Decellularised matrices from force loaded periodontal ligament stem cells support osteogenic differentiation

Abstract

Periodontal ligament stem cells (hPDLSCs) are mechanosensing cells responding to mechanical forces. This study investigates the impact of decellularised extracellular matrix (dECM) derived from intermittent compressive force (ICF)-treated hPDLSCs on osteogenic differentiation. hPDSCLs were subjected to ICF loading at 1.5 g/cm² for 24 h and then maintained with normal medium (N) or osteogenic induction medium (OM) followed by a decellularisation process. dECMs derived from ICF (dECM-ICF) were characterised using a scanning electron microscope, energy-dispersive X-ray spectroscopy, and proteomic analysis. hPDLSCs were re-seeded on dECM-ICF. Cell proliferation and viability were examined by resazurin and LIVE/DEAD assays. Mineralisation was determined by Alizarin Red S staining. Results demonstrated that dECM-ICF-derived from OM (dECM-ICF-OM) exhibited a fibrillar network structure and showed no cellular component while preserving fibronectin and type I collagen. dECM-ICF exhibited biocompatibility, as indicated by the absence of cytotoxic effects and the ability of hPDLSCs to attach, spread, and proliferate. dECM-ICF-OM significantly enhanced mineral deposition compared to dECM-ICF from normal conditions (dECM-ICF-N). Proteomic analysis of dECM-ICF demonstrated the upregulated proteins in the PI3K-Akt, Ras, MAPK, mTOR, ErbB, TNF, and VEGF signallings. In conclusion, dECM-ICF supports hPDLSCs growth and osteogenic differentiation. dECM-ICF is a promising cell-free natural scaffold to promote periodontal regeneration.

Keywords: Decellularisation; Extracellular matrix; Intermittent compressive force; Periodontal ligament stem cells.

Fig. 1

Mesenchymal stem cell characterisation. Surface marker expression was determined using flow cytometry analysis (A). Cells were maintained in a growth medium or osteogenic induction medium for 14 days, and the mineralisation was examined using Alizarin Red S staining (B). Cells were maintained in a growth medium or adipogenic induction medium for 16 days, and the intracellular lipid accumulation was examined using Oil Red O staining (C). Cells were treated with intermittent compressive force (ICF) for 24 h prior to maintaining in a normal medium or osteogenic induction medium, and the mineralisation was examined using Alizarin Red S staining (D). The decellularised process was performed and the mineralisation was examined using Alizarin Red S staining (E).

Fig. 2

Characterisation of decellularised ECM (dECM) and dECM-derived from intermittent compressive force-loaded hPDLSCs (dECM-ICF). Fibronectin and type I-collagen were identified in ECM and dECM derived from ICF treatment using immunofluorescence staining (A and B). The ultrastructure of dECM was observed using scanning electron microscopy (C and D). The chemical elements of dECMs were examined using energy-dispersive X-ray spectrometry (E).

Fig. 3

Biological responses of hPDLSCs on dECM-ICF. Cell proliferation of hPDLSC on dECM-ICF was observed using a resazurin assay in days 1, 3, and 7 (A). Cell viability was evaluated using live/dead assay at 24 h; live cells are shown in green (calcein-AM), and dead cells appeared in red (EthD-1) (B). Cell attachment and spreading were analysed using scanning electron microscopic analysis at 30 min, 24 h, and 7 days (C). TCP = Tissue culture plate.

Fig. 4

dECM-ICF promotes the mineralisation of hPDLSCs. hPDLSCs were re-seeded on dECM-ICF-N and dECM-ICF-OM and cultured in either a growth medium (GM) or an osteogenic induction medium (OM). Mineralisation was determined using Alizarin Red S staining (A). The mineral deposition was quantified by solubilised ARS-positive nodules and the relative absorbance at 570 nm (B). The mRNA expression levels of the osteogenic maker genes, including BMP2, OSX, OPN, DMP1, ALP, COL1A1, DSPP, RUNX2, BSP, and OCN, were analysed using real-time qPCR (C-L, respectively). Bars indicate the statistically significant differences between groups. * p < 0.05, **p < 0.01.

Fig. 5

Protein profiling and pathway analysis of dECM-ICF in N and OM conditions. The top 50 differentially regulated proteins of dECM-ICF in N compared to OM condition, gene lists were selected by p-values and analysed using Heatmapper (A). Venn diagram of the protein in of dECM-ICF in N and OM conditions (B), based on raw protein counts.

Fig. 6

KEGG pathway analysis of the top 20 protein expressions of dECM-ICF maintained in N and OM media conditions, conducted using WebGestalt (A-B).

Fig. 7

The metascape gene list analysis of differentially expressed proteins of dECM-ICF in N and OM conditions. demonstrated the bar graph of the top 20 enriched genes in the Pathway and Process Enrichment Analysis categories of differentially expressed proteins of dECM-ICF in N compared to dECM-ICF in OM condition, colour by p-values upregulated (A) and downregulated (C). The protein-protein interaction (PPI) network of dECM-ICF in OM condition shows clustered enrichment categories and the relationship among these clusters (cluster ID) for upregulated proteins (B), and PPI network of downregulated proteins in dECM-ICF in OM condition (D). All analyses were performed using Metascape.

Fig. 8

KEGG pathway analysis of dECM-ICF using Metascape gene list analysis. The KEGG-enriched pathways of dECM-ICF in OM condition compared to dECM-ICF in N condition were analyzed using WebGestalt. Upregulated protein expression in the KEGG-enriched pathways (A). Downregulated protein expression in the KEGG-enriched pathways (B). The analysis was conducted by assessing protein expression levels as log2 values, with significantly regulated proteins identified at an FDR-corrected p-value of < 0.05, representing pathway-associated protein expression.