EDTA). (A) Representative images of rat ASCs immunolabeled for (top row) nuclei (blue) and F-actin (green), and (bottom row) nuclei (blue) and α-smooth muscle actin (αSMA, magenta). (scale bar = 50 μm for all groups, except Stiff-to-Stiff (2): scale bar = 75 μm). Quantitative measures computed from fluorescent images: (B) cell area, (C) actin coherency (alignment), and (D) αSMA positive cells. Data are shown as mean ± standard deviation. ** p < 0.01. **** p < 0.0001.

Figure 2.

(A) Representative images of rat adipose-derived stem cells (ASCs) immunolabeled for (top row) nuclei (blue outline), cell membrane (magenta outline), and extracellular matrix (white), and (bottom row) intensity heatmap of extracellular matrix deposition. (scale bar = 100 μm). Quantitative measures computed from fluorescent images for extracellular matrix intensity profile for (B) Soft and Stiff groups, and (C) Soft-to-Stiff (1) and Stiff-to-Stiff (1) groups. (dark line = mean, and light lines = standard deviation). (D) Area under the curve for the extracellular matrix intensity profiles for each group. Data are shown as mean ± standard deviation. ** p < 0.01. *** p < 0.001.

Figure 3.

Schematic of the in vivo experimental method timeline. Adipose-derived stem cells (ASCs) were isolated from rat subcutaneous adipose tissue then cultured on either fibronectin-coated 1 kPa polyacrylamide gels for two weeks or tissue culture plastic (TCP) for one week before injection. (lightning bolt = surgery, syringe = injection). Representative sagittal histology (toluidine blue) illustrate joint anatomy (top row scale bar = 1 mm) and general characteristics of the anterior capsule (bottom row scale bar = 400 μm) after 21 days of immobilization. (dotted square = region of interest shown at higher magnification in the bottom row, C = capsule, H = humerus, R = radius, M = muscle, INJ-TCP = injured and injected with cells cultured on tissue culture plastic (TCP), INJ-GEL = injured and injected with cells cultured on fibronectin-coated 1 kPa polyacrylamide gels).

Figure 4.

Quantitative results are shown for flexion-extension: (A) total range-of-motion (ROM), (B) neutral zone length and (C) neutral zone stiffness. Limbs were evaluated after 21 days of immobilization. Data are shown as mean ± standard deviation. # p < 0.05, ### p < 0.001, and #### p < 0.0001 different from control. * p < 0.05. Data for control and INJ were published previously (7). (INJ-TCP = injured and injected with cells cultured on tissue culture plastic (TCP), INJ-GEL = injured and injected with cells cultured on fibronectin-coated 1 kPa polyacrylamide gels).

Figure 5.

Adipokine array results: Heatmap of normalized intensity ratio for Soft/Stiff and Soft-to-Stiff (1)/Stiff-to-Stiff (1) with statistical results from two-way analysis of variance (ANOVA) for stiffness and priming with post-hoc Bonferroni corrected with False Discovery Rate using the Benjamini-Hochberg method. * p < 0.05. ** p < 0.01.

Figure 6.

(A) Representative images of human adipose-derived stem cells (ASCs) immunolabeled for nuclei (blue) and F-actin (green). (scale bar = 50 μm). Quantitative measures computed from fluorescent images: (B) cell area and (C) actin coherency (alignment). (D) Monocyte chemoattractant protein-1 (MCP-1) expression in human ASC conditioned media. Data are shown as mean ± standard deviation. * p < 0.05. *** p < 0.001. **** p < 0.0001.

Figure 7.

While YAP does not drive long-term mechanical memory in vitro, cell memory may potentially be regulated by nuclear reprogramming (e.g., altered epigenetics and/or microRNA expression) (41, 59). Increased pro-regenerative cytokine secretion (e.g., MCP-1, IGFBP-6, Endocan) after soft priming may signal to the resident cell population in vivo to mitigate the fibrotic wound healing response in our rat elbow contracture model which subsequently improved joint motion.