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Self-assembled Collagen-Fibrin Hydrogel Reinforces Tissue Engineered Adventitia Vessels Seeded with Human Fibroblasts.

ABSTRACT: Efforts for tissue engineering vascular grafts focuses on the tunica media and intima, although the tunica adventitia serves as the primary structural support for blood vessels. In surgery, during endarterectomies, surgeons can strip the vessel, leaving the adventitia as the main strength layer to close the vessel. Here, we adapted our recently developed technique of forming vascular tissue rings then stacking the rings into a tubular structure, to accommodate human fibroblasts to create adventitia vessels in 8 days. Collagen production and fibril cross-linking was augmented with TGF-? and ascorbic acid, significantly increasing tensile strength to 57.8?±?3.07 kPa (p?=?0.008). Collagen type I gel was added to the base fibrin hydrogel to further increase strength. Groups were: Fibrin only; 0.7?mg/ml COL; 1.7?mg/ml COL; and 2.2?mg/ml COL. The 0.7?mg/ml collagen rings resulted in the highest tensile strength at 77.0?±?18.1 kPa (p?=?0.015). Culture periods of 1-2 weeks resulted in an increase in extracellular matrix deposition and significantly higher failure strength but not ultimate tensile strength. Histological analysis showed the 0.7?mg/ml COL group had significantly more, mature collagen. Thus, a hydrogel of 0.7?mg/ml collagen in fibrin was ideal for creating and strengthening engineered adventitia vessels.

SUBMITTER: Patel B

PROVIDER: S-EPMC5818609 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Self-assembled Collagen-Fibrin Hydrogel Reinforces Tissue Engineered Adventitia Vessels Seeded with Human Fibroblasts.

Patel Bijal B Xu Zhengfan Z Pinnock Cameron B CB Kabbani Loay S LS Lam Mai T MT

Scientific reports 20180219 1

Efforts for tissue engineering vascular grafts focuses on the tunica media and intima, although the tunica adventitia serves as the primary structural support for blood vessels. In surgery, during endarterectomies, surgeons can strip the vessel, leaving the adventitia as the main strength layer to close the vessel. Here, we adapted our recently developed technique of forming vascular tissue rings then stacking the rings into a tubular structure, to accommodate human fibroblasts to create adventi ...[more]

PMID: 29459640

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Project description:PurposeThe aim of this study was to identify the presence of collagen-binding integrin subunits in human scleral fibroblasts (HSFs) and investigate their actual functions in maintaining the mechanical creep properties of the HSFs-seeded collagen matrix.MethodsPrimary HSFs were cultured in vitro. Reverse- transcription PCR was used to detect mRNA expression of integrin α1, α2, and β1 subunits in HSFs. In addition, western blot analysis and immunofluorescence were used to detect their protein in HSFs. Monoclonal antibodies were applied directly against the extracellular domains of integrin subunits in HSFs cultured in the three-dimensional collagen gels to block the interaction between HSFs and the extracellular collagen matrix. The effects of anti-integrin antibodies on HSFs morphology in collagen gel were observed. The effects of the added antibodies on fibroblast-mediated collagen gels' contraction were evaluated. Furthermore, the changes in mechanical creep properties of collagen gel were measured by a biomechanics test instrument.ResultsThe mRNA and protein expressions of collagen-binding integrin α1, α2, and β1 subunits were present in HSFs. The elongated bipolar cells converted to spherical shapes after 6 h after the addition of integrin α1β1 and α2β1 antibody. The blocking of integrin α1β1 and α2β1 subunits noticeably decreased the contraction in the collagen gels. In addition, all samples were subjected to a constantly applied load of 0.03 N for 600 s. The blocking of integrin α1β1 and α2β1 subunits also induced increases in the values of final extension, creep extension, and creep rate, compared to those of the controls (p<0.01). Furthermore, the creep elements were significantly increased with the augmentation of the integrin antibody dose (p<0.01). The final extension of the integrin α2β1 antibody (1 μg/ml or 4 μg/ml) group was significantly higher compared to that of the integrin α1β1 antibody (1 μg/ml or 4 μg/ml) group (p<0.01). However, the creep extension and creep rate of the integrin α2β1 antibody (1 μg/ml or 4 μg/ml) group were not significantly different from those in the integrin α1β1 antibody (1 μg/ml or 4 μg/ml) group (p>0.05).ConclusionsOur findings suggested that HSF integrin α1β1 and α2β1 participated in maintaining the mechanical creep properties of the HSFs-seeded collagen matrix. Furthermore, integrin α2β1 might play a more crucial role in maintaining the mechanical creep properties of the collagen matrix than does integrin α1β1.

Project description:Architecture and microstructure of type I collagen fibers constitute central regulators of tumor invasion with aligned fibers providing a route for migration of stromal and cancer cells. Several different aspects of fibrillar collagen, such as stiffness, density, thickness, and pore size, may regulate migration of cancer cells, but determining effects of any one parameter requires clear decoupling of physical properties of collagen networks. The objective of this work is to develop and apply an in vitro three-dimensional (3D) tumor-extra cellular matrix (ECM) model with tunable physical parameters to define how stromal fibroblasts modulate collagen microstructure to control migration of breast cancer cells. We incorporated two different types of polyhedral oligomeric silsesquioxane (POSS) nano-molecules into a collagen/alginate matrix to induce different mechanisms of gelling. The resultant biomimetic, nanocomposite hydrogels show different collagen fibrillar microstructures while maintaining constant overall matrix stiffness, density, and porosimetry. Spheroids of human mammary fibroblasts embedded in these 3D matrices remodel the collagen network to varying extents based on differences in underlying matrix microstructures. The remodeled collagen matrix shows oriented, thicker fibrillar tracks, facilitating invasion of tumor cells. By decoupling effects of matrix stiffness and architecture, our nanocomposite hydrogels serve as robust platforms to investigate how biophysical properties of tumor environments control key processes regulating tumor progression in breast cancer and other malignancies. STATEMENT OF SIGNIFICANCE: Our manuscript demonstrates a new type of nanocomposite hydrogel with two different gelling mechanisms, produced by incorporating two types of polyhedral oligomeric silsesquioxane (POSS) nano-molecules into a collagen/alginate matrix. The resultant biomimetic hydrogels show different fibrillar collagen microstructures while maintaining constant overall matrix stiffness, density, and porosimetry. These gels allow us to uncouple effects of matrix stiffness versus architecture on migration and invasion of breast cancer cells and stromal fibroblasts. Upon embedding spheroids of human mammary fibroblasts (HMFs) and dissociated 231 breast cancer cells, we showed that HMFs remodeled the collagen network to differing extents dependent on starting matrix microstructures in each hydrogel. The remodeled collagen matrix showed aligned collagen fibers perpendicular to the surface of a spheroid with migrating HMFs following these fibers as occurs in tumors in vivo. To our knowledge, this is the first study showing significant different fibrillar collagen microstructures with constant collagen density and gel stiffness. This study establishes a new type of nanocomposite 3D hydrogels for studies of biophysical and cellular interactions in engineered tumor environments.

Dataset Information

Self-assembled Collagen-Fibrin Hydrogel Reinforces Tissue Engineered Adventitia Vessels Seeded with Human Fibroblasts.

Publications

Self-assembled Collagen-Fibrin Hydrogel Reinforces Tissue Engineered Adventitia Vessels Seeded with Human Fibroblasts.

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