Significant dentoalveolar bone defects, often resulting from trauma, infection, or surgery, necessitate effective treatment options beyond spontaneous healing. Current treatments include bone grafts (autografts, allografts, xenografts), each with limitations such as donor site morbidity, cost, immunogenicity, and disease transmission risks. Synthetic bone substitutes offer a promising alternative, and 3D printing allows for the creation of personalized scaffolds tailored to individual defect volumes using imaging data. Calcium phosphate derivatives, particularly biphasic calcium phosphates (BCP) combining hydroxyapatite (HA) and tricalcium phosphate (TCP), are attractive materials due to their biocompatibility, osteoconductive properties, and ability to be 3D-printed. The combination of 70-80% TCP and 20-30% HA enhances angiogenesis and promotes bone regeneration. Further improvements in bone regeneration can be achieved by incorporating bioactive molecules that enhance cell migration and attachment. MicroRNAs (miRNAs), such as miR-302a-3p, are post-transcriptional regulators with potential to stimulate osteoblast function by downregulating COUP-TFII (a repressor of RUNX2, crucial for osteogenesis) and activating RANKL (involved in osteoclastogenesis). However, miRNAs are susceptible to degradation, necessitating delivery systems like hydroxyapatite nanoparticles (HA-NPs). Surface modification of HA-NPs with 3-aminopropyltriethoxysilane (APTES) enhances miRNA condensation and cellular uptake. This study aimed to develop and evaluate a 3D-printed TCP/HA scaffold delivering miR-302a-3p via HA-NPs-APTES to stimulate bone regeneration in a mouse calvarial model.

The literature extensively supports the use of bone grafts for critical-sized bone defects, but limitations regarding donor site morbidity, cost, and potential for disease transmission have driven the development of synthetic bone substitutes. Studies comparing block versus particulate bone grafts suggest superior outcomes with block substitutes in craniofacial defects, emphasizing the advantage of pre-shaped scaffolds. 3D printing technology further enhances this by enabling the fabrication of patient-specific scaffolds based on CT or MRI data. Biphasic calcium phosphates (BCP), combining HA and TCP, have been widely studied for bone regeneration due to their structural, biological, and mechanical properties. The optimal HA/TCP ratio (around 70-80% TCP and 20-30% HA) is known to enhance angiogenesis and facilitate bone regeneration comparable to autografts. The addition of bioactive molecules, including growth factors (BMP-2, VEGF, FGF) and miRNAs, has been explored to further improve bone regeneration. miR-302a-3p's role in stimulating osteoblast function through COUP-TFII downregulation and RUNX2 upregulation has been demonstrated in vitro. The use of HA-NPs as a delivery system for miRNAs provides biocompatibility, osteoconductive properties, and a large surface area for miRNA condensation. APTES modification of HA-NPs further improves miRNA stability and cellular uptake, enhancing the therapeutic potential of this combination.

The study employed both in vitro and in vivo methods. In vitro experiments involved synthesizing HA-NPs and modifying them with APTES. Two methods (M1 and M2) were used to incorporate HA-NPs-APTES and miR-302a-3p into 3D-printed TCP/HA scaffolds. M1 involved incorporating the components into the cement during hardening, while M2 involved applying the mixture to the scaffold surface after hardening. Biocompatibility was assessed using a resazurin assay and fluorescent microscopy (FITC-labeled HA-NPs-APTES). The delivery of miR-302a-3p and its effect on gene expression (COUP-TFII, RUNX2, ALP, OCN, OSX) were evaluated using qPCR. For in vivo studies, 4 mm critical-sized calvarial defects were created in mice (n=4 per group). Four groups were established: control (no scaffold), bare scaffold, scaffold with HA-NPs-APTES (without miR), and scaffold with HA-NPs-APTES-miR (using M2 method). Bone regeneration was assessed at 2, 4, and 6 weeks post-surgery using micro-CT (BV/TV, unfilled pores) and histomorphometry (new bone formation). Statistical analysis (one-way ANOVA, Tukey's post hoc test) was performed.

In vitro, both M1 and M2 scaffolds showed biocompatibility. M2 demonstrated significantly enhanced miR-302a-3p delivery compared to M1 and direct miRNA conjugation (S-Mi), resulting in COUP-TFII downregulation and RUNX2 upregulation in MG63 and HmOBs cells. In vivo, micro-CT analysis revealed significantly higher BV/TV and fewer unfilled pores in the HA-NPs-APTES-miR group at all time points (2, 4, and 6 weeks) compared to the control and HA-NPs-APTES groups. Histomorphometry demonstrated earlier new bone formation in the center of the defect in the HA-NPs-APTES-miR group at 2 weeks. At 4 and 6 weeks, the HA-NPs-APTES-miR group showed significantly higher total new bone formation, particularly in the central region of the defect. The difference in bone formation was most pronounced in the central region of the defect. The proportion of new bone in the center of the scaffold modified with HA-NPs-APTES-miR increased from approximately 50% at 4 weeks to 60% at 6 weeks, indicating significant osseoconduction and osseoinduction.

This study successfully demonstrated the enhanced bone regeneration potential of a 3D-printed TCP/HA scaffold modified with HA-NPs-APTES delivering miR-302a-3p. The superior performance of M2, involving surface application, likely stems from improved accessibility of the nanoparticles to cells for miR release. The in vivo results strongly support the osteoinductive role of miR-302a-3p, as evidenced by accelerated new bone formation in the central region of the defect. The observed increase in BV/TV and decrease in unfilled pores further underscore the effectiveness of this approach. The findings highlight the synergistic effect of the 3D-printed TCP/HA scaffold's osteoconductive properties and the osteoinductive capacity of miR-302a-3p delivered via HA-NPs-APTES. This approach offers a promising strategy for treating critical-sized bone defects, potentially leading to improved clinical outcomes.

This study successfully demonstrated that a 3D-printed TCP/HA scaffold modified with HA-NPs-APTES for miR-302a-3p delivery significantly enhances bone regeneration in a mouse calvarial model. The method M2 proved superior for miR delivery and bone regeneration. Future research could focus on optimizing the HA-NPs-APTES concentration and exploring the application of this strategy in larger animal models and eventually clinical trials. Investigating other miRNAs with bone regenerative potential and exploring different scaffold designs could further enhance the therapeutic efficacy.

The study used a mouse calvarial model, which may not fully reflect the complexities of human bone regeneration. The sample size (n=4 per group) is relatively small, limiting the statistical power. Long-term effects and the complete resorption of the scaffold were not fully evaluated. Further investigations are needed to determine the optimal dose of miRNA and the long-term effects on bone regeneration.