Architecture mechanics mediated osteogenic progression in bone regeneration of artificial scaffolds

Abstract

Scaffold architecture exerts a considerable influence on the osteogenic effect through stress transmission, as the deformation of scaffolds alters the mechanical microenvironment of cells adhering to scaffold surface. Despite extensive research on bone regeneration influenced by scaffold architecture, present studies have not addressed the biological mechanism underlying scaffold architecture-induced stress stimulation (SASS) on cells yet, posing a great challenge in revealing the biomechanical cues between scaffold architecture and osteogenic progression. Therefore, graphite, fullerene, and diamond scaffolds with gradient stress stimulation to cells after deformation were prepared. The cellular biomechanical mechanisms of SASS through single-cell RNA sequencing indicated that architectures providing SASS can induce the enrichment of focal adhesion and osteogenic differentiation pathways of bone mesenchymal stem cells and balance bone resorption of osteoclasts and bone formation of osteoblasts. Besides, SASS enhances bone regeneration for repairing critical-sized defects in vivo. These results provide insights for artificial bone scaffold design and clarify the biomechanical cues between SASS and osteogenic progression.

Fig. 1.. The architecture design of artificial scaffolds with different SASS.

(A) Schematic illustration of the structure design of GP, FL, and DM scaffolds based on the crystal structures of carbon. (B) SEM images showing the structure of GP, FL, and DM scaffolds. Scale bars, 500 μm. (C) Porosity and surface-volume ratio of GP, FL, and DM scaffolds. (D) Diagram of cell stretching stimulated by scaffold compression while lifting the barbell and the stress-time curve of a scaffold. (E) Contour plot of strain for GP, FL, and DM scaffolds and Bone when pressed to deform to 10%. (F) Element proportion-strain curves at four time points (the end of steps 1 to 4) for GP, FL, and DM scaffolds and Bone. (G to J) Strain analysis on the elements proportion-strain curves of the mode, the average, the SD, and the peak width at half-height for GP, FL, DM scaffolds, and Bone at the end of steps 1 to 4. (K and L) Compressive strength and modulus of GP, FL, and DM scaffolds. (M) SEM images of compressive fracture location. Scale bar, 500 μm.

Fig. 2.. SASS-mediated osteogenic commitment of MSCs.

(A) t-distributed stochastic neighbor embedding (TSNE) visualization of osteocytes, OBs, OCs, MSCs, hematopoietic stem and progenitor cells (HSPCs), embryonic stem cells (ESCs), and multipotential progenitor cells (MPCs) clusters after subgroup regrouping. (B) Cell ratio in the MSCs, OCs, OBs and osteocytes subtypes from DM, FL, and GP scaffolds. (C to E) KEGG pathway enrichment analyses of all differentially expressed genes by scRNA-seq in DM versus GP scaffolds, DM versus FL scaffolds, and FL versus GP scaffolds. (F to H) Heatmap of mechanical, functional, and ion balance expression for MSCs in GP, FL, and DM scaffolds. (I to K) Boxplots showing the transcript levels of related genes in GP (n = 166), FL (n = 951), and DM (n = 399) scaffolds. Boxplots display the following parameters: the median (middle line), the mean (“+” marks within “boxes”), the 10 and 90 percentile (lower and upper edges of the “boxes”), the largest/smallest values no further than 1.5 times the distance between the 10 and 90 percentile (upper/lower whiskers), and data beyond the end of the whiskers (individually plotted dots).

Fig. 3.. SASS balances the bone resorption of OCs and bone formation of OBs.

(A) Uniform manifold approximation and projection (UMAP) visualization of OCs (n = 719 cells) after subgroup regrouping, GP (red), FL (blue), and DM (green) scaffolds. (B to D) KEGG pathway enrichment analyses of all differentially expressed genes by scRNA-seq in DM versus GP scaffolds, DM versus FL scaffolds, and FL versus GP scaffolds. (E) Heatmap of related genes for OCs in GP, FL, and DM scaffolds. (F) Boxplots showing the mechanical expression and functional expression levels of related genes in GP (n = 110), FL (n = 315), and DM (n = 294) scaffolds. (G) UMAP visualization of OBs (n = 1046 cells) after subgroup regrouping, GP (red), FL (blue), and DM (green) scaffolds. (H to J) KEGG pathway enrichment analyses of all differentially expressed genes by scRNA-seq in DM versus GP scaffolds, DM versus FL scaffolds, and FL versus GP scaffolds. (K) Heatmap of related genes for OBs in GP, FL, and DM scaffolds. (L) Boxplots showing the mechanical expression and functional expression levels of related genes for OBs in GP (n = 51), FL (n = 629), and DM (n = 366) scaffolds. Boxplots display the following parameters: the median (middle line), the mean (+ marks within boxes), the 10 and 90 percentile (lower and upper edges of the boxes), the largest/smallest values no further than 1.5 times the distance between the 10 and 90 percentile (upper/lower whiskers), and data beyond the end of the whiskers (individually plotted dots).

Fig. 4.. SASS promotes repair of the half-segmental diaphyseal bone defect of rats with critical size.

(A) Footprint photo, 2D pressure heatmap, pressure-time map, and footprint 3D pressure heatmap of the rats. (B and C) Micro–computed tomography (Micro-CT)–based representative 2D coronal plane slices (top), sagittal plane slices (middle), and 3D reconstructions (bottom) evaluation of critical defect healing 4 and 12 weeks after surgery for GP, FL, and DM groups. Scale bars, 1 mm. (D to G) Micro-CT quantification of critical defect femur healing 4 and 12 weeks after surgery for GP, FL, and DM groups. BV/TV, BMD, Tb.N., and Tb.Th. Data are means ± SD (n = 5 per group). (H) Bending strength of Bone, blank, GP, FL, and DM groups critical defect femur healing 4 and 12 weeks after surgery. Statistical analysis was performed using two-way analysis of variance (ANOVA) test with the Tukey’s post-test. (I) H&E histological images, Masson trichrome staining images, and Sirius red of critical defect healing 4 weeks after surgery of GP, FL, and DM groups. Scale bars, 1 mm for low magnification and 200 μm for high magnification. (J) H&E histological images and Masson trichrome staining images of critical defect healing 12 weeks after surgery of GP, FL, and DM groups. Scale bars, 1 mm for low magnification and 200 μm for high magnification. (K) SEM images of surfaces and fracture surfaces showing the structure evaluation of critical defect healing 4 and 12 weeks after surgery in groups GP, FL, and DM groups. Scale bars, 1 mm.

Fig. 5.. Relative protein expression of bone reconstruction after implantation and the difference in repair effect.

(A) Representative immunohistochemistry staining for common bone markers OPN, BMP2, BSP, and OPG of critical defect femur healing 4 weeks after surgery for GP, FL, and DM groups. Scale bars, 200 μm for low magnification and 50 μm for high magnification. (B) Semi-quantitative mean optical density results of immunohistochemistry staining (n = 10 per group). (C and D) Immunofluorescence staining for the cell markers’ 4′,6-diamidino-2-phenylindole (DAPI), thy-1 membrane glycoprotein (THY1), alkaline phosphatase (ALPL), acid phosphatase 5 (ACP5), and merge of critical defect femur healing 4 weeks after surgery for GP, FL, and DM groups. Scale bars, 500 μm for low magnification and 200 μm for high magnification. (E) Semi-quantitative positive area results of immunofluorescence staining (n = 10 per group).

Fig. 6.. In vivo bone reconstruction in Bama pig femur defect model brought by SASS.

(A) Schematic diagram of the Bama pig femur defect model. (B) Photos of Bama pig’s surgical process, including critical femur defect modeling, scaffold implantation, plate fixation, and wound suture. (C) Radiographic follow-up coronal plane slices (top), sagittal plane slices (middle), and 3D reconstructions (bottom) images of one Bama pig in GP group and DM group at 4 and 8 months after surgery. Scale bar, 25 mm. (D) Micro-CT quantification of BV/TV, BMD, Tb.N., and Tb.Th. in GP (n = 3) and DM (n = 3) groups at 4 and 8 months after surgery. (E) Photos of the femur defect after incision along the coronal plane after healing 12 months. Scale bar, 10 mm. (F) Micro-CT representative 2D transverse plane slices, sagittal plane slices, coronal plane slices, and 3D reconstructions evaluation of critical defect healing 4 and 12 weeks after surgery in GP and DM groups. Scale bars, 10 mm. (G) Micro-CT quantification of BV/TV, BS/TV, BMD, Tb.N., and Tb.Th. of critical defect femur healing 12 months after surgery in group GP and group DM. (H) H&E and Masson trichrome staining images in DM and GP groups at 12 months after surgery. Scale bars, 3 mm for low magnification and 1 mm for high magnification. (I) SEM and EDS mapping images showing the fracture defect of critical defect healing 12 months after surgery in GP and DM groups. Scale bars, 1 mm.

Fig. 7.. Schematic diagram illustrating the biological mechanism of SASS in promoting osteogenic differentiation of MSCs, bone resorption of OCs, and bone formation of OBs.

It demonstrates that SASS stimulates the mechanical pathway and regulates actin cytoskeleton to enhance each functional pathway enrichment. This ultimately leads to intensified osteogenic differentiation of MSCs and a coupling between bone resorption of OCs and bone formation of OBs.