Final Report Summary - HYDROZONES (Bioactivated hierarchical hydrogels as zonal implants for articular cartilage regeneration)
Executive Summary:
Degeneration of cartilage is a major cause of chronic pain, lost mobility and reduced quality of life for millions of European citizens. No clinical therapy is available that leads to healing of cartilage defects. Current cartilage implants cannot establish the hierarchical tissue organisation that appears critical for normal cartilage function. HydroZONES hypothesised that a biomimetic zonal organisation is critical for implants to achieve cartilage regeneration. It thus represented an interdisciplinary consortium that adopted a strategy to regenerate articular cartilage based on recapitulation of the tissues zonal structure employing 3D bioprinting technology.
Therefore, the HydroZONES project pursued the following 2 main objectives:
1. To develop a hierarchically structured hydrogel based mechanically stable scaffold that recapitulates the zonal distribution of the native articular cartilage.
2. To develop and validate a predictive in vitro test system for future evaluation of products for cartilage regeneration in order to reduce the amount of necessary animal experiments in the future.
Regarding main objective 1, three hydrogel platforms were developed. Materials were designed and optimised in such a way that they could be printed in highly defined 3D scaffolds also in double printing with thermoplasts to achieve the required mechanical stability of the printed scaffolds. Cell compatibility was evaluated for the different hydrogels and showed that cell viability was retained during hydrogel cross-linking reactions for MSC’s and chondrocytes. Hydrogel compositions were optimised regarding cell differentiation by investigating the effect of hyaluronic acid content and heparin content. Zonal organisation was obtained by layer-by-layer deposition of hydrogel materials of different composition. In this way, zonal constructs with zones of different mechanical properties, glycosaminoglycan content, degradation rate and cell type have been prepared. Finally, 2 different optimised 3D printed scaffolds were assessed in large animals. In the equine model, the use of composite osteochondral scaffolds with a zonal design for cartilage repair achieved partial tissue repair. Despite the absence of a full chondral matrix within the defects, the biomechanical properties were substantially better (showing higher stiffness) in the zonal constructs with both biomaterials. Good lateral integration with adjacent cartilage as well as integration of the scaffolds with the surrounding bone tissue was achieved with both hydrogels. Even though the strategy is currently not mature enough to achieve production of hyaline-like regenerated tissue, the evidence gathered in the equine studies suggest that this is a promising approach that, after optimisation, may lead to significant improvements in the quality of matrix production in cartilage repair.
For main objective 2, a 3D in-vitro/ex-vivo osteochondral platform was developed, consisting of hardware, operating procedures and related assays for (osteo)chondral implants was designed and developed. It was shown that the system allows for a more realistic and native-like environment resulting in a better cartilage regeneration response in comparison to conventional in-vitro studies without having the native tissue around, which increases the predictive value of in-vitro testing. Mechanical loading was included in the system, to make the environment even more realistic and comparable to the situation in the native joint. In addition, the development of the in-silico model allowed for more focussed design of experimental studies, obviating the need for trial and error studies. The results of the modelling were used to select optimal parameters. In particular, this predicted the growth factor concentrations and co-culture settings required to induce MSC chondrogenesis in vitro. Further in-silico modelling predicts the bulk mechanical properties of fibre-reinforced scaffolds, so that these can be designed to withstand the demanding loading environment in the joint. This unique combination of predictive in-silico and in-vitro models is a very valuable tool for evaluation the regenerative capacity of implant materials prior to in vivo testing, reducing the amount of animal experiments and related costs, time and effort. Importantly, this has led to the development of a commercially available product.
Project Context and Objectives:
Degeneration of cartilage is a major cause of chronic pain, lost mobility and reduced quality of life for millions of European citizens. From a clinical point of view treatment to achieve cartilage regeneration (hyaline) and not only repair (fibrous) remains a great challenge. No clinical therapy is available that leads to healing of cartilage defects.
Current cartilage implants cannot establish the hierarchical tissue organisation that appears critical for normal cartilage function. HydroZONES hypothesised that a biomimetic zonal organisation is critical for implants to achieve cartilage regeneration. It thus represented an interdisciplinary consortium that adopted a strategy to regenerate, rather than repair, articular cartilage based on the tissues zonal structure and function.
HydroZONES used advanced bioprinting technology for fabrication of 3D biofunctional hydrogel constructs, eventually mechanically reinforced by degradable polymer scaffolds, as biomimetic reconstitution of the zonal organisation of natural cartilage. Constructs have been evaluated for cell-free application and also for combination with chrondrogenic cells (chondrocytes and/or MSC). Stringent in vitro and long term in vivo testing of the constructs has been employed.
Cutting edge 3D tissue models and bioreactor technology were used together with in silico modelling to develop an in vitro assay and test system.
Installation of a quality and regulatory affair management system, preparation of GMP production, accredited in vitro testing and involvement of clinical partners and companies with experience in clinical trials was combined with the aim to ensure that the best performing construct is brought into an optimal position for entering clinical trials after project end.
HydroZONES aimed to advance the European Union as world leader in the field of joint cartilage regeneration. Thus, generating economic impact for different markets along the product developmental line and advance European biomaterials industry on several levels. It aimed to generate the basis for a dramatic added value to OA patients and increase the quality of life for millions of EU citizens.
HydroZONES overreaching aim was to balance the combination of advancing European biomaterials industry with scientific excellence and verify or falsify a radical new strategy for implant design, the recapitulation of the hierarchical tissue organisation for improved performance, at the example of cartilage.
In order to achieve this overall mission, the HydroZONES project pursued the following objectives:
3. To develop a hierarchically structured hydrogel based mechanically stable scaffold that recapitulates the zonal distribution of the native articular cartilage.
4. To develop and validate a predictive in vitro test system for future evaluation of products for cartilage regeneration in order to reduce the amount of necessary animal experiments in the future.
These objectives were pursued by establishing a step wise material development process, divided up into three levels of materials, including:
1. Non-hierarchical but mechanically reinforced biofunctional hydrogels
HydroZONES reinforced biofunctional (cell laden) hydrogels by polymer scaffolds and compared scaffolds made of the proprietary polymer of the partner #12 CCT to the clinically used thermoplast poly(caprolactone) (PCL) to obtain best mechanical reinforcement. HydroZONES thus facilitated advancement of the therapeutic approach developing hydrogel carriers that may be used with or without scaffold to ensure cell retention and early mechanical support, and, ultimately, improve stable cartilage regeneration for the patient.
2. Pre-defined “favourite” layered materials with zonal arrangement
The three hydrogel systems were assessed to identify the optimal material combination for one cell-free and one cell-containing construct, with least complexity possible but as much bioactivation as needed according to in vitro and in vivo evaluation. This yielded selected scaffolds which, together with fibrin as clinically used hydrogel-control, have been long-term tested in vivo in mini pigs and horses (WP6).
3. Advanced layered and mechanically reinforced constructs
A third aim of the project was the development of multiphase printing process of clinically used thermoplastic polymers with biofunctional hydrogels (“double-printing”) for generation of zonally arranged mechanically reinforced hydrogels. This enabled the development of advanced layered constructs and the optimisation of the hydrogel-soaked thermoplast scaffolds with more biomimetic non-layered hydrogel materials that outperform the current standard. Mechanical strength and stability were designed to be maintained by the thermoplast-component 12 to 18 months, while hydrogels were designed to degrade more rapidly (3-6 months) to allow cell and tissue ingrowth.
Degradation of the scaffolds in vivo was checked histologically. The constructs underwent a well-defined and stringent in vitro (WP3), in vivo biocompatibility and biofunctionality (WP5) and long-term in vivo testing in the joint at the place where the implant is supposed to be used in humans (“orthotopic”; WP6). Advanced scaffold integration methods were assessed in pilot studies.
AIM AND CONCEPT OF HYDROZONES
HydroZONES has formed a consortium that supported the aims set in an innovative and excellent means. The consortium was formed in view of the objectives described above to enable the development of implants with anatomical reproduction of articular cartilage based on biocompatible and biofunctional hydrogels that mimic cartilage ECM (cellular level) and the natural tissue organisation and zonal structure of natural cartilage (tissue-level).
HydroZONES comprised three leading internationally recognised European laboratories in biomaterials development and hydrogel technology (Partners #06 UU, #07 IPF and #01 UKW) that provided established but innovative hydrogel technology comprising usability in printing devices, specific functionalisation with peptides, GAGs and growth factors and finally the option to load the hydrogels with cells during printing due to cytocompatible cross-linking reactions. Scaffolding was intended to be addressed in HydroZONES by the application and development of 3D bioprinting technologies by Partner #08 PIL and of commercial bio-printers.
HydroZONES used chondrocytes and BMSCs for initial in vitro assessment of the scaffolds. Detailed studies were performed with human cells as well but also with the respective porcine (minipig) and equine (horse) cells for correlation with the in vivo experiments. HydroZONES installed a thorough testing and selection procedure through in vitro testing (Partners #05 UMCU & #01 UKW (conform to ISO 10993-5)), biocompatibility and biofunctionality screening in nude mice (Partners #02 UNAV & #09 QUT) and evaluation in long-term pre-clinical minipig and horse models, the best current large animal model for cartilage (Partners #03 HD & #06 UU). Establishment of a consortium wide quality and regulatory affairs management system according to EN ISO 13485:2007 (QM/RAM; Partner #11 HCS) allowed to set a new internationally acceptable standard for pre-clinical testing of (osteo-) chondral implants. Development of the QM/RAM system ensured that developments within HydroZONES complied with all relevant preclinical regulatory affairs. Several partners - clinicians (partners #01 UKW, #02 UNAV & #05 UMCU) and SME (partner #12 CCT) had experience with clinical trials, which ensured that the developments within HydroZONES stay focused on the defined project endpoint and were performed according to relevant regulatory affairs (partner #11 HCS).
Moreover, cutting edge 3D tissue models, bioreactor technology and automatisation know-how (Partners #01 UKW, #08 PIL, #15 LifeTec & #13 PROSPA) were integrated, as well as state of the art in silico modelling capacity (Partner #04 UOXF). These competencies were combined to achieve the second major objective of developing a 3D in vitro assay for chondral implants, together with the prototype hardware to perform the assay, which has then been developed into a commercial product.
At project start, HydroZONES encompassed within the consortium five SME partners, out of which three exploitation partners ideally distributed along the developmental chain of HydroZONES to prevent colliding exploitation interests:
• Partner #13 PROSPA (printing technology) exploiting the advanced bioprinter
• Partner #11 HCS (QM support and consultancy) exploiting the QM/RAM system
• Partner #12 CCT (clinical product) exploited the HydroZONES constructs for clinical application – but has withdrawn after 4 years due to financial difficulties.
• Partner #15 LifeTec (bioreactor technology) exploiting the test-system
• Partner #16 POVA (GMP production) exploiting the GMP produced biomaterial
For scale-up and production of the devices, the consortium incorporated partners with experience regarding software (Partner #13 PROSPA) and manufacture (Partner #15 LifeTec). It also included partners with experience in material production and scale up under cGMP (Partner #16 POVA) and manufacture and production of scaffolds for clinical use (Partner #12 CCT). Partner #12 CCT furthermore served as the main exploitation partner with focus on human clinical use. Until their termination Partner #12 CCT was providing an exploitation manager (EM) to the project to ensure that the technical know-how and intellectual property developed in the project is evaluated and properly protected. CCT had the scientific, technical, quality control and regulatory experience of bringing HydroZONES technology to the market. After CCTs termination this function was taken over by Partner #06 UU (Prof. René van Weeren).
The work programme was divided into 9 work packages. WP1 concernced materials and scaffolds in close interaction with WP2 that dealt with automatization of assembly. Cytocompatibility and in vitro assessment was performed in WP3, while in WP4, the in vitro assay and test system was developed. Biocompatibility and biofunctionality screening was performed in WP5, and long term pre-clinical in vivo testing was performed in WP6. In WP7, a management system for quality & regulatory affairs (QM/RAM) was developed, WP8 dealt with training, dissemination and exploitation, and WP9 concerned project management.
In order to optimise the accountability and flow of results, the following methodological choices have been made:
• Partners have concentrated their person-months-resources in those work packages where they deliver results. As part of these resources they also followed the results of the other work packages and contributed advice and support to other work packages as needed. Overall technical coordination was covered by WP9.
• Deliverables have been organised to optimise individual accountability.
• Deliverables have been aligned with major project milestones to provide a natural way to measure progress.
The next chapter will present work development and achievements by WP.
Project Results:
WP 1: Materials and scaffolds
WP Objective
The objective of WP1 was to develop and characterise printable and biofunctionalisable hydrogels that degrade between 3-6 months and use them to generate 3D hydrogel scaffolds and constructs. Advanced printers that allow the printing of cell-containing gels and the double printing of hydrogels and thermoplasts were available at month 24. The materials, scaffolds and constructs were transferred to WP3 for in vitro assessment, prepared for in-vivo biocompatibility and biofunctionality screening in WP5 and for long-term pre-clinical in-vivo testing in WP6. Scaffolds and constructs were also prepared for testing in WP4 and used for validation of the 3D in-vitro assay and test system. Prototypes were tested throughout the course of the project and feedback from WPs 3, 4, 5 and 6 was used to optimise and fine-tune the properties of the materials.
Summary of WP results (linked to the WP objectives)
Synthesis of the Hydrogels
Synthesis and physicochemical characterisation of three distinctively different hydrogel systems as biofunctional cell carrier and scaffold materials was established in the three biomaterial groups.
At IPF, an in-situ assembly scheme for multicomponent hydrogel materials was established on the basis of a Michael addition reaction of starPEG-peptide conjugates with maleimide-functionalised heparin. The matrix platform covers key features of ECM including adhesion ligand functions, cell-driven degradation, modulated stiffness and sustainable cytokine delivery.
UU developed thermosensitive and photo-cross-linkable hydrogels based on hyaluronic acid (HA). The thermosensitive properties of the gels warrant a material, which behaves as a liquid at 4 ºC and therefore enables flow through the needle of a printer head, and subsequently undergoes gelation around 37°C (temperature of the heated plate of the printer). A thermosensitive and highly biocompatible construct was developed by using a combination of partially methacrylated poly(N-(2-hydroxypropyl) methacrylamide-mono-di-lactate-poly (ethylene glycol) triblock co-polymer and methacrylated hyaluronic acid (HAMA).
UKW developed UV-cross linkable hydrogels based on polyglycidols (PG) and modified natural hyaluronic acid (HA). The UV-crosslinking mechanism which is used for those hydrogels is the thiol-ene-click reaction, which can be done in presence of cells. For the hydrogel formation different thiol-modified polymers (HA-SH and PG-SH) and polymers with a double bond in the side chain have been synthesised.
Establishing of the printing Process
Printing processes for the different hydrogels were established and improved for the different physical and chemical crosslinking steps. Cell survival was improved via changed formulations and printing steps.
UU and UKW used a printing process followed by a short UV-light exposure to obtain stable and highly defined hydrogel scaffolds with a zonal architecture. Cells have been incorporated and showed a good cell survival. Importantly, labelled cells printed in different layers showed that a highly defined printing process was obtained in which specific cells can be precisely deposited at required locations within the scaffold.
At IPF, a different plotting process was developed for starPEG-heparin and pure starPEG hydrogels as the gel component immediately starts to react upon mixing. A new printing head for simultaneous dispensing of both gel components was developed together with GeSiM and installed at the NanoplotterTM. After extensive optimisation of the process parameters (pH and solid contend of the hydrogel solutions, spot pattern, plotting speed, droplet volume, target temperature and humidity) homogeneous hydrogels have been printed that show similar mechanical properties as casted hydrogels of similar composition. Highly defined and layered 3D constructs have been developed and good cell survival of cells within has been shown.
Printing in combination with thermoplasts
Printing in combination with mechanical stable PCL scaffolds allowed for the creation of robust cell carriers (→ WP2)
Double printing of hydrogels and PCL has been performed by two partners of the HydroZONES consortium (UKW and UU) as these partners have developed hydrogels that have suitable properties to be deposited in a double printing process. Hybrid 3D printed constructs were prepared using a 3DDiscovery robotic dispensing system using two print heads that suspended PCL and hydrogel filaments respectively in a layer by layer method. Highly defined scaffolds have been prepared. Importantly, gels were loaded with chondrocytes and showed a good cell viability during and after the printing process.
GMP Production of a selected hydrogel material
An up-scaled manufacturing route towards allyl-modified polyglycidol and thiol-modified hyaluronic acid is available and GMP-ready. Establishment of different synthesis and purification methods for anionic polymerisation of poly(glycidols) and modification of natural polymers was established and improved.
PoVa optimised and provided a scale-up strategy for the synthesis route towards the two polymers developed by UKW ready for cGMP manufacturing. The first polymer is an allyl-functional polyglycidol (abbreviated PG-01) and the second a thiol-modified hyaluronic acid (abbreviated HA 01). First, a user requirements document was written and based on this document, in close consultation with a supplier of reactor equipment a large scale reactor set-up was designed. IQ-OQ-PQ test protocols were written, tested and qualified. Manufacturing instructions were written for the PG-01 process. Via this instruction protocol, two test batches were manufactured using the qualified reactor set-up. The products of the two test batches were analyzed via the analytical methods that were developed. The results showed that the PG-01 manufacturing process provides a product that meets all of the set specifications in a reproducible fashion. In a similar way, a synthesis and scale up strategy was developed for the complementary polymer HA-01. Although the first test batch met most quality criteria, optimisation of the final drying step in the second batch led to a batch that met all the specified criteria.
Main Results linked to the general objectives of HydroZONES
WP 1 developed and synthesised three hydrogel platforms for the regeneration of articular cartilage.
Materials were designed and optimised in such a way that they could be printed in highly defined 3D scaffolds. Required mechanical stability of the printed scaffolds was realised by using advanced hybrid printing technology using reinforcing thermoplastic fibers to match the mechanical properties of native cartilage and retain the scaffold for extended periods of time needed for regenerating the damaged tissue. Hydrogel filaments were double printed together with the thermoplastic fibers resulting in custom designed cell containing scaffolds.
Cell compatibility was evaluated for the different hydrogels and showed that cell viability was retained during hydrogel cross-linking reactions for MSC’s and chondrocytes. Hydrogel compositions were optimised regarding cell differentiation by investigating the effect of hyaluronic acid content and heparin content. Heparin is well-known to entrap growth factors and hyaluronic acid microgels were embedded as proteins (e.g. growth factors) delivery particles. The best performing hydrogel compositions were further evaluated in vivo in WP5.
Zonal organisation was obtained by layer-by-layer deposition of hydrogel materials of different composition. In this way, zonal constructs with zones of different mechanical properties, glycosaminoglycan content, degradation rate and cell type have been prepared to show the versatility of the printing process. Finally, optimised 3D printed scaffolds were transferred to WP6 for in vivo studies in large animals.
WP2: Automatisation of assembly
WP Objective
Develop a multi-material bioprinter system for the printing of 3D zonal cartilage constructs with functionalised bioactive hydrogels (WP1) and embedded cells (WP3). In collaboration with Partner #13 PROSPA, Partner #08 PIL will tailor the hydrogel-printers for the application needs of Partners #01UKW and #05 UMCU.
Summary of WP results (linked to the WP objectives)
Two first-generation state of the art bioprinter devices, equipped with one printing head, were designed and produced by Partner #08 PIL. The system enables the automated printing of hydrogel-based constructs with or without encapsulated cells for subsequent evaluation and optimisation. Dedicated control software was developed by Partner #08 PIL and #17 PROSPA for the automated production of 3D layered structures and it was fully integrated and synchronised with the hardware of the bioprinter. After a first set of morphological screening tests, carried out at Partner #08 PIL using Scanning Electron Microscopy (SEM) with the aim of determining the accuracy and reproducibility of the bioprinter, the devices were delivered to Partners #05 UMCU and #01 UKW for further evaluation and optimisation.
The development of single-head first generation bioprinters suffered significant alterations. In consequence, more complex printer functions were pursued, in order to fit the adjusted requirements for Partners #01UKW and #05 UMCU printing devices. Adjusted printing equipment already included multi-material printing solutions (i.e. thermoplastic and hydrogel printing heads) and these specific requirements were taken into consideration in the planning and development of these.
Despite of severe delays occurred in Task 2.1. All the main requisites were addressed in the developed printers (second generation). Therefore, and regarding the printer requirements being fulfilled by the current equipment (Bioprinter I, and Bioprinter II) and the alternative solution encountered by the consortium through the acquisition of commercially available for simpler hydrogel printing processes, efforts were pursued towards multi-head/ multi-material printer’s development. In fact, it could be considered that focus has been transferred and effective work has been undertaken towards the delivery of functional multi-material devices fulfilling second-generation bioprinters criteria.
Based on the previous experience, the first generation bioprinter devices were upgraded and advanced according to the feedback of Partners #01 UKW and #05 UMCU. The second-generation devices were equipped accordingly, for example regarding speed (at least 2000mm/min) and precision of printhead movement in X and Y direction, temperature control with high accuracy between 2°C and 37°C through the development and integration of heating/cooling circulating batch. An irradiation system composed of a light source and an optical fibre were implemented at the printing heads enabling an additional cross-linking mechanism of the extruded hydrogels. The collector/baseplate system was redesigned for allow levelling. Control software was further developed by Partners #08 PIL and #13 PROSPA enabling full and user friendly control over the respective printers. After a preliminary morphological evaluation of 3D multi-material constructs, Partner #08 PIL sent a researcher to Partners #05 UMCU and #01 UKW to collaborate on the in vitro assessment of the constructs and subsequent optimisation of the bioprinting system. Importantly, these advanced bioprinters were designed considering the possibility to fit and work independently inside a laminar hood (guarantying sterile environment during optimisation stage).
Main Results linked to the general objectives of HydroZONES
The automated design and production of 3D constructs capable of mimicking the functional hierarchy of native tissues represents a paramount challenge for Tissue Engineering (TE). HydroZONES aimed to develop a multi-material bioprinter system suitable to 3D print zonal cartilage constructs (WP2). That was obtained by the assembly of extrusion-head and multi-head hydrogel dispensing module holding up to three different hydrogels (WP1) with embedded
cells (WP3).
Two multi-material Printing systems were effectively developed and delivered to Partners #01 UKW and #05 UMCU for assessment of the produced constructs and subsequent optimisation of the bioprinting system. Following preliminary testing, improvements were deemed necessary and were mostly added to the equipment before shipment to the consortium partners (Bioprinter II - #05 UMCU and Bioprinter II - #01 UKW).
Therefore, two variations of the Bioprinter systems were developed and improved to allow the extrusion of thermoplastic materials (e.g. PCL) and the printing of multiple hydrogels towards the generation of layered mechanically stable implants through the hybrid printing of thermoplastic (for structural support) and hydrogel loaded with cells and/or other molecules (for functionalisation).
The development and implementation of a software (algorithm and interface), HydroZONES G-code Generator Software, was done in close cooperation with Partner #16 PROSPA. The software allows for the definition of specific production parameters. These include: profile geometry (rectangular, elliptical, or custom (hand drawn) forms), layer composition (PCL and/or Hydrogel), thickness and pore size, independent definitions for each biomaterial head (thermoplastic and hydrogel(s)). It also considers adjustment for the nozzles distance to the baseplate and collision avoidance functions (of the printing heads with the building structure). Accordingly, the SOP manual for the use of second-generation bioprinters was produced, containing all the specifications and procedures (both hardware and software) for its correct operation.
Following the tailoring of the systems, after its completing, some more issues were addressed by Partners #01 UKW and #05 UMCU. For instance, in month #40 meeting was discussed the suitability of having thinner (0.25 mm) needles for the thermoplastic extrusion, as well as testing other manometers that would increase the precision in pressure adjustment. Although, the former request did not provide a reliable filaments extrusion, higher precision manometers were used inducing higher control of the applied pressure.
WP3: In vitro assessment
WP Objective
The overall objective of WP3 was to evaluate the chondrogenic potential of the developed materials that have been developed in WP1 and, as such, fulfil the criteria in terms of mechanical properties and gelation kinetics for 3D printing. In WP3 materials were selected for further in vivo evaluation in WP5 and WP6. Accredited testing according to DIN EN ISO/IEC 17025 and DIN ISO 10993-5 were performed according to the QM/RAM system and product development plan.
Summary of WP results (linked to the WP objectives)
WP3 established within the consortium SOPs for harvesting and culturing human, equine and porcine chondrocytes and MSCs, and optimised and unified histological procedures and analysing techniques (characterisation of cell population, analysis of specific markers such as CD105, CD90, CD73) for homogeneous processing of samples across the different laboratories within the consortium. This allowed different groups to culture and evaluate the chondrogenic capacity of the different hydrogels developed by WP1 partners and compare results across laboratories.
Each biomaterial was cultured with different cell types and concentration of polymers to establish optimal conditions for cell viability, material degradation and chondrogenic potential with regards to GAG and collagen production.
Different alternative biomaterials were also investigated as potential candidates, such as a collagen II based hydrogel, however due to solubility issues, this was abandoned to concentrate efforts on more promising materials.
P(AGE/G)-HA-SH
Firstly, the different components of the PAGE hydrogel were assessed for viability and cultured with hMSCs for evaluation of chondrogenic differentiation, followed by the evaluation of the P(AGE/G)-HA-SH system with equine and human MSCs. The materials composition was thus adjusted for optimal degradation. Furthermore, the influence of TGF-b on hMSCs behaviour was assessed, followed by the effect of zonal configuration and 3D printing on chondrogenic differentiation of both human and equine MSCs. Briefly, constructs with complex organisation based on PAGE hydrogels biofunctionalised with hyaluronic acid sulphated derivatives (HA-SH) were generated and chondrogenic differentiation of MSC was evaluated. Suitability of standard HA/PG hydrogels and cast HA/PG hydrogels supported by printed PCL scaffolds for chondrogenesis of MSC was demonstrated and, additionally, redifferentiation of chondrocytes. Consequently, these constructs were advanced to the large animals studies. Furthermore, 3D printed constructs utilizing an HA/PG formulation adapted to the bioprinting process were produced. The general printability of MSC within these systems could be successfully demonstrated.
pHPMA
The pHPMA optimal formulation for chondrogenic differentiation with respect to components concentration and cell concentration was assessed with equine chondrocytes and MSCs. This showed that the M10P10HA20 combination with equine chondrocytes displayed the most promising results and was advanced to further studies, after multiple optimisation steps in preparation for the next cultures (UV cross-linking methods, molecular weight change to increase hydrogel stability in culture). This was followed by the testing of different compositions for optimal 3D printing and assessment of the influence of the process which showed severe inhibition of chondrogenic potential on equine chondrocytes and MSCs.
The pHPMA-HAMA system was then shown to be a suitable platform for cartilage matrix deposition by encapsulated chondrocytes. HAMA affected chondrogenesis in a dose-dependent fashion, and an optimal HAMA concentration was identified between 0.25 and 0.5% v/v. Concentrations higher than 0.5% resulted in no improvement of chondrogenic differentiation. Even though addition of HAMA improved the printability of the gel and even if the hydrogels could be printed (both with and without HAMA) supporting cell viability, long-term differentiation studies indicated that the printing process with these hydrogels is not suitable for cartilage formation, possibly due to the high viscosity and consequently from the high shear stresses experience by the cells during extrusion.
GelMA
Parallel to this, a back-up alternative material based on gelatin methacryloyl (gelMA) was also evaluated for stability, chondrogenic capacity with and without 3D printing with equine chondrocytes and MSCs. Printability and chondrogenesis were optimised by adding rheology modifiers such as gellan gum and bioactive molecules like hyaluronic acid.
StarPEG/heparin
Initially, hMSCs were embedded to assess chondrogenic differentiation in PEGh hydrogels, with different degrees of incorporated enzymatically cleavable peptidic linkers (0%, 50%, 100% peptidic crosslinkers) and cultured under chondrogenic conditions. These initial materials did not provide a stable scaffold to build up a cartilaginous matrix from porcine articular chondrocytes in vitro, therefore it was decided to proceed with hydrogels in which only 50% of crosslinks are MMP degradable. Therefore, thiolated hyaluronic acid and acrylated PEG based hydrogels were tested for viability with chondrocytes and BMSCs, showing chondrogenic capacity only for chondrocytes.
StarPEG/heparin different formulations were tested for chondrogenic differentiation, with and without the functionalisation of RGD and other specific cartilage peptides. In particular, StarPEG/heparin degradability was established, along with the effects of adhesion peptides on matrix production and distribution with different cell types. Furthermore, cell behaviour in printed constructs was assessed, showing that while porcine chondrocytes and human MSCs were not affected by this procedure, pMSCs lost functionality.
Based on these evaluations, optimal formulations, compositions and cell combination for each material were established for in vivo studies, leading to the decision to eliminate pHPMA from long term large animal studies based on the poor chondrogenic capacity when printed.
Main Results linked to the general objectives of HydroZONES
As a first approximation of the natural anatomic situation, the effects of a zonal configuration of the cultures was evaluated extensively in vitro. Biocompatibility of all three developed materials was found to be satisfactory in vitro and biofunctionality of one of the gels (pHPMA-gel in printed, but not in cast form) turned out to be insufficient, for which reason this gel was not elected for further in vivo testing.
WP4: 3D in vitro assay and test system
WP Objective
The objective of WP4 is to establish and modify a 3D in vitro model and bioreactor system to evaluate chondral regeneration, as well as cell-material interaction and material induced cell migration and differentiation under physiological conditions in a bioreactor system. Thereafter, based on this bioreactor system, a validated standardised ex vivo throughput platform for commercial contract R&D services has been developed. In addition, a suite of new mathematical models have been developed and validated, together with robust and efficient computational tools that allow simulation of chondral regeneration in vitro and in vivo. The in silico and in vitro models developed were standardised and validated with the in vivo results of WP5 and WP6. Finally, the theoretical models were adopted to identify those aspects of material and construct design that have the greatest influence on in vivo integration and long-term functionality.
Summary of WP results (linked to the WP objectives)
The main result related to the first objective was the development of the 3D in vitro model for evaluation of (osteo)chondral regeneration, cell-material interactions and material induced cell migration and differentiation (LTG). The osteochondral culture platform was developed in stages, at first a static platform technology was realised, without incorporation of mechanical loading. In parallel, in the first phase of the project, a set of tools and standard operation procedures were developed to isolate osteochondral explants from different species in a standardised and feasible manner, such that experiments and methods between the different labs could be easily aligned and compared. In addition, a standard operation procedure was developed for the culture of osteochondral explants in the osteochondral culture platform. The osteochondral culture platform consists of a customised insert in which osteochondral explants can be cultured such that cartilage and bone compartments are separated externally, allowing for specific culture conditions for the specific tissues. WP4 partners have proved that this design is critical for maintaining osteochondral explant metabolic activity, cartilage ECM composition and mechanical properties in ex vivo culture up to 12 weeks (Schwab et al, ALTEX, 2017). To the best of its knowledge, this is much longer than any other study in literature has ever reported. Subsequently, all partners in WP4 have obtained the culture platform and have performed several studies with it, in which different chondral implants developed within the consortium have been evaluated with respect to cartilage regeneration. In these studies, it has been shown that cartilage matrix production is dependent on hydrogel material, supplementation of TGF-β1 in cartilage media and encapsulated cell type. At the same time, the culture system was further developed by designing and developing a mechanical loading modality that was then incorporated in the existing static system. The mechanical loading modality allows for controlled loading of the cultured osteochondral explants with adjustable (physiological) loading parameters, such as duration, frequency, displacement and force in a sterile manner. In addition, the system allows for online measurement of the mechanical properties of the tissue, without needing to sacrifice the samples, being an online monitoring tool for the quality and effectiveness of the applied treatment.
In silico modelling was performed alongside the experimental work. The partners developed an in-silico model of the regeneration of a hydrogel inserted into an ex vivo osteochondral explant allowing the investigation of different cell seeding strategies as well as the role of growth factors (Kimpton et al, Math. Biosciences, 2017). This predicted that cell infiltration alone will not generate the layered architecture seen in healthy cartilage, and that this could only be achieved by zonal seeding of chondrocytes within the construct. This is broadly in line with experimental work performed on cell infiltration and seeding strategies at UMCU. WP4 also developed an in-silico model for the interaction between TGF-β, MSCs and chondrocytes (Chen et al., J Theor Bio, submitted) . This enables to predict a critical concentration of TGF-β to be added to a culture medium to induce chondrogenesis of a population of stem cells: this prediction was in line with the 10 ng/mL used in the standard operating procedures of the HydroZONES core experiments. This model also predicts that a population of MSCs will differentiate when co-cultured with chondrocytes; the predicted initial chondrocyte density required to achieve this is approximately 20%, in line with experiments performed at Partner #01 UKW. Finally, WP4 partners developed a phenomenological in-silico model of fibre-reinforced hydrogel construct which describes the synergistic increase in stiffness obtained by combining these materials (Visser et al, Nature Comms, 2015). In order to resolve discrepancy between this simple model and experimental observations we developed a continuum in-silico model of fibre-reinforced hydrogel construct via the technique of mathematical homogenisation, which takes into account the small-scale features of the fibres on the bulk mechanical properties of the construct (Chen et al., Eur J Appl Math, submitted). This was validated using experimental data from Partner #05 UMCU.
Main Results linked to the general objectives of HydroZONES
The main results mentioned in the section above are directly linked to the second general objective of the HydroZONES project. A 3D in-vitro/ex-vivo osteochondral platform was developed, consisting of hardware, operating procedures and related assays for (osteo)chondral implants was designed and developed in the first phase of the project. It was shown that the system allows for a more realistic and native-like environment resulting in a better cartilage regeneration response in comparison to conventional in-vitro studies without having the native tissue around, which makes the translation to in-vivo results better and increases the predictive value of in-vitro testing. Mechanical loading was included in the system, to make the environment even more realistic and comparable to the situation in the native joint which further increased the predictive value of the system. In order to validate the system, chondral implants developed within HydroZONES were evaluated both in the ex-vivo osteochondral platform and in in-vivo animal studies and results were compared. In addition, the development of the in-silico model allowed for more focussed design of experimental studies, obviating the need for trial and error studies. The results of the modelling were used to select optimal parameters. In particular, this predicted the growth factor concentrations and co-culture settings required to induce MSC chondrogenesis in vitro. Further in-silico modelling predicts the bulk mechanical properties of fibre-reinforced scaffolds, so that these can be designed to withstand the demanding loading environment in the joint. This unique combination of predictive in-silico and in-vitro models is a very valuable tool for evaluation the regenerative capacity of implant materials prior to in vivo testing, reducing the amount of animal experiments and related costs, time and effort.
WP5: Biocompatibility and biofunctionality Screening
WP Objective
The overall objective of WP5 was to develop an in-vivo screening system to determine the biocompatibility and biofunctionality of constructs previously selected in vitro by WP3. By ectopic implantation into immunocompetent mice we want to determine the immunological response elicited by new materials. In addition, by developing the ectopic system into immunodeficient mice we evaluated the chondrogenic properties of cell-free materials as well as materials loaded with chondrocytes and BMSCs of different origin (human, horse, pig). Finally, in order to determine the chondrogenic properties of optimised zonal cell-laden constructs an ectopic osteochondral plug implantation model was developed.
Summary of WP results (linked to the WP objectives)
During the time of the project, a system for immunotolerance analysis was established in mice. All materials demonstrate good immunotolerance and low fibrosis invasion and no acute inflammatory response were detected, allowing us a secure translation of materials to large animal models. First generation (pre-casted) and second-generation (zonal pre-casted) hydrogels did not yield significant extracellular matrix production into immunodeficient animals. On the other hand, by establishing a more chondrogenic environment through our osteochondral plug implantation model we demonstrated a reparative response mediated by the PCL reinforcement together with an essential role of the subchondral bone in the cartilage repair process. Furthermore, using this model, our results demonstrated a superior repair capacity of reinforced zonal constructs mimicking the native structure of the articular cartilage.
Main Results linked to the general objectives of HydroZONES
WP5 results using materials mimicking the zonal structure of native cartilage, with chondrocytes on the top layer and BMSCs on the bottom layer showed greater integration with native surrounding tissue as well as enhanced reparative response and superior ECM deposition by gel-embedded cells.
WP6: Long-term pre-clinical in vivo evaluation
WP Objective
Long-term testing of the three favourite materials developed in WP1 by the devices provided from WP2 and evaluated in-vitro (WP3) and in-vivo (WP5) to set new standards of pre-clinical testing in minipigs (Task 6.3) and in the biomechanically challenging equine model (Task 6.4). Therefore, development of enhanced methods for scaffold fixation and durable integration by validation of a tissue glue and durable cell-support via an anabolic stimulus (GDF-5) in minipigs (Task 6.1) and horses (Task 6.2). Validation whether PDGF enhances chemoattraction of host cells to cell-free scaffolds (Task 6.5) and selection of the best layered cell-containing HydroZONES construct among 2 material options (Task 6.6). Determination of in-vivo performance of four improved, second generation layered HydroZONES constructs. (Task 6.7)
Summary of WP results (linked to the WP objectives)
Generally summarising the work done in WP 6 it was discovered that there was no cell-invasion in all 3 materials in an ectopic mouse model. Cell-invasion cannot be rescued by 150 ng PDGF per construct of PAGE or starPEG: No large animal experiments with PDGF.
There is high potential of GDF-5 augmented fibrin glue to stimulate calcified cartilage formation in a new ectopic mouse model. No enhanced fixation of CCT-enforced hydrogel scaffolds by GDF-5 augmented fibrin glue in minipig defects.
Partner #12 CCT-enforced hydrogel has to be abended since neither gluing nor suturing fixed CCT-enforced hydrogel scaffolds in minipig cartilage defects and development of a new PCL enforcement in WP1 was necessary.
There was a need for adaption of degradation of first generation hydrogel materials in minipig joints. Lack of cell-invasion of all 3 materials in minipig cartilage defects led to abandonment of cell-free approaches with these hydrogels.
Due to premature loss of constructs from cartilage defects in minipigs and horses, cancellation of non-enforced hydrogel constructs was performed.
It was also discovered that localisation and defect preparation have major impact on integration of reinforced hydrogel scaffolds: Use of a drill required for press-fit fixation of enforced constructs without fibrin glue into trochlear defects of minipigs.
After establishment of the best integration strategy, performance of the two most promising cell-seeded hydrogel materials starPEG and PAGE/HASH was compared in trochlear cartilage defects in minipigs for 12 weeks. The established osteochondral defect model in minipigs was successful in retaining PCL-enforced starPEG and PAGE/HASH constructs in the medial trochlear groove. No adverse effects were observed during the trial and at explantation. Defect treatment with PCL-enforced constructs tended to induce more bone loss (µCT). The starPEG group was even significantly worse than empty controls after 12 weeks. No significant differences in regeneration outcome between starPEG, PAGE/HASH and empty defects (histological scoring) after 12 weeks demonstrating no significant benefit of defect treatment with the chosen design.
A zonal versus non-zonal design of starPEG hydrogel constructs with polycaprolactone (PCL) enforcement was compared in minipigs over 6 months:
• Zonal and non-zonal PCL-enforced starPEG constructs were retained in all osteochondral defects and the printed PCL architecture remained intact. Unfortunately, loading pressed 16/18 constructs beneath the cartilage level.
• Defect treatment with enforced constructs induced significant bone loss according to µCT analysis compared to empty controls.
• In most cases hydrogel material disintegrated, was flushed out of the enforcement into the underlying bone, with persisting fragments undergoing degradation in the subchondral bone.
• Safranin-O-positive regeneration tissue at cartilage level often seemed host-derived. In 9/18 defects Safranin-O-positive regeneration tissue was connected to transplanted hydrogel/PCL. In 5 cases implanted cells persisted in hydrogel fragments and contributed to the Safranin-O-positive, but mostly collagen-type-II-negative regeneration tissue. No benefit of the non-zonal architecture with chondrocytes versus the zonal architecture with MSC and chondrocytes was evident.
• The quality of cartilage repair judged by modified O´Driscoll score was very heterogeneous in both treatment groups and results were overall significantly worse than in untreated controls with no indication of a benefit of the zonal design.
• Overall, the strategy is yet insufficiently mature for in vivo application in cartilage defects since non-treated cartilage defects performed better than treated defects.
As for the horse trial testing of materials (in cast, reinforced and printed form) was developed in the consortium ectopically in the horse model (safety testing). As well as testing and development of fixation methods for the biomaterials: assessment of commercial versus autologous fibrin glue and the development of the 3D-printed osteochondral PCL anchor.
Short-term orthotopic testing of all materials in the horse was carried out and optimisation of the production zonal constructs for in vivo orthotopic implantation in the equine model (2 materials plus back-up material) was performed.
Finally, long-term (6 months) assessment of the two finally selected materials (starPEG and PAGE/HASH) in zonal and non-zonal form in the equine model was carried out. Comparing zonal versus non-zonal design of starPEG and PAGE/HASH hydrogel constructs with polycaprolactone (PCL) enforcement in the equine model over 6 months with the following conclusions:
• Zonal and non-zonal PCL-enforced starPEG and PAGE/HASH constructs were retained in all osteochondral defects and the printed PCL architecture remained intact.
• Reinforced constructs showed good lateral integration with surrounding cartilage and bone, with new bone formation in the osteal anchor. It was consistently observed that the centre of the osteal anchor was not calcified, suggesting a longer time may be needed to achieve full calcification in the anchor.
• In most cases hydrogel material persisted in fragments and within the reinforcements, independently of whether the design was zonal or not. StarPEG showed overall a higher persistence than PAGE/HASH based constructs.
• The quality of cartilage repair judged by a modified O´Driscoll score was very heterogeneous in both treatment groups, however semi-quantitative scoring suggested a higher performance of the zonal group in the starPEG based constructs.
• Despite absence of full chondral matrix formation, stiffness was higher in the zonal group in both hydrogels.
• Within the hydrogel fragments cells were visible, along with isolated areas positive to collagen II staining, suggesting that at the endpoint of the experiment the chondral matrix formation was still going on within the (surviving) hydrogel.
• Overall, the findings suggest that the approach as taken in our studies is promising, warranting further investigations. Implantation of osteochondral constructs of which the cartilage layer is hydrogel-based, has a zonal organisation and is reinforced with a stiff material seems a promising avenue that, after optimisation, may lead to significant improvements in the quality of matrix production in cartilage repair.
Main Results linked to the general objectives of HydroZONES
Based on µCT and histological scoring of tissue regeneration, treatment with PCL-enforced starPEG or PCL-enforced PAGE/HASH constructs did not improve defect healing at 3 months in minipigs. Zonal design showed no benefit versus non-zonal design at 6 months and both PCL-enforced starPEG groups had a negative impact on defect healing compared to untreated controls based on quantitative µCT analysis and semiquantitative histological scoring of a 6-month minipig study. Overall, the strategy is yet insufficiently mature for in vivo application in cartilage defects.
The use of composite osteochondral scaffolds with a zonal design for cartilage repair achieved partial tissue repair. Despite the absence of a full chondral matrix within the defects, the biomechanical properties were substantially better (showing higher stiffness) in the zonal constructs with both biomaterials, suggesting that the combination of MSCs and chondroprogenitor cells in a layered hydrogel construct is beneficial for the biomechanical properties of repair tissue. Good lateral integration with adjacent cartilage was achieved with both hydrogels, as was good integration of the scaffolds with the surrounding bone tissue.
The strategy is currently not mature enough to achieve production of hyaline-like regenerated tissue, however the evidence gathered in the equine studies suggest that this is a promising approach that, after optimisation, may lead to significant improvements in the quality of matrix production in cartilage repair.
WP7: Management system for quality & regulatory affairs (QM/RAM)
WP objective
Medical devices and tissue engineering products have to fulfil various legal regulations and standards already during their design phase if those are developed to be marketed inside of the EU. Processes have to be defined and performed conformingly, for the design phase as well as for the later manufacturing, and the conformity of those has to be made evident by standardised means of documentation. The objective of WP7 was to provide in-processly the means and structures which allow best management of resources and processes and which guarantees that at the end of the project the fulfilment of the legal regulations and of the relevant standards can be proven. At the closure of the project’s funding there is a Master Design History File and a Master Design Dossier for functional materials/TE devices was presented and issued and serves as a basis not only for the developments in HydroZONES but for future developer’s, manufacturer’s or distributor’s Design Dossiers according to Annex II.4 of the Medical Device Directive 93/42/EWG + 2007/47/EG of the EU for later CE-marking and for attaining the 510k premarket approval of the US Food and Drug Administration FDA. Connecting ports for modules for the efficient proof of the fulfilment of specific requirements of additional regulatory areas like Australia, Japan, Canada shall be implemented.
The developed generic quality management system shall thereby guarantee
• a general standardisation of documentation
• transparency and comparability of parameters / results / decisions / costs
• a common understanding of the objectives and the actual status of the project
Summary of WP results (linked to the WP objectives)
WP7 has contributed to an increase of the quality and safety of the HydroZONES processes and developments. Safety and quality in the realisation of TE/ATMP products is generated by management during the following phases:
1. research
2. development
3. verification
4. validation / clinical evaluation
5. translation to manufacturing / scale-up
6. manufacturing
7. in-process testing
8. final testing
9. post marketing follow-up
HydroZONES was covering the phases 1 to 3. Partner #11 HCS has provided knowledge and consultancy for the conforming planning and performance of the critical activities of these phases. As defined in the objectives, Partners #11 HCS has paid attention that the QM and the regulatory requirements have been known and understood in time by the related consortium partners to assure, that the development of HydroZONES Tissue Engineering products was done and documented in anticipation and in respect of the QM and regulatory requirements.
WP7 has contributed to design conforming proceedings and conforming documentations of the HydroZONES processes and results which may be of high value for industrial partners (as spin-off companies) for the following issues:
• compelling risk management of designs and processes
• efficient scale-up and translation process to manufacturing
• comprehensive validation, testing and documentation of the final products
• probably successful evaluation and approval process by the specific European authorities and institutions in a survivable period of time.
Main Results linked to the general objectives of HydroZONES
WP 7 provided support of the Consortium partners in planning and in implementation of adequate quality management and Regulatory Affairs Management Systems. Thus enabling innovative R&D work with a clear aim of industrial application and clinical trials, setting a new standard for tissue engineering research and biofabrication.
Potential Impact:
Damage of articular cartilage or intervertebral disc cartilage occurs frequently and is followed by a process of OA, ultimately leading to pain and joint malfunction. OA causes severe loss in quality of life in approximately 40 million European Citizens. As such, it is the leading cause of disability, a more frequent cause of activity limitation than heart disease, cancer or diabetes. It accounts for more disability among the elderly in Europe than any other disease. It is in 6.6% of cases associated with severe psychological distress and a major cause of the high work disability benefits. While the total direct annual costs of OA in the US are estimated at $89.1 billion, the indirect costs are also high, largely a result of work-related losses and home-care costs. For example, the impact of arthritic diseases on earnings increased in recent years, with $108 billions of earnings being lost in 2003 in the United States alone. In France, direct costs of OA exceeded €1.6 billion in 2002 and accounted for 13 million physician visits. That year’s figures represented a 156% increase in costs over 1993, which was for more than 90% due to an increase in the number of patients, rather than to an increase of costs per patient.
With the above-mentioned challenges, HydroZONES major results and research achievements help to reduce costs for European healthcare systems by the generation of cheaper, potentially cell-free, alternatives based on instructive biomaterials to regenerate the complex tissue organisation at the defect site. It might lead to a breakthrough in the way OA is treated today and reduce the burden for the patients and their surrounding community.
Scientific impact of WP:
Throughout the lifetime of the project the different WP have achieved significant progress in their scientific research bringing the solution several steps closer in an innovative way that supports the project’s objectives.
WP1 developed newly designed and fully characterised biofunctional hydrogel systems, which are available for 3D culture of MSCs and chondrocytes for the regeneration of articular cartilage and were subsequently optimised for the 3D printing process. Crosslinking steps are proven to be cyto-compatible and provide good cellular survival in the hydrogels. Important parameters regarding the design and synthesis of biomaterials for cartilage regeneration implants have been defined balancing the need for mechanical stability and biological performance. The establishment of the printing process created insights and experience in the 3D printing of cell-laden hydrogels with new printing techniques (extrusion based bioprinting and reactive inkjet printing). Together with the application of double printing together with thermoplasts, the generation of mechanically stable composite constructs allows the production of robust cartilage constructs. The established GMP-production of one of the hydrogel systems provides a good starting point for initial clinical studies and aims at a possible commercialisation of this developed hydrogel.
According to the established objectives for WP2, significant achievements were obtained in the development of customised bioprinters. As exposed in the deliverables, 3 bioprinting platforms with novel technology led the production of equipment in a form ready for large-scale production. Innovative hybrid hardware was accomplished according to the requirements of optimal scaffolds design.
G-code proved to be a reliable approach for the control of production settings and integrated definition of general scaffold characteristics.
In addition, the multidisciplinary requirements to achieve the novel bioprinters, enhanced the high-quality training of a set of engineers, who are now in the forefront of equipment development.
WP3 carried out Establishment and optimisation of fundamental SOPs for harvesting, culturing and analysis of chondrocytes and MSCs across different species. It performed first in vitro testing and optimisation of three new semi-synthetic biomaterials. As well as in vitro demonstration of the potential of bioprinting as a strategy to engineer articular cartilage, and advancement of 3-D printing techniques for cell-laden hydrogels for cartilage repair.
Finally, biofabrication of 3D printed, zonal-like constructs with mechanically reinforcing structures was developed.
New mathematical techniques were developed in the course of the in-silico modelling to address the core requirements of WP4. These abstract methods have potential to address similar problems in other fields of applied scientific research.
WP5 has established and validated an in vivo screening system in the framework of HydroZONES, that allow us to test the behaviour of cells and new materials to be used in regenerative medicine. In the field of articular cartilage, this would speed up the translation to the clinic of promising materials and implant designs reducing the cost of testing. Besides, the osteochondral ectopic in vivo model demonstrated that it is a feasible and versatile system capable of evaluate and predict the role of different combination of factors in the repair process.
Through its research, WP6 has carried out the first testing of zonal constructs for cartilage repair in large animal models. Performed optimisation of both the porcine and the equine model and completed the development of an effective fixation method for osteochondral scaffolds that due to its design allows the assessment of the chondral part without influence from the osteal part that only serves as the bone anchor.
WP7 enabled the funding, sponsoring and non-financial support by external partners. This can be gained more easily by start-ups, spin-offs or global players, as regulatory risks are identified and managed during the research and development phase already.
Translation of the HZ results to industry is accelerated – and thus the supply of the patients may be possible earlier - because needs of verification and validation are anticipated in the development phase already thorough the close support of WP7. Thus, number of failures of RegMed enterprises can be reduced.
By the WP7 Quality Management and Regulatory Management Package - which enforces that a conforming development process and a conforming documentation of it is built up 'in-processly' and in time - all parties which are concerned may be able to better calculate and manage resources and to better estimate times to market.
Success rate of innovations and of enterprises in the European Union in the global field of Regenerative Medicine shall be increased by this remarkably.
Leading the cutting end research in osteochondral reconstruction, representatives of the consortium took place in InoovaBone stakeholders day and workshops resulting in direct influence on the selected topics for research in the next NMBP work programme 2018 – 2020 placing the emphasis of the EU on the direct continuation the work carried out in HydroZONES.
Socio-economic impact and the wider societal implications
As HydroZONES targets a common medical challenge that is affecting millions of people around the world, directing affecting their longevity and quality of life – progress in the research in the field and promising result can have effect not only on the scientific community but also direct effect on the public lowering the personal and economic burden.
Hydrogels, which were successfully tested in long term studies, could improve the therapy of difficult to treat cartilage defects and provide new therapeutic options. The collaboration in the groups of WP1 provided an excellent scientific training and education of the participating PhD students and scientific co-workers in a highly interdisciplinary and international research environment. This led to the creation of an international network of researchers in the field of biomaterials and biofabrication.
WP2 gathered scientists, engineers and physicians in interdisciplinary teams using a variety of methods to construct biological substitutes, boosting an effective synergy between mechanical engineering and biomedical sciences; providing revolutionary steps in manufacturing at industry 4.0. 3D bioprinting market was valued at $98.6 million in 2015, and an annual growth of 36% for the next 6 years is expected . The work developed in WP2 supported the position of Europe in the front end for adequate technology development.
WP3 achieved establishment of relevance of zonal character of implants as a guideline for further research. It carried out optimisation of culture conditions and 3D printing techniques of biomaterials for cartilage repair as advancement for future research towards in vivo applications in animal models.
The osteochondral culture platform that has been developed within WP4 is now used by Partner #15 LifeTec Group to perform contract research for clients in order to evaluate their newly developed bone and cartilage repair studies. Companies that develop cartilage or bone repair strategies can significantly reduce the number of animals needed, by using the developed osteochondral platform within HydroZONES as a screening tool for testing arrays of different material compositions, implant coatings and drug dose-response studies. This way, the number of animals (ethical considerations) and associated costs can be significantly reduced with 30-60k€ per product development. Moreover, the time-to-market can be significantly reduced, with estimated 1-2 years per developed product. As the osteochondral culture platform is easy and affordable and can be produced in a fast and customised manner, this allows both industrial and academic researchers to use the system for their research purposes. Already quite a number of academic and industrial clients (>10) are making use of their system to answer their (fundamental) research questions or to evaluate their newly developed treatments and therapies.
The predictive, validated in silico models developed within WP4 allow for more focussed experimental design in future studies. The in-silico models predict the optimal choices of culture and implant parameters, as well as guide the design of the fibre-reinforced hydrogel scaffolds, significantly reducing the trial-and-error character of these kind of studies. This will significantly reduce time (6-12 months for a typical implant material study) needed for produced development and in vitro screening and prior to in vivo studies. The new mathematical techniques that were developed in the course of the in-silico modelling to address the core requirements of WP4 have wide application in other fields of scientific research.
Due to the unique composition of WP4 (mathematical and experimental researchers; academic and industrial partners), there was a lot of interaction and knowledge transfer between the different partners, even beyond this specific work package. Researchers spent some time at each other’s institutes (academic vs industry) to experience other working environment. Mathematical and experimental researchers joined forces in several meetings, allowing for a much better understanding of each other’s disciplines and needs, which had a clear synergistic effect on the outcomes of this work package. Moreover, these collaborations will exist also beyond the HydroZONES funding period, already shown by new collaboration initiatives between the different partners (#04 UOXF – #15 LifeTec Group; #05 UMCU – #15 LifeTec Group; #04 UOXF– #05 UMCU; #04 UOXF – #01 UKW).
From a Socio-economic point of view the developed model of small animal trials by WP5 reduces the number of large animals used in research, decreasing the ethic and social concerns in the use of animals in research. In consequence, implies the reduction of costs in translational medicine by an early identification of non-optimal materials.
WP6 in its research contributed to the establishment of relevance of zonal character of implants as a guideline for further research. Leading to reduction of unnecessary spillage of animals by drawing attention to the unsuitability of (allogeneic) fibrin glue for the attachment of chondral scaffolds in the equine model and possibly in the porcine model.
WP7 enabled through its support in the project to reduce regulatory risks and prepared adequate mitigation plan during the research phase. By developing regulations and QA system, translations of the project’s results to the industry is much faster due to reduced need of verification.
Main dissemination activities and exploitation of your WP
The developed hydrogel systems provide an excellent basis for further application in different fields, the gels provide the basis for further research in drug delivery, tissue engineering and 3D printing. Besides, the established printing protocols and procedures can also be applied for other hydrogel systems.
Newly developed printing systems (reactive dual pipet inkjet printing) evolved from the collaboration of a WP1 Partner #07 IPF with the company Gesim in order to allow printing of the dual component hydrogel system.
The obtained achievements on the bioprinting systems have proven to be suitable to be exploited by Partner #13 PROSPA. Furthermore, the technology advances are already being used in other research projects and were considered for research proposals (e.g. RIA H2020-FETOPEN-2017, RegMedXB Programme (NL), Gravitation Programme Materials Driven Regeneration (NL)).
The GMP prepared hydrogel system is applicable for other projects and already led to an intended participation with the established hydrogel platform in another research consortium by Partner #16 PolyVation. The experience with the newly established synthesis schemes and purification methods applied for the GMP production have already been applied for other research projects.
Partner #05 UMCU organised and hosted the HydroZONES symposium “Paving the path for biofabrication in Europe” (described D8.3). It also published in Science Impact – Joint Endeavours. (http://www.ingentaconnect.com/content/sil/impact/2017/00002017/00000008/art00026?crawler=true&mimetype=application/pdf)
Partner #05 UMCU Participated in activities organised jointly with other H2020 projects and was represented by Jos Malda: visit Researcher.
Last but not least, it promoted the education of young researchers such as the PhD defence Vivian Mouser carried out on the 15th of June 2017 in Utrecht
Partner #15 LifeTec has developed an exploitable product of Osteochondral Platform oriented for orthopaedic and pharmaceutical research, testing and screening. Testing the performance of an implant, device or therapy in a cost-efficient manner in living and functional ex-vivo cultured osteochondral biopsies. The platform is already in use for evaluation of material and cell-based cartilage regeneration strategies (TRL-9) in the context of small scale projects with academic as well as industry partners.
An QM/RAM system has been created in project by Partner #11 HCS and proposal of consultancy in the building up of similar QM/RAM systems was offered to institutions doing ATMP research.
Outlook and future research:
For a significant improvement and adjustment of the hydrogels systems developed in WP1, a more efficient control over hydrogel degradability appeared to be necessary in order to control cell ingrowth and migration as well as differentiation and balance this with the necessary scaffold integrity to ensure the implant stability and filling of the defect. Therefore, the following items are recommended for follow-up studies:
• Improvement and adjustment of hydrogel degradability (including e.g. triggered degradation by external stimulus) to enhance cell ingrowth/migration and differentiation and balance that with necessary scaffold integrity.
• Incorporation of biological cues to enhance differentiation especially in vivo.
• Improvement of printing resolution and speed to obtain personalised implants.
• Optimise printing process to obtain the same level of cell differentiation in printed cell-laden constructs as in casted constructs.
Starting from the zonal cartilage constructs developed in WP3, further refinement of hydrogels and bioinks for cartilage printing will be performed. Moreover, the next step will be the fabrication of fully bioprinted osteochondral constructs comprising also a cellularised engineered bone compartment.
Additional activities would be refinement and convergence of multiple biofabrication technologies to engineer large joint grafts, toward the bioprinting of a fully regenerated joint replacement.
The osteochondral platform developed in WP4 will be taken into a next generation by Partner #15 LifeTec Group, by developing disease models in a controlled way in order to test therapies and treatments in a relevant (pathological) environment, mimicking the disease situation in the patients. This work will be performed within the CarBon project, which has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 721432.
In addition, the osteochondral platform is already in use by different national and international research consortia to evaluate their developed (osteo)chondral implants. For example in RegMedXB (https://www.regmedxb.com/projects/taking-steps-towards-a-bioengineered-joint) BIOGEL (http://www.biogel-mariecurie.eu/; Marie Sklodowska-Curie grant agreement No 642687) and Wiliam Hunter Revisited (Dutch national funded research project; http://www.stw.nl/nl/content/p15-23-william-hunter-revisited-activating-intrinsic-cartilage-repair-restore-joint).
In addition, the osteochondral platform is used by industrial partners to test their materials and tissue engineering strategies for cartilage and bone regeneration. For example, the Dutch company OsteoPharma (http://www.osteo-pharma.com/) is using the ex vivo platform to evaluate their bone therapeutics and select most promising compositions for in vivo studies.
Future in silico work is planned to evaluate the role of mechanical loading and the remodelling of the extracellular matrix. Funding for this research will be pursued through the EPSRC and the Australian Research Council (via a new collaboration with the University of Adelaide).
The possibility to further in-depth characterisation of this advanced ectopic model developed in WP5 would allow to determine the mechanisms and pathways implicated the natural processes of articular repair. By identifying the key mediators of natural repair, it would be possible to develop novel treatments to slow down the progression of articular cartilage damage progression and OA appearance. In addition, the proposed in vivo model allows in vivo testing of different materials and implants designs.
WP6 development in long term trials will thrive in the future to use the improved models for further testing of other materials. As well as pursue further development along the lines set in the project for meeting the need for cartilage repair in the veterinary market.
Partners #11 HCS will continue the investigation of the administrative processes of applications for getting release on TE products inside of the EU to derive recommendations to the EU Commission for regulatory and institutional improvements. As well as investigation of the effectiveness and the efficiency of communication and for sharing of experiences in larger consortia for research & development (e.g. of medical devices, medicinal product and combinations of those/ATMPs).
List of Websites:
https://www.hydrozones.eu/
Degeneration of cartilage is a major cause of chronic pain, lost mobility and reduced quality of life for millions of European citizens. No clinical therapy is available that leads to healing of cartilage defects. Current cartilage implants cannot establish the hierarchical tissue organisation that appears critical for normal cartilage function. HydroZONES hypothesised that a biomimetic zonal organisation is critical for implants to achieve cartilage regeneration. It thus represented an interdisciplinary consortium that adopted a strategy to regenerate articular cartilage based on recapitulation of the tissues zonal structure employing 3D bioprinting technology.
Therefore, the HydroZONES project pursued the following 2 main objectives:
1. To develop a hierarchically structured hydrogel based mechanically stable scaffold that recapitulates the zonal distribution of the native articular cartilage.
2. To develop and validate a predictive in vitro test system for future evaluation of products for cartilage regeneration in order to reduce the amount of necessary animal experiments in the future.
Regarding main objective 1, three hydrogel platforms were developed. Materials were designed and optimised in such a way that they could be printed in highly defined 3D scaffolds also in double printing with thermoplasts to achieve the required mechanical stability of the printed scaffolds. Cell compatibility was evaluated for the different hydrogels and showed that cell viability was retained during hydrogel cross-linking reactions for MSC’s and chondrocytes. Hydrogel compositions were optimised regarding cell differentiation by investigating the effect of hyaluronic acid content and heparin content. Zonal organisation was obtained by layer-by-layer deposition of hydrogel materials of different composition. In this way, zonal constructs with zones of different mechanical properties, glycosaminoglycan content, degradation rate and cell type have been prepared. Finally, 2 different optimised 3D printed scaffolds were assessed in large animals. In the equine model, the use of composite osteochondral scaffolds with a zonal design for cartilage repair achieved partial tissue repair. Despite the absence of a full chondral matrix within the defects, the biomechanical properties were substantially better (showing higher stiffness) in the zonal constructs with both biomaterials. Good lateral integration with adjacent cartilage as well as integration of the scaffolds with the surrounding bone tissue was achieved with both hydrogels. Even though the strategy is currently not mature enough to achieve production of hyaline-like regenerated tissue, the evidence gathered in the equine studies suggest that this is a promising approach that, after optimisation, may lead to significant improvements in the quality of matrix production in cartilage repair.
For main objective 2, a 3D in-vitro/ex-vivo osteochondral platform was developed, consisting of hardware, operating procedures and related assays for (osteo)chondral implants was designed and developed. It was shown that the system allows for a more realistic and native-like environment resulting in a better cartilage regeneration response in comparison to conventional in-vitro studies without having the native tissue around, which increases the predictive value of in-vitro testing. Mechanical loading was included in the system, to make the environment even more realistic and comparable to the situation in the native joint. In addition, the development of the in-silico model allowed for more focussed design of experimental studies, obviating the need for trial and error studies. The results of the modelling were used to select optimal parameters. In particular, this predicted the growth factor concentrations and co-culture settings required to induce MSC chondrogenesis in vitro. Further in-silico modelling predicts the bulk mechanical properties of fibre-reinforced scaffolds, so that these can be designed to withstand the demanding loading environment in the joint. This unique combination of predictive in-silico and in-vitro models is a very valuable tool for evaluation the regenerative capacity of implant materials prior to in vivo testing, reducing the amount of animal experiments and related costs, time and effort. Importantly, this has led to the development of a commercially available product.
Project Context and Objectives:
Degeneration of cartilage is a major cause of chronic pain, lost mobility and reduced quality of life for millions of European citizens. From a clinical point of view treatment to achieve cartilage regeneration (hyaline) and not only repair (fibrous) remains a great challenge. No clinical therapy is available that leads to healing of cartilage defects.
Current cartilage implants cannot establish the hierarchical tissue organisation that appears critical for normal cartilage function. HydroZONES hypothesised that a biomimetic zonal organisation is critical for implants to achieve cartilage regeneration. It thus represented an interdisciplinary consortium that adopted a strategy to regenerate, rather than repair, articular cartilage based on the tissues zonal structure and function.
HydroZONES used advanced bioprinting technology for fabrication of 3D biofunctional hydrogel constructs, eventually mechanically reinforced by degradable polymer scaffolds, as biomimetic reconstitution of the zonal organisation of natural cartilage. Constructs have been evaluated for cell-free application and also for combination with chrondrogenic cells (chondrocytes and/or MSC). Stringent in vitro and long term in vivo testing of the constructs has been employed.
Cutting edge 3D tissue models and bioreactor technology were used together with in silico modelling to develop an in vitro assay and test system.
Installation of a quality and regulatory affair management system, preparation of GMP production, accredited in vitro testing and involvement of clinical partners and companies with experience in clinical trials was combined with the aim to ensure that the best performing construct is brought into an optimal position for entering clinical trials after project end.
HydroZONES aimed to advance the European Union as world leader in the field of joint cartilage regeneration. Thus, generating economic impact for different markets along the product developmental line and advance European biomaterials industry on several levels. It aimed to generate the basis for a dramatic added value to OA patients and increase the quality of life for millions of EU citizens.
HydroZONES overreaching aim was to balance the combination of advancing European biomaterials industry with scientific excellence and verify or falsify a radical new strategy for implant design, the recapitulation of the hierarchical tissue organisation for improved performance, at the example of cartilage.
In order to achieve this overall mission, the HydroZONES project pursued the following objectives:
3. To develop a hierarchically structured hydrogel based mechanically stable scaffold that recapitulates the zonal distribution of the native articular cartilage.
4. To develop and validate a predictive in vitro test system for future evaluation of products for cartilage regeneration in order to reduce the amount of necessary animal experiments in the future.
These objectives were pursued by establishing a step wise material development process, divided up into three levels of materials, including:
1. Non-hierarchical but mechanically reinforced biofunctional hydrogels
HydroZONES reinforced biofunctional (cell laden) hydrogels by polymer scaffolds and compared scaffolds made of the proprietary polymer of the partner #12 CCT to the clinically used thermoplast poly(caprolactone) (PCL) to obtain best mechanical reinforcement. HydroZONES thus facilitated advancement of the therapeutic approach developing hydrogel carriers that may be used with or without scaffold to ensure cell retention and early mechanical support, and, ultimately, improve stable cartilage regeneration for the patient.
2. Pre-defined “favourite” layered materials with zonal arrangement
The three hydrogel systems were assessed to identify the optimal material combination for one cell-free and one cell-containing construct, with least complexity possible but as much bioactivation as needed according to in vitro and in vivo evaluation. This yielded selected scaffolds which, together with fibrin as clinically used hydrogel-control, have been long-term tested in vivo in mini pigs and horses (WP6).
3. Advanced layered and mechanically reinforced constructs
A third aim of the project was the development of multiphase printing process of clinically used thermoplastic polymers with biofunctional hydrogels (“double-printing”) for generation of zonally arranged mechanically reinforced hydrogels. This enabled the development of advanced layered constructs and the optimisation of the hydrogel-soaked thermoplast scaffolds with more biomimetic non-layered hydrogel materials that outperform the current standard. Mechanical strength and stability were designed to be maintained by the thermoplast-component 12 to 18 months, while hydrogels were designed to degrade more rapidly (3-6 months) to allow cell and tissue ingrowth.
Degradation of the scaffolds in vivo was checked histologically. The constructs underwent a well-defined and stringent in vitro (WP3), in vivo biocompatibility and biofunctionality (WP5) and long-term in vivo testing in the joint at the place where the implant is supposed to be used in humans (“orthotopic”; WP6). Advanced scaffold integration methods were assessed in pilot studies.
AIM AND CONCEPT OF HYDROZONES
HydroZONES has formed a consortium that supported the aims set in an innovative and excellent means. The consortium was formed in view of the objectives described above to enable the development of implants with anatomical reproduction of articular cartilage based on biocompatible and biofunctional hydrogels that mimic cartilage ECM (cellular level) and the natural tissue organisation and zonal structure of natural cartilage (tissue-level).
HydroZONES comprised three leading internationally recognised European laboratories in biomaterials development and hydrogel technology (Partners #06 UU, #07 IPF and #01 UKW) that provided established but innovative hydrogel technology comprising usability in printing devices, specific functionalisation with peptides, GAGs and growth factors and finally the option to load the hydrogels with cells during printing due to cytocompatible cross-linking reactions. Scaffolding was intended to be addressed in HydroZONES by the application and development of 3D bioprinting technologies by Partner #08 PIL and of commercial bio-printers.
HydroZONES used chondrocytes and BMSCs for initial in vitro assessment of the scaffolds. Detailed studies were performed with human cells as well but also with the respective porcine (minipig) and equine (horse) cells for correlation with the in vivo experiments. HydroZONES installed a thorough testing and selection procedure through in vitro testing (Partners #05 UMCU & #01 UKW (conform to ISO 10993-5)), biocompatibility and biofunctionality screening in nude mice (Partners #02 UNAV & #09 QUT) and evaluation in long-term pre-clinical minipig and horse models, the best current large animal model for cartilage (Partners #03 HD & #06 UU). Establishment of a consortium wide quality and regulatory affairs management system according to EN ISO 13485:2007 (QM/RAM; Partner #11 HCS) allowed to set a new internationally acceptable standard for pre-clinical testing of (osteo-) chondral implants. Development of the QM/RAM system ensured that developments within HydroZONES complied with all relevant preclinical regulatory affairs. Several partners - clinicians (partners #01 UKW, #02 UNAV & #05 UMCU) and SME (partner #12 CCT) had experience with clinical trials, which ensured that the developments within HydroZONES stay focused on the defined project endpoint and were performed according to relevant regulatory affairs (partner #11 HCS).
Moreover, cutting edge 3D tissue models, bioreactor technology and automatisation know-how (Partners #01 UKW, #08 PIL, #15 LifeTec & #13 PROSPA) were integrated, as well as state of the art in silico modelling capacity (Partner #04 UOXF). These competencies were combined to achieve the second major objective of developing a 3D in vitro assay for chondral implants, together with the prototype hardware to perform the assay, which has then been developed into a commercial product.
At project start, HydroZONES encompassed within the consortium five SME partners, out of which three exploitation partners ideally distributed along the developmental chain of HydroZONES to prevent colliding exploitation interests:
• Partner #13 PROSPA (printing technology) exploiting the advanced bioprinter
• Partner #11 HCS (QM support and consultancy) exploiting the QM/RAM system
• Partner #12 CCT (clinical product) exploited the HydroZONES constructs for clinical application – but has withdrawn after 4 years due to financial difficulties.
• Partner #15 LifeTec (bioreactor technology) exploiting the test-system
• Partner #16 POVA (GMP production) exploiting the GMP produced biomaterial
For scale-up and production of the devices, the consortium incorporated partners with experience regarding software (Partner #13 PROSPA) and manufacture (Partner #15 LifeTec). It also included partners with experience in material production and scale up under cGMP (Partner #16 POVA) and manufacture and production of scaffolds for clinical use (Partner #12 CCT). Partner #12 CCT furthermore served as the main exploitation partner with focus on human clinical use. Until their termination Partner #12 CCT was providing an exploitation manager (EM) to the project to ensure that the technical know-how and intellectual property developed in the project is evaluated and properly protected. CCT had the scientific, technical, quality control and regulatory experience of bringing HydroZONES technology to the market. After CCTs termination this function was taken over by Partner #06 UU (Prof. René van Weeren).
The work programme was divided into 9 work packages. WP1 concernced materials and scaffolds in close interaction with WP2 that dealt with automatization of assembly. Cytocompatibility and in vitro assessment was performed in WP3, while in WP4, the in vitro assay and test system was developed. Biocompatibility and biofunctionality screening was performed in WP5, and long term pre-clinical in vivo testing was performed in WP6. In WP7, a management system for quality & regulatory affairs (QM/RAM) was developed, WP8 dealt with training, dissemination and exploitation, and WP9 concerned project management.
In order to optimise the accountability and flow of results, the following methodological choices have been made:
• Partners have concentrated their person-months-resources in those work packages where they deliver results. As part of these resources they also followed the results of the other work packages and contributed advice and support to other work packages as needed. Overall technical coordination was covered by WP9.
• Deliverables have been organised to optimise individual accountability.
• Deliverables have been aligned with major project milestones to provide a natural way to measure progress.
The next chapter will present work development and achievements by WP.
Project Results:
WP 1: Materials and scaffolds
WP Objective
The objective of WP1 was to develop and characterise printable and biofunctionalisable hydrogels that degrade between 3-6 months and use them to generate 3D hydrogel scaffolds and constructs. Advanced printers that allow the printing of cell-containing gels and the double printing of hydrogels and thermoplasts were available at month 24. The materials, scaffolds and constructs were transferred to WP3 for in vitro assessment, prepared for in-vivo biocompatibility and biofunctionality screening in WP5 and for long-term pre-clinical in-vivo testing in WP6. Scaffolds and constructs were also prepared for testing in WP4 and used for validation of the 3D in-vitro assay and test system. Prototypes were tested throughout the course of the project and feedback from WPs 3, 4, 5 and 6 was used to optimise and fine-tune the properties of the materials.
Summary of WP results (linked to the WP objectives)
Synthesis of the Hydrogels
Synthesis and physicochemical characterisation of three distinctively different hydrogel systems as biofunctional cell carrier and scaffold materials was established in the three biomaterial groups.
At IPF, an in-situ assembly scheme for multicomponent hydrogel materials was established on the basis of a Michael addition reaction of starPEG-peptide conjugates with maleimide-functionalised heparin. The matrix platform covers key features of ECM including adhesion ligand functions, cell-driven degradation, modulated stiffness and sustainable cytokine delivery.
UU developed thermosensitive and photo-cross-linkable hydrogels based on hyaluronic acid (HA). The thermosensitive properties of the gels warrant a material, which behaves as a liquid at 4 ºC and therefore enables flow through the needle of a printer head, and subsequently undergoes gelation around 37°C (temperature of the heated plate of the printer). A thermosensitive and highly biocompatible construct was developed by using a combination of partially methacrylated poly(N-(2-hydroxypropyl) methacrylamide-mono-di-lactate-poly (ethylene glycol) triblock co-polymer and methacrylated hyaluronic acid (HAMA).
UKW developed UV-cross linkable hydrogels based on polyglycidols (PG) and modified natural hyaluronic acid (HA). The UV-crosslinking mechanism which is used for those hydrogels is the thiol-ene-click reaction, which can be done in presence of cells. For the hydrogel formation different thiol-modified polymers (HA-SH and PG-SH) and polymers with a double bond in the side chain have been synthesised.
Establishing of the printing Process
Printing processes for the different hydrogels were established and improved for the different physical and chemical crosslinking steps. Cell survival was improved via changed formulations and printing steps.
UU and UKW used a printing process followed by a short UV-light exposure to obtain stable and highly defined hydrogel scaffolds with a zonal architecture. Cells have been incorporated and showed a good cell survival. Importantly, labelled cells printed in different layers showed that a highly defined printing process was obtained in which specific cells can be precisely deposited at required locations within the scaffold.
At IPF, a different plotting process was developed for starPEG-heparin and pure starPEG hydrogels as the gel component immediately starts to react upon mixing. A new printing head for simultaneous dispensing of both gel components was developed together with GeSiM and installed at the NanoplotterTM. After extensive optimisation of the process parameters (pH and solid contend of the hydrogel solutions, spot pattern, plotting speed, droplet volume, target temperature and humidity) homogeneous hydrogels have been printed that show similar mechanical properties as casted hydrogels of similar composition. Highly defined and layered 3D constructs have been developed and good cell survival of cells within has been shown.
Printing in combination with thermoplasts
Printing in combination with mechanical stable PCL scaffolds allowed for the creation of robust cell carriers (→ WP2)
Double printing of hydrogels and PCL has been performed by two partners of the HydroZONES consortium (UKW and UU) as these partners have developed hydrogels that have suitable properties to be deposited in a double printing process. Hybrid 3D printed constructs were prepared using a 3DDiscovery robotic dispensing system using two print heads that suspended PCL and hydrogel filaments respectively in a layer by layer method. Highly defined scaffolds have been prepared. Importantly, gels were loaded with chondrocytes and showed a good cell viability during and after the printing process.
GMP Production of a selected hydrogel material
An up-scaled manufacturing route towards allyl-modified polyglycidol and thiol-modified hyaluronic acid is available and GMP-ready. Establishment of different synthesis and purification methods for anionic polymerisation of poly(glycidols) and modification of natural polymers was established and improved.
PoVa optimised and provided a scale-up strategy for the synthesis route towards the two polymers developed by UKW ready for cGMP manufacturing. The first polymer is an allyl-functional polyglycidol (abbreviated PG-01) and the second a thiol-modified hyaluronic acid (abbreviated HA 01). First, a user requirements document was written and based on this document, in close consultation with a supplier of reactor equipment a large scale reactor set-up was designed. IQ-OQ-PQ test protocols were written, tested and qualified. Manufacturing instructions were written for the PG-01 process. Via this instruction protocol, two test batches were manufactured using the qualified reactor set-up. The products of the two test batches were analyzed via the analytical methods that were developed. The results showed that the PG-01 manufacturing process provides a product that meets all of the set specifications in a reproducible fashion. In a similar way, a synthesis and scale up strategy was developed for the complementary polymer HA-01. Although the first test batch met most quality criteria, optimisation of the final drying step in the second batch led to a batch that met all the specified criteria.
Main Results linked to the general objectives of HydroZONES
WP 1 developed and synthesised three hydrogel platforms for the regeneration of articular cartilage.
Materials were designed and optimised in such a way that they could be printed in highly defined 3D scaffolds. Required mechanical stability of the printed scaffolds was realised by using advanced hybrid printing technology using reinforcing thermoplastic fibers to match the mechanical properties of native cartilage and retain the scaffold for extended periods of time needed for regenerating the damaged tissue. Hydrogel filaments were double printed together with the thermoplastic fibers resulting in custom designed cell containing scaffolds.
Cell compatibility was evaluated for the different hydrogels and showed that cell viability was retained during hydrogel cross-linking reactions for MSC’s and chondrocytes. Hydrogel compositions were optimised regarding cell differentiation by investigating the effect of hyaluronic acid content and heparin content. Heparin is well-known to entrap growth factors and hyaluronic acid microgels were embedded as proteins (e.g. growth factors) delivery particles. The best performing hydrogel compositions were further evaluated in vivo in WP5.
Zonal organisation was obtained by layer-by-layer deposition of hydrogel materials of different composition. In this way, zonal constructs with zones of different mechanical properties, glycosaminoglycan content, degradation rate and cell type have been prepared to show the versatility of the printing process. Finally, optimised 3D printed scaffolds were transferred to WP6 for in vivo studies in large animals.
WP2: Automatisation of assembly
WP Objective
Develop a multi-material bioprinter system for the printing of 3D zonal cartilage constructs with functionalised bioactive hydrogels (WP1) and embedded cells (WP3). In collaboration with Partner #13 PROSPA, Partner #08 PIL will tailor the hydrogel-printers for the application needs of Partners #01UKW and #05 UMCU.
Summary of WP results (linked to the WP objectives)
Two first-generation state of the art bioprinter devices, equipped with one printing head, were designed and produced by Partner #08 PIL. The system enables the automated printing of hydrogel-based constructs with or without encapsulated cells for subsequent evaluation and optimisation. Dedicated control software was developed by Partner #08 PIL and #17 PROSPA for the automated production of 3D layered structures and it was fully integrated and synchronised with the hardware of the bioprinter. After a first set of morphological screening tests, carried out at Partner #08 PIL using Scanning Electron Microscopy (SEM) with the aim of determining the accuracy and reproducibility of the bioprinter, the devices were delivered to Partners #05 UMCU and #01 UKW for further evaluation and optimisation.
The development of single-head first generation bioprinters suffered significant alterations. In consequence, more complex printer functions were pursued, in order to fit the adjusted requirements for Partners #01UKW and #05 UMCU printing devices. Adjusted printing equipment already included multi-material printing solutions (i.e. thermoplastic and hydrogel printing heads) and these specific requirements were taken into consideration in the planning and development of these.
Despite of severe delays occurred in Task 2.1. All the main requisites were addressed in the developed printers (second generation). Therefore, and regarding the printer requirements being fulfilled by the current equipment (Bioprinter I, and Bioprinter II) and the alternative solution encountered by the consortium through the acquisition of commercially available for simpler hydrogel printing processes, efforts were pursued towards multi-head/ multi-material printer’s development. In fact, it could be considered that focus has been transferred and effective work has been undertaken towards the delivery of functional multi-material devices fulfilling second-generation bioprinters criteria.
Based on the previous experience, the first generation bioprinter devices were upgraded and advanced according to the feedback of Partners #01 UKW and #05 UMCU. The second-generation devices were equipped accordingly, for example regarding speed (at least 2000mm/min) and precision of printhead movement in X and Y direction, temperature control with high accuracy between 2°C and 37°C through the development and integration of heating/cooling circulating batch. An irradiation system composed of a light source and an optical fibre were implemented at the printing heads enabling an additional cross-linking mechanism of the extruded hydrogels. The collector/baseplate system was redesigned for allow levelling. Control software was further developed by Partners #08 PIL and #13 PROSPA enabling full and user friendly control over the respective printers. After a preliminary morphological evaluation of 3D multi-material constructs, Partner #08 PIL sent a researcher to Partners #05 UMCU and #01 UKW to collaborate on the in vitro assessment of the constructs and subsequent optimisation of the bioprinting system. Importantly, these advanced bioprinters were designed considering the possibility to fit and work independently inside a laminar hood (guarantying sterile environment during optimisation stage).
Main Results linked to the general objectives of HydroZONES
The automated design and production of 3D constructs capable of mimicking the functional hierarchy of native tissues represents a paramount challenge for Tissue Engineering (TE). HydroZONES aimed to develop a multi-material bioprinter system suitable to 3D print zonal cartilage constructs (WP2). That was obtained by the assembly of extrusion-head and multi-head hydrogel dispensing module holding up to three different hydrogels (WP1) with embedded
cells (WP3).
Two multi-material Printing systems were effectively developed and delivered to Partners #01 UKW and #05 UMCU for assessment of the produced constructs and subsequent optimisation of the bioprinting system. Following preliminary testing, improvements were deemed necessary and were mostly added to the equipment before shipment to the consortium partners (Bioprinter II - #05 UMCU and Bioprinter II - #01 UKW).
Therefore, two variations of the Bioprinter systems were developed and improved to allow the extrusion of thermoplastic materials (e.g. PCL) and the printing of multiple hydrogels towards the generation of layered mechanically stable implants through the hybrid printing of thermoplastic (for structural support) and hydrogel loaded with cells and/or other molecules (for functionalisation).
The development and implementation of a software (algorithm and interface), HydroZONES G-code Generator Software, was done in close cooperation with Partner #16 PROSPA. The software allows for the definition of specific production parameters. These include: profile geometry (rectangular, elliptical, or custom (hand drawn) forms), layer composition (PCL and/or Hydrogel), thickness and pore size, independent definitions for each biomaterial head (thermoplastic and hydrogel(s)). It also considers adjustment for the nozzles distance to the baseplate and collision avoidance functions (of the printing heads with the building structure). Accordingly, the SOP manual for the use of second-generation bioprinters was produced, containing all the specifications and procedures (both hardware and software) for its correct operation.
Following the tailoring of the systems, after its completing, some more issues were addressed by Partners #01 UKW and #05 UMCU. For instance, in month #40 meeting was discussed the suitability of having thinner (0.25 mm) needles for the thermoplastic extrusion, as well as testing other manometers that would increase the precision in pressure adjustment. Although, the former request did not provide a reliable filaments extrusion, higher precision manometers were used inducing higher control of the applied pressure.
WP3: In vitro assessment
WP Objective
The overall objective of WP3 was to evaluate the chondrogenic potential of the developed materials that have been developed in WP1 and, as such, fulfil the criteria in terms of mechanical properties and gelation kinetics for 3D printing. In WP3 materials were selected for further in vivo evaluation in WP5 and WP6. Accredited testing according to DIN EN ISO/IEC 17025 and DIN ISO 10993-5 were performed according to the QM/RAM system and product development plan.
Summary of WP results (linked to the WP objectives)
WP3 established within the consortium SOPs for harvesting and culturing human, equine and porcine chondrocytes and MSCs, and optimised and unified histological procedures and analysing techniques (characterisation of cell population, analysis of specific markers such as CD105, CD90, CD73) for homogeneous processing of samples across the different laboratories within the consortium. This allowed different groups to culture and evaluate the chondrogenic capacity of the different hydrogels developed by WP1 partners and compare results across laboratories.
Each biomaterial was cultured with different cell types and concentration of polymers to establish optimal conditions for cell viability, material degradation and chondrogenic potential with regards to GAG and collagen production.
Different alternative biomaterials were also investigated as potential candidates, such as a collagen II based hydrogel, however due to solubility issues, this was abandoned to concentrate efforts on more promising materials.
P(AGE/G)-HA-SH
Firstly, the different components of the PAGE hydrogel were assessed for viability and cultured with hMSCs for evaluation of chondrogenic differentiation, followed by the evaluation of the P(AGE/G)-HA-SH system with equine and human MSCs. The materials composition was thus adjusted for optimal degradation. Furthermore, the influence of TGF-b on hMSCs behaviour was assessed, followed by the effect of zonal configuration and 3D printing on chondrogenic differentiation of both human and equine MSCs. Briefly, constructs with complex organisation based on PAGE hydrogels biofunctionalised with hyaluronic acid sulphated derivatives (HA-SH) were generated and chondrogenic differentiation of MSC was evaluated. Suitability of standard HA/PG hydrogels and cast HA/PG hydrogels supported by printed PCL scaffolds for chondrogenesis of MSC was demonstrated and, additionally, redifferentiation of chondrocytes. Consequently, these constructs were advanced to the large animals studies. Furthermore, 3D printed constructs utilizing an HA/PG formulation adapted to the bioprinting process were produced. The general printability of MSC within these systems could be successfully demonstrated.
pHPMA
The pHPMA optimal formulation for chondrogenic differentiation with respect to components concentration and cell concentration was assessed with equine chondrocytes and MSCs. This showed that the M10P10HA20 combination with equine chondrocytes displayed the most promising results and was advanced to further studies, after multiple optimisation steps in preparation for the next cultures (UV cross-linking methods, molecular weight change to increase hydrogel stability in culture). This was followed by the testing of different compositions for optimal 3D printing and assessment of the influence of the process which showed severe inhibition of chondrogenic potential on equine chondrocytes and MSCs.
The pHPMA-HAMA system was then shown to be a suitable platform for cartilage matrix deposition by encapsulated chondrocytes. HAMA affected chondrogenesis in a dose-dependent fashion, and an optimal HAMA concentration was identified between 0.25 and 0.5% v/v. Concentrations higher than 0.5% resulted in no improvement of chondrogenic differentiation. Even though addition of HAMA improved the printability of the gel and even if the hydrogels could be printed (both with and without HAMA) supporting cell viability, long-term differentiation studies indicated that the printing process with these hydrogels is not suitable for cartilage formation, possibly due to the high viscosity and consequently from the high shear stresses experience by the cells during extrusion.
GelMA
Parallel to this, a back-up alternative material based on gelatin methacryloyl (gelMA) was also evaluated for stability, chondrogenic capacity with and without 3D printing with equine chondrocytes and MSCs. Printability and chondrogenesis were optimised by adding rheology modifiers such as gellan gum and bioactive molecules like hyaluronic acid.
StarPEG/heparin
Initially, hMSCs were embedded to assess chondrogenic differentiation in PEGh hydrogels, with different degrees of incorporated enzymatically cleavable peptidic linkers (0%, 50%, 100% peptidic crosslinkers) and cultured under chondrogenic conditions. These initial materials did not provide a stable scaffold to build up a cartilaginous matrix from porcine articular chondrocytes in vitro, therefore it was decided to proceed with hydrogels in which only 50% of crosslinks are MMP degradable. Therefore, thiolated hyaluronic acid and acrylated PEG based hydrogels were tested for viability with chondrocytes and BMSCs, showing chondrogenic capacity only for chondrocytes.
StarPEG/heparin different formulations were tested for chondrogenic differentiation, with and without the functionalisation of RGD and other specific cartilage peptides. In particular, StarPEG/heparin degradability was established, along with the effects of adhesion peptides on matrix production and distribution with different cell types. Furthermore, cell behaviour in printed constructs was assessed, showing that while porcine chondrocytes and human MSCs were not affected by this procedure, pMSCs lost functionality.
Based on these evaluations, optimal formulations, compositions and cell combination for each material were established for in vivo studies, leading to the decision to eliminate pHPMA from long term large animal studies based on the poor chondrogenic capacity when printed.
Main Results linked to the general objectives of HydroZONES
As a first approximation of the natural anatomic situation, the effects of a zonal configuration of the cultures was evaluated extensively in vitro. Biocompatibility of all three developed materials was found to be satisfactory in vitro and biofunctionality of one of the gels (pHPMA-gel in printed, but not in cast form) turned out to be insufficient, for which reason this gel was not elected for further in vivo testing.
WP4: 3D in vitro assay and test system
WP Objective
The objective of WP4 is to establish and modify a 3D in vitro model and bioreactor system to evaluate chondral regeneration, as well as cell-material interaction and material induced cell migration and differentiation under physiological conditions in a bioreactor system. Thereafter, based on this bioreactor system, a validated standardised ex vivo throughput platform for commercial contract R&D services has been developed. In addition, a suite of new mathematical models have been developed and validated, together with robust and efficient computational tools that allow simulation of chondral regeneration in vitro and in vivo. The in silico and in vitro models developed were standardised and validated with the in vivo results of WP5 and WP6. Finally, the theoretical models were adopted to identify those aspects of material and construct design that have the greatest influence on in vivo integration and long-term functionality.
Summary of WP results (linked to the WP objectives)
The main result related to the first objective was the development of the 3D in vitro model for evaluation of (osteo)chondral regeneration, cell-material interactions and material induced cell migration and differentiation (LTG). The osteochondral culture platform was developed in stages, at first a static platform technology was realised, without incorporation of mechanical loading. In parallel, in the first phase of the project, a set of tools and standard operation procedures were developed to isolate osteochondral explants from different species in a standardised and feasible manner, such that experiments and methods between the different labs could be easily aligned and compared. In addition, a standard operation procedure was developed for the culture of osteochondral explants in the osteochondral culture platform. The osteochondral culture platform consists of a customised insert in which osteochondral explants can be cultured such that cartilage and bone compartments are separated externally, allowing for specific culture conditions for the specific tissues. WP4 partners have proved that this design is critical for maintaining osteochondral explant metabolic activity, cartilage ECM composition and mechanical properties in ex vivo culture up to 12 weeks (Schwab et al, ALTEX, 2017). To the best of its knowledge, this is much longer than any other study in literature has ever reported. Subsequently, all partners in WP4 have obtained the culture platform and have performed several studies with it, in which different chondral implants developed within the consortium have been evaluated with respect to cartilage regeneration. In these studies, it has been shown that cartilage matrix production is dependent on hydrogel material, supplementation of TGF-β1 in cartilage media and encapsulated cell type. At the same time, the culture system was further developed by designing and developing a mechanical loading modality that was then incorporated in the existing static system. The mechanical loading modality allows for controlled loading of the cultured osteochondral explants with adjustable (physiological) loading parameters, such as duration, frequency, displacement and force in a sterile manner. In addition, the system allows for online measurement of the mechanical properties of the tissue, without needing to sacrifice the samples, being an online monitoring tool for the quality and effectiveness of the applied treatment.
In silico modelling was performed alongside the experimental work. The partners developed an in-silico model of the regeneration of a hydrogel inserted into an ex vivo osteochondral explant allowing the investigation of different cell seeding strategies as well as the role of growth factors (Kimpton et al, Math. Biosciences, 2017). This predicted that cell infiltration alone will not generate the layered architecture seen in healthy cartilage, and that this could only be achieved by zonal seeding of chondrocytes within the construct. This is broadly in line with experimental work performed on cell infiltration and seeding strategies at UMCU. WP4 also developed an in-silico model for the interaction between TGF-β, MSCs and chondrocytes (Chen et al., J Theor Bio, submitted) . This enables to predict a critical concentration of TGF-β to be added to a culture medium to induce chondrogenesis of a population of stem cells: this prediction was in line with the 10 ng/mL used in the standard operating procedures of the HydroZONES core experiments. This model also predicts that a population of MSCs will differentiate when co-cultured with chondrocytes; the predicted initial chondrocyte density required to achieve this is approximately 20%, in line with experiments performed at Partner #01 UKW. Finally, WP4 partners developed a phenomenological in-silico model of fibre-reinforced hydrogel construct which describes the synergistic increase in stiffness obtained by combining these materials (Visser et al, Nature Comms, 2015). In order to resolve discrepancy between this simple model and experimental observations we developed a continuum in-silico model of fibre-reinforced hydrogel construct via the technique of mathematical homogenisation, which takes into account the small-scale features of the fibres on the bulk mechanical properties of the construct (Chen et al., Eur J Appl Math, submitted). This was validated using experimental data from Partner #05 UMCU.
Main Results linked to the general objectives of HydroZONES
The main results mentioned in the section above are directly linked to the second general objective of the HydroZONES project. A 3D in-vitro/ex-vivo osteochondral platform was developed, consisting of hardware, operating procedures and related assays for (osteo)chondral implants was designed and developed in the first phase of the project. It was shown that the system allows for a more realistic and native-like environment resulting in a better cartilage regeneration response in comparison to conventional in-vitro studies without having the native tissue around, which makes the translation to in-vivo results better and increases the predictive value of in-vitro testing. Mechanical loading was included in the system, to make the environment even more realistic and comparable to the situation in the native joint which further increased the predictive value of the system. In order to validate the system, chondral implants developed within HydroZONES were evaluated both in the ex-vivo osteochondral platform and in in-vivo animal studies and results were compared. In addition, the development of the in-silico model allowed for more focussed design of experimental studies, obviating the need for trial and error studies. The results of the modelling were used to select optimal parameters. In particular, this predicted the growth factor concentrations and co-culture settings required to induce MSC chondrogenesis in vitro. Further in-silico modelling predicts the bulk mechanical properties of fibre-reinforced scaffolds, so that these can be designed to withstand the demanding loading environment in the joint. This unique combination of predictive in-silico and in-vitro models is a very valuable tool for evaluation the regenerative capacity of implant materials prior to in vivo testing, reducing the amount of animal experiments and related costs, time and effort.
WP5: Biocompatibility and biofunctionality Screening
WP Objective
The overall objective of WP5 was to develop an in-vivo screening system to determine the biocompatibility and biofunctionality of constructs previously selected in vitro by WP3. By ectopic implantation into immunocompetent mice we want to determine the immunological response elicited by new materials. In addition, by developing the ectopic system into immunodeficient mice we evaluated the chondrogenic properties of cell-free materials as well as materials loaded with chondrocytes and BMSCs of different origin (human, horse, pig). Finally, in order to determine the chondrogenic properties of optimised zonal cell-laden constructs an ectopic osteochondral plug implantation model was developed.
Summary of WP results (linked to the WP objectives)
During the time of the project, a system for immunotolerance analysis was established in mice. All materials demonstrate good immunotolerance and low fibrosis invasion and no acute inflammatory response were detected, allowing us a secure translation of materials to large animal models. First generation (pre-casted) and second-generation (zonal pre-casted) hydrogels did not yield significant extracellular matrix production into immunodeficient animals. On the other hand, by establishing a more chondrogenic environment through our osteochondral plug implantation model we demonstrated a reparative response mediated by the PCL reinforcement together with an essential role of the subchondral bone in the cartilage repair process. Furthermore, using this model, our results demonstrated a superior repair capacity of reinforced zonal constructs mimicking the native structure of the articular cartilage.
Main Results linked to the general objectives of HydroZONES
WP5 results using materials mimicking the zonal structure of native cartilage, with chondrocytes on the top layer and BMSCs on the bottom layer showed greater integration with native surrounding tissue as well as enhanced reparative response and superior ECM deposition by gel-embedded cells.
WP6: Long-term pre-clinical in vivo evaluation
WP Objective
Long-term testing of the three favourite materials developed in WP1 by the devices provided from WP2 and evaluated in-vitro (WP3) and in-vivo (WP5) to set new standards of pre-clinical testing in minipigs (Task 6.3) and in the biomechanically challenging equine model (Task 6.4). Therefore, development of enhanced methods for scaffold fixation and durable integration by validation of a tissue glue and durable cell-support via an anabolic stimulus (GDF-5) in minipigs (Task 6.1) and horses (Task 6.2). Validation whether PDGF enhances chemoattraction of host cells to cell-free scaffolds (Task 6.5) and selection of the best layered cell-containing HydroZONES construct among 2 material options (Task 6.6). Determination of in-vivo performance of four improved, second generation layered HydroZONES constructs. (Task 6.7)
Summary of WP results (linked to the WP objectives)
Generally summarising the work done in WP 6 it was discovered that there was no cell-invasion in all 3 materials in an ectopic mouse model. Cell-invasion cannot be rescued by 150 ng PDGF per construct of PAGE or starPEG: No large animal experiments with PDGF.
There is high potential of GDF-5 augmented fibrin glue to stimulate calcified cartilage formation in a new ectopic mouse model. No enhanced fixation of CCT-enforced hydrogel scaffolds by GDF-5 augmented fibrin glue in minipig defects.
Partner #12 CCT-enforced hydrogel has to be abended since neither gluing nor suturing fixed CCT-enforced hydrogel scaffolds in minipig cartilage defects and development of a new PCL enforcement in WP1 was necessary.
There was a need for adaption of degradation of first generation hydrogel materials in minipig joints. Lack of cell-invasion of all 3 materials in minipig cartilage defects led to abandonment of cell-free approaches with these hydrogels.
Due to premature loss of constructs from cartilage defects in minipigs and horses, cancellation of non-enforced hydrogel constructs was performed.
It was also discovered that localisation and defect preparation have major impact on integration of reinforced hydrogel scaffolds: Use of a drill required for press-fit fixation of enforced constructs without fibrin glue into trochlear defects of minipigs.
After establishment of the best integration strategy, performance of the two most promising cell-seeded hydrogel materials starPEG and PAGE/HASH was compared in trochlear cartilage defects in minipigs for 12 weeks. The established osteochondral defect model in minipigs was successful in retaining PCL-enforced starPEG and PAGE/HASH constructs in the medial trochlear groove. No adverse effects were observed during the trial and at explantation. Defect treatment with PCL-enforced constructs tended to induce more bone loss (µCT). The starPEG group was even significantly worse than empty controls after 12 weeks. No significant differences in regeneration outcome between starPEG, PAGE/HASH and empty defects (histological scoring) after 12 weeks demonstrating no significant benefit of defect treatment with the chosen design.
A zonal versus non-zonal design of starPEG hydrogel constructs with polycaprolactone (PCL) enforcement was compared in minipigs over 6 months:
• Zonal and non-zonal PCL-enforced starPEG constructs were retained in all osteochondral defects and the printed PCL architecture remained intact. Unfortunately, loading pressed 16/18 constructs beneath the cartilage level.
• Defect treatment with enforced constructs induced significant bone loss according to µCT analysis compared to empty controls.
• In most cases hydrogel material disintegrated, was flushed out of the enforcement into the underlying bone, with persisting fragments undergoing degradation in the subchondral bone.
• Safranin-O-positive regeneration tissue at cartilage level often seemed host-derived. In 9/18 defects Safranin-O-positive regeneration tissue was connected to transplanted hydrogel/PCL. In 5 cases implanted cells persisted in hydrogel fragments and contributed to the Safranin-O-positive, but mostly collagen-type-II-negative regeneration tissue. No benefit of the non-zonal architecture with chondrocytes versus the zonal architecture with MSC and chondrocytes was evident.
• The quality of cartilage repair judged by modified O´Driscoll score was very heterogeneous in both treatment groups and results were overall significantly worse than in untreated controls with no indication of a benefit of the zonal design.
• Overall, the strategy is yet insufficiently mature for in vivo application in cartilage defects since non-treated cartilage defects performed better than treated defects.
As for the horse trial testing of materials (in cast, reinforced and printed form) was developed in the consortium ectopically in the horse model (safety testing). As well as testing and development of fixation methods for the biomaterials: assessment of commercial versus autologous fibrin glue and the development of the 3D-printed osteochondral PCL anchor.
Short-term orthotopic testing of all materials in the horse was carried out and optimisation of the production zonal constructs for in vivo orthotopic implantation in the equine model (2 materials plus back-up material) was performed.
Finally, long-term (6 months) assessment of the two finally selected materials (starPEG and PAGE/HASH) in zonal and non-zonal form in the equine model was carried out. Comparing zonal versus non-zonal design of starPEG and PAGE/HASH hydrogel constructs with polycaprolactone (PCL) enforcement in the equine model over 6 months with the following conclusions:
• Zonal and non-zonal PCL-enforced starPEG and PAGE/HASH constructs were retained in all osteochondral defects and the printed PCL architecture remained intact.
• Reinforced constructs showed good lateral integration with surrounding cartilage and bone, with new bone formation in the osteal anchor. It was consistently observed that the centre of the osteal anchor was not calcified, suggesting a longer time may be needed to achieve full calcification in the anchor.
• In most cases hydrogel material persisted in fragments and within the reinforcements, independently of whether the design was zonal or not. StarPEG showed overall a higher persistence than PAGE/HASH based constructs.
• The quality of cartilage repair judged by a modified O´Driscoll score was very heterogeneous in both treatment groups, however semi-quantitative scoring suggested a higher performance of the zonal group in the starPEG based constructs.
• Despite absence of full chondral matrix formation, stiffness was higher in the zonal group in both hydrogels.
• Within the hydrogel fragments cells were visible, along with isolated areas positive to collagen II staining, suggesting that at the endpoint of the experiment the chondral matrix formation was still going on within the (surviving) hydrogel.
• Overall, the findings suggest that the approach as taken in our studies is promising, warranting further investigations. Implantation of osteochondral constructs of which the cartilage layer is hydrogel-based, has a zonal organisation and is reinforced with a stiff material seems a promising avenue that, after optimisation, may lead to significant improvements in the quality of matrix production in cartilage repair.
Main Results linked to the general objectives of HydroZONES
Based on µCT and histological scoring of tissue regeneration, treatment with PCL-enforced starPEG or PCL-enforced PAGE/HASH constructs did not improve defect healing at 3 months in minipigs. Zonal design showed no benefit versus non-zonal design at 6 months and both PCL-enforced starPEG groups had a negative impact on defect healing compared to untreated controls based on quantitative µCT analysis and semiquantitative histological scoring of a 6-month minipig study. Overall, the strategy is yet insufficiently mature for in vivo application in cartilage defects.
The use of composite osteochondral scaffolds with a zonal design for cartilage repair achieved partial tissue repair. Despite the absence of a full chondral matrix within the defects, the biomechanical properties were substantially better (showing higher stiffness) in the zonal constructs with both biomaterials, suggesting that the combination of MSCs and chondroprogenitor cells in a layered hydrogel construct is beneficial for the biomechanical properties of repair tissue. Good lateral integration with adjacent cartilage was achieved with both hydrogels, as was good integration of the scaffolds with the surrounding bone tissue.
The strategy is currently not mature enough to achieve production of hyaline-like regenerated tissue, however the evidence gathered in the equine studies suggest that this is a promising approach that, after optimisation, may lead to significant improvements in the quality of matrix production in cartilage repair.
WP7: Management system for quality & regulatory affairs (QM/RAM)
WP objective
Medical devices and tissue engineering products have to fulfil various legal regulations and standards already during their design phase if those are developed to be marketed inside of the EU. Processes have to be defined and performed conformingly, for the design phase as well as for the later manufacturing, and the conformity of those has to be made evident by standardised means of documentation. The objective of WP7 was to provide in-processly the means and structures which allow best management of resources and processes and which guarantees that at the end of the project the fulfilment of the legal regulations and of the relevant standards can be proven. At the closure of the project’s funding there is a Master Design History File and a Master Design Dossier for functional materials/TE devices was presented and issued and serves as a basis not only for the developments in HydroZONES but for future developer’s, manufacturer’s or distributor’s Design Dossiers according to Annex II.4 of the Medical Device Directive 93/42/EWG + 2007/47/EG of the EU for later CE-marking and for attaining the 510k premarket approval of the US Food and Drug Administration FDA. Connecting ports for modules for the efficient proof of the fulfilment of specific requirements of additional regulatory areas like Australia, Japan, Canada shall be implemented.
The developed generic quality management system shall thereby guarantee
• a general standardisation of documentation
• transparency and comparability of parameters / results / decisions / costs
• a common understanding of the objectives and the actual status of the project
Summary of WP results (linked to the WP objectives)
WP7 has contributed to an increase of the quality and safety of the HydroZONES processes and developments. Safety and quality in the realisation of TE/ATMP products is generated by management during the following phases:
1. research
2. development
3. verification
4. validation / clinical evaluation
5. translation to manufacturing / scale-up
6. manufacturing
7. in-process testing
8. final testing
9. post marketing follow-up
HydroZONES was covering the phases 1 to 3. Partner #11 HCS has provided knowledge and consultancy for the conforming planning and performance of the critical activities of these phases. As defined in the objectives, Partners #11 HCS has paid attention that the QM and the regulatory requirements have been known and understood in time by the related consortium partners to assure, that the development of HydroZONES Tissue Engineering products was done and documented in anticipation and in respect of the QM and regulatory requirements.
WP7 has contributed to design conforming proceedings and conforming documentations of the HydroZONES processes and results which may be of high value for industrial partners (as spin-off companies) for the following issues:
• compelling risk management of designs and processes
• efficient scale-up and translation process to manufacturing
• comprehensive validation, testing and documentation of the final products
• probably successful evaluation and approval process by the specific European authorities and institutions in a survivable period of time.
Main Results linked to the general objectives of HydroZONES
WP 7 provided support of the Consortium partners in planning and in implementation of adequate quality management and Regulatory Affairs Management Systems. Thus enabling innovative R&D work with a clear aim of industrial application and clinical trials, setting a new standard for tissue engineering research and biofabrication.
Potential Impact:
Damage of articular cartilage or intervertebral disc cartilage occurs frequently and is followed by a process of OA, ultimately leading to pain and joint malfunction. OA causes severe loss in quality of life in approximately 40 million European Citizens. As such, it is the leading cause of disability, a more frequent cause of activity limitation than heart disease, cancer or diabetes. It accounts for more disability among the elderly in Europe than any other disease. It is in 6.6% of cases associated with severe psychological distress and a major cause of the high work disability benefits. While the total direct annual costs of OA in the US are estimated at $89.1 billion, the indirect costs are also high, largely a result of work-related losses and home-care costs. For example, the impact of arthritic diseases on earnings increased in recent years, with $108 billions of earnings being lost in 2003 in the United States alone. In France, direct costs of OA exceeded €1.6 billion in 2002 and accounted for 13 million physician visits. That year’s figures represented a 156% increase in costs over 1993, which was for more than 90% due to an increase in the number of patients, rather than to an increase of costs per patient.
With the above-mentioned challenges, HydroZONES major results and research achievements help to reduce costs for European healthcare systems by the generation of cheaper, potentially cell-free, alternatives based on instructive biomaterials to regenerate the complex tissue organisation at the defect site. It might lead to a breakthrough in the way OA is treated today and reduce the burden for the patients and their surrounding community.
Scientific impact of WP:
Throughout the lifetime of the project the different WP have achieved significant progress in their scientific research bringing the solution several steps closer in an innovative way that supports the project’s objectives.
WP1 developed newly designed and fully characterised biofunctional hydrogel systems, which are available for 3D culture of MSCs and chondrocytes for the regeneration of articular cartilage and were subsequently optimised for the 3D printing process. Crosslinking steps are proven to be cyto-compatible and provide good cellular survival in the hydrogels. Important parameters regarding the design and synthesis of biomaterials for cartilage regeneration implants have been defined balancing the need for mechanical stability and biological performance. The establishment of the printing process created insights and experience in the 3D printing of cell-laden hydrogels with new printing techniques (extrusion based bioprinting and reactive inkjet printing). Together with the application of double printing together with thermoplasts, the generation of mechanically stable composite constructs allows the production of robust cartilage constructs. The established GMP-production of one of the hydrogel systems provides a good starting point for initial clinical studies and aims at a possible commercialisation of this developed hydrogel.
According to the established objectives for WP2, significant achievements were obtained in the development of customised bioprinters. As exposed in the deliverables, 3 bioprinting platforms with novel technology led the production of equipment in a form ready for large-scale production. Innovative hybrid hardware was accomplished according to the requirements of optimal scaffolds design.
G-code proved to be a reliable approach for the control of production settings and integrated definition of general scaffold characteristics.
In addition, the multidisciplinary requirements to achieve the novel bioprinters, enhanced the high-quality training of a set of engineers, who are now in the forefront of equipment development.
WP3 carried out Establishment and optimisation of fundamental SOPs for harvesting, culturing and analysis of chondrocytes and MSCs across different species. It performed first in vitro testing and optimisation of three new semi-synthetic biomaterials. As well as in vitro demonstration of the potential of bioprinting as a strategy to engineer articular cartilage, and advancement of 3-D printing techniques for cell-laden hydrogels for cartilage repair.
Finally, biofabrication of 3D printed, zonal-like constructs with mechanically reinforcing structures was developed.
New mathematical techniques were developed in the course of the in-silico modelling to address the core requirements of WP4. These abstract methods have potential to address similar problems in other fields of applied scientific research.
WP5 has established and validated an in vivo screening system in the framework of HydroZONES, that allow us to test the behaviour of cells and new materials to be used in regenerative medicine. In the field of articular cartilage, this would speed up the translation to the clinic of promising materials and implant designs reducing the cost of testing. Besides, the osteochondral ectopic in vivo model demonstrated that it is a feasible and versatile system capable of evaluate and predict the role of different combination of factors in the repair process.
Through its research, WP6 has carried out the first testing of zonal constructs for cartilage repair in large animal models. Performed optimisation of both the porcine and the equine model and completed the development of an effective fixation method for osteochondral scaffolds that due to its design allows the assessment of the chondral part without influence from the osteal part that only serves as the bone anchor.
WP7 enabled the funding, sponsoring and non-financial support by external partners. This can be gained more easily by start-ups, spin-offs or global players, as regulatory risks are identified and managed during the research and development phase already.
Translation of the HZ results to industry is accelerated – and thus the supply of the patients may be possible earlier - because needs of verification and validation are anticipated in the development phase already thorough the close support of WP7. Thus, number of failures of RegMed enterprises can be reduced.
By the WP7 Quality Management and Regulatory Management Package - which enforces that a conforming development process and a conforming documentation of it is built up 'in-processly' and in time - all parties which are concerned may be able to better calculate and manage resources and to better estimate times to market.
Success rate of innovations and of enterprises in the European Union in the global field of Regenerative Medicine shall be increased by this remarkably.
Leading the cutting end research in osteochondral reconstruction, representatives of the consortium took place in InoovaBone stakeholders day and workshops resulting in direct influence on the selected topics for research in the next NMBP work programme 2018 – 2020 placing the emphasis of the EU on the direct continuation the work carried out in HydroZONES.
Socio-economic impact and the wider societal implications
As HydroZONES targets a common medical challenge that is affecting millions of people around the world, directing affecting their longevity and quality of life – progress in the research in the field and promising result can have effect not only on the scientific community but also direct effect on the public lowering the personal and economic burden.
Hydrogels, which were successfully tested in long term studies, could improve the therapy of difficult to treat cartilage defects and provide new therapeutic options. The collaboration in the groups of WP1 provided an excellent scientific training and education of the participating PhD students and scientific co-workers in a highly interdisciplinary and international research environment. This led to the creation of an international network of researchers in the field of biomaterials and biofabrication.
WP2 gathered scientists, engineers and physicians in interdisciplinary teams using a variety of methods to construct biological substitutes, boosting an effective synergy between mechanical engineering and biomedical sciences; providing revolutionary steps in manufacturing at industry 4.0. 3D bioprinting market was valued at $98.6 million in 2015, and an annual growth of 36% for the next 6 years is expected . The work developed in WP2 supported the position of Europe in the front end for adequate technology development.
WP3 achieved establishment of relevance of zonal character of implants as a guideline for further research. It carried out optimisation of culture conditions and 3D printing techniques of biomaterials for cartilage repair as advancement for future research towards in vivo applications in animal models.
The osteochondral culture platform that has been developed within WP4 is now used by Partner #15 LifeTec Group to perform contract research for clients in order to evaluate their newly developed bone and cartilage repair studies. Companies that develop cartilage or bone repair strategies can significantly reduce the number of animals needed, by using the developed osteochondral platform within HydroZONES as a screening tool for testing arrays of different material compositions, implant coatings and drug dose-response studies. This way, the number of animals (ethical considerations) and associated costs can be significantly reduced with 30-60k€ per product development. Moreover, the time-to-market can be significantly reduced, with estimated 1-2 years per developed product. As the osteochondral culture platform is easy and affordable and can be produced in a fast and customised manner, this allows both industrial and academic researchers to use the system for their research purposes. Already quite a number of academic and industrial clients (>10) are making use of their system to answer their (fundamental) research questions or to evaluate their newly developed treatments and therapies.
The predictive, validated in silico models developed within WP4 allow for more focussed experimental design in future studies. The in-silico models predict the optimal choices of culture and implant parameters, as well as guide the design of the fibre-reinforced hydrogel scaffolds, significantly reducing the trial-and-error character of these kind of studies. This will significantly reduce time (6-12 months for a typical implant material study) needed for produced development and in vitro screening and prior to in vivo studies. The new mathematical techniques that were developed in the course of the in-silico modelling to address the core requirements of WP4 have wide application in other fields of scientific research.
Due to the unique composition of WP4 (mathematical and experimental researchers; academic and industrial partners), there was a lot of interaction and knowledge transfer between the different partners, even beyond this specific work package. Researchers spent some time at each other’s institutes (academic vs industry) to experience other working environment. Mathematical and experimental researchers joined forces in several meetings, allowing for a much better understanding of each other’s disciplines and needs, which had a clear synergistic effect on the outcomes of this work package. Moreover, these collaborations will exist also beyond the HydroZONES funding period, already shown by new collaboration initiatives between the different partners (#04 UOXF – #15 LifeTec Group; #05 UMCU – #15 LifeTec Group; #04 UOXF– #05 UMCU; #04 UOXF – #01 UKW).
From a Socio-economic point of view the developed model of small animal trials by WP5 reduces the number of large animals used in research, decreasing the ethic and social concerns in the use of animals in research. In consequence, implies the reduction of costs in translational medicine by an early identification of non-optimal materials.
WP6 in its research contributed to the establishment of relevance of zonal character of implants as a guideline for further research. Leading to reduction of unnecessary spillage of animals by drawing attention to the unsuitability of (allogeneic) fibrin glue for the attachment of chondral scaffolds in the equine model and possibly in the porcine model.
WP7 enabled through its support in the project to reduce regulatory risks and prepared adequate mitigation plan during the research phase. By developing regulations and QA system, translations of the project’s results to the industry is much faster due to reduced need of verification.
Main dissemination activities and exploitation of your WP
The developed hydrogel systems provide an excellent basis for further application in different fields, the gels provide the basis for further research in drug delivery, tissue engineering and 3D printing. Besides, the established printing protocols and procedures can also be applied for other hydrogel systems.
Newly developed printing systems (reactive dual pipet inkjet printing) evolved from the collaboration of a WP1 Partner #07 IPF with the company Gesim in order to allow printing of the dual component hydrogel system.
The obtained achievements on the bioprinting systems have proven to be suitable to be exploited by Partner #13 PROSPA. Furthermore, the technology advances are already being used in other research projects and were considered for research proposals (e.g. RIA H2020-FETOPEN-2017, RegMedXB Programme (NL), Gravitation Programme Materials Driven Regeneration (NL)).
The GMP prepared hydrogel system is applicable for other projects and already led to an intended participation with the established hydrogel platform in another research consortium by Partner #16 PolyVation. The experience with the newly established synthesis schemes and purification methods applied for the GMP production have already been applied for other research projects.
Partner #05 UMCU organised and hosted the HydroZONES symposium “Paving the path for biofabrication in Europe” (described D8.3). It also published in Science Impact – Joint Endeavours. (http://www.ingentaconnect.com/content/sil/impact/2017/00002017/00000008/art00026?crawler=true&mimetype=application/pdf)
Partner #05 UMCU Participated in activities organised jointly with other H2020 projects and was represented by Jos Malda: visit Researcher.
Last but not least, it promoted the education of young researchers such as the PhD defence Vivian Mouser carried out on the 15th of June 2017 in Utrecht
Partner #15 LifeTec has developed an exploitable product of Osteochondral Platform oriented for orthopaedic and pharmaceutical research, testing and screening. Testing the performance of an implant, device or therapy in a cost-efficient manner in living and functional ex-vivo cultured osteochondral biopsies. The platform is already in use for evaluation of material and cell-based cartilage regeneration strategies (TRL-9) in the context of small scale projects with academic as well as industry partners.
An QM/RAM system has been created in project by Partner #11 HCS and proposal of consultancy in the building up of similar QM/RAM systems was offered to institutions doing ATMP research.
Outlook and future research:
For a significant improvement and adjustment of the hydrogels systems developed in WP1, a more efficient control over hydrogel degradability appeared to be necessary in order to control cell ingrowth and migration as well as differentiation and balance this with the necessary scaffold integrity to ensure the implant stability and filling of the defect. Therefore, the following items are recommended for follow-up studies:
• Improvement and adjustment of hydrogel degradability (including e.g. triggered degradation by external stimulus) to enhance cell ingrowth/migration and differentiation and balance that with necessary scaffold integrity.
• Incorporation of biological cues to enhance differentiation especially in vivo.
• Improvement of printing resolution and speed to obtain personalised implants.
• Optimise printing process to obtain the same level of cell differentiation in printed cell-laden constructs as in casted constructs.
Starting from the zonal cartilage constructs developed in WP3, further refinement of hydrogels and bioinks for cartilage printing will be performed. Moreover, the next step will be the fabrication of fully bioprinted osteochondral constructs comprising also a cellularised engineered bone compartment.
Additional activities would be refinement and convergence of multiple biofabrication technologies to engineer large joint grafts, toward the bioprinting of a fully regenerated joint replacement.
The osteochondral platform developed in WP4 will be taken into a next generation by Partner #15 LifeTec Group, by developing disease models in a controlled way in order to test therapies and treatments in a relevant (pathological) environment, mimicking the disease situation in the patients. This work will be performed within the CarBon project, which has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 721432.
In addition, the osteochondral platform is already in use by different national and international research consortia to evaluate their developed (osteo)chondral implants. For example in RegMedXB (https://www.regmedxb.com/projects/taking-steps-towards-a-bioengineered-joint) BIOGEL (http://www.biogel-mariecurie.eu/; Marie Sklodowska-Curie grant agreement No 642687) and Wiliam Hunter Revisited (Dutch national funded research project; http://www.stw.nl/nl/content/p15-23-william-hunter-revisited-activating-intrinsic-cartilage-repair-restore-joint).
In addition, the osteochondral platform is used by industrial partners to test their materials and tissue engineering strategies for cartilage and bone regeneration. For example, the Dutch company OsteoPharma (http://www.osteo-pharma.com/) is using the ex vivo platform to evaluate their bone therapeutics and select most promising compositions for in vivo studies.
Future in silico work is planned to evaluate the role of mechanical loading and the remodelling of the extracellular matrix. Funding for this research will be pursued through the EPSRC and the Australian Research Council (via a new collaboration with the University of Adelaide).
The possibility to further in-depth characterisation of this advanced ectopic model developed in WP5 would allow to determine the mechanisms and pathways implicated the natural processes of articular repair. By identifying the key mediators of natural repair, it would be possible to develop novel treatments to slow down the progression of articular cartilage damage progression and OA appearance. In addition, the proposed in vivo model allows in vivo testing of different materials and implants designs.
WP6 development in long term trials will thrive in the future to use the improved models for further testing of other materials. As well as pursue further development along the lines set in the project for meeting the need for cartilage repair in the veterinary market.
Partners #11 HCS will continue the investigation of the administrative processes of applications for getting release on TE products inside of the EU to derive recommendations to the EU Commission for regulatory and institutional improvements. As well as investigation of the effectiveness and the efficiency of communication and for sharing of experiences in larger consortia for research & development (e.g. of medical devices, medicinal product and combinations of those/ATMPs).
List of Websites:
https://www.hydrozones.eu/