A novel process was developed to fabricate core/shell-structured 3D scaffolds, made of calcium-deficient hydroxyapatite (CDHA) and alginate laden with pre-osteoblast MC3T3-E1 cells, through a combination of cement chemistry, dual paste-extruding deposition (PED), and cell printing. The cement reaction of calcium phosphates replaced the typical sintering process of the ceramic scaffold fabrication after the simultaneous printing of the ceramics and cell-laden hydrogel. The alginate crosslinking process was divided into two steps using different concentrations of CaCl₂, during and after 3D printing, in order to obtain a stable 3D core/shell structure and high cell viability. The whole process was carried out under conditions (neutral pH and a temperature between room temperature and 37 °C) that were gentle to the cells, so the cells incorporated into the shell remained alive throughout the 3D scaffold for the entire culture period (35 days). The core/shell structured scaffold significantly enhanced the mechanical properties when compared with a hydrogel that uses a typical cell-printing process or with a ceramic scaffold, due to the co-operative effect of each material. The compressive strength of the CDHA/alginate scaffolds in the wet state was 3.2 MPa, whereas the compressive strength of alginate could not be determined in the wet state. The 3D structural morphology of CDHA/alginate scaffolds was well retained, even after a compression test, and showed less deformation because the CDHA ceramic-core was encapsulated within the elastic alginate. The process developed in this study suggests a new cell printing model that has excellent potential for application in the field of bone tissue regeneration.

3D porous calcium deficient hydroxyapatite (CDHA) scaffolds with phytoestrogens were fabricated for osteoporotic bone tissue regeneration through a combination of 3D printing techniques and cement chemistry as a room temperature process. Quercetin, one of the major phytoestrogens isolated from onions and apples, was directly incorporated into CDHA for local administration in place of bisphosphonates (BPs) (which are recognized as standard treatment in osteoporosis), to avoid drug side effects. The CDHA scaffolds with quercetin (QC–CDHA) showed favorable mechanical properties (compressive strength < 21 MPa) as well as pore morphology. Quercetin was steadily released with the biodegradation of CDHA scaffolds in vitro without any initial burst. The QC–CDHA scaffolds greatly influenced both osteoblast and osteoclast cell activities. The QC–CDHA scaffolds significantly increased pre-osteoblast cell (MC3T3-E1) proliferation, differentiation, and mineralization, whereas osteoclast cell (RANK treated RAW 264.7) proliferation and differentiation were dramatically suppressed. The influence of quercetin on bone tissue regeneration was superior to alendronate, which is one of the most commonly administered BPs. All results indicated that quercetin in CDHA scaffolds plays an important role in both enhancing bone formation and suppressing bone resorption. Consequently, this technology promises great potential in osteoporotic bone tissue regeneration.

Multifunctional bone substitute materials (BSM) have gained considerable attention with the exponential increase in aging populations. The development of hybrid materials for diagnosis and therapy of bone-related diseases and dysfunctions, especially, has been a significant challenge in the biological and the biomedical field, due to the shortage of agents with specificity and selectivity toward bone. In this study, a hybrid material, referred as Alen-CDs@CDHA, fabricated from alendronate-conjugated carbon dots (Alen-CDs) and calcium-deficient hydroxyapatite (CDHA, the mineral component of bones) scaffolds is offered as a novel multifunctional BSM for in vivo osteoclasts deactivation and fluorescence imaging. The fluorescent Alen-CDs were hydrothermally prepared using phytic acid as carbon source, followed by conjugating alendronate, for controlled alendronate release and fluorescent imaging under acidic conditions. As-prepared fluorescent Alen-CDs were consecutively immobilized on surfaces of CDHA scaffolds, exhibiting high affinity by bisphosphonate group, easily fabricated from α-tricalcium phosphate (α-TCP) paste using three-dimensional (3D) printing system. The resultant Alen-CDs@CDHA caused a significant decrease (> 50%) in viability of osteoclasts at 7 days after in vitro treatment. Furthermore, when Alen-CDs@CDHA was implanted in balb/c nude mice for in vivo evaluation, we found Alen-CDs@CDHA to be suitable for bone imaging through fluorescence signals, without necrosis or inflammatory symptoms in the epidermal tissues. Thus, these observations offer new opportunities for a novel and revolutionary use of Alen-CDs@CDHA as highly specific multifunctional BSM for bone diagnosis and imaging, and as bone-specific drug delivery materials, eventually providing anti-osteoclastogenic treatments solution for degenerative bone disorders. STATEMENT OF SIGNIFICANCE: Alen-CDs@CDHA significantly reduced the viability of osteoclasts and fluorescently imaged in vivo after transplantation, releasing drug via pH modulation. The development of fluorescence materials for bone imaging remains still a major challenge in the biomedical field owing to the shortage of selectivity and specificity. The results could lead to improvements in bone treatment strategies, as it could reduce the invasiveness of procedures and the associated negative outcomes, and increase the precision of strategies. Further, we believe that this study will be of interest to the readership of your journal as clearly focuses on the advancement of a biomaterial, where we have engineered a substance to substitute bone and integrate with a living system.