Large bone-implants for osteochondral regeneration, fabrication and ex-vivo testing

Abstract

The high frequency of osteoarthritis in those aged above 60 years combined with the observing trend of overpopulation leads to increased healthcare costs. To date, clinical treatments seem to be insufficient to restore cartilage and underlying bone degeneration. Bioprinting fabrication techniques constitute a promising approach to mimic and anchor both the biological and mechanical properties of the two different tissues. Moreover, the simultaneous development and junction of an osseous and a cartilaginous compartment would be beneficial to provide mechanical fixation to the latter. Each tissue, osseous tissue and articular cartilage, presents different physical and mechanical properties. Hence, different biomaterials and bioprinting techniques are commonly used in each case. Therefore, the fusion of an osseous (HB via pneumatic-driven dispensing) and a cartilaginous (PCL1 via Melt Electro-writing) compartment was investigated in the study. Currently, pneumatic-driven dispensing systems combined with Melt Electro-writing are used to fabricate reinforced hydrogels as a cartilage equivalent. The used techniques are generating constructs using a layer by layer fashion on a flat surface. The possibility to generate the respective constructs on top of a non-smooth and concave surface, such as an osseous implant, was studied in the present work. Using two different bioprinting techniques, it was possible to fabricate clinically relevant-size bone-implants for osteochondral defects. Firstly, the osseous compartment of the implant was constructed by Fused Method Deposition (FDM) using Polycaprolactone (PCL1). Secondly, Hyperelastic “bone” (HB) was chosen based on the osteoinductive properties it shows. Inversely, the HB-implants were fabricated using a pneumatic-driven dispensing system. Apart from the superior osteoinductive properties of HB over PCL1, the bulk stiffness of the two materials was measured under uniaxial compression. Following the same protocol, HB-scaffolds with specific internal architecture were evaluated. The different groups of porous HB-scaffolds presented different design details (outer periphery loop and/or closed top layer) to evaluate the impact of each element on the final stiffness. Also, the effect of an in-vitro pre-culture period in chondrogenic differentiation medium on the stiffness of the osseous scaffolds was examined. The gross shape of the osseous compartment may contribute to the fixation of the osteochondral implant. Therefore, multiple types of osseous defects were introduced in the current work. The corresponding implants were fabricated as previously described. The size of the implants and the extent of confinement from the surrounding material were set as the parameters of the study. To evaluate the joint integrity, a Digital Light Processed ex-vivo model was fabricated. Particularly, the stifle joint of sheep was chosen as an ex-vivo model to replicate the human knee physiology and anatomy. By inserting the fabricated implants into the sites of defects and testing them under the employment of physiological loading conditions, displacement data were acquired. A Python-code that generates a G-code allowing printing in the transverse plane to that of the building plate was successfully developed. The particular Python-code creates a G-code that enables the print-head to move on top of a concave and non-smooth surface with high accuracy. The results indicate that the Young’s modulus elasticity of the osteoinductive HB is lower compared to that of the native trabecular bone. Also, neither the periphery loop nor the closed top layer of the porous HB-scaffolds seem to have a significant impact on the stiffness. The same stands for the pre-culture period in chondrogenic differentiation medium. The ex-vivo testing of the fabricated implants indicates that a width of 10 mm might be crucial for the joint integrity. Inversely, the confinement of the implant by the surrounding material seems not to affect it. Finally, the fusion of the osseous and cartilaginous compartment appears to be dependent on the material inconsistencies of the bone equivalent. However, porous structures were demonstrated on top of the osseous-surface.