Organs-on-Chip as advanced models of osteoarthritis and mechanically active body districts

The development of a new drug is estimated to require $ 2.8 billion [1, 2] and a time comprised between 10 and 15 years [3]. Moreover, only 10% of compounds considered in phase I clinical trials reach the market [3]. This inefficiency correlates with the lack of predictive preclinical disease models [4].
Classic 2D plasticware-based models were demonstrated to be over-simplified and ineffective drug response predictors [4]. Conversely, costly and time expensive animal models, beside presenting ethical concerns, cannot be adopted in the initial, high throughput, phases of chemical entities screenings [5].
Organs-on-chip (OoC) are microfluidic based devices aiming at recapitulating organ and tissue level functions in vitro [6]. Allowing an increased control over the experimental environment, OoCs are gaining increasing consensus as disease modelling tools. The ability to model diseases affecting mechanically active body districts, such as the musculoskeletal apparatus or the myocardium remains however an open challenge. This aspect becomes particularly relevant when proper disease mimicry requires the incorporation of multiple tissues. Moreover, most OoCs are currently limited to single chamber devices thus impairing their applicability as high throughput screening tools [7].
Case in point, osteoarthritis (OA) is a degenerative disease mainly affecting load bearing joints such as knees and hips. Exact estimates of OA prevalence vary depending on the considered definition of OA, the joint of interest, and the studied population [8]. Knee osteoarthritis was deemed to effect roughly 10% of men and 13% of women over the age of 60 [9]. Despite this, a successful OA treatment is currently missing [10] being existing therapeutic choices palliatives aimed at relieving symptoms rather than reversing the degenerative processes.
OA is recognized as a multifactorial and complex disorder affecting the joint as a whole. OA pathological alterations include hyaline cartilage focal damage and degradation; chondrocyte assumption of a hypertrophic phenotype; vascular invasion, expansion towards the upper layers, and increase of the mineral content of calcified cartilage; and sclerosis and hypomineralization of subchondral bone. Indeed, the whole osteochondral unit (OCU) is affected [11, 12].
While no consensus is present regarding OA origin, a clear correlation between the pathology and mechanical risk factors such as trauma, joint misalignment and obesity has been demonstrated [13][14].
The lack of disease modifying anti-OA drugs was associated to the absence of relevant preclinical OA models [15]. The generation of representative OA models is however challenging given the difficulty of representing the multitude of tissues involved as well as the natural mechanically active joint environment.
In this framework, the present PhD work aimed at exploiting OoCs principles and microfabrication techniques together with tissue engineering, to develop disease relevant models of mechanically active tissues with a particular focus on OA.
The final aim was broke-down into five subsections, each aimed at delivering a specific technological or biological advancement.
i) Initially, a mechanically active OoC platform capable of providing three dimensional (3D) constructs with physiological (i.e. 10%) or hyperphysiological (i.e. 30%) compressive levels was designed, functionally validated, and exploited to achieve (i) a healthy cartilage on chip model (CoC) positive for the deposition of cartilage extracellular matrix constituents (e.g. collagen type II and aggrecan) and to (ii) induce OA traits in the CoC through the sole application of hyperphysiological compression. Specifically, it was feasible to recapitulate the imbalance of catabolic and anabolic processes, the increased inflammatory state, and the hypertrophic phenotype characterizing cartilage in OA. The model was also exploited to evaluate the response to known and innovative anti OA compounds.
ii) The adoption of OoCs as possible testing systems in drug screening campaigns depends on the achievement of a satisfactory experimental throughput. A platform capable of subjecting multiple independent 3D constructs to cyclical mechanical stimulation was engineered and functionally validated. Appropriately designed valves positioned between the different culture chambers allowed to inject multiple compartments with a single operation while assuring their independence during the culture period. As a proof of concept, the device was adopted to apply cyclical strain to cardiomyocytes and fibroblasts co-cultures recapitulating some traits of cardiac fibrosis. The device was then modified to make it suitable for the study of OA, shifting its mechanical stimulation from the stretching to the compression of hosted tissues.
iii) Cartilage and bone are examples of tissues with a hierarchical organization. During OA the whole OCU is affected leading to altered tissues mechanical properties.
A direct assessment of the mechanical, compositional and morphological alterations characterizing the different OCU tissues of OA patients was performed focusing on a dimensional scale relevant for developing future complex OA models based on the OoC technology.
Specifically, indentation type atomic force microscopy (IT-AFM) was adopted to study OCU tissues mechanical properties at a sub-micrometre scale and differentiating between hyaline cartilage zones, and calcified cartilage and subchondral layers. The technique was then translated to the determination of the mechanical properties of cartilaginous microconstructs.
iv) While the whole OCU is affected by OA, most in vitro models are limited to the study of cartilage. Modelling physiologically relevant mechanical stimulation in other OCU layers remains a challenge.
To fill this gap, a new OoC platform enabling the provision of well-defined and discrete levels of mechanical compression to directly interfaced superimposed 3D microconstructs was designed. Specifically, a new microfluidic concept (namely Vertical Capillary burst Valve, VBV) was introduced, consenting the vertical superimposition of two 3D cell-laden hydrogels and the spatially precise modulation of the strain field experienced by the two tissues. The device was validated demonstrating that different layers of cartilaginous constructs can be subjected to various levels of compression resulting in distinct mechanotransduction signalling. The platform was then exploited to demonstrate that local perturbations in the composition of acellular subchondral layers resulted in modulations of the loading response of cartilage-like constructs.
v) Finally, the culture conditions to obtain cellular microconstructs recapitulating multiple OCU layers were investigated. Specifically, primary human articular chondrocytes (hACs) and mesenchymal stromal cells (MSCs) were differentiated, respectively, in hyaline cartilage and calcified cartilage. A biphasic microconstruct integrating the two tissues was then achieved through a specifically designed OoC platform allowing a direct interface between two adjacent cell-laden hydrogels. The model was further integrated with a vascular compartment, used to preliminary assess how endothelial cells influence MSCs differentiation. Finally, providing spatially discrete mechanical compression to the obtained biphasic constructs, a preliminary evaluation of the effect of loading was performed.
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