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Wound healing with cross-linked small intestine submucosa (SIS) sponge through stem cell recruiting by Substance P1

  • 김신아 (Shina Kim, 아주대학교 일반대학원)

초록/요약

Repairing skin tissue is the most important regenerative component of wound healing. In this study, we used a scaffold with similar composition and human- derived mesenchymal stem cells that autologously secrete cytokines, growth factors, and cells to induce tissue regeneration. Therefore, we first prepared a wound covering sponge using porcine small intestinal submucosa (SIS), which is characterized as a natural material that is biocompatible, biodegradable, and non- toxic. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N- Hydroxysuccinimide (NHS) were used to prepare SIS as a wound covering material. Substance P1 (SP1) is a analog peptide of substance P, that promotes stem cell migration to tissue. SP1 has non-toxic properties and smoothly attracted hMSCs to specific sites in vitro, and these results were evaluated by toxicity assessment, wound healing assay and trans-well plate migration assay. In vivo, hMSCs fluorescence imaging showed that SP1, which was physically contained in SP1-SIS sponge attached to the wound site, was released early in wound healing and migrated to the stem cells. This was confirmed not only by real-time observation of fluorescence tagging of hMSCs, but also by CD29 and CD44 staining, which are antibodies present on the surface of stem cells. In addition, the synthesis of collagen, an essential factor for skin tissue regeneration, was confirmed by Masson trichrome staining (MTS), and the formation of blood vessels was confirmed by CD31 staining, and it was found that the experimental group with SP1-SIS was the best when compared with other experimental groups. Thus, we found that SP1-SIS wound covering material designed and manufactured by us can attract endogenous stem cells to a specific area, attach to the scaffold and skin tissue, and interact with them to proceed with wound healing through tissue regeneration.

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목차

1. Introduction 1
2. Experimental 7
2.1. Materials 7
2.2. Culturing human mesenchymal stem cells (hMSCs) in vitro 7
2.3. Preparation of fluorescence labeled hMSCs 8
2.4. Preparation of C-SIS and SP1 loaded C-SIS 9
2.5. Preparation of near-infrared (NIR) fluorescence-labeled SIS sponges 10
2.6. SEM analysis 11
2.7. Measurement of contact angle of SIS sponges 11
2.8. In vitro degradation assay of SIS sponges 12
2.9. In vitro peptide release assay of SP1 loaded SIS sponges 12
2.10. In vitro cytotoxicity test of SP and SP1 with hMSCs 13
2.11. In vitro migration of hMSCs with SP and SP1 14
2.12. In vitro migration of hMSCs with SP and SP1 loaded SIS sponges 14
2.13. Animal experiments 16
2.14. In vivo degradation assay of SIS sponges 16
2.15. In vivo peptide release assay of SP1 loaded SIS sponges 17
2.16. In vivo migration of ICG-labeled hMSCs toward SIS sponges 17
2.17. In vivo wound healing assay with SIS sponges 18
2.18. In vivo histological analysis by hematoxylin & eosin (H & E) staining / Masson trichrome staining (MTS) 19
2.19. In vivo immunofluorescence staining of migrated and vascularized hMSCs by CD29 / CD44 and CD31 staining 20
2.20. Statistical analysis 21
3. Results & Discussion 22
3.1. Characterization of manufactured SIS sponges 22
3.2. In vitro peptide release assay of SP1 loaded SIS sponges 25
3.3. In vitro cytotoxicity test of SP and SP1 with hMSCs 27
3.4. In vitro migration of hMSCs with SP and SP1 28
3.5. In vitro migration of hMSCs with SP and SP1 loaded SIS sponges 30
3.6. In vivo degradation assay of SIS sponges 33
3.7. In vivo peptide release assay of SP1 loaded SIS sponges 33
3.8. In vivo migration of ICG-labeled hMSCs toward SIS sponges 36
3.9. In vivo wound healing assay with SIS sponges 40
3.10. In vivo histological analysis by hematoxylin & eosin (H & E) staining / Masson trichrome staining (MTS) 43
3.11. In vivo immunofluorescence staining of migrated and vascularized hMSCs by CD29 / CD44 and CD31 staining 46
4. Conclusion 51
REFERENCES 52
LIST OF PUBLICATIONS 57
LIST OF AWARDS 59

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