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Osteogenic differentiation of migrated hMSCs using an injectable, Substance P1-loaded BMP-2 mimetic peptide modified click-crosslinked hyaluronic acid hydrogel

  • 김희은 (Hee Eun Kim, 아주대학교 일반대학원)

초록/요약

Bone tissue engineering is a promising approach for treating damaged bone tissues. In this study, the scaffold for bone tissue engineering was designed with the appropriate combination of biological factors. We used hyaluronic acid (HA) based hydrogel scaffold for bone tissue engineering because HA is biodegradable, biocompatible, and nontoxic. To form an injectable click-crosslinked hyaluronic acid hydrogel (Cc-HA), trans-cyclooctene-modified HA (HA-TCO) and tetrazine-modified HA (HA-Tet) were prepared. Substance P1 (SP1) is a substance P peptide analog, that promotes stem cell migration. SP1 effectively induced the migration of human mesenchymal stem cells (hMSCs) in vitro, observed in a wound healing assay and trans-well plate migration assay. In vivo fluorescence imaging results confirmed that SP1 released from the injected SP1-Cc-HA hydrogel into mouse body effectively promoted the migration of ICG-labeled hMSCs. Bone morphogenetic protein-2 (BMP-2) mimicking peptide (BmP) induced in vitro osteogenic differentiation of hMSCs compared to that in the control group. Additionally, BmP treatment showed a greater increase in expression of RUNX2, osteonectin, and osteopontin mRNA levels compared to those in the control group. In animal experiments, SP1 attracted a large number of MSCs toward the Cc-HA-BmP hydrogel, after which BmP induced osteogenic differentiation, as demonstrated in Von Kossa staining, Alizarin Red S staining, and qRT-PCR. Thus, our injectable formulation of SP1-Cc-HA-BmP hydrogel can induce the migration of endogenous stem cells to the scaffold and osteogenic differentiation of migrated stem cells for efficient clinical treatment of damaged bone tissue.

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목차

1. Introduction 1
2. Experimental 7
2.1. Materials 7
2.2. hMSCs culture 7
2.3. Labeling of hMSCs 8
2.4. Preparation of Cc-HA 9
2.5. Preparation of Cc-HA-BmP 10
2.6. Preparation of near-infrared (NIR) fluorescence-labeled HA, HA-Tet and HA-TCO 11
2.7. Rheological measurements 12
2.8. Measurement of injection force for injectable HA hydrogel formulations 12
2.9. In vitro cytotoxicity of peptide and hydrogel scaffold 13
2.10. In vitro migration of hMSCs 14
2.11. Osteogenic differentiation of hMSCs in the presence of BMP-2 or BmP in vitro 16
2.12. Osteogenic differentiation of hMSCs in Cc-HA, Cc-HA(+BmP) and Cc-HA-BmP hydrogel in vitro 18
2.13. Animal Experiments 19
2.14. In vivo imaging of injectable formulations 19
2.15. In vivo migration of MSCs in the hydrogel scaffolds 20
2.16. In vivo release profiles of SP1-F from SP1-F-loaded hydrogels 20
2.17. In vivo osteogenic differentiation of MSCs in the hydrogel scaffolds 21
2.18. Immunofluorescence staining of migrated hMSCs in Cc-HA, SP1-Cc-HA, SP1-Cc-HA(+BmP) and SP1-Cc-HA-BmP hydrogel in vivo (CD31) 22
2.19. Statistical analysis 23
3. Results & Discussion 24
3.1. Characterization of injectable click-crosslinked HA hydrogels 24
3.2. In vivo persistence of HA and Cc-HA hydrogel 27
3.3. In vitro cytotoxicity of peptide and hydrogel scaffold 29
3.4. Migration of hMSCs in the presence of SP or SP1 in vitro 31
3.5. In vitro hMSCs migration toward SP or SP1-loaded Cc-HA hydrogels 31
3.6. Osteogenic differentiation of hMSCs in the presence of BMP-2 or BmP in vitro 35
3.7. Osteogenic differentiation of hMSCs in Cc-HA, Cc-HA(+BmP) and Cc-HA-BmP hydrogel in vitro 36
3.8. In vivo hMSCs migration toward SP1-loaded Cc-HA hydrogels 39
3.9. In vivo real-time images of SP1-F from hydrogels 41
3.10. Osteogenic differentiation of hMSCs in Cc-HA, Cc-HA(+BmP) and Cc-HA-BmP hydrogel with SP1 in vivo 43
4. Conclusion 52
REFERENCES 53

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