Design and optimization of 3D Bioplotted scaffolds with a multi-layered architecture for osteochondral tissue regeneration

Knee osteoarthritis (OA) affects about 300 million people worldwide. In its early phases, OA involves the joint cartilage only; however, as it progresses the subchondral bone is more obviously involved, creating an osteochondral (OC) disease pattern. In recent years, 3D tissue engineered substitutes have emerged as promising alternative to traditional strategies (i.e autografts transplantation, subchondral drilling, etc..). Although successful in many aspects, these 3D scaffolds lack in mimicking the complex architecture of the natural stratified OC tissue. To address this hurdle, in this study we investigated the development and optimization of a multi-layered scaffold with incorporated vertical porosity and ceramic content gradients, by using a 3D Bioplotter system. A tri-layered structure based on a composite of poly (ε-caprolactone) (PCL) and strontium-doped nanohydroxyapatite (Sr-nHA) was designed. The overall architecture was made of three phases, printed continuously to mimic the native stratified OC tissue. Specifically, the first six layers (bottom portion) were made of PCL/Sr-nHA (20% w/w) in order to reproduce the bone counterpart, whereas to mimic the top cartilaginous tissue the upper portion was based on five layers made of PCL/Sr-nHa, with a lower ceramic content (10% w/w ). The distance between the strands for both these portions was set to 0.8 mm. To simulate the harder mechanical properties of the calcified cartilage, the strands’ distance of this intermediate layer was reduced to 0.7 mm. Bare PCL scaffolds were used as control for all the experiments performed. Physico-chemical characterization was conducted in order to prove the presence of both materials in the concentrations designed as well as to examine the surfaces’ topography, porosity and wettability; while compressive tests were performed to assess the mechanical properties of the resulting tri-layer architecture. U2OS cells were used in order to assess the biological properties of the novel developed scaffolds. MTT colorimetric assay and DAPI qualitative assay showed an increased cells proliferation during the first week of seeding. Furthermore, The ALP activity showed osteoblast differentiation starting from 14 days of seeding. Alizarin Red staining evidenced calcium deposits after 7 day of seeding indicating cell-mediated scaffold’s mineralization. In conclusion, based on the physico-chemical and biological characterization results, the as produced PCL/SrHA scaffold, both in terms of material composition and architecture, demonstrated to be a promising candidate towards the development of a functional implant for OC tissue regeneration.