Vascularized bone grafts based on gelatin-heparin conjugated cryogels

다국어 초록 (Multilingual Abstract)

A cryogel is a one of the scaffolds which have high porosity with interconnected macropores providing cell compatible microenvironment. In addition, cryogels can be utilized in minimally invasive surgery due to its sponge-like properties, including rapid shape recovery and injectability. Herein, we developed an injectable cryogel by conjugating heparin to gelatin as a vascular endothelial growth factor (VEGF) and fibroblasts carrier for the treatment of ischemic hind limb mouse model. Our gelatin/heparin cryogel showed a mesh-like network dependent on the gelatin concentration, having a different mechanical properties in swelling ratio, interconnected porosity and elasticity. In addition, integrating heparin into cryogel allowed controlled release of VEGF for a long period, leading to a therapeutic effect demonstrated in in vitro endothelial cell angiogenesis study. Also, its sponge-like properties enabled cryogels to be applied as an injectable system with desirable maintenance of cells and proteins after injection. And, as gelatin is one of the natural polymers that have a high biocompatibility, gelatin/heparin cryogel showed high cell loading efficiency and viability without any other treatment. As a result, overall in this study, we optimized the combination of the concentration of gelatin and heparin in aspect of sustainable release of VEGF and injectability. Finally, optimized gelatin/heparin cryogel integrated with VEGF and NIH-3T3 fibroblasts was applied to in vivo ischemic hind limb model and demonstrated its angiogenic potential by improving neovascularization into the cryogel. As utilizing our gelatin/heparin cryogels with human mesenchymal stem cell (hMSCs), we expect to see the therapeutic effect for bone remodeling.

목차 (Table of Contents)

• 1. Introduction 1
• 2. Experimental section 3
• 2.1 Preparation of gelatin-heparin cryogels 3
• 2.2 Characterization of gelatin-heparin cryogels 4
• 2.3 Rheology test 5
• 2.4 Heparin detection via alcian blue assay 6
• 2.5 Synthesis of RITC-heparin conjugated cryogel 6
• 2.6 Heparin mediated protein release kinetics 7
• 2.7 HUVEC migration and tube formation assay 7
• 2.8 Injecatability test 8
• 2.9 Preparation of cell seeded scaffolds 9
• 2.10 PicoGreen and live/dead assay 9
• 2.11 In vivo ischemic hinlimb mouse model 10
• 2.12 OCT embedding 10
• 2.13 Histology & immunostaining 11
• 3. Results and discussion 13
• 3.1 Fabrication of gelatin/heparin cryogel 13
• 3.2 Characterization of gelatin/heparin cryogels 14
• 3.3 Rheological properties of gelatin/heparin cryogels 16
• 3.4 Heparin conjugated microarchitecture of gelatin/heparin cryogels according to the heparin concentration 17
• 3.5 Heparin mediated VEGF release kinetics of gelatin/heparin cryogels according to the heparin concentration 18
• 3.6 In vitro angiogenic response of HUVEC on VEGF dependent on heparin concentration of gelatin/heparin cryogels 20
• 3.7 Injectability of gelatin/heparin cryogels 21
• 3.8 Injectable gelatin/heparin cryogel as an effective carrier of NIH-3T3 fibroblasts 23
• 3.9 Effect of gelatin/heparin cryogels containing VEGF or/and NIH-3T3 fibroblasts on in vivo ischemic hind limb mouse model for neovascularization 24
• 4. Conclusion 28
• REFERENCES 39
• 요약 (국문초록) 47