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SP1-functionalized SIS scaffolds driving auto-regenerative wound repair through endogenous stem cell migration

  • 김예진 (Yejin Kim, 아주대학교 일반대학원)

초록/요약

Recent advances in regenerative medicine have shifted focus toward strategies that activate endogenous repair mechanisms rather than relying on exogenous cell transplantation, which remains limited by cell availability, immune complications, low engraftment efficiency, and regulatory constraints. In this thesis, a bioactive scaffold based on chemically crosslinked small intestinal submucosa (Cx-SIS) was developed and functionalized with the synthetic chemotactic peptide SP1 to promote endogenous mesenchymal stem cell (MSC) recruitment during wound healing. The scaffold provided structural support and controlled SP1 release through modulation of crosslinking density. In vitro studies confirmed that SP1 was non- cytotoxic and significantly enhanced MSC migration. In a murine full-thickness skin wound model, the SP1-loaded Cx-SIS scaffold accelerated wound closure and improved tissue remodeling compared with control groups. Histological and immunofluorescence analyses demonstrated enhanced MSC recruitment, angiogenesis, and regulated inflammatory responses, as evidenced by altered CD29, CD31, CD68, and NK1R expression. Overall, this study demonstrates a scaffold-mediated, cell-free wound healing strategy that harnesses endogenous stem cell migration and immune modulation, providing a promising framework for regenerative biomaterials based on intrinsic tissue repair. Keywords: SP1 peptide, Small intestinal submucosa, endogenous mesenchymal stem cell recruitment, regenerative wound repair.

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목차

1. Introduction 1
2. Experimental 3
2.1. Materials 3
2.2. Culturing mouse mesenchymal stem cells (mMSCs) in vitro 3
2.3. Preparation of fluorescence labeled mMSCs 4
2.4. In vitro cytotoxicity evaluation of SP and SP1 in mMSCs 5
2.5. In vitro recruitment analysis using PKH-mMSCs by SP and SP1. 5
2.6. Preparation of Cx-SIS scaffolds 6
2.7. Characterization of Cx-SIS scaffolds 6
2.8. Preparation of SIS scaffolds conjugated with Texas Red (Tex) fluorescent dye 7
2.9. Preparation of Cx-SIS scaffolds incorporating SP1 (SP1+Cx-SIS) 8
2.10. In vitro analysis of SP1-F release behavior in N-SIS and Cx-SIS scaffolds loaded with SP1-F. 8
2.11. Animal experiments 9
2.12. Wound healing assessment using Cx-SIS and SP1+Cx-SIS scaffolds 9
2.13. Degradation of Tex-Cx-SIS scaffolds 10
2.14. Analysis of SP1-F release characteristics in SP1-F–incorporated N-SIS and Cx- SIS scaffolds. 10
2.15. Migration analysis of ICG-mMSCs in response to Cx-SIS and SP1+Cx-SIS scaffolds 11
2.16. Histological evaluation of wound tissues using hematoxylin and eosin (H&E) staining and MTS assay 12
2.17. Immunofluorescence analysis of CD68, CD31, CD29, and NK1R expres sion in wound tissue 13
3. Results & Discussion 14
3.1. Fabrication and physicochemical characterization of Cx-SIS scaffolds 14
3.2. In vitro assessment of SP1-F release kinetics in SIS scaffolds 17
3.3. In vitro assessment of SP- and SP1-induced cytotoxicity in mMSCs 18
3.4. In vitro wound healing assay performed to analyze mMSC migration in response to SP and SP 1 20
3.5. degradation assay of N-SIS and Cx-SIS scaffolds 21
3.6. Evaluation of SP1-F release behavior from N-SIS and Cx-SIS scaffolds 22
3.7. Tracking of ICG-labeled mMSC migration in response to control, Cx-SIS, and SP1+Cx-SIS scaffolds 24
3.8. Evaluation of wound regeneration using Cx-SIS and SP1+Cx-SIS scaffolds 26
3.9. Histological evaluation of regenerated tissue using H&E and Masson's trichrome staining 28
3.10. Evaluation of macrophage infiltration using CD68 immunofluorescence staining in wound tissue 31
3.11. Evaluation of neovascularization using CD31 immunofluorescence staining in wound tissue 33
3.12. Evaluation of endogenous mMSC recruitment using CD29 immunostaining in wound tissue 35
3.13. Evaluation of NK1R expression in wound tissue 37
4. Conclusion 38
Reference. 41
List of Publications 47
List of Presentations 48

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