Within WP1, three hydrogel systems were developed as basis for hierarchical for 3D printing. Hyaluronic acid based gels based on non-thiolated and thiolated hyaluronic acid, which can be subsequently cross-linked using allyl modified polyglycidols together with thiol modified polyglycidol through UV mediated thiol-ene click reaction, have been established. 3D constructs were achieved, which exhibited sufficient shape fidelity to prepare several layers of gel with appropriate resolution. Also thermosensitive and photo-cross-linkable hydrogels based on partially methacrylated poly(N-(2-hydroxypropyl) methacrylamide-mono-di-lactate-poly (ethylene glycol) triblock co-polymer and methacrylated hyaluronic acid (high and low molecular weight HAMA) or methacrylated chondroitin sulfate (ChSMA) were synthesized and 3D printed to scaffolds with well-defined shapes. Thermosensitive gels containing ChSMA have been printed up to 20 layers yielding a porous scaffold which retained its porous 3D shape also after swelling in buffer. Finally, customized multicomponent starPEG-peptide-heparin hydrogels that are cross-linked via Michael-type reaction and are degradable through enzymatically cleavable peptides at the distal ends of the starPEG molecules were established and are under evaluation for 3D printing.

For cell culture, a HydroZONES SOPs for cell isolation, culture and differentiation experiments was established in WP3. These SOPs are now applied within all WP3 partner labs for HydroZONES related experiments. All laboratories acquired FBS and growth factors of the same batch and origin in order to facilitate comparison of outcomes between the partners. Viability and chondrogenic differentiation of BMScs was confirmed within the mentioned hydrogels according to this SOP.

Regarding the development of an in-vitro test system for osteochondral plugs in WP4, vital osteochondral plugs are available, and several methods are established to analyze metabolic state of the cartilage as well as bone compartment. A definitive design prototype of the bioreactor has been developed and tested, and the separation of cartilage and bone compartments is established. It is possible to perfuse a porcine 8.5 mm plug completely with 48h of perfusion at 1 ml/min. Also the in vivo test model for osteochondral plugs was established in WP5 and the first hydrogels have been assessed for biocompatibility using the ectopic subcutaneous murine model. Long term in vivo testing within WP6 has not yet started, but WP6 partners are in active discussion with the consortium.

The HydroZONES Quality Management System and the HydroZONES Regulatory Affairs Management System have been built up in WP7 and the related documentations have been validated, trained and edited to the consortium. Each consortium member has been invited and enabled to apply and modify the tools which these systems provide for structuring and standardisation of their activities for success of the project.

Within WP8, A Consortium Agreement was developed and signed by all partners. A patentability assessment form was developed by the Exploitation Manager and made available to the consortium. Also a dissemination plan was set up in the first project months including various dissemination activities. A project homepage (www.hydrozones.eu) has been set up, a project logo, project flyer, a project poster and a project exhibition stand were generated. Several press releases and presentations of the project at scientific meetings and exhibitions were performed. A plan for training courses was developed, and 2 training courses have already been held. An SOP on HydroZONES research fellowships was generated to support the exchange of PhD students and Post docs between research labs and also between academia and industry within the consortium members, and 2 fellowships have so far been awarded.