1. We optimized and selected nanocarriers. This includes MultiCaps and EnzyCaps, nanocarriers composed of novel biomaterials that can release their cargo based on the presence of enzymatic activity. We also developed further 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. Nanocarriers were loaded with biologically active compounds.
2. We developed targeting approaches for a variety of joint and IVD disease conditions. For cartilage defects and IVD degeneration, injection of hydrogels containing biologically active compounds either or not loaded in nanocarriers were developed. For cartilage defects and osteoarthritis, specific proteins called antibodies, capable of recognizing specific tissues are coupled to nanocarriers.
3. We developed and applied a toolbox of imaging methods to monitor whether nanocarriers actually reach the targeted tissues, stay there and have an effect. We used 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 of delivery efficenty at both the molecular and tissue level.
In more detail:
Loaded Nanocarriers
The release of the compounds (such as drugs) can be tailored during preparation of MultiCaps and EnzyCaps. For EnzyCaps the release of compounds out of the nanocarrier is tuned by enzymes present in the environment.
Different compounds such as LinkN and FGF18 have been incorporated in Nanoghosts, as well as small molecules that can influence gene expression of the cells (so-called siRNA, miRNA and antimiRNA) to influence cartilage formation. Protocols have been established to incorporate these compounds in Nanoghosts with good efficiency and the suitability of the nanoghost to bring the compound in the endogenous cells was demonstrated.
Targeting approaches
Antibodies that specifically recognise degraded joint tissues and inflammatory macrophages were developed and produced. In laboratory experiments, it was demonstrated that these antibodies are capable of recognizing their epitopes. The generated antibodies were linked to nanocarriers and used in the first in vivo studies with longitudinal tracking.
Hydrogels were prepared out of materials that naturally occur in the body. 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 were compared for their ability to attract cells into the defect area. The performance of the materials was further improved by incorporation of chemotactic factors as well as factors that could stimulate formation of cartilage tissue. One of the gels was found particulary suitable to repair cartilage defects.
Development of model systems
Assays have been developed to screen for the release and functionality of compounds that aim to stimulate cartilage regeneration. We have made genetically modified cells that transmit light when they produce cartilage. Also a system was developed to test whether anti-inflammatory compounds after release from nanocarriers are still active.
An existing system to evaluate IVD tissue in the lab was adjusted to make it suitable for simultaneous tests to evaluate hydrogels and nanocarriers. Entire IVDs were cultured in a bioreactor system and 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 mechanically load osteochondral units that contain a defect that was filled with hydrogels. The effect of mechanical loading on the attraction of endogenous cells in the hydrogel filled defect and its effect on cartilage repair can be studied in this model system.
Imaging methods
A method was established to visualize retention of nanocarriers in cartilage tissue by inclusion of a fluorescent tag. Nanocarriers (with and without antibodies) have been tracked in time after injection into a knee joint.
A protocol has been set-up, validated and tested for local detection of Triamcinolone in cartilage tissue using MSI. Novel methods have been established to analyse the effect of drugs on specific constituents of cartilage tissue.
Dissemination
The first results have been published in peer reviewed scientific journal. Many abstracts were published based on work presented by the ESR on conferences. Next to scientific publications we have disseminated to the general public via newspapers, newsletters as well as EuroNews (
https://www.euronews.com/2018/03/12/finding-new-cures-for-osteoarthritis(opens in new window)) and Youtube (
https://www.youtube.com/watch?v=ugZsKFWKrmA(opens in new window)).
