Periodic Reporting for period 2 - TargetCaRe (Targeting Cartilage Regeneration in joint and intervertebral disc diseases)
Période du rapport: 2017-03-01 au 2019-02-28
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 was 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 in the future.
We aim for regeneration of damaged and degenerated tissues in joint or IVD diseases. For this purpose we have developed strategies that specifically target the diseases and affected tissues by using hydrogels and so-called nanocarriers (tiny hollow structures) 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. We have developed assays, including imaging techniques as well as optimized bioreactors that simulate the mechanical and inflammatory environment in the body, to test the release and functionality of the drugs and factors. The nanocariers showed promising results for delivery of drugs to target cells. One of the gels was found particularly suitable to repair cartilage defects.
We aim for regeneration of damaged and degenerated tissues in joint or IVD diseases. For this purpose we have developed strategies that specifically target the diseases and affected tissues by using hydrogels and so-called nanocarriers (tiny hollow structures) 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. We have developed assays, including imaging techniques as well as optimized bioreactors that simulate the mechanical and inflammatory environment in the body, to test the release and functionality of the drugs and factors. The nanocariers showed promising results for delivery of drugs to target cells. One of the gels was found particularly suitable to repair cartilage defects.
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) and Youtube (https://www.youtube.com/watch?v=ugZsKFWKrmA).
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) and Youtube (https://www.youtube.com/watch?v=ugZsKFWKrmA).
TargetCaRe has studied and developed the concept of targeted stimulation of the body’s intrinsic capacity to regenerate joint tissues. Essential steps have been taken to enable techniques in the future to regenerate joint tissues affected by diseases. A series of highly innovative techniques, models and tools has been developed. These newly developed technologies are available for research now and for commercialisation in the future and 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. Moreover, a network of young scientists has evolved that is currently joining novel institutions and companies, thereby implementing the obtained knowledge and furthering biomedical science in regenerative medicine by applying the tools and technologies acquired in TargetCaRe.