The possible use of nanoparticles to treat osteoporosis has been tested during the recent Italian mission Futura on the ISS. The project coordinator, Livia Visai, tells Pharma World Magazine about this challenging experience and the future steps of sperimentation

NATO team with the Experiment containers ready for the Space Alessandro Mariani (Kayser Italia srl, Livorno), Luca Serafini (Kayser Italia srl, Livorno), Fabio Creati (Kayser Italia srl, Livorno), Derek Duflo (Nasa, KSC, Cape Canaveral, Florida, USA), Marco Vukich (Kayser Italia srl, Livorno), Barbara Pascucci (CNR-IC, Roma), e Francesco Cristofaro (UniversitĂ  di Pavia, Pavia); Giuseppina Rea (CNR-IC, Roma), Livia Visai (UniversitĂ  di Pavia, Pavia), Giuseppe Pani (UniversitĂ  di Milano, Milano)
NATO team with the Experiment containers ready for the Space Alessandro Mariani (Kayser Italia srl, Livorno), Luca Serafini (Kayser Italia srl, Livorno), Fabio Creati (Kayser Italia srl, Livorno), Derek Duflo (Nasa, KSC, Cape Canaveral, Florida, USA), Marco Vukich (Kayser Italia srl, Livorno), Barbara Pascucci (CNR-IC, Roma), e Francesco Cristofaro (UniversitĂ  di Pavia, Pavia); Giuseppina Rea (CNR-IC, Roma), Livia Visai (UniversitĂ  di Pavia, Pavia), Giuseppe Pani (UniversitĂ  di Milano, Milano).

It was just one year ago when the SpX-CRS-6 spacecraft was launched – on April 14, 2015 – from the Kennedy Space Center (KSC) to the International Space Station (ISS) carrying on board a very precious load for the Italian team of scientists coordinated by biologist Livia Visai of the University of Pavia. The Nanoparticles and Osteoporosis (NATO) experiment has been designed to test nano-based countermeasures for treatment of microgravity-induced osteoporosis, a condition typically affecting crew members during spaceflight. The microgravity experienced in the outer space makes the living environment on the ISS completely different from the planet Earth, with the major consequence of metabolic changes that cause the body to lose calcium and the resulting decrease of bone density. The NATO was part of the Futura project, a wide set of different experiments selected by the Italian Space Agency and aimed to investigate key aspects of man flight into the outer space. The project was jointly developed by the University of Pavia, the Pharmacological and Biomolecular Science Department of the University of Milan and the Crystallography institute of the Consiglio Nazionale delle Ricerche (CNR) in Rome in order to develop new approaches to prevent bone loss during future space missions as well as osteoporosis on Earth. «Only people who lived such a charged-up experience can fully understand its meaning for a scientist», tells Livia Visai about the moment the spacecraft leaved the Kennedy Space Center in Cape Canaveral.

Microgravity and osteoporosis

The human being is the only biped animal, standing and moving using two feet instead of four. The vertical arrangement of the human vertebral column causes a stronger interaction with gravity, a fundamental force deeply affecting bones and muscular tone as the body needs to optimize usage of its structure and energy to maintain the correct posture. The microgravity conditions experienced by the living tissues during spaceflights activate bone remodelling, a consequence of the loss of the physiological balance among the production of new bone tissue and the absorption of the old one, a process involving the action – respectively – of osteoblasts and osteoclasts cell types. The bone mass of astronauts decreases about 2% each month during spaceflights: once back on Earth, they have to undergo physical rehabilitation to restore the physiological conditions of the body structural tissues.

Osteoporosis is a pathological condition typical of the old age characterised by calcium loss from the bones, which therefore can fracture more easily. No definitive pharmacological treatment for the disease has yet been found. The NATO project studied the use of hydroxyapatite nanoparticles – the mineral substance forming the 3D inorganic, structural core of human bones – as a potential therapeutic approach to treat bone loss.

«Bone remodelling is ten-fold accelerated in the absence of gravity. Our project aims to study the in vitro effect of the nanoparticles on the viability and morphology of mature osteoblasts and osteoclasts. The experiment on the orbital station was run on osteoblasts. We tested osteoclasts in parallel studies on Earth under simulated microgravity conditions. Preliminary data seem to indicate that our hypothesis is very interesting. More conclusive data will be obtained from the analysis of samples tested on the ISS. Just one experiment on the orbital station is not enough to draw a statistical conclusion: we shall run new experiments to fully confirm the validity of our hypothesis and we hope to do it in a time not too far», tells Visai.

The Italian team hope that its results might represent a viable hypothesis for a new approach to target osteoporosis, an approach which might also find the interest of pharmaceutical companies. «The sequencing of RNA allows to identify therapeutic targets in terms of not yet identified genes and proteins – adds the project coordinator. – We currently have no active collaborations with pharmaceutical companies, but these processes are always very long. If we can identify something useful to treat osteoporosis, there could be a considerable economic interest».

A multidisciplinary approach

The team of professor Angela Rizzo of the University of Milan used its random positioning machine to run parallel experiments on Earth under simulated microgravity conditions, while the CNR’s group led by Giuseppina Rea characterised the genetic profile of the samples using genomic sequencing RNA techniques. The contribution of industrial partner of the project, Kayser Italy, was crucial to develop the hardware needed to ship the experiment to space and run it on the orbital station. The design of all lab equipment to be used on the ISS is very different from the standard one typical of experiments on Earth: all containers must be sealed to prevent spilling and must ensure the right conditions for the cellular growth, which has to be activated using a dedicated electronic systems. Kayser Italy has already participated to 63 space missions with 93 different loads.

«Just after the selection of the project by the Italian Space Agency in 2013, we started a very close collaboration with the NASA offices responsible for the a priori evaluation of all potential problems that might arise during the flight, and which need to be resolved before departure», tells Livia Visai. The scientists joined the Kennedy Space Center laboratories in late March 2015 to run the final pre-flight experiments. «On April 11, two days before the scheduled departure date, we set up the in vitro experiment inside the hardware created by Kayser. We then transferred it to the people responsible for the positioning of all materials in the cargo craft». The delayed departure due to adverse weather conditions caused great concerns among scientists waiting for their experiments to leave Earth for space. The delay has a particular impact on biological experiments, which – in the case the delay had extended more than two days – should be completely reassembled to be ready for the next window for launch.

«Captain Cristoforetti transferred the experiment to the European Space Agency’s incubator, already present on board of the space station», says Visai. The Italian astronaut interacted with NATO project team starting from the very preliminary stages of planning of the experiment. in order to fully understand and manage all relevant scientific and technological details. The special containers for cellular culture shipped to the space contained human mesenchymal stem cells, which once on the ISS were exposed to nano-particles of hydroxyapatite. The experimental unit also contained five tanks for the reagents, a fluidic system for their dispensation and a micro-control panel for activation of the experiment, whose parameters were set before launch. The unit was designed to fit into the Experiment Container (KIC-SL), which was then inserted into the incubator of the KUBIC Columbus Lab on the space Station. The incubator, built by the European Space Agency, allows to set the temperature from + 6° to + 38°C and can host different types of experiments, from cell cultures to seeds and small animals. At the end of the experiment, the cell cultures were frozen at -100 ° C to crystallize the situation and prevent damages during the return on Earth.

The next steps

Cells cultivated on the ISS during the NATO experiment returned on Earth on May 26, 2015. The materials collected from the Dragon capsule splashed down in the Pacific Ocean have been first transferred to the Johnson Space Center in Houston to check the hardware conditions before their shipment to Italy. «We opened the six experimental containers on June 9 in MIlan, just two days before the return of Captain Cristoforetti on Earth – tells Livia Visai -. We checked if the electronic systems maintained the correct temperature ​​and whether the biological samples were extractable. We are now conducting all post-flight analysis to assess the sustainability of our hypothesis». The effect induced in vitro by the hydroxyapatite nanoparticles in cell culture is being evaluated through the identification of the global gene expression profile and X-ray microdiffraction analysis. Cell cultures which underwent parallel treatment on Earth, under normal conditions and simulated microgravity, are used as controls. Captain Cristoforetti also conducted a second Italian experiment on osteoporosis (Stem Cell Differentiation, SCD) during her stay on the ISS, which was aimed to study the differentiation of human mesenchymal stem cells into osteoblasts.

According to the final results of the NATO project, expected by the end of 2016, a new in-depth mission into space might be planned. The next mission of an Italian astronaut will occur in 2017, when Paolo Nespoli will return for the third time on the ISS. It could represent a good occasion to further explore the possible use of hydroxyapatite nanoparticles to treat osteoporosis and similar conditions.

What is osteoporosis

Human joints concept with the skeleton anatomy of the body with a group of panels of sore joints glowing as a pain and injury or arthritis illness symbol for health care and medical symptoms.

Osteoporosis causes the reduction of the bone mass and changes in the micro-architecture of the bones. The direct consequence is an increased risk of fractures due to increased bone fragility. The strong reduction of oestrogens that occurs in women in post-menopause directly affects the activity of osteoblasts and osteoclasts, the cells responsible, respectively, for the production of the organic matrix of bone mineralization and of its reabsorption. Physical activity is important to prevent the disease, along with the dietary consumption of calcium, phosphorus and vitamin D.

According to the World Health Organisation, osteoporosis causes more than 8.9 million fractures worldwide each year, more than 4.5 million in the Americas and Europe (Table 1). Healthcare costs related to the disease should also consider complications and long periods of hospitalisation for patients at high risk, such as the post-menopausal female population.

WHO Scientific Group on the assessment of osteoporosis at primary health care level, Summary Meeting Report, May 2004

Space and Technology. Table 1