Periodic Reporting for period 1 - uKNEEversal (uKNEEversal: a miniaturized 3D in vitro model of human joint to gain new knowledge on Osteoarthritis pathophysiology)
Reporting period: 2019-09-01 to 2021-08-31
uKNEEversal aims at generating a comprehensive in vitro human model of Osteoarthritis (OA) to fill lacks of existing pre-clinical tools and open perspectives to investigate OA mechanisms and therapies. Degenerative joint diseases, among which OA is the most prevalent, represent the second cause of disability worldwide. Available pharmacological options are limited and focus on the reduction of symptoms, being unable to factually reverse the pathology. One of the major obstacles towards the development of disease-reversing therapies is the gap of knowledge on OA mechanisms, linked to the lack of reliable OA pre-clinical models. Existing pre-clinical tools are currently not able to dissect OA complexity in terms of cross talks among compartments and molecular pathways affected. Although animal models may offer a complex enough model, they often fail in predicting typical human responses and recent legislation about 3R principle is pushing Pharma towards methods alternative to animals. In this regards, one ground-breaking goal in the field is the generation of an OA model able to reflect the complexity of the disease in vitro. To date, most in vitro OA models, based on traditional 2D and macroscale culture systems, are affected by sampling sites, poor scalability, and are too simplistic to recapitulate joint 3D architecture. As an alternative, human organs-on-chips (OOC) have been claimed able to transform many areas of basic research and drug development. OOC are in vitro tools that can recapitulate a human organ’s function better than traditional culture systems, in a miniaturized environment. Miniaturization has indeed showed an enhanced control leading to in vitro models recapitulating the native organ functions with accuracy hardly achievable at the macroscale.
In this scenario, uKNEEversal aims at filling the gap of existing poorly predictive pre-clinical models generating an in vitro human osteoarthritic joint model through organs-on-chip technology. To this aim, uKNEEversal overall objectives are three: 1) technical development of a 3D micro-platform able to host the functional unit of human joint including a cartilage construct, a bone layer and a tidemark and integrating a compartment for mechanical stimulation; 2) application of this platform to generate a healthy joint model starting from primary cells from patients; 3) triggering OA traits within the generated model by taking advantage of clinical observations in OA patients and analyzing the pathways activated during OA onset in different compartments.
In this scenario, uKNEEversal aims at filling the gap of existing poorly predictive pre-clinical models generating an in vitro human osteoarthritic joint model through organs-on-chip technology. To this aim, uKNEEversal overall objectives are three: 1) technical development of a 3D micro-platform able to host the functional unit of human joint including a cartilage construct, a bone layer and a tidemark and integrating a compartment for mechanical stimulation; 2) application of this platform to generate a healthy joint model starting from primary cells from patients; 3) triggering OA traits within the generated model by taking advantage of clinical observations in OA patients and analyzing the pathways activated during OA onset in different compartments.
First, we focused on the development of a 3D microfluidic platform enabling for compartmentalized culture and mechanical stimulation of 3D joint constructs in vitro. To achieve this aim, we started from a cartilage-on-chip device previously developed by our research group in POLIMI, and demonstrated able to culture 3D cartilaginous microconstructs and subject them to physiological or hyperphysiological mechanical overload (i.e. 10% or 30% confined compression). To better capture the complexity of human joint and develop an enabling tool to elucidate cartilage and subchondral bone crosstalk, a first co-culture module was designed in POLIMI enabling for compartmentalized 3D cell co-culturing, through generation of flanked double-layers 3D microscale constructs. In vivo, cartilage strains were demonstrated to vary deeply within tissue layers with an overall compression of -5% resulting in strain levels of -35% in the superficial zone, -5% in the transitional zone and -1% in the deep zone. The realization of models with different superimposed tissues, as those characterizing the osteochondral unit (OCU), and capable of applying compartment specific physiologically relevant mechanical stimuli is however impaired by limitations affecting available OoC technologies. To fill these gaps, we developed an Organ on Chip platform based on a new technological concept, enabling to provide well-defined and discrete levels of pathophysiological mechanical compression to bi-phasic microconstructs whom spatial organization mimics OCU elements functional anatomy. The newly introduced VBV was combined with a methodology previously developed by our group in POLIMI to provide 3D microconstructs with well-defined confined compression levels.
The developed miniaturized platforms were exploited to generate a healthy model of articular joint by using healthy articular chondrocytes (ACs) and mesenchymal stromal cells (MSCs) obtained from the routine cell banking performed at USB. Culture conditions promoting individual cartilage and calcified cartilage constructs formation were then optimized at POLIMI within a simplified version of the device (i.e. hosting a single 3D construct). Upon optimization of a culture medium permissive for single constructs culture conditions, bi-phasic OCU constructs have been generated.
The generated miniaturized OCU model were finally stressed by mimicking an OA-like microenvironment to induce the pathology onset. The effect of different compression levels applied to the constructs in the two compartments was first evaluated assessing the modulation of a set of genes associated with mechanotransduction signaling. The obtained in vitro OA model was then exploited to investigate over the influence of a cross talk between cartilage and bone/calcified cartilage during OA triggering. Moving to the complete OCU model, we focused on effect of bone mechanical alteration on cartilage, with a specific focused on the role of TGFβ- BMP and Wnt-βcatenin pathways. Localized stiffening of the area immediately adjacent to the tidemark was indeed previously correlated to increased stresses in cartilage low layers upon loading. The developed OCU model was thus exploited to test the hypothesis that alterations and inhomogeneities in the local mechanical properties of subchondral layers may cause a pathological response to loading in the cartilage compartment.
The developed miniaturized platforms were exploited to generate a healthy model of articular joint by using healthy articular chondrocytes (ACs) and mesenchymal stromal cells (MSCs) obtained from the routine cell banking performed at USB. Culture conditions promoting individual cartilage and calcified cartilage constructs formation were then optimized at POLIMI within a simplified version of the device (i.e. hosting a single 3D construct). Upon optimization of a culture medium permissive for single constructs culture conditions, bi-phasic OCU constructs have been generated.
The generated miniaturized OCU model were finally stressed by mimicking an OA-like microenvironment to induce the pathology onset. The effect of different compression levels applied to the constructs in the two compartments was first evaluated assessing the modulation of a set of genes associated with mechanotransduction signaling. The obtained in vitro OA model was then exploited to investigate over the influence of a cross talk between cartilage and bone/calcified cartilage during OA triggering. Moving to the complete OCU model, we focused on effect of bone mechanical alteration on cartilage, with a specific focused on the role of TGFβ- BMP and Wnt-βcatenin pathways. Localized stiffening of the area immediately adjacent to the tidemark was indeed previously correlated to increased stresses in cartilage low layers upon loading. The developed OCU model was thus exploited to test the hypothesis that alterations and inhomogeneities in the local mechanical properties of subchondral layers may cause a pathological response to loading in the cartilage compartment.
This study underlined the importance to study the pathology at a cell-relevant size-scale using organs-on-chip technology and it defines a starting point for the establishment of a functional in vitro OA model. However, this work was still limited to a single compartment and lacked the ability to reproduce the 3D architecture of the joint. In this scenario, uKNEEversal aims at filling the gap of existing poorly predictive pre-clinical models by generating an in vitro human OA joint model through organs-on-chip technology. In this contest, I strongly believe that my research has led to the released of an unprecedented tool able to accelerate the finding of a therapy, also embracing another key principle of EU being the reduction and potential replacement of animal model in research and drug discovery pipeline (Directive 2010/63/EU). The solutions identified in this project will thus benefit pharmaceutical companies and Biotech directly active in the development of innovative treatments for OA. Project outcomes have been disseminated to such stakeholders via publication of scientific papers in high impact journals and participation to conferences as detailed in the next paragraph.