Additively manufactured bioceramic scaffolds based on triply periodic minimal surfaces for bone regeneration

Abstract

The study focused on the effects of a triply periodic minimal surface (TPMS) scaffolds, varying in porosity, on the repair of mandibular defects in New Zealand white rabbits. Four TPMS configurations (40%, 50%, 60%, and 70% porosity) were fabricated with β-tricalcium phosphate bioceramic via additive manufacturing. Scaffold properties were assessed through scanning electron microscopy and mechanical testing. For proliferation and adhesion assays, mouse bone marrow stem cells (BMSCs) were cultured on these scaffolds. In vivo, the scaffolds were implanted into rabbit mandibular defects for 2 months. Histological staining evaluated osteogenic potential. Moreover, RNA-sequencing analysis and RT-qPCR revealed the significant involvement of angiogenesis-related factors and Hippo signaling pathway in influencing BMSCs behavior. Notably, the 70% porosity TPMS scaffold exhibited optimal compressive strength, superior cell proliferation, adhesion, and significantly enhanced osteogenesis and angiogenesis. These findings underscore the substantial potential of 70% porosity TPMS scaffolds in effectively promoting bone regeneration within mandibular defects.

Keywords: 3D printer/additive manufacture; TPMS bone scaffold; bone regeneration; mandibular defect; osteogenesis.

Figure 1.

(a) Digital photos of TPMS bone scaffold with porosities of 40%, 50%, 60%, and 70%, (b) observing the micro-morphology of scaffolds with different porosities under scanning electron microscope, (c) the compressive strength of the scaffolds with different porosities, (d) the elastic modulus of the scaffolds with different porosities, and (e) the biodegradability of scaffolds with different porosities. *p < 0.05. **p < 0.01.

Figure 2.

(a) Live-Dead staining of TPMS bone scaffold with 40%, 50%, 60%, and 70% porosities, (b) column analysis diagram of CCK-8 experiment, and (c) histogram of average fluorescence intensity. *p < 0.05. **p < 0.01. ****p < 0.0001.

Figure 3.

(a) Laser confocal images of TPMS bone scaffolds with porosity of 40%, 50%, 60%, and 70% under a 40-fold microscope (DAPI shows the nucleus, F-actin is stained with Phalloidin) and (b) scanning electron microscope images of TPMS bone scaffold with porosity of 40%, 50%, 60%, and 70%. Blue arrows refer to mouse bone marrow mesenchymal stem cells.

Figure 4.

(a) Flow chart of mandibular defect modeling and TPMS-loaded bone scaffold in New Zealand white rabbits, (b) Micro-CT images of TPMS bone scaffolds with porosities of 40%, 50%, 60%, and 70%, (c) BV: ROI area bone tissue volume, (d) TV: The total volume of the ROI area, and (e) BV/TV: The ratio of bone tissue to tissue volume can reflect changes in bone mass. (f) Tb.Th: Average thickness of bone trabeculae. **p < 0.01. ***p < 0.001. ****p < 0.0001.

Figure 5.

Histological and immunohistochemical analysis of bone regeneration induced by TPMS scaffolds. Note: The yellow asterisks indicate mature bone tissue, the red arrows indicate new blood vessels, “T” indicates scaffolds.

Figure 6.

OPG immunofluorescent staining of bone regeneration induced by TPMS scaffolds. Note: DAPI staining blue represents the nucleus, green represents the OPG protein.

Figure 7.

(a) Principal components (PC) analysis on tissues from TPMS scaffolds and tissues from control group, (b and c) Heatmaps of osteoblast differentiation-related genes, Angiogenesis-related factors and Hippo signaling regulation pathways genes expression after implant the scaffold for 2 weeks (red: high expression; blue: low expression), (d and e) GSEA enrichment analysis, (f) differential alternative splicing events between TPMS and control samples, (g) GO enrichment analysis. (h) KEGG pathway analysis of DEGs, (i) differentially expressed transcription factors family, and (j) RT-qPCR analysis of five genes (VEGFA, S100A4, YAP1, CSF1R, and HES1) related osteoblast differentiation, angiogenesis and Hippo signaling regulation pathways.