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N-acetylcysteine and ascorbic acid-2-phosphate incorporatedhydrogels as autologous stem cell delivery system for the treatment of diabetic wounds.
Thesis (PhD)--Stellenbosch University, 2023.
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Stellenbosch : Stellenbosch University
2025
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| _version_ | 1867613781952036865 |
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| access_status_str | Open Access |
| author | Meeser, Michelle |
| author2 | Van de Vyver, Mari |
| author_browse | Meeser, Michelle Van de Vyver, Mari |
| author_facet | Van de Vyver, Mari Meeser, Michelle |
| author_sort | Meeser, Michelle |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description |
Thesis (PhD)--Stellenbosch University, 2023. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/131830 |
| institution | Stellenbosch University (South Africa) |
| last_indexed | 2026-06-10T12:41:36.774Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2025 |
| publishDateRange | 2025 |
| publishDateSort | 2025 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/131830 N-acetylcysteine and ascorbic acid-2-phosphate incorporatedhydrogels as autologous stem cell delivery system for the treatment of diabetic wounds. Meeser, Michelle Van de Vyver, Mari Vlok, Mare Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine. Division of Clinical Pharmacology. Mesenchymal stem cells Colloids -- Therapeutic use Bone marrow cells Diabetes -- Pathophysiology Stem cells UCTD Thesis (PhD)--Stellenbosch University, 2023. ENGLISH 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. AFRIKAANSE OPSOMMING: Agtergrond: Beenmurg inwonende mesenchimale stamselle (MSCs) is sensitief vir veranderinge in die mikro omgewing en kwesbaar vir glukose toksisiteit. Onder diabetiese toestande, benadeel blootstelling aan hiperglukemie, inflammasie en oksidatiewe stres binne die mikro omgewing die regeneratiewe kapasiteit van MSCs en disreguleer hul immuunmodulerende funksies. Daar is getoon dat die antioksidante Nasetielsisteïen (NAC) en askorbiensuur 2-fosfaat (AAP) die lewensvatbaarheid en groeitempo van diabetiese MSCs ex vivo verbeter en oormatige pro-inflammatoriese sitokien vrystelling onderdruk. Die presiese werkingsmeganisme is egter nog onduidelik en moet verduidelik word. Die doel van hierdie studie was dus om a) die doeltreffendheid van ex vivo NAC/AAP-voorbehandeling te evalueer om diabetiese BM-MSCs disfunksie deur proteomiese analise te gebruik teë te werk en om b) die terapeutiese doeltreffendheid van voorbehandelde BM-MSCs wat in 'n hidrogel afleweringstelsel om weefselregenerasie van diabetiese wonde in vivo te bevorder. Metodes: Die sellyn C3H10T1/2 is in ‘n hidrogel-optimaliseringstudie gebruik om selinkapseling, -migrasie en antioksidantdiffusie vanaf die hidrogel te bepaal. Beenmurg MSCs was geïsoleer vanuit vetsugtige diabetiese muise (B6.Cg-lepob/J (ob/ob); >40g, 6 weke oud, n=8) en die selgetal was in kultuur vir 'n tydperk van 13 dae met of sonder antioksidant aanvulling gegroei. Antioksidant behandeling het bestaan uit 7.5mM NAC + 0.6mM AAP met medium wat elke 4 dae verander was. Nadat 70% samevloeing bereik is, was gekondisioneerde media versamel, en die selle was gelys om die intrasellulêre proteïeninhoud te oes. Proteïenmonsters was verwerk deur die gebruik making van gestandaardiseerde prosedures en geanaliseer met die gebruik van etiketvrye LC-MS/MS. Statistiese analise is in Scaffold uitgevoer en funksionele padanalise en proteïeninteraksies was vir die differensiële proteïene van belang (p < 0.05) (geïdentifiseer deur biostatistiese analise van die LC-MS/MS data) gekarakteriseer deur van die program STRING gebruik te maak. Die sekretoom van voorbehandelde en onbehandelde BM-MSCs was in 'n hidrogel afleweringsvoertuig geïnkorporeer en aan wonde in 'n muis diabetiese wondmodel (n=5 diere/behandelingsgroep met 2 wonde per dier) toegedien. Die persentasie wondsluiting was vir oppervlakkige wondgenesing geassesseer en histologiese (Masson trichroom en Hematoksilien & Eosien) analise was uitgevoer op die genesende eienskappe van die wondbed om behandelingseffektiwiteit te bepaal. Resultate: Hidrogel-kapsulering van antioksidante en selle (sellyn C3H10T1/2) het bewys dat die metode versoenbaar is. N-asetielsisteïen (NAC) en AAP kon uit die hidrogel diffundeer en die sellyn se funksionaliteit was by konsentrasies van 15-18% hidrogel ondersteun. In ooreenstemming met vorige bevindinge, kon NAC/AAP voorbehandeling die ex vivo groeitempo van MSCs gedeeltelik verbeter en hulle teen sel dood beskerm. LC-MS/MS het 5602 proteïene geïdentifiseer waarvan 199 uniek was aan die onbehandelde groep en 52 uniek was aan die anti-oksidant-voorbehandelde groep. Die differensieel uitgedrukte proteïene in die onbehandelde groep het in terme van biologiese prosesse prominente sel struktuur (matriks proteïene) gedemonstreer, en geprogrammeerde sel dood (Fas, RIPK3) in vergelyking met immunomodulasie (MRC1, STAT1) in die voorbehandeling groep gedemonstreer was. Histologie-analise het aangedui dat ongeag die behandelingsgroepe (histologietelling: Kontrole 6±2, Onbehandeld 6±2, Voorbehandeld 6±3) die wonde almal in die proliferatiewe fase van genesing op dag 14 was. Spesifieke wondgenesing uitkomste in die groepe het inflammatoriese fase genesing gedemonstreer in kontrole (1 wond) en onbehandelde (1 wond) groepe met voorbehandeling hoofsaaklik in proliferasie fase genesing (4 wonde). Hermodellering fase genesing was bereik in voorbehandeling (1 wond) en oorwegend onbehandelde wonde (3 wonde). Afsluiting: Die antioksidant-voorbehandeling word beskou as voordelig vir MSCs, maar was nie in staat om die volle funksionaliteit van die selle te herstel om weefselewewig in 'n diabetiese nie-genesende wond te verbeter nie. 'n Multifaktoriale benadering tot diabetiese wondgenesing word in toekomstige navorsing vereis. Doctoral 2025-04-01T06:59:09Z 2025-04-01T06:59:09Z 2024-12 Thesis https://scholar.sun.ac.za/handle/10019.1/131830 Stellenbosch University xviii, 145 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Mesenchymal stem cells Colloids -- Therapeutic use Bone marrow cells Diabetes -- Pathophysiology Stem cells UCTD Meeser, Michelle N-acetylcysteine and ascorbic acid-2-phosphate incorporatedhydrogels as autologous stem cell delivery system for the treatment of diabetic wounds. |
| title | N-acetylcysteine and ascorbic acid-2-phosphate incorporatedhydrogels as autologous stem cell delivery system for the treatment of diabetic wounds. |
| title_full | N-acetylcysteine and ascorbic acid-2-phosphate incorporatedhydrogels as autologous stem cell delivery system for the treatment of diabetic wounds. |
| title_fullStr | N-acetylcysteine and ascorbic acid-2-phosphate incorporatedhydrogels as autologous stem cell delivery system for the treatment of diabetic wounds. |
| title_full_unstemmed | N-acetylcysteine and ascorbic acid-2-phosphate incorporatedhydrogels as autologous stem cell delivery system for the treatment of diabetic wounds. |
| title_short | N-acetylcysteine and ascorbic acid-2-phosphate incorporatedhydrogels as autologous stem cell delivery system for the treatment of diabetic wounds. |
| title_sort | n acetylcysteine and ascorbic acid 2 phosphate incorporatedhydrogels as autologous stem cell delivery system for the treatment of diabetic wounds |
| topic | Mesenchymal stem cells Colloids -- Therapeutic use Bone marrow cells Diabetes -- Pathophysiology Stem cells UCTD |
| url | https://scholar.sun.ac.za/handle/10019.1/131830 |
| work_keys_str_mv | AT meesermichelle nacetylcysteineandascorbicacid2phosphateincorporatedhydrogelsasautologousstemcelldeliverysystemforthetreatmentofdiabeticwounds |