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

Abstract

Current in vitro thrombosis models utilise simplistic 2D surfaces coated with purified components of the subendothelial matrix. The lack of a realistic humanised model has led to greater study of thrombus formation in in vivo tests in animals. Here we aimed to develop 3D hydrogel-based replicas of the medial and adventitial layers of the human artery to produce a surface that can optimally support thrombus formation under physiological flow conditions. These tissue-engineered medial- (TEML) and adventitial-layer (TEAL) hydrogels were developed by culturing human coronary artery smooth muscle cells and human aortic adventitial fibroblasts within collagen hydrogels, both individually and in co-culture. Platelet aggregation upon these hydrogels was studied using a custom-made parallel flow chamber. When cultured in the presence of ascorbic acid, the medial-layer hydrogels were able to produce sufficient neo-collagen to support effective platelet aggregation under arterial flow conditions. Both TEML and TEAL hydrogels possessed measurable tissue factor activity and could trigger coagulation of platelet-poor plasma in a factor VII-dependent manner. Biomimetic hydrogel replicas of the subendothelial layers of the human artery are effective substrates for a humanised in vitro thrombosis model that could reduce animal experimentation by replacing current in vivo models.

Developing a Biomimetic 3D Neointimal Layer as a Prothrombotic Substrate for a Humanized In Vitro Model of Atherothrombosis

“…The prothrombin time measurements of the M1- and foam cell-containing hydrogels demonstrate that they are both capable of triggering blood coagulation. To investigate whether this activity could be due to enhanced tissue factor in the foam cells in the 3D neointimal biomimetic hydrogel, we used a previously developed microplate assay of extrinsic clotting factor activity [ 24 ]. This assay involves incubating the constructs with a solution containing inactive Factor VII (the target of tissue factor), and a fluorescent indicator of activated factor VII (FVIIa) activity, SN-17a.…”

“…When digested by FVIIa, SN-17a becomes more fluorescent, and so the rate of fluorescence increase can be used to assess the tissue factor activity contained within each construct in the absence of any endogenously produced FVIIa. Previous experiments on samples containing human coronary artery smooth muscle cells have demonstrated that this fluorescence increase is dependent upon both the presence of a tissue factor-bearing construct and the presence of inactive Factor VII, demonstrating that the fluorescence increases are due to the presence of tissue factor in the sample [ 24 ]. However, previous studies have demonstrated that macrophages in atherosclerotic plaques are also able to produce FVII [ 45 ], indicating that fluorescent increases will be sensitive to the total activity of the extrinsic clotting factors, tissue factor, and factor VIIa, within this sample ( Figure 6 C).…”

“…Previously, we have developed a humanized in vitro arterial thrombosis model [ 22 , 23 , 24 ]. This model was produced using tissue-engineering techniques to produce a 3D human arterial construct.…”

“…Simultaneously, a medial layer construct was grown by culturing human coronary artery smooth muscle cells (HCASMCs) within a 3D type I collagen hydrogel. When complete, the intima and medial layer constructs can then be assembled to create the complete tissue-engineered human arterial construct (TEAC) that has both the anti- and pro-thrombotic properties of the intimal and medial layers of the native artery [ 22 , 23 , 24 ]. This layer-by-layer fabrication method used to produce the TEAC allows for each layer of the construct to be independently cultured prior to being brought together to produce the complete structure.…”

Modelling arterial thrombus formation in vitro

“…Advances towards more vessel like in-vitro models have now progressed beyond the inclusion of endothelial cells to vascular constructs in an attempt to include other vascular components and avoid the use of rigid chambers which are much stiffer than the vascular wall [ 58 ▪▪ ]. To achieve this, bioprinting has been adopted.…”