Comparing to other conventional fabrication techniques, 3D printing is advantageous in producing bone tissue engineering scaffolds with customized shape, tailored pore size/porosity, required mechanical properties and even desirable biomolecule delivery capability. However, towards scaffolds with a large volume, it is highly difficult to enable seed cells to migrate to the central region of the scaffolds, resulting in an inhomogeneous cell distribution, and hence lowering the bone forming ability. To address this problem, in this investigation, cell-laden bone tissue engineering scaffolds consisting of osteogenic peptide loaded β-tricalcium phosphate/poly(lactic-co-glycolic acid) (OP/TCP/PLGA) nanocomposite scaffolds and rat bone marrow derived mesenchymal stem cells (rBMSCs)-laden Gelatin/GelMA hydrogel fillers were produced through a “dual-nozzle” cryogenic hybrid 3D printing. The cell-laden scaffolds exhibited a bi-phasic structure and were mechanically similar to human cancellous bone. OP can be released from the hybrid scaffolds in a sustained manner. rBMSCs laden in the hydrogel patterns exhibited a high viability during and after cryogenic hybrid 3D printing process and can be further released from the hydrogel struts and achieve cell anchorage on the surface of adjacent OTP struts. The OP released from OTP struts enhanced rBMSCs proliferation. Comparing to rBMSC-laden hybrid scaffolds without OP incorporation, the rBMSC-laden hybrid scaffolds incorporated with OP significantly up-regulated the osteogenic differentiation of rBMSCs, by showing a higher level of alkaline phosphatase (ALP) expression and calcium deposition. This “proof-ofconcept” study provided a facile method to form cell-laden bone tissue engineering scaffolds with not only required mechanical strength, biomimetic structure and prolonged biomolecule release but also excellent cell delivery capability with uniform cell distribution.

How to fabricate bone tissue engineering scaffolds with excellent antibacterial and bone regeneration ability has attracted increasing attention. Herein, we produced a hierarchical porous β-tricalcium phosphate (β-TCP)/poly(lactic-co-glycolic acid)-polycaprolactone composite bone tissue engineering scaffold containing tetracycline hydrochloride (TCH) through a micro-extrusion-based cryogenic 3D printing of Pickering emulsion inks, in which the hydrophobic silica (h-SiO2) nanoparticles were used as emulsifiers to stabilize composite Pickering emulsion inks. Hierarchically porous scaffolds with desirable antibacterial properties and bone-forming ability were obtained. Grid scaffolds with a macroscopic pore size of 250.03 ± 75.88 μm and a large number of secondary micropores with a diameter of 24.70 ± 15.56 μm can be fabricated through cryogenic 3D printing, followed by freeze-drying treatment, whereas the grid structure of scaffolds printed or dried at room temperature was discontinuous, and fewer micropores could be observed on the strut surface. Moreover, the impartment of β-TCP in scaffolds changed the shape and density of the micropores but endowed the scaffold with better osteoconductivity. Scaffolds loaded with TCH had excellent antibacterial properties and could effectively promote the adhesion, expansion, proliferation, and osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells afterward. The scaffolds loaded with TCH could realize the strategy to “kill bacteria first, then induce osteogenesis”. Such hierarchically porous scaffolds with abundant micropores, excellent antibacterial property, and improved bone-forming ability display great prospects in treating bone defects with infection.