Anti-Aging β-Klotho Gene-Activated Scaffold Promotes Rejuvenative Wound Healing Response in Human Adipose-Derived Stem Cells

Abstract

Wound healing requires a tight orchestration of complex cellular events. Disruption in the cell-signaling events can severely impair healing. The application of biomaterial scaffolds has shown healing potential; however, the potential is insufficient for optimal wound maturation. This study explored the functional impact of a collagen-chondroitin sulfate scaffold functionalized with nanoparticles carrying an anti-aging gene β-Klotho on human adipose-derived stem cells (ADSCs) for rejuvenative healing applications. We studied the response in the ADSCs in three phases: (1) transcriptional activities of pluripotency factors (Oct-4, Nanog and Sox-2), proliferation marker (Ki-67), wound healing regulators (TGF-β3 and TGF-β1); (2) paracrine bioactivity of the secretome generated by the ADSCs; and (3) regeneration of basement membrane (fibronectin, laminin, and collagen IV proteins) and expression of scar-associated proteins (α-SMA and elastin proteins) towards maturation. Overall, we found that the β-Klotho gene-activated scaffold offers controlled activation of ADSCs’ regenerative abilities. On day 3, the ADSCs on the gene-activated scaffold showed enhanced (2.5-fold) activation of transcription factor Oct-4 that was regulated transiently. This response was accompanied by a 3.6-fold increase in the expression of the anti-fibrotic gene TGF-β3. Through paracrine signaling, the ADSCs-laden gene-activated scaffold also controlled human endothelial angiogenesis and pro-fibrotic response in dermal fibroblasts. Towards maturation, the ADSCs-laden gene-activated scaffold further showed an enhanced regeneration of the basement membrane through increases in laminin (2.1-fold) and collagen IV (8.8-fold) deposition. The ADSCs also expressed 2-fold lower amounts of the scar-associated α-SMA protein with improved qualitative elastin matrix deposition. Collectively, we determined that the β-Klotho gene-activated scaffold possesses tremendous potential for wound healing and could advance stem cell-based therapy for rejuvenative healing applications.

The Rejuvenation and Functional Restoration of Aged Adipose Stem Cells by DUXAP10 Knockdown via the Regulation of the miR-214-3p/RASSF5 Axis

“… 43 , 44 However, the regenerative capabilities of these cells diminish with donor aging, diabetes, and prolonged in vitro culture, thereby limiting their clinical utility. 45 Previous research has explored several strategies to enhance the wound healing properties of ASCs, including the upregulation of miR-1248 46 or an anti-aging gene β-Klotho, 47 cultivation in a 3D coculture system, 48 and pre-treatment with a NOX1/4 inhibitor GKT137831 49 to mitigate reactive oxygen species production. In the current investigation, we have identified a novel approach wherein the knockdown of DUXAP10 enhances the therapeutic potential of aged ASCs in skin wound healing, as evidenced by improved wound collagen deposition, neovascularization, and accelerated wound closure.…”

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

“…GAS was developed using a two-step process as defined previously [ 35 , 36 , 37 , 38 ]. Bovine tendon type 1 collagen and shark cartilage chondroitin-6-sulfate (Sigma, Edinburgh, UK) scaffolds were prepared through an optimized freeze-drying process [ 37 ], then treated at 105 °C under vacuum for sterilization and scaffold crosslinking [ 38 ], followed by further chemical cross-linking with 14 mM N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride and 5.5 mM N-Hydroxysuccinimide (EDC/NHS, Sigma, UK) solution to enhance their mechanical stability.…”

“…The cross-linked scaffolds were then washed with PBS (Gibco, London, UK) before soak-loading polyplex nanoparticles on to the scaffolds for 40 min. Based on previous studies [ 35 , 36 , 39 ], polyplex particles were formulated by mixing a specified amount of branched cationic 25 kDa polyethyleneimine (PEI) (Sigma-Aldrich, Dublin, Ireland) for anionic pDNA (fixed at a dose of 2 μg) delivery [ 28 , 36 , 40 ] and given an N/P ratio of 10. Three scaffold groups are prepared: (1) gene-free scaffold (GFS, no pDNA), (2) β-klotho gene-activated scaffold and (3) Glyoxalase-1/β-Klotho gene-activated scaffold (dual GAS).…”

An Up-to-Date Review of Biomaterials Application in Wound Management

“…A different example is offered by Laiva et al [ 123 ], who have developed a collagen-chondroitin sulfate scaffold functionalized with nanoparticles carrying an anti-aging gene, β-Klotho, on human adipose-derived stem cells (ADSCs). By studying this biomaterial, it was established that it ensures controlled activation of ADSC’s regenerative abilities, enhances activation of transcription factor Oct-4, increases the expression of the anti-fibrotic gene TGF-β3, controls human endothelial angiogenesis and pro-fibrotic response in dermal fibroblasts, enhances regeneration of the basement membrane, and decreases the levels of scar-associated α-SMA protein with improved qualitative elastin matrix deposition.…”

“… Classification and examples of polymers used for fabricating biomaterial scaffolds. Created based on information from [ 115 , 118 , 120 , 121 , 122 , 123 , 124 , 125 , 126 ]. Abbreviations: PCL—poly (ε-caprolactone), PLA—polylactic acid, PLGA—poly(lactic-co-glycolic acid), PAM—polyacrylamide, PHB—poly-3-hydroxybutyrate, PUR—polyurethane.…”