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The newly discovered chemokine-mimicking peptide-loaded cross-linked hyaluronan scaffold to accelerate wound healing through endogenous mesenchymal stem cell recruitment

  • 김영훈 (아주대학교 일반대학원)

초록/요약

Endogenous stem cells are tissue-specific adult stem cells that are can self-renewal and differentiate into specific cells. These cells migrate to the specific regions in the body where tissue healing is required. Because endogenous stem cells are involved in the healing and regeneration of wounded or diseased tissues or organs, in situ tissue regeneration using endogenous stem cells may overcome the limitations of ex vivo stem cell manipulation in conventional tissue engineering. Chemoattractants such as substance-p, and stromal-derived factor 1, are powerful endogenous stem cell activating factors and can be used to prolong and improve endogenous stem cell assembly. However, the number of chemoattractant-guided endogenous stem cells that migrate during the endogenous repair is generally too low to achieve full in situ tissue regeneration. Thus, new chemoattractants are needed to increase the migration of endogenous stem cells to the impaired area. Among these factors, stromal cell-derived factor 1 (SDF-1) binds to the stem cell CXCR4 receptor. We used stromal cell-derived factor 1-mimic peptide-4 (SMP-4), which strengthened the binding force of stem cell receptors using the motif search package of the molecular operating environment (MOE) suite by mimicking the peptide sequence used for stem cell migration. We analyzed wound healing after adding SDF-1 and SMP-4 drugs to a hyaluronic acid scaffold in the skin tissue damaged up to the subcutaneous fat layer. The results showed that collagen synthesis was promoted by fibroblast proliferation, differentiation, and neovascularization. We also observed rapid skin regeneration by angiogenesis and vascular endothelial progenitor cell migration.

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목차

1. Introduction 1
2. Experimental 3
2.1. Materials 4
2.2. Proliferation process of human mesenchymal stem cells (hMSCs) 4
2.3.PKH-Labeling of human mesenchymal stem cells (hMSCs) 4
2.4. ICG-Labeling of human mesenchymal stem cells (hMSCs) 4
2.5. Preparation of SMP-4-loaded click-crosslinked HA (Cx-HA) scaffold 5
2.6. Preparation of near-infrared (NIR) fluorescence-labeled HA, HA-DBCO and HA-Azide 6
2.7. Rheological measurements 6
2.8. SEM analysis 7
2.9. Displacement of ethanol analysis 7
2.10. Nano-indenter analysis 7
2.11. In vitro cytotoxicity of peptides or Cx-HA scaffold 8
2.12. In vitro migration of hMSCs towards Cx-HA, SMP-4-loaded Cx-HA, and SDF-1-loaded Cx-HA 8
2.13. In vitro release profiles of SMP-4-loaded Cx-HA and HA scaffold 9
2.14. In vitro degradation profiles of SMP-4-loaded Cx-HA and HA scaffold 9
2.15. Animal experiment 9
2.16. In vivo degradation image and release profile of SMP-4-loaded Cx-HA and HA scaffold 10
2.17. In vivo migration of ICG-labeling MSCs toward the SMP-4-loaded Cx-HA and HA scaffold 10
2.18 In vivo wound healing assessment via wound size analysis 11
2.19. In vivo wound healing assessment via analysis 11
2.20. In vivo Immunofluorescence staining of migrated hMSCs in SMP-4-loaded Cx-HA, SDF-1-loaded Cx-HA, or Cx-HA scaffold at skin tissue (CD29, CD31, CD44) 11
2.21. In vivo RT-PCR 13
3. Results&Discussion 15
3.1. Physical property measurement of click-crosslinking HA (Cx-HA) scaffold 15
3.2. In vitro Wound healing assay to hMSCs in the presence of SDF-1 or SMP-4 16
3.3. In vitro cytotoxicity to hMSCs in the presence of SDF-1 or SMP-4 scaffold 17
3.4. In vitro hMSCs migration toward SMP-4 or SDF-1 loaded Cx-HA scaffold 18
3.5. In vitro release profile of SMP-4-d-F from SMP-4-d-F-loaded HA or Cx-HA scaffold and degradation of HA or Cx-HA scaffold 20
3.6. In vivo release profile of SMP-4-d-F from SMP-4-d-F-loaded HA or Cx-HA scaffold and degradation of HA or Cx-HA scaffold 21
3.7. In vivo ICG-labeled hMSCs migration toward SMP-4 or SDF-1 loaded Cx-HA scaffold 22
3.8. In vivo evaluation of wound healing effects through MSC migration 24
3.9. In vivo masson's trichrome-stained histological images of wound healing effects to MSC migration 26
3.10. In vivo migration analysis via the surface (CD29 and CD44) staining in injected hMSCs skin tissue 30
3.11. In vivo migration analysis via the surface (CD31) staining in injected hMSCs skin tissue 33
3.12. In vivo migration analysis via the surface (CD29 and CD44) staining in endogenous mMSCs skin tissue 35
3.13. In vivo migration analysis via the surface (CD31) staining in endogenous mMSCs skin tissue 38
3.14. In vivo CD29, CD31, CD44, and CXCR4 RT-PCR of mouse wounded skin tissue 39
4. Conclusion 51
REFERENCES 52

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