3D-printed biochemical–biomechanical gradient scaffolds from high internal phase Pickering emulsion with dual gelatin-modulated gelling kinetics for enhanced osteochondral regeneration

Abstract

Three-dimensional (3D) printing enables the precise design of biomaterials at predefined spatial locations, representing a promising strategy for constructing highly organized osteochondral tissue with multiple morphogen gradients. However, most current 3D printing strategies limit resolution to the microscale, lacking the nanoscale resolution necessary to replicate the organization of natural bone extracellular matrix. Herein, we present a 3D printing strategy that employs high internal phase Pickering emulsions to fabricate osteochondral scaffolds with continuous biochemical–biomechanical gradients. We developed a single-step fabrication process that compartmentalizes emulsion stabilization using gelatin nanoparticles (GNPs) exclusively for cartilage layers, and GNPs/hydroxyapatite co-stabilization for subchondral bone layers. The extrudability, printing window, and shape fidelity were simply adjusted by blending two types of gelatin (fish and bovine) with different temperature responsiveness in the continuous phase. The nozzle path defined the macropores, while the emulsion droplets determined pore size at the microscale and nanoscale levels. The scaffold effectively modulated bone marrow stromal cells’ fate towards osteogenic and chondrogenic lineages by spatially differentially regulating distinct gene and protein markers, leading to synchronous regeneration of both cartilage and subchondral bone. Furthermore, the hierarchical gradient scaffold significantly accelerated osteochondral regeneration compared to single cartilage and subchondral bone layers following implantation into the osteochondral defect in rats, with seamless tissue integration and restored zonal architecture. Overall, this approach offers a versatile platform that enables simultaneous adjustment of pore dimensions across macro-, micro-, and nanoscales through a single-step fabrication process, potentially eliminating the need for complex pore-forming and post-treatments for designing highly organized tissue scaffolds.