ERC PROJECT 3D-JOINT (647426) JOS MALDA 2015-2020

01/09/2020

OVERALL OBJECTIVES OF THE PROJECT

The world has a significant medical challenge in repairing injured or diseased joints. Joint degeneration and its related pain is a major socio-economic burden that will increase over the next decade and is currently addressed by implanting a metal prosthesis. For the long term, the ideal solution to joint injury is to successfully regenerate rather than replace the damaged cartilage with synthetic implants. Recent advances in key technologies are now bringing this “holy grail” within reach; regenerative approaches, based on cell therapy, are already clinically available albeit only for smaller focal cartilage defects.

One of these key technologies is three-dimensional (3D) bio-printing, which provides a greatly controlled placement and organization of living constructs through the layer-by-layer deposition of materials and cells. These tissue constructs can be applied as tissue models for research and screening. However, the lack of biomechanical properties of these tissue constructs has hampered their application to the regeneration of damaged, degenerated or diseased tissue.

The overall goal of 3D-JOINT was to generate a durable, mechanically stable, biological joint replacement by simultaneous (bio-)printing of resorbable thermoplastic polymers and cell-laden hydrogels that can aid in and steer the regeneration of the congruent articulating surface.

The core aims of this project were to:

• Generate a biological joint replacement of clinically relevant size using advanced biofabrication technology, and

• Mature the generated constructs in a bioreactor system, yielding mechanically stable implants that survive in the in vivo joint environment.

• Produce proof-of-principle in a challenging large animal model.

MAIN RESULTS

Cylindrical constructs were designed and generated (first generation) that contained integrated bone and cartilage phases to establish the converged biofabrication approaches and assess the mechanical integrity of the implants. Subsequently, construct size was scaled-up (second and third generations) and implants with more intricate shapes were fabricated.

Advances in the complexity of the design were achieved at both macro- and microscales. On macroscale, we developed an approaches to assess the shape fidelity of bioinks. Further, we developed a converged printing process, allowing for improved integration of the cartilage and bone components. Moreover, this convergence allowed for control over the microfibre deposition and cell deposition simultaneously. Additionally, convergence was also achieved at the level of the interface between cartilage and bone by fusing the PCL microfibers in a calcium phosphate-based bone compartment and at the level of the fibre reinforcement of the hydrogels. This led to an improved strength between the engineered cartilage-to-bone interface.

Furthermore, we focused on further improving the osteochondral plug on a micro-scale level for orthotopic implantation in a long-term large animal model, while still improving the technology to fabricate larger fibre reinforced implants by converged printing of anatomically relevant structures. Computational models and advances in microfibre design, inspired by the orientation of collagen fibres in native tissue, significant increased both the compressive as shear properties. Focusing on the micro-scale resulted in the fabrication of osteochondral implants that showed hierarchy in fibre architecture (including a superficial zone) and cell density that were able to withstand the challenging in vivo environment of the stifle joint in the equine model. Eventually, personalized larger osteochondral constructs, that were based on CT scans and consisted of a fibre reinforced cartilage layer, were fabricated along the converge printing approach and mechanically tested in an ex vivo model.

PROGRESS BEYOND THE STATE OF THE ART

The cartilage part of the developed implants, consists of a reinforced composite hydrogel structure containing regenerative cells. Gel-MA based hydrogels were already proven to be highly performant both as bioinks for printing  and for abundant neo-cartilage synthesis from encapsulated cells. The optimal bioprinting window for GelMA, unmodified or supplemented with the rheology-modifier gellan gum, was identified, while preserving abundant and homogenous cartilage deposition into the hydrogel matrix .

Additionally, the key role of the yield behavior of the hydrogel as determinant of printability and capability to mix cells was elucidated. Chondrocytes, MSCs, and the recently identified Articular Cartilage-derived Progenitor Cells (ACPCs) were embedded into the bioink, and chondrogenic differentiation confirmed, underscoring the potential of these materials to print zonal-like constructs.

To understand the observed mechanical performance and reinforcement mechanism of the manufactured micro-fibre reinforced hydrogels, two FE models were developed. We revealed that the reinforcement mechanism of the composite constructs is governed by the fibres being pulled in tension by the lateral expansion of the hydrogel, as well as by the fibre cross-section interconnections. The model has been used to predict the response of the 3D-JOINT constructs in other relevant mechanical situations that are difficult to mimic in laboratory.

A printable bone substitute was generated from a printable calcium phosphate cement based on osteoconductive materials that is able to set at room temperature and neutral pH; compatible with labile materials and biological components. Several porous scaffold designs have been produced, with increasing range of porosity. We have demonstrated the ability of the material to support attachment, proliferation and osteogenic differentiation of MSCs in vitro. Additionally, a first in vivo study was performed to assess the osteoconductive performance of these porous scaffolds.

In the effort the generate and print full osteochondral constructs (and eventually larger joint structures), an approach to enhance integration of the biomaterials intended for the cartilage region (microfiber reinforced, cell-laden GelMA bioinks), and the underlying subchondral bone substitutes was developed. We demonstrated the necessity of providing stable anchoring into the subchondral bone for tissue engineered cartilage constructs. As such, the MEW-fabricated microfibers used to reinforce the GelMA hydrogels have been used as anchoring system between the engineered bone-cartilage compartments in osteochondral constructs.

One of the technologies developed during this project is the converged printing of microfibres and extrusion based printing. Further understanding of the underlying physical principles of the MEW process allowed for accurate fibre deposition on anatomical relevant structures and materials. This study established the groundwork for translating MEW from planar to anatomically relevant geometries. This resurfacing approach allowed us to translate from the relatively small plug to larger and clinically more relevant (patient-specific) implants.