Critical bone defects are considered one of the major clinical challenges in reconstructive bone surgery. The combination of 3D printed conductive scaffolds and exogenous electrical stimulation (ES) is a potential favorable approach for bone tissue repair. In this study, 3D conductive scaffolds made with biocompatible and biodegradable polycaprolactone (PCL) and multi-walled carbon nanotubes (MWCNTs) were produced using the extrusion-based additive manufacturing to treat large calvary bone defects in rats. Histology results show that the use of PCL/MWCNTs scaffolds and ES contributes to thicker and increased bone tissue formation within the bone defect. Angiogenesis and mineralization are also significantly promoted using high concentration of MWCNTs (3 wt%) and ES. Moreover, scaffolds favor the tartrate-resistant acid phosphatase (TRAP) positive cell formation, while the addition of MWCNTs seems to inhibit the osteoclastogenesis but present limited effects on the osteoclast functionalities (receptor activator of nuclear factor κβ ligand (RANKL) and osteoprotegerin (OPG) expressions). The use of ES promotes the osteoclastogenesis and RANKL expressions, showing a dominant effect in the bone remodeling process. These results indicate that the combination of 3D printed conductive PCL/MWCNTs scaffold and ES is a promising strategy to treat critical bone defects and provide a cue to establish an optimal protocol to use conductive scaffolds and ES for bone tissue engineering.

The development of advanced biomaterials and manufacturing processes to fabricate biologically and mechanically appropriate scaffolds for bone tissue is a significant challenge. Polycaprolactone (PCL) is a biocompatible and degradable polymer used in bone tissue engineering, but it lacks biofunctionalization. Bioceramics, such as hydroxyapatite (HA) and β tricalcium phosphate (β-TCP), which are similar chemically to native bone, can facilitate both osteointegration and osteoinduction whilst improving the biomechanics of a scaffold. Carbon nanotubes (CNTs) display exceptional electrical conductivity and mechanical properties. A major limitation is the understanding of how PCL-based scaffolds containing HA, TCP, and CNTs behave in vivo in a bone regeneration model. The objective of this study was to evaluate the use of three-dimensional (3D) printed PCL-based composite scaffolds containing CNTs, HA, and β-TCP during the initial osteogenic and inflammatory response phase in a critical bone defect rat model. Gene expression related to early osteogenesis, the inflammatory phase, and tissue formation was evaluated using quantitative real-time PCR (RT-qPCR). Tissue formation and mineralization were assessed by histomorphometry. The CNT+HA/TCP group presented higher expression of osteogenic genes after seven days. The CNT+HA and CNT+TCP groups stimulated higher gene expression for tissue formation and mineralization, and pro- and anti-inflammatory genes after 14 and 30 days. Moreover, the CNT+TCP and CNT+HA/TCP groups showed higher gene expressions related to M1 macrophages. The association of CNTs with ceramics at 10wt% (CNT+HA/TCP) showed lower expressions of inflammatory genes and higher osteogenic, presenting a positive impact and balanced cell signaling for early bone formation. The association of CNTs with both ceramics promoted a minor inflammatory response and faster bone tissue formation.