Liposomal Delivery of Demineralized Dentin Matrix for Dental Tissue Regeneration

Abstract

Current dental restorations have short longevity, and consequently, there is a need for novel tissue engineering strategies that aim to regenerate the dentin-pulp complex. Dentin matrix contains a myriad of bioactive growth factors and extracellular matrix proteins associated with the recruitment, proliferation, and differentiation of dental pulp progenitor cells. In this study, we show that demineralized dentin matrix (DDM), from noncarious dentine, can be encapsulated into liposomes for delivery to dental tissue to promote regeneration. Liposomes were formulated to encapsulate 0-100 μg/mL DDM, lysed with Triton X, and used in vascular endothelial growth factor (VEGF) and transforming growth factor-β1 (TGF-β1) enzyme-linked immunosorbent assays to quantify release. The encapsulation efficiencies were calculated to be 25.9% and 28.8% (VEGF/TGF-β1) for 50 μg/mL DDM liposomes and 39% and 146.7% (VEGF/TGF-β1) for 100 μg/mL DDM liposomes. All liposome formulations had no cytotoxic effects on a dental pulp stem cell (DPSC) clone, as shown by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolium bromide), Caspase 3/7 assays, and cell counts. The ability of the liposomes to stimulate DPSC chemotactic recruitment was tested by Boyden chamber chemotaxis assays. Unloaded liposomes alone stimulated significant progenitor cell recruitment, while DDM-loaded liposomes further promoted chemotactic recruitment in a dose-dependent manner. DDM liposomes promoted the upregulation of "osteodentin" markers osteocalcin and RUNX2 (Runt-related transcription factor 2) in DPSCs after 9 days of treatment, determined by real-time quantitative PCR. Furthermore, Alizarin Red S staining showed that unloaded liposomes alone induced biomineralization of DPSCs, and DDM liposomes further increased the amount of mineralization observed. DDM liposomes were more effective than free DDM (10 μg/mL) at activating recruitment and osteogenic differentiation of DPSC, which are key events in the endogenous repair of the dentin-pulp complex. The study has highlighted the therapeutic potential of bioactive DDM liposomes in activating dental tissue repair in vitro, suggesting that liposomal delivery from biomaterials could be a valuable tool for reparative dentistry and hard-tissue engineering applications.

Keywords: demineralized dentin matrix; dental pulp stem cells; dental tissue engineering; liposomes; odontogenesis; reparative dentinogenesis; restorative materials.

FIG. 1.

DDM liposomes had no cytotoxic effects in hDPSCs. DPSCs were treated with different concentrations of DDM liposomes (0–100 μg/mL; liposome concentration of 10 μg/mL) for 3, 9, and 21 days and then used in a MTT assay (A) to assess cell viability. The cells were then counted with a hemocytometer (B) or used in a Caspase-Glo 3/7 assay (C), which measures apoptotic caspase-3 activity using a bioluminescence substrate. n = 3 ± SEM significance is indicated by *p < 0.05 and **p < 0.01 (unpaired Student's t-test compared to control). DDM, demineralized dentin matrix; DPSCs, dental pulp stem cells; hDPSCs, human dental pulp stem cells; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolium bromide; RLU, relative luminescence unit; SEM, standard error of the mean; TBS, Tris-buffered saline.

FIG. 2.

DDM liposomes promoted the chemotaxis of hDPSCs in a dose-dependent manner. DPSCs were seeded into Boyden transwell inserts, placed on liposome treatments (100 μg/mL), SFM, free DDM (10 μg/mL), or control (basal α-MEM) and allowed to pass through a 0.8 μm membrane for 48 h. Membranes were then fixed and stained with 0.1% (w/v) crystal violet, respective photos were taken (B) and recruited cells counted (A). n = 3 ± SEM significance indicated by *p < 0.05, **p < 0.01, and ****p < 0.0001 (unpaired Student's t-test compared to control). SFM, serum-free media. Color images available online at www.liebertpub.com/tea

FIG. 3.

DDM liposomes promoted the osteogenic differentiation of hDPSC progenitor cells. DPSCs were treated with DDM liposomes (0–100 μg/mL DDM, 10 μg/mL liposome), free 10 μg/mL, or control for DDM for 3, 9, and 21 days. qRT-PCR was performed using primers to amplify RUNX2, an early osteodentine marker, at days 3 and 9 (A) and osteocalcin, a late osteodentine marker, at days 9 and 21 (B), their expression compared to endogenous control GAPDH. The data are shown relative to day 3 control (A) and day 9 control (B). After 35 days of treatment, the amount of calcium deposition was assessed by Alizarin Red S staining using 2% (w/v) Alizarin Red S, cultures treated with 100 μg/mL DDM liposomes, and representative pictures taken at 20× magnification (C). n = 3 ± SEM significance indicated by *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (unpaired Student's t-test compared to control). GAPDH, glyceraldehyde 3-phosphate dehydrogenase; qRT-PCR, quantitative real-time PCR; RUNX2, Runt-related transcription factor 2. Color images available online at www.liebertpub.com/tea