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Targeting Cartilage Regeneration in joint and intervertebral disc diseases

Periodic Reporting for period 1 - TargetCaRe (Targeting Cartilage Regeneration in joint and intervertebral disc diseases)

Reporting period: 2015-03-01 to 2017-02-28

The worldwide burden of chronic musculoskeletal diseases, in particular osteoarthritis (OA) and chronic low back pain caused by intervertebral disc disease (IVD), has incited considerable scientific efforts to finding regenerative treatments that would cure or slow the progression of these diseases. In particular the use of specific proteins (growth factors) and stem cells has been in focus. However, none of these have translated to the clinic as a routine treatment yet.

The objective of TargetCaRe is to train a group of researchers skilled in a variety of innovative technologies that go beyond the current state-of-art and make clinical application of regenerative medicine for joint and intervertebral disc diseases feasible and successful.
We aim for regeneration of damaged and degenerated tissues in joint or IVD diseases. For this purpose we develop strategies that specifically target the diseases and affected tissues. We aim for regeneration of these tissues by using so-called nanocarriers: tiny vesicles that can be filled with e.g. anti-inflammatory drugs or factors that stimulate regeneration. This system will stimulate and exploit the body’s own capacity for regeneration.
To achieve this we will:
Optimize and select nanocarriers. This includes MultiCaps and EnzyCaps, nanocarriers composed of novel biomaterials, and so-called Nanoghosts: nanocarriers prepared out of the plasma membrane of mesenchymal stromal cells (MSCs) in such way that the contents and machinery of the cells are removed, but the specific capacity that allows the MSCs to localize and move to injured tissues in the body is retained. All nanocarriers will be loaded with biologically active compounds.
Develop targeting approaches for a variety of joint and IVD disease conditions. For cartilage defects and IVD degeneration, injection of hydrogels containing biologically active compounds loaded or not in nanocarriers will be developed. For cartilage defects and osteoarthritis, specific proteins called antibodies, capable of recognizing specific tissues, will be coupled to nanocarriers.
Develop and apply a toolbox of imaging methods to monitor whether nanocarriers actually reach the targeted tissues, stay there and have an effect. We use advanced in vivo imaging techniques, such as highly advanced Magnetic Resonance Imaging (19F MRI) and near-infrared imaging as well as Mass Spectrometry Imaging (MSI) for evaluation at both molecular and tissue level.

TargetCaRe has now been ongoing for two years, we provide an update of the promising results that have been achieved so far.
Loaded Nanocarriers
Different bioactive compounds are being considered for their ability to attract cells that can regenerate damaged tissue, to promote production of cartilage or to prevent degenerative processes in joint cartilage. The effects of different compounds are tested on human tissues and cells in established laboratory systems. A first selection has been made to start incorporating compounds into the different types of nanocarriers.
Significant progress has been made on the synthesis of MultiCaps and EnzyCaps. The release of the drugs can be tailored during preparation of the nanocarriers. For EnzyCaps the release of compounds out of the nanocarrier will be tuned by enzymes present in the environment. Two initial cargoes have been selected which will now be incorporated in the nanocarriers: Triamcinolone, a known anti-inflammatory drug, and LinkN, which has gained interest in the scientific community for its capacity to stimulate regeneration of IVD tissue.
Research on Nanoghosts has also made substantial progress. It has been found that Nanoghosts retain the original MSC capacities to bind specific compounds to their surface; Nanoghosts can localize and move towards sites of inflammation. LinkN will be incorporated in Nanoghosts, as well as small molecules that can influence gene expression of the cells (so-called siRNA and miRNA) to influence cartilage formation. Protocols have been established to incorporate these compounds in Nanoghosts with good efficiency.

Targeting approaches
Hydrogels were prepared out of materials that naturally occur in the body using specialized protocols. Hyaluronic acid is a constituent of cartilage and other connective tissues, and fibrin is a protein that is involved in wound healing. Hydrogels formed using these materials can be used as a substrate for growing cartilage cells. A hydrogel made of another constituent of cartilage, collagen, was used as a model system to develop analysis methods to study migration of cells in hydrogels.
Antibodies specifically recognising degraded joint tissues were developed and produced. In laboratory experiments, it was demonstrated that these antibodies are capable of recognizing cartilage and binding to its surface. To be able to use these in further applications, the attachment of antibodies onto the MultiCaps is now being optimized.

Development of model systems
Assays have been developed and tested to screen for the release and functionality of compounds that aim to stimulate cartilage regeneration. We have made a genetically modified cell type that transmits light when it starts to produce cartilage. Also a system was developed to test whether anti-inflammatory compounds are released from nanocarriers and whether they are still active. These assays are now available for the consortium to evaluate release and activity of compounds from nanocarriers.
An existing system to evaluate bovine IVD tissue in the lab has been adjusted to make it suitable for simultaneous tests with small pieces of human IVD tissue to evaluate hydrogels and nanocarriers. A custom made bioreactor was used to simulate an inflammatory or degenerative environment. In this bioreactor, entire IVDs from tails of young calves were cultured. To further mimic the degenerative process, IVDs were injected with inflammation-inducing compounds. This model has been validated and is now ready to use for testing of release and activity of compounds from nanocarriers.

A method has been established to create tissue samples consisting of bovine cartilage and bone in a standardised way. These samples can also be placed in a mechanical loading device, which simulates mechanical joint loading, to evaluate resistance of hydrogels to mechanical loading. Also the behaviour of cells within hydrogels can be studied.

Imaging methods
A method was established to visualize retention of nanocarriers in cartilage tissue by inclusion of a fluorescent tag, which enabled detection of nanocarriers by opti
TargetCaRe studies and develops the concept of stimulation of the body’s intrinsic capacity to regenerate joint tissues. In the first two years of the project, essential steps have been taken in order to eventually achieve regeneration of joint tissues affected by joint diseases. A series of highly innovative techniques, models and tools has been developed. These newly developed technologies will have a large impact on the progression of development of new treatments, not only for OA and IVD disease, but for regenerative medicine in general.
Images of nanocarrier synthesis and osteochondral biopsy model
Schematic representation of the work flow of TargetCaRe
Targeted local delivery of regenerative / anti-degenerative compounds using nanocarriers