Decellularized pulp matrix as scaffold for mesenchymal stem cell mediated bone regeneration

Abstract

Scaffolds that are used for bone repair should provide an adequate environment for biomineralization by mesenchymal stem cells (MSCs). Recently, decellularized pulp matrices (DPM) have been utilized in endodontics for their high regenerative potential. Inspired by the dystrophic calcification on the pulp matrix known as pulp stone, we developed acellular pulp bioscaffolds and examined their potential in facilitating MSCs mineralization for bone defect repair. Pulp was decellularized, then retention of its structural integrity was confirmed by histological, mechanical, and biochemical evaluations. MSCs were seeded and proliferation, osteogenic gene expression, and biomineralization were assessed to verify DPM's osteogenic effects in vitro. MicroCT, energy-dispersive X-ray (EDX), and histological analyses were used to confirm that DPM seeded with MSCs result in greater mineralization on rat critical-sized defects than that without MSCs. Overall, our study proves DPM's potential to serve as a scaffolding material for MSC-mediated bone regeneration for future craniofacial bone tissue engineering.

Keywords: Decellularized pulp matrix; biomineralization; critical sized defect; dystrophic calcification; mesenchymal stem cells.

Figure 1.

Schematic of the study. Pulps isolated from third molars were decellularized to prepare DPM. MSCs isolated from the rat femur were seeded with DPM and implanted on the rat CSD.

Figure 2.

Gross images of decellularized pulp matrix (DPM, a: Left) and natural pulp matrix (NPM, a: Right) (Scale bar 5 mm), DNA analysis to confirm acellularity by agarose gel electrophoresis after decellularization (b), DAPI staining of both coronal and apical region of the natural and decellularized pulp (scale bar: 50 µm) (c), DNA quantification by nanodrop (d), and Histological and scanning electron microscopic (SEM) pictures of the DPM and NPM (e).

Figure 3.

Mechanical and biochemical properties of dental pulp before and after decellularization process. Tensile strength was tested on NPM and DPM (n = 5, *p < 0.05). Stress-versus-strain analysis (a) and the peak failure force (b) indicated no significant differences between NPM and DPM (*p > 0.05). Soluble protein level was significantly decreased (*p < 0.05) (c), but total collagen contents remained at a similar level (*p > 0.05) after decellularization process.

Figure 4.

Characterization of MSCs isolated from rat bone marrow and their in vitro toxic, proliferative and osteogenic effect by DPM. MSCs were stained with CD44 and CD90, differentiated to chondrogenic, adipogenic, and osteogenic linage, and showed colony-forming capability (a), the Live/Dead Assay was performed to measure DPM’s toxicity, with viable cells staining as green by Calcein-AM and dead cells staining as red by EtD-1 (b), MSC proliferation with and without DPM (control) was assessed by MTS on 1, 3, 5, and 7 days (c), the osteogenic gene expression was evaluated by real-time PCR analysis after culturing with and without DPM. All genes were normalized with GAPDH expression (d), microscopic images of mineralization by MSCs with and without DPM at 14, 21, and 28 days under osteogenic induction media (scale bar: 50 µm) (e), and CPC extraction of Alizarin Red S–stain was quantified by measuring the absorbance at 570 nm (f).

Figure 5.

Micro-CT images of explanted calvaria containing critical sized defect after 12 weeks of implantation with DPM versus DPM seeded with MSCs (a), both bone volume (b), and new bone area (c) were calculated using Image J software. Red circle: defect site (8 mm in diameter). n = 4, *p < 0.05.

Figure 6.

Evaluation of pulp matrices after in vivo biomineralization after 12 weeks of implantation in the CSD. H&E staining showed overall newly formed tissues in the medial-sagittal area on the defect site regenerated with DPM + MSCs, DPM, and calvaria, respectively. Trichrome staining revealed transition from pulp matrices to new bony collagen matrices, represented by the blue color (red arrows), Na: natural bone, N: newly formed bone, and S: DPM scaffold (a). EDX analysis for calcium and phosphate composition on three randomly selected areas on the CSD sites in each group (b), histological assessment of the vascular trait of newly formed bone with DPM + MSCs compared to calvaria, DPM, and NPM. Black arrows indicate vasculature trace (c), and in vitro measurement of vascular endothelial cell growth factor (VEGF) in both NPM and DPM (d).