Exploring a Chemotactic Role for EVs from Progenitor Cell Populations of Human Exfoliated Deciduous Teeth for Promoting Migration of Naïve BMSCs in Bone Repair Process.

Mobilization of naïve bone marrow mesenchymal stromal cells (BMSCs) is crucial to desired bone regeneration in both orthopedic and dental contexts. In such conditions, mesenchymal progenitor cell populations from human exfoliated deciduous teeth (SHEDs) present advantageous multipotent properties with easy accessibility which makes them a good candidate in both bone and periodontal tissue regeneration. Extracellular vesicles (EVs) are a functional membranous structure which could participate in multiple cell interactions and imitate the biological functions of their parenting cells largely. To assess their ability to mobilize naïve BMSCs in the bone repair process, Nanosight Tracking Analysis (NTA) and Enzyme-Linked Immunosorbent Assays (ELISA) were performed to illustrate the composition and functional contents of EV samples derived from SHEDs with different culturing time (24 h, 48 h, and 72 h). Afterwards, the Boyden chamber assay was performed to compare their capacity for mobilizing naïve BMSCs. One-way analysis of variance (ANOVA) with a post hoc Turkey test was performed for statistical analysis. SHEDs-derived EVs collected from 24 h, 48 h, and 72 h time points, namely, EV24, EV48, and EV72, were mainly secreted as exosomes and tended to reform into smaller size as a result of sonication indicated by NTA results. Moreover, different EV groups were found to be abundant with multiple growth factors including transforming growth factor-ß1 (TGF-ß1), platelet-derived growth factor (PDGF), insulin-like growth factor-1 (IGF-1), and fibroblast growth factor-2 (FGF-2) given the detections through ELISA. Boyden chamber assays implied the migratory efficiency of BMSCs driven by EVs at varying concentrations. However, the results showed that migration of BMSCs driven by different EV groups was not statistically significant even with chemotactic factors contained (P > 0.05). Taken together, these data suggest that EVs derived from SHEDs are secreted in functional forms and present a potential of mobilizing naïve BMSCs, which may propose their relevance in assisting bone regeneration.

Efficacy of copolymer scaffolds delivering human demineralised dentine matrix for bone regeneration.

Poly(L-lactide-co-ε-caprolactone) scaffolds were functionalised by 10 or 20 µg/mL of human demineralised dentine matrix. Release kinetics up to 21 days and their osteogenic potential on human bone marrow stromal cells after 7 and 21 days were studied. A total of 390 proteins were identified by mass spectrometry. Bone regeneration proteins showed initial burst of release. Human bone marrow stromal cells were cultured on scaffolds physisorbed with 20 µg/mL and cultured in basal medium (DDM group) or physisorbed and cultured in osteogenic medium or cultured on non-functionalised scaffolds in osteogenic medium. The human bone marrow stromal cells proliferated less in demineralised dentine matrix group and activated ERK/1/2 after both time points. Cells on DDM group showed highest expression of IL-6 and IL-8 at 7 days and expressed higher collagen type 1 alpha 2, SPP1 and bone morphogenetic protein-2 until 21 days. Extracellular protein revealed higher collagen type 1 and bone morphogenetic protein-2 at 21 days in demineralised dentine matrix group. Cells on DDM group showed signs of mineralisation. The functionalised scaffolds were able to stimulate osteogenic differentiation of human bone marrow stromal cells.

Liposomal Delivery of Demineralized Dentin Matrix for Dental Tissue Regeneration.

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-ß1 (TGF-ß1) enzyme-linked immunosorbent assays to quantify release. The encapsulation efficiencies were calculated to be 25.9% and 28.8% (VEGF/TGF-ß1) for 50 µg/mL DDM liposomes and 39% and 146.7% (VEGF/TGF-ß1) 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.