Calcium pyrophosphate crystal formation and osteoarthritis: in vitro and in vivo studies – CAPYROSIS

CAPYROSIS is a basic science research project based on a pluridisciplinary approach combining the study of the formation of synthetic calcium pyrophosphate (CPP) crystals in vitro, the characterisation of synthetic and biological crystals and also biological cell and animal studies to progress in the identification and the knowledge of these crystals and the mechanisms associated to the development of the disease. The originality of this project relies on the pooling of clinical considerations and physico-chimical fundamental studies involving : i) chemists and chemical engineers for the synthesis, the physico-chemical study of CPP in solution (synthesis, stability, transformation, nucleation and crystal growth kinetics, influence of additives (inhibitors,…)) and the conception and use of a system that could mimic the volume and interfaces encountered in a joint, ii) physicists for the fine characterisation of synthetic and biological crystals (use of laboratory and large scale (SOLEIL synchrotron) equipments), iii) biologists and clinicians for the in vitro and in vivo study of the inflammatory properties of the various CPP crystals, and the expression of proteins known to be associated to the formation of CPP crystals within the osteoarthritic cartilage and cartilage calcification.

A new fast and reproducible method of synthesis of the four CPP pure phases (amorphous CPP, m-CPPD, t-CPPD, m-CCPTß) has been developed. We resolved the m-CPPD structure, synthesised a new monoclinic monohydrated CPP phase and pointed out the inhibitory effect of some ionic additives on the crystallisation of m-CPPD and t-CPPD.
We demonstrated that CPP crystals have a different inflammatory potential (m-CPPD more inflammatory than t-CPPD ; m-CPPTß and amorphous CPP are non-inflammatory) and activated the NLRP3 inflammasome and IL-1ß (central cytokine for inflammatory responses) production by monocytes and inflammatory gene expression. Preliminary results suggest that the effect of CPPD crystals are modulated by adsorbed proteins on their surface.

The constitution of the bank of meniscii with and without calcification associated with precise histological and physico-chemical data of the types of crystals will be continued beyond this project. It is a unique bank. Its exploitation will make it possible to better understand in vivo the crystal formation mechanisms.
The demonstration of the different inflammatory potentials for the different crystallised phases of hydrated calcium pyrophosphate helps better understand the broad clinical spectrum associated with CPP crystal deposition in humans. These results open new trends of research by studying, for example, the clinical phenotype as a function of the phases of the CPP crystals. These studies may be conducted using the characterisation tools used in this project such as FTIR and Raman vibrational spectroscopies.
The understanding of the mechanisms of cell activation induced by the different phases of CPP crystals and in particular the production of IL-1b will allow the targeting of either this cytokine or its activation pathways in the acute inflammatory reactions triggered by these crystals. Similarly, the possibility of modulating the cellular response by modifying the proteins adsorbed on the surface of crystals opens interesting therapeutic trends that will be worth testing in vivo models.
Finally, the demonstration in vitro of the evolution of the different CPP phases formed suggests that it will be possible in vivo to modify the formation of crystals towards less inflammatory phases.

Scientific production (in december 2017) :
6 international papers in journal with review commitee, 2 international and 2 national proceedings with review commitee, 11 international and 9 national oral communications among wich 6 are invited conferences (5 international et 1 national) and 11 poster communications (1 national et 10 international congresses). Other papers (at least 5) will be written by the end of 2018 and the results of this project will be presented in other upcoming international conferences: Eurocrystals workshop 2018, EULAR 2018,… Two PhD students involved in the project contributed to wide audience scientific diffusion action in the form of a workshop on biomineralisations at the Muséum d’Histoire Naturelle de Toulouse within the framework of the « Année de la cristallographie » (february 2014). Finally a CAPYROSIS international workshop has been organised by CIRIMAT at the end of CAPYROSIS project (14/11/2017 at the Maison de la Recherche et de la Valorisation of the University of Toulouse). We invited two external international experts and 29 persons attended this workshop.

Prices related to the project :
2015 Léopold Escande PhD thesis price for Pierre Gras (price attributed to the best PhD theses of the year by the Institut National Polytechnique de Toulouse) and best poster price for K Ley-Ngardigal at the Ecole Doctorale Science de la Matière annual meeting (Toulouse, 2015).

Osteoarthritis (OA) is the most common form of chronic rheumatic diseases and among the most costly diseases (both in terms of direct and indirect costs). In 2007, it has been estimated that around 35 to 40 million European people suffer from OA and current treatments are chiefly designed to relieve symptoms until joint replacement becomes necessary. Aggravating factors identified to date include a family history of OA, mechanical overload (overweight), older age, and the presence of calcium-containing microcrystals in the joint. The two main types of calcium-containing crystals found in synovial fluid and cartilage are hydrated calcium pyrophosphates (CPP: Ca2P2O7, nH2O) and basic calcium phosphates (BCP, such as octacalcium phosphate, carbonated apatite,…) crystals. Beside OA, CPP deposition (CPPD) can also give rise to destructive arthropathies but current treatments are solely symptomatic and are not able to dissolve/prevent the formation of CPP crystals. Therefore, there is a need to understand how CPP crystals are formed and how they induce joint destruction.
If physico-chemical reactivity of synthetic and biological BCP crystals are largely studied in the literature, the formation and dissolution of CPP crystals is much less studied and not fully understood. Indeed, the actual scientific knowledge on CPP phases is rather poor.
CAPYROSIS is a basic research project combining a pluridisciplinary approach including material science, engineering and modelisation, and living science in order to understand the mechanisms of CPP formation and CPP crystal-related arthropathies.
Several steps/tasks will be involved: i) to better understand the causal relationship of the appearance of such mineral phases and the possible role of cartilage aging and OA in these processes, ii) to develop new methods to make correct and early diagnosis (including method of detection of amorphous CPP in vivo) and, iii) to propose drugs for treatment or prevention of CPP crystal-related arthropathies.
The originality of the present project lies in merging clinical considerations and fundamental physical-chemical studies. The interdisciplinary approach that will be developed, involving chemists, chemical engineers, physicists, biologists and clinicians, is based on investigations on synthetic compounds and biological specimen (hydrated calcium pyrophosphates) and on the use of a system modeling the effect of volume and interfaces that can be found in a joint which should allow a significant improvement of our understanding of the formation of CPP phases in vitro and in vivo.
The project stretches over 48 months, includes four main tasks and gathers four French academic laboratories internationally renowned: the Institut Carnot CIRIMAT and the Laboratoire de Génie Chimique in Toulouse, the Laboratoire de Physique des Solides in Orsay, and the UMR 606 Inserm in Paris, providing complementary specialities, domains of knowledge and expertise for the good development of this inter- and multidisciplinary project: chemists and chemical engineers for the synthesis, the physico-chemical study of CPP in solution and the study of their nucleation and dissolution kinetics and of the influence of additives (inhibitors/promoters), physicists for the fine characterisation of synthetic and biological CPP (including the use of laboratory and synchrotron facilities), biologists and clinicians for investigating both in vivo and in vitro the inflammatory properties of the different CPP crystalline and amorphous phases, and the expression of the four proteins known to be involved in CPP crystals formation in OA cartilage along with cartilage calcification.
This project will highlight mechanisms of CPP crystal formation in vitro as well as mechanisms of CPP crystal-related disease and joint destruction. It will identify new therapeutic targets capable to dissolve CPP crystals and to inhibit disease progression.