Development of a total atherosclerotic occlusion with cell-mediated calcium in animal arteries using tissue engineered scaffolds

The most risky atherosclerosis includes the presence of calcium deposits in lumen of totally occluded arteries. Most animal models of total atherosclerotic occlusion do not mimic gradual occlusion process of the vessel and is often missing physiological calcium deposits in occlusion sites. The aim of this dissertation is to create chronic atherosclerotic occlusion that contains calcium in animal arteries using tissue engineered scaffolds. The overall project is composed of the following four aspects of work: (1) calcification ability of human aortic smooth muscle cells (HASMCs) on polylactic acid (PLA) films, (2) calcification of human primary osteoblast (HOB) cultures on polycaprolactone (PCL) scaffolds with TGF-â1 loading, (3) the effect of flow on the calcification of HOB/PCL construct, and (4) establishment of a total atherosclerotic occlusion in an animal artery by the implantation of HOB/PCL construct.

(1) HASMCs were cultured on PLA films with three calcification induction techniques (BMP-2, Ca/Pi, and beta-GP treatments). Calcium staining, BMP-2 production of cells, and pH condition in media has been investigated. BMP-2 treatment did not show calcification in HASMC cultures. HASMC cultures are capable of depositing calcium through Ca/Pi stimulation, but not through beta-GP treatment. PLA films may hinder mineralization of cell cultures. Gas plasma treatment had no effect on cell culture calcification.

(2) HOBs were first treated with TGF-beta1 and dexamethasone (Dex) in media on bare wells. Calcification was visualized under light microscope. Next, HOBs were cultured on PCL scaffolds with TGF-beta1 loading in the presence or absence of Dex in media. DNA content, ALP activity, amount and distribution of calcium were examined. On bare wells, highest calcification was observed in groups with both TGF-beta1 (0.02 ng/ml) and Dex (10 -10 M) in media. When cultured on scaffolds in Dex supplemented media, TGF-beta1 appeared to have an inhibitory effect on scaffold calcification. When HOBs were cultured without Dex in media, the lower amount of TGF-beta1 loading (5 ng) showed the most calcification, high DNA synthesis, and high ALP activity on scaffolds.

(3) HOBs were cultured on PCL scaffolds with TGF-beta1 loading under dynamic flows. DNA content, amount of calcium, visualization of calcium on scaffolds, and percentage weight loss of PCL scaffolds was tested. Dynamic flow improves the intensity and enlarges the distribution of calcium on polycaprolactone scaffolds. Calcification in HOB cultures can occur early under flow conditions.

(4) HOB/PCL constructs were implanted into rabbit femoral arteries after 28 days of in vitro initiation of calcification treatment. Angiograms, gross histology of arteries were captured to examine the occlusion of arteries. Fluorescent stain of calcium and EDS detection of calcium element was performed. Femoral arteries stayed totally occluded over 28 days. Physiologically deposited calcium was observed in chronic total occlusion (CTO) sites at 3, 10, and 28 days with the day-10 specimens showing the maximum calcium. The animal work showed a successful CTO model was developed using osteoblast seeded PCL scaffolds with TGF-beta1 loading.

This dissertation demonstrated the successful creation of a novel calcified CTO model in an animal artery using tissue-engineering strategies. This CTO model can be used to develop new devices and therapies to treat total atherosclerotic occlusion.