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This work investigated a new three-dimensional (3D) printing methodology to prepare porous scaffolds containing horizontal pore and composition gradients. To achieve that, a multimaterial printing technology developed in our laboratory was adapted to incorporate pore gradients. Fibers were printed by welding segments with unique material compositions and fiber diameters. Particularly, we focused on the preparation of model composite poly(ɛ-caprolactone)-based scaffolds with radial gradients of particulate hydroxyapatite (HA) content (higher concentrations in the outer region of the scaffold) and porosity (higher in the inner region). The morphology of the scaffolds revealed that the methodology allowed the fabrication of discrete regions with compressive mechanical properties similar to human trabecular bone while maintaining structural integrity. HA distribution was homogeneous within individual regions and no particle aggregation was detected by microCT analysis and Alizarin Red S staining. Finally, the incubation of the scaffolds in simulated body fluid resulted in the deposition of significantly higher amounts of calcium deposits in the regions of the scaffolds with higher HA content. This work provides a new tool for the preparation of porous scaffolds containing porosity and composition gradients for complex tissue engineering applications. IMPACT STATEMENT: In this study, we report the development of a novel multimaterial segmented three-dimensional printing methodology to fabricate porous scaffolds containing discrete horizontal gradients of composition and porosity. This methodology is particularly beneficial to preparing porous scaffolds with intricate structures and graded compositions for the regeneration of complex tissues. The technique presented is compatible with many commercially available bioprinters commonly used in biofabrication, and can be adapted to better replicate the architectural and compositional requirements of individual tissues compared with traditional scaffold printing methods.

In this work, we present a printing method to fabricate scaffolds consisting of multimaterial segmented fibers. Particularly, we developed a reproducible printing process to create single fibers with multiple discrete compositions and control over the distribution of particulate ceramics—namely hydroxyapatite (HA) and β-tricalcium phosphate (TCP)—within poly(ɛ-caprolactone)-based composite scaffolds. Tensile testing revealed that the mechanical integrity of individual segmented fibers was preserved compared with nonsegmented fibers, and microcomputed tomography and thermal analysis confirmed the homogeneous distribution of ceramics incorporated in the fiber compositions. Moreover, we printed and characterized composite scaffolds containing model inverse radial gradients of HA and TCP that could serve as a tunable platform to control the degradation rate of the scaffolds and match bone tissue ingrowth. The morphology of the gradient scaffolds was assessed, and their bulk compressive mechanical properties were found to be in the same range as human trabecular bone. Finally, scaffold degradation was monitored for up to 10 weeks in phosphate-buffered saline pH 7.4 and 0.1 M HCl solution, and scaffolds containing TCP in their composition showed increased degradation compared with those containing HA. This work provides a new methodology for the fabrication and characterization of porous scaffolds containing designer composition gradients that could serve as a platform for the preparation of complex scaffolds for tissue engineering applications. IMPACT STATEMENT: This study introduces a segmented three-dimensional printing methodology to create multimaterial porous scaffolds with discrete gradients and controlled distribution of compositions. This methodology can be adapted for the preparation of complex, multimaterial scaffolds with hierarchical structures and mechanical integrity useful in tissue engineering.

A critical challenge to the success of biodegradable vascular grafts is the establishment of a healthy endothelium. To establish this monolayer of endothelial cells (ECs), a variety of techniques have been developed, including cell seeding. Vascular grafts may be seeded with relevant cell types and allowed to mature before implantation. Due to the low proliferative ability of adult ECs and issues with donor site morbidity, there has been increasing interest in using endothelial progenitor cells (EPCs) for vascular healing procedures. In this work, we combined the proliferative and differentiation capabilities of a commercial cell line of early EPCs with an established bioreactor system to support the maturation of cell-seeded vascular grafts. All components of the vascular graft and bioreactor setup are commercially available and allow for complete customization of the scaffold and culturing system. This bioreactor setup enables the control of flow through the graft, imparting fluid shear stress on EPCs and affecting cellular proliferation and differentiation. Grafts cultured with EPCs in the bioreactor system demonstrated greatly increased cell populations and neotissue formation compared with grafts seeded and cultured in a static system. Increased expression of markers for mature endothelial tissues were also observed in bioreactor-cultured EPC-seeded grafts. These findings suggest the distinct advantages of a customizable bioreactor setup for the proliferation and maturation of EPCs. Such a strategy may be beneficial for utilizing EPCs in vascular tissue engineering applications.