N-acetylcysteine and ascorbic acid-2-phosphate incorporatedhydrogels as autologous stem cell delivery system for the treatment of diabetic wounds
Date
2024-12
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Stellenbosch : Stellenbosch University
Abstract
Background: Bone marrow resident mesenchymal stem cells (MSCs) are sensitive to changes in the micro-environment and vulnerable to glucose toxicity. Under diabetic conditions, exposure to hyperglycaemia, inflammation and oxidative stress within the micro-environment impairs the regenerative capacity of MSCs and dysregulate their immunomodulatory functions. The antioxidants N-acetylcysteine (NAC) and ascorbic acid 2-phosphate (AAP) has been shown to improve the viability and growth rate of diabetic MSCs ex vivo and suppress excessive pro-inflammatory cytokine release. The exact mechanism of action is however still unclear and needs elucidation. The aim of this study was thus to a) assess the efficacy of ex vivo NAC/AAP pre-treatment to counteract diabetic BM-MSCs dysfunction using proteomic analysis and to b) investigate the therapeutic efficacy of pre-treated BM-MSCs incorporated into a hydrogel delivery system to promote tissue regeneration of diabetic wounds in vivo.
Methods: The cell line C3H10T1/2 was utilised in hydrogel optimisation studies to assess cell encapsulation, migration and antioxidant diffusion from the hydrogel. Bone marrow MSCs were isolated from obese diabetic mice (B6.Cg-lepob/J (ob/ob); >40g, 6 weeks, n=8) and the cell number expanded in culture with or without antioxidant supplementation for a period of 13 days. Antioxidant treatment consisted of 7.5mM NAC + 0.6mM AAP with media changed every 4 days. Upon reaching 70% confluence, conditioned media was collected, and the cells lysed to harvest the intracellular protein content. Protein samples were processed using standardized procedures and analysed using label free LC-MS/MS. Statistical analysis was performed in Scaffold and functional pathway analysis and protein interactions were mapped for the differential proteins of interest (p < 0.05) (identified through biostatistical analysis of the LC-MS/MS data) using STRING. The secretome of pretreated and untreated BM-MSCs were incorporated in a hydrogel delivery vehicle and administered to wounds in a murine diabetic wound model (n=5 animals/treatment group with 2 wounds per animal). The percentage wound closure was assessed for superficial wound healing and histological (Masson trichrome and Haematoxylin & Eosin) analysis was performed on the healing characteristics of the wound bed to determine treatment effectiveness.
Results: Hydrogel encapsulated antioxidants and cells (cell line C3H10T1/2) proved to be compatible. The antioxidant NAC was able to diffuse out of the hydrogel and the cell line’s functionality was supported at concentrations of 15-18% hydrogel. Consistent with previous findings, NAC/AAP pretreatment was able to partially improve the ex vivo growth rate of MSCs and protect them against cell death. LC-MS/MS identified 5602 proteins of which 199 was unique to the non-treatment group and 52 was unique to the antioxidant pretreated group. The differentially expressed proteins in the untreated group demonstrated prominent cell structure (matrix proteins), and programmed cell death (Fas, RIPK3) biological processes compared to immunomodulation (MRC1, STAT1) demonstrated in the pretreatment group. Histology analysis indicated that irrespective of the treatment groups (histology score: Control 6±2, Untreated 6±2, Pretreated 6±3) the wounds were all in the proliferative phase of healing on day 14. Specific wound healing outcomes in the groups demonstrated inflammatory phase healing in control (1 wound) and untreated (1 wound) groups with pretreatment predominantly in proliferation phase healing (4 wounds). Remodelling phase healing was achieved in pretreatment (1 wound) and predominantly untreated wounds (3 wounds).
Conclusion: The antioxidant pretreatment deems beneficial to MSCs however was not able to restore full functionality of the cells to promote regeneration of a diabetic wound. A multifactorial approach to diabetic wound healing is required in future research.
Description
Thesis (PhD)--Stellenbosch University, 2023.