Dual Glyoxalase-1 and β-Klotho Gene-Activated Scaffold Reduces Methylglyoxal and Reprograms Diabetic Adipose-Derived Stem Cells: Prospects in Improved Wound Healing

Abstract

Tissue engineering approaches aim to provide biocompatible scaffold supports that allow healing to progress often in healthy tissue. In diabetic foot ulcers (DFUs), hyperglycemia impedes ulcer regeneration, due to complications involving accumulations of cellular methylglyoxal (MG), a key component of oxidated stress and premature cellular aging which further limits repair. In this study, we aim to reduce MG using a collagen-chondroitin sulfate gene-activated scaffold (GAS) containing the glyoxalase-1 gene (GLO-1) to scavenge MG and anti-fibrotic β-klotho to restore stem cell activity in diabetic adipose-derived stem cells (dADSCs). dADSCs were cultured on dual GAS constructs for 21 days in high-glucose media in vitro. Our results show that dADSCs cultured on dual GAS significantly reduced MG accumulation (-84%; p < 0.05) compared to the gene-free controls. Similar reductions in profibrotic proteins α-smooth muscle actin (-65%) and fibronectin (-76%; p < 0.05) were identified in dual GAS groups. Similar findings were observed in the expression of pro-scarring structural proteins collagen I (-62%), collagen IV (-70%) and collagen VII (-86%). A non-significant decrease in the expression of basement membrane protein E-cadherin (-59%) was noted; however, the dual GAS showed a significant increase in the expression of laminin (+300%). We conclude that dual GAS-containing Glo-1 and β-klotho had a synergistic MG detoxification and anti-fibrotic role in dADSC's. This may be beneficial to provide better wound healing in DFUs by controlling the diabetic environment and rejuvenating the diabetic stem cells towards improved wound healing.

Keywords: adipose-derived stem cells; anti-fibrotic; gene-activated scaffold; glyoxalase-1; matrix deposition; methylglyoxal; stem cells rejuvenation; wound healing; β-klotho.

Figure 1

GAS demonstrated significant suppression of methylglyoxal on day 21, especially the expression of dual GAS which was notably reduced by 84% compared to GFS. Scale bar 50 µm. Data represent mean ± standard deviation (n = 3), * indicates p < 0.05.

Figure 2

GAS expressed decreasing trend of fibronectin and α-smooth muscle actin extracellular depositions compared to GFS on day 21. The fibronectin and α-SMA expressions on dual GAS were reduced by 76% and 65%, respectively, compared to GFS. Scale bar 50 µm. Data represent mean ± standard deviation (n = 3) * indicates p < 0.05.

Figure 3

GAS showed reduced collagen expressions on day 21. Dual GAS expressed the least of Collagen IV and Collagen VII which were decreased by 70% and 86% (p < 0.05), respectively, compared to GFS. Also, 62% drop in Collagen I was observed on dual GAS compared to GFS, * indicates p < 0.05 and ** indicates p < 0.01.

Figure 4

GFS expressed minimal cellular laminin, whereas dual GAS robustly enhanced 300% deposition of extracellular laminin matrix compared to that of GFS. Contrary, dual GAS showed 59% downregulations of cell–cell adhesion dimer E-cadherin deposition. Scale bar 50 µm. Data represent mean ± standard deviation (n = 3). * indicates p < 0.05.

Figure 4

GFS expressed minimal cellular laminin, whereas dual GAS robustly enhanced 300% deposition of extracellular laminin matrix compared to that of GFS. Contrary, dual GAS showed 59% downregulations of cell–cell adhesion dimer E-cadherin deposition. Scale bar 50 µm. Data represent mean ± standard deviation (n = 3). * indicates p < 0.05.