Nanoindentation-based biomechanics drive engineering of a 3D bioprinted human heart valve disease model


Abstract


INTRODUCTION

In Calcific Aortic Valve Disease (CAVD), microcalcifications disrupt the structural integrity of the aortic valve (AV) leaflets, which consist of three (mechanically) distinct layers: fibrosa, spongiosa, and ventricularis. Currently, there is no therapeutic treatment. Adequate in vitro models to study the disease pathobiology are lacking due to the difficulty in recreating the complex valvular mechanical properties required to prevent spontaneous pathological differentiation of the mechanosensitive valvular interstitial cells (VICs) populating the leaflets. To overcome this difficulty, we engineered a human 3D bioprinted model of CAVD that recapitulates the mechanical environment of the AV leaflet and enables biological study of CAVD.


METHODS

Human AV leaflets were obtained from patients undergoing AV replacement surgery for the treatment of CAVD. The individual layers of the human aortic valves were separated by microdissection techniques, and the Young’s modulus was measured by nanoindentation. To mimic the layer’s mechanical properties, we developed a 3D hybrid hydrogel model incorporating methacrylated gelatin (GelMA) and methacrylated hyaluronic acid (HAMA). By encapsulating human VICs within the hydrogels and employing 3D bioprinting techniques, we simulated the mechanical properties and structural organization of the native AV tissue extracellular matrix. The hydrogel constructs were then exposed to normal growth media (NM) supplemented with osteogenic factors (osteogenic media, OM) to stimulate the formation of early microcalcification. Microcalcifications were visualized with a near infrared fluorescent calcium tracer.


RESULTS

The median Young’s moduli of the fibrosa, spongiosa, and ventricularis layer were 37.1 kPa,

15.4 kPa, and 26.9 kPa, respectively. Hydrogels composed of 5%GelMa and 1%HAMA, crosslinked for 90 or 30 seconds mimicked the Young’s moduli of the fibrosa and spongiosa layers respectively (Figure 1). We further showed that OM stimulation of VIC-laden 3D bioprinted hydrogels with these moduli mimics calcification processes observed in native valves with low levels of apoptosis (Figure 2).


CONCLUSIONS

We have created a novel 3D bioprinted CAVD model that mimics the mechanical properties of the CAVD valve leaflet and drives rapid microcalcification in culture without apoptosis. This model can accelerate research into disease mechanisms and serve as a first step for developing a high-throughput drug-screening model.


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Figure 1 . Mechanical characterisation of the individual AV leaflet layers and hybrid hydrogels crosslinked for 90 and 30 seconds. Results show distinct stiffness of the individual layers. The fibrosa and spongiosa layer mechanics are best recapitulated by the 90 respectivally 30 seconds crosslinked hydrogels.

Photocrosslinking: 365nm UV light at 2.5 mW/cm 2. Median of 9 measurements per sample shown; n=5-8 samples per condition; Median; *: p<0.05.


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Figure 2 . The fibrosa-like, and spongiosa- like hydrogel 3D bioprinted CAVD models show high cell viability and the formation of microcalcifications after 14 days of cell culture. Blue: Hoechst staining for nucleus, red: microcalcifications visualized by near infrared imaging agent, green: TUNEL apoptosis assay.

(a) Human VICs cultured in NM in fibrosa- like and spongiosa-like hydrogels. (b) haVICs cultured in OM fibrosa-like and spongiosa-like hydrogels show an increase in calcification while maintaining high cell viability. Bar 200μm.