Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - 3D-JOINT (3D Bioprinting of JOINT Replacements)

Teaser

\"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...

Summary

\"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.

Having established a cartilage-focused research laboratory in the University Medical Center Utrecht, I have addressed this biomechanical limitation of hydrogels through the use of hydrogel composites. Specifically, I have pioneered a 3D bio-printing technology that combines accurately printed small diameter thermoplastic filaments with cell invasive hydrogels to form strong fibre-reinforced constructs. This, in combination with bioreactor technology, is the key to the generation of larger, complex tissue constructs with cartilage-like biomechanical resilience. With 3D-JOINT I will use my in-depth bio-printing and bioreactor knowledge and experience to develop a multi-phasic 3D-printed biological replacement of the joint.

The overall goal of 3D-JOINT is 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 are 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.
\"

Work performed

Initially cylindrical constructs contained integrated bone and cartilage phases to establish the converged biofabrication approaches and assess the mechanical integrity of the implants. Subsequently, construct size will be scaled-up and the more intricate shapes of the were fabricated.

Advances in the complexity of the design is achieved at both macro and microscales. On macroscale we developed an approach to assess the shapefidelity of bioinks. Further, we designed the printing process in a one-step approach (convergence), allowing for improved integration of the polycaprolactone (PCL) microfibers for the cartilage component with the porous PCL bone component that was made with fused deposition modeling (FDM). Furthermore, fabrication transitioned from cylindrical constructs towards humeral head implants (for New Zealand white rabbits), where the design was based on CT images. Moreover, on the microscale, convergence was also achieved both 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.

Final results

Cartilage compartment
The cartilage part, 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 (Schuurman et al. Macromol Biosci 2012; Visser et al. Biofabrication 2013; Levato et al. Biofabrication 2014) and for abundant neo-cartilage synthesis from encapsulated cells (Levett et al., Acta Biomater 2014, Visser et al. Biomaterials 2015). Moreover, our recent research further unraveled key materials properties of this bioink and its potential in cartilage biofabrication. The optimal bioprinting window for GelMA, unmodified or supplemented with the rheology-modifier gellan gum, was identified, highlighting the range of polymer concentration and temperatures necessary to print this material, while preserving abundant and homogenous cartilage deposition into the hydrogel matrix (Mouser et al., Biofabrication 2016).

Additionally, the key role of the yield behavior of the hydrogel as determinant of printability and capability to mix cells was elucidated. Next, GelMA-based hydrogels were further investigated to thoroughly characterize the chondrogenic response of multiple cell types with cartilage-forming ability that were encapsulated in these matrices. Chondrocytes, MSCs, and the recently identified Articular Cartilage-derived Progenitor Cells (ACPCs) were embedded into the bioink, and chondrogenic differentiation was elucidated at a gene and protein expression level. While GelMA supported satisfactory cartilage matrix production from all these cell types, different bioink-cell pairs exhibited distinct cartilage biomarkers expression, with MSCs producing the highest amount of GAGs, associated with mild presence of hypertrophic/deeper zone markers (COL X), and ACPCs assuming a profile more affine to the superficial zone of cartilage (with elevated PRG4 expression). Hence, adequate cell-geMA bioink compositions can be used to recapitulate specific features of cartilage ECM, and potentially combined to create and print zonal-like constructs (Levato et al. Acta Biomater 2017).

To understand the observed mechanical performance and reinforcement mechanism of the manufactured micro-fibre reinforced hydrogels, two FE models, implementing material properties measured experimentally, were developed. A continuum- FE model was based on an idealized scaffold geometry to capture the linear reinforcement mechanisms, while a second micro-FE model based on micro-CT images of the real constructs was developed to capture the non-linear reinforcement mechanisms. We demonstrated that the reinforcement mechanism of the composite constructs is governed by two different mechanisms. The first contribution is created by the fibres being pulled in tension by the lateral expansion of the hydrogel. This mechanism dominates the reinforcement of composite constructs with low fibre volume fraction. While the second contribution comes from the fibre cross-section interconnections. These locations dominate the reinforcing mechanism above a certain fibre volume fraction (Figure 1). The micro-FE model also gives important fundamental insights about the real structural deformation of these novel reinforced hydrogel during axial compression (Castilho et al, Scientific Reports 2018). The model is currently being used to predict the response of the 3D-JOINT constructs in other relevant mechanical situations, i.e. combined compression and shear loading, that are difficult to mimic in laboratory.

Moreover, in collaboration with Oxford University, we developed an additional theoretical framework using the homogenization approach to describe the mechanical performance of these composite constructs. Here, the fibre scaffolds were treated as a linear elastic material and the hydrogel as a poro-elastic material. The homogenized description reflected well the orthotropic nature of the composites and will enable us to complement the previous FE mode