Developing Biomimetic Hydrogels of the Arterial Wall as a Prothrombotic Substrate for In Vitro Human Thrombosis Models

Figures

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  Figure 1. Ascorbic acid supplementation increases the production of neo-collagen by HCASMCs in the tissue-engineered medial layer hydrogels. (A) Immunofluorescent imaging of the TEMLs cultured with (ASC) and without (NASC) daily supplementation of 50 µg/mL ascorbic acid. ASC- and NASC-treated TEMLs with mouse anti-human collagen I alpha 1 antibody. Images were taken from the most fluorescent slice within the z-stack. A primary-free negative control is shown in which the TEML was incubated with only the fluorescently labelled secondary antibody (scale bars are 200 µm). (B) Mean slice fluorescence of the ASC- and NASC-treated TEMLs. * shows significance of p < 0.05 compared to NASC control.

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  Figure 2. Ascorbic acid supplementation of the TEML hydrogels enhances its pro-aggregatory properties under physiological flow. (A,B) Washed human platelets were exposed to NASC-treated and ASC-treated TEMLs, and their respective acellular controls, under magnetic stirring for 7 min at 37 °C. Samples were then transferred to a light transmission aggregometer (A) A representative aggregometry trace. (B) A bar chart summarising the extent of aggregation elicited by the TEML hydrogels and collagen hydrogel following the 7-min incubation period. (C) 3D printed chamber utilised to perfuse washed DiOC6-labelled human platelets at physiological arterial shear stresses (14 dynes/cm2) over the ASC-treated and NASC-treated TEMLs, as well as an acellular collagen hydrogel control. Representative images are shown from samples perfused with blood from three different donors.—Scale bar = 5 mm. (D) A summary bar chart showing the mean fluorescent intensity of the TEML surface after platelet perfusion. * indicates p < 0.05 compared to acellular control, † indicates p < 0.05 compared to NASC.

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  Figure 3. The tissue-engineered medial layer hydrogel can trigger blood coagulation. (A) Diagram illustrating the prothrombin time capture for TEML. (i) TEML hydrogels or acellular controls are placed onto sodium acetate frames and placed into contact with the surface of prewarmed, recalcified human platelet-poor plasma (PPP). (ii) The sample is kept warm in a 37 °C water bath. (iii) The time taken between TEML contact with PPP and fibrin formation is recorded. (B) A representative photograph of PPP clotting on the surface of the TEML hydrogel post-incubation. (C) Bar chart showing the mean prothrombin times recorded for ASC- and NASC-treated TEML hydrogels. * indicates p < 0.05 compared to NASC control. Created with BioRender.com.

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  Figure 4. Tissue-engineered medial layer hydrogels trigger blood coagulation through the extrinsic coagulation pathway. (A) Prothrombin times were measured for TEML samples which were exposed to prewarmed, recalcified PPP samples pre-treated with either 50 µg/mL Corn Trypsin Inhibitor (CTI) or an equivalent volume of its vehicle, PBS. (B) Prothrombin times for TEML hydrogels which were exposed to either PPP from our donor stock, commercially acquired factor VII-depleted plasma (FVII-D), and the same factor VII-depleted plasma which had been exogenously supplemented with of 10 nM inactive factor VII (FVII-D+). * indicates p < 0.05 compared to the PPP control. † indicates p < 0.05 compared to the FVII-D sample.

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  Figure 5. The tissue-engineered medial layer hydrogel possesses measurable tissue factor activity. (A) Diagram illustrating a novel method of measuring tissue factor activity in TEMLs cultured on 48-well microplates. Culture media was removed and an HBS solution containing 10 nM inactive factor VII, 1 mM CaCl2, and 100 µM SN-17a was added to either TEML hydrogels (1) or an empty well (2; FVII control). Additionally, SN-17a was added to a well of HBS alone (3; HBS control). The well plate was then immediately inserted into the microplate reader and measured fluorometrically. The tissue factor (if any) within the wells will immediately activate the available FVII forming FVIIa. (B) Graph showing the mean response observed at all time points across the 5 individual experiments for ASC- and NASC-treated TEMLs as well as experiments in which either the TEML gel (HBS; empty well) or inactive factor VII (FVII) was omitted from the experiment. Note the initial increase in fluorescence in the ASC and NASC samples due to the time delay between adding SN-17a into the wells and the starting of the fluorescent readings. (C) A bar chart summarising the change in SN-17a fluorescence observed between the…

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  Figure 6. The adventitial layer is not required to effectively trigger blood coagulation in the tissue-engineered blood vessel construct. (A) Diagram illustrating the method of TEML-TEAL co-culture fabrication. (i) After fabrication of TEML and TEAL hydrogels, rat-tail collagen gel solution was added to the adluminal surface of the TEML hydrogels. (ii) TEAL hydrogels were then placed atop the TEML. (iii) The hydrogels were then placed into an incubator for an additional 30 min to allow the collagen in between the layers to gelate and bond them together. Co-cultures were then transferred to 24-well plates and cultured in a 65:35 ratio of HCASMC: HAoAF culture media, both supplemented with 50 µg/mL ascorbic acid. (B) Mean prothrombin times measure on the TEML layer alone, TEAL layer alone, and TEML-TEAL co-culture. Please note that no significant difference was found between any of the groups. Created with BioRender.com.