A unique biomimetic modification endows polyetherketoneketone scaffold with osteoinductivity by activating cAMP/PKA signaling pathway

Abstract

Osteoinductivity of a biomaterial scaffold can notably enhance the bone healing performance. In this study, we developed a biomimetic and hierarchically porous polyetherketoneketone (PEKK) scaffold with unique osteoinductivity using a combined surface treatment strategy of a sulfonated process and a nano bone-like apatite deposition. In a beagle intramuscular model, the scaffold induced bone formation ectopically after 12-week implantation. The better bone healing ability of the scaffold than the original PEKK was also confirmed in orthotopic sites. After culturing with bone marrow-derived mesenchymal stem cells (BMSCs), the scaffold induced osteogenic differentiation of BMSCs, and the new bone formation could be mainly depending on cell signaling through adenylate cyclase 9, which activates the cyclic adenosine monophosphate/protein kinase A signaling cascade pathways. The current work reports a new osteoinductive synthetic polymeric scaffold with its detailed molecular mechanism of action for bone repair and regeneration.

Fig. 1.. Preparation and characterization of the biomimetic PEKK-B materials.

(A) Schematic illustration of the preparation of biomimetic PEKK scaffold. (B) In situ self-assembly of bone-like apatite nanoparticles on the microporous network of sulfonated PEKK scaffold. (C) Microcomputed tomography (μ-CT) reconstruction and (D) scanning electron microscopy (SEM) observation for various PEKK scaffolds and natural trabecular bone. (E) Typical atomic force microscopy (AFM) images and (F) corresponding surface roughness of PEKK scaffolds and natural trabecular bone. (G) Compressive strength of PEKK scaffolds and natural trabecular bone (error bars, means ± SD; n = 3 per group). All analyses were done using one-way analysis of variance (ANOVA) with Tukey’s post hoc test.

Fig. 2.. Evaluation of the bone formation in heterotopic and orthotopic sites induced by the PEKK scaffold.

(A) Timeline and arrangement of the in vivo experiments. (B) Implantation site of the scaffold and the μ-CT evaluation of new bone (white) formation within the scaffold. (C) Histological analysis for new bone formation within the scaffold (M, material; B, bone tissue). EDS, energy-dispersive spectrometry. (D) Bone induction incidence of the scaffold. (E) Quantitative analysis of the volume and (F) area fraction of new bone formation within the scaffold. (G) Hematoxylin and eosin staining of histological sections and quantitative analysis of the area fraction of new bone formation within different PEKK scaffolds at week 12 postoperatively (M, material; B, bone tissue). (H) μ-CT evaluation and quantitative analysis of the volume fraction of new bone formation (gray) within different PEKK scaffolds at week 12 postoperatively (error bars, means ± SD; n = 10 per group; all analyses were done using unpaired Student’s t test). ***P < 0.001.

Fig. 3.. Effect of the scaffolds on osteogenic differentiation of BMSCs in vitro.

(A) SEM and CLSM observations and (B) the corresponding quantitative assay of the cell area and (C) cell number of BMSCs cultured on the scaffolds for 1 day (actin filament is stained red, while the cell nuclei are stained blue). (D) Cell viability of BMSCs cultured on the scaffolds at days 1, 3, and 5, and several osteogenic gene expressions of BMSCs cultured on the scaffolds at days 3 and 5 (error bars, means ± SD; n = 6 per group for cell viability test; n = 3 per group for qRT-PCR analysis). All analyses were done using one-way ANOVA with Tukey’s post hoc test. *P < 0.05 and ***P < 0.001.

Fig. 4.. Analysis of cell signaling pathways mediated by the scaffolds during the osteogenic differentiation of BMSCs.

(A) Principal components analysis and (B) t-distributed stochastic neighbor embedding (tSNE) cluster analysis of BMSCs cultured on the scaffolds at day 3. (C) Volcano plot showing differentially regulated genes in the PEKK-B as compared to the PEKK group. Genes with an absolute fold change of >1.5 and a P value of <0.05 are highlighted in green and red, denoting down- and up-regulated genes, respectively. (D) Circular visualization of the results of gene-annotation enrichment analysis. (E) Heatmap of genes that were differentially expressed in PEKK-B versus PEKK group with a fold change of >1.5 and a P value of <0.05. (F) qRT-PCR validation for representative genes (error bars, means ± SD; n = 6 per group; all analyses were done using unpaired Student’s t test). *P < 0.05 and **P < 0.01.

Fig. 5.. Regulation of ADCY9/cAMP/PKA signaling activated by the scaffolds on BMSCs osteogenic differentiation.

(A) Schematic of the scaffolds cultured with BMSCs treated by PAK inhibitor H89 or ADCY inhibitor SQ22536 for 3 days. (B) Western blot analysis of ADCY9, PKA, MEK/p-MEK, ERK/p-ERK, ALP, and β-actin and (C to G) the corresponding quantitative analysis of band intensities. (H) Relative expression of cAMP in BMSCs as assessed by enzyme-linked immunosorbent assay kit (error bars, means ± SD; n = 3 per group; all analyses were done using one-way ANOVA with Tukey’s post hoc test). *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 6.. The scaffolds induce bone formation ectopically by cAMP/PKA-activated BMSCs.

(A) Schematic illustration of the implanted scaffolds loaded with BMSCs treated with different inhibitors. (B) Histological analysis for new bone formation within the scaffold at day 90 postoperatively (M, material; B, blood vessel; NB, newly formed bone). (C) Immunohistochemical staining for the osteogenic marker OCN (brown, black arrow). (D) Schematic diagram of the molecular mechanism for BMSCs osteogenic differentiation induced by the PEKK-B scaffold (error bars, means ± SD; n = 5 per group; all analyses were done using one-way ANOVA with Tukey’s post hoc test). **P < 0.01 and ***P < 0.001.