Fabrication and mechanical characterization of 3D printed vertical uniform and gradient scaffolds for bone and osteochondral tissue engineering.

Recent developments in 3D printing (3DP) research have led to a variety of scaffold designs and techniques for osteochondral tissue engineering; however, the simultaneous incorporation of multiple types of gradients within the same construct remains a challenge. Herein, we describe the fabrication and mechanical characterization of porous poly(ε-caprolactone) (PCL) and PCL-hydroxyapatite (HA) scaffolds with incorporated vertical porosity and ceramic content gradients via a multimaterial extrusion 3DP system. Scaffolds of 0 wt% HA (PCL), 15 wt% HA (HA15), or 30 wt% HA (HA30) were fabricated with uniform composition and porosity (using 0.2 mm, 0.5 mm, or 0.9 mm on-center fiber spacing), uniform composition and gradient porosity, and gradient composition (PCL-HA15-HA30) and porosity. Micro-CT imaging and porosity analysis demonstrated the ability to incorporate both vertical porosity and pore size gradients and a ceramic gradient, which collectively recapitulate gradients found in native osteochondral tissues. Uniaxial compression testing demonstrated an inverse relationship between porosity, ϕ, and compressive modulus, E, and yield stress, σ, for uniform porosity scaffolds, however, no differences were observed as a result of ceramic incorporation. All scaffolds demonstrated compressive moduli within the appropriate range for trabecular bone, with average moduli between 86 ± 14-220 ± 26 MPa. Uniform porosity and pore size scaffolds for all ceramic levels had compressive moduli between 205 ± 37-220 ± 26 MPa, 112 ± 13-118 ± 23 MPa, and 86 ± 14-97 ± 8 MPa respectively for porosities ranging between 14 ± 4-20 ± 6%, 36 ± 3-43 ± 4%, and 54 ± 2-57 ± 2%, with the moduli and yield stresses of low porosity scaffolds being significantly greater (p < 0.05) than those of all other groups. Single (porosity) gradient and dual (composition/porosity) gradient scaffolds demonstrated compressive properties similar (p > 0.05) to those of the highest porosity uniform scaffolds (porosity gradient scaffolds 98 ± 23-107 ± 6 MPa, and 102 ± 7 MPa for dual composition/porosity gradient scaffolds), indicating that these properties are more heavily influenced by the weakest section of the gradient. The compression data for uniform scaffolds were also readily modeled, yielding scaling laws of the form E ∼ (1 - ϕ) and σ ∼ (1 - ϕ), which demonstrated that the compressive properties evaluated in this study were well-aligned with expectations from previous literature and were readily modeled with good fidelity independent of polymer scaffold geometry and ceramic content. All uniform scaffolds were similarly deformed and recovered despite different porosities, while the large-pore sections of porosity gradient scaffolds were significantly more deformed than all other groups, indicating that porosity may not be an independent factor in determining strain recovery. Moving forward, the technique described here will serve as the template for more complex multimaterial constructs with bioactive cues that better match native tissue physiology and promote tissue regeneration. STATEMENT OF SIGNIFICANCE: This manuscript describes the fabrication and mechanical characterization of "dual" porosity/ceramic content gradient scaffolds produced via a multimaterial extrusion 3D printing system for osteochondral tissue engineering. Such scaffolds are designed to better address the simultaneous gradients in architecture and mineralization found in native osteochondral tissue. The results of this study demonstrate that this technique may serve as a template for future advances in 3D printing technology that may better address the inherent complexity in such heterogeneous tissues.