title: Engineering a Dual-Layer Chitosan-Lactide Hydrogel To Create Endothelial Cell Aggregate-Induced Microvascular Networks In Vitro and Increase Blood Perfusion In Vivo.
abstract: Here, we report the use of chemically cross-linked and photo-cross-linked hydrogels to engineer human umbilical vein endothelial cell (HUVEC) aggregate-induced microvascular networks to increase blood perfusion in vivo. First, we studied the effect of chemically cross-linked and photo-cross-linked chitosan-lactide hydrogels on stiffness, degradation rates, and HUVEC behaviors. The photo-cross-linked hydrogel was relatively stiff (E = ∼15 kPa) and possessed more compact networks, denser surface texture, and lower enzymatic degradation rates than the relatively soft, chemically cross-linked hydrogel (E = ∼2 kPa). While both hydrogels exhibited nontoxicity, the soft chemically cross-linked hydrogels expedited the formation of cell aggregates compared to the photo-cross-linked hydrogels. Cells on the less stiff, chemically cross-linked hydrogels expressed more matrix metalloproteinase (MMP) activity than the stiffer, photo-cross-linked hydrogel. This difference in MMP activity resulted in a more dramatic decrease in mechanical stiffness after 3 days of incubation for the chemically cross-linked hydrogel, as compared to the photo-cross-linked one. After determining the physical and biological properties of each hydrogel, we accordingly engineered a dual-layer hydrogel construct consisting of the relatively soft, chemically cross-linked hydrogel layer for HUVEC encapsulation, and the relatively stiff, acellular, photo-cross-linked hydrogel for retention of cell-laden microvasculature above. This dual-layer hydrogel construct enabled a lasting HUVEC aggregate-induced microvascular network due to the combination of stable substrate, enriched cell adhesion molecules, and extracellular matrix proteins. We tested the dual-layer hydrogel construct in a mouse model of hind-limb ischemia, where the HUVEC aggregate-induced microvascular networks significantly enhanced blood perfusion rate to ischemic legs and decreased tissue necrosis compared with both no treatment and nonaggregated HUVEC-loaded hydrogels within 2 weeks. This study suggests an effective means for regulating hydrogel properties to facilitate a stable, HUVEC aggregate-induced microvascular network for a variety of vascularized tissue applications.
authors.forename: Sungwoo
authors.initials: S
authors.lastname: Kim
authors.affiliation: Department of Orthopedic Surgery, Kyoto University , 54 Kawaharacho, Shogoin, Sakyo-ku Kyoto 606-8507, Japan.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Kawai
authors.forename: Derek
authors.initials: D
authors.lastname: Wang
authors.forename: Yunzhi
authors.initials: Y
authors.lastname: Yang
mesh_terms: D000818:Animals; D048271:Chitosan / Q000737:chemistry*; D004146:Dioxanes / Q000737:chemistry*; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D020100:Hydrogels / Q000138:chemical synthesis* / Q000494:pharmacology*; D055806:Microvessels / Q000187:drug effects* / Q000254:growth & development; D023822:Tissue Engineering
chemical_list: D004146:Dioxanes; D020100:Hydrogels; D048271:Chitosan; C091880:dilactide
keywords: HUVECs; aggregates; angiogenesis; chitosan; fibrinogen; hind-limb ischemia; hydrogel; lactide; prevascularization