Canonical and Noncanonical WNT-Loaded Hydrogel for Dentin-Pulp Regeneration

Canonical and noncanonical WNT signaling pathways are tissue specific and play roles in mineralized tissue development and repair. Here we characterize the activity of noncanonical WNT5B and canonical WNT10B in dental pulp stem cells (DPSCs) in osteo-/odontogenesis and target dentin-pulp complex regeneration using rWNT-loaded hydrogels. DPSCs in monolayers were treated with recombinant WNT10B (rWNT10B, 50 ng/mL), rWNT5B (50 ng/mL), or the canonical WNT pan-activator CHIR 99021 (10 nM). Gene expression of osteo-/odontogenic markers and Alizarin Red assay were performed. Collagen-based hydrogels were prepared from bovine type I collagen and loaded with rWNT10B, rWNT5B, or CHIR. A control group with no treatment was included. WNT release from hydrogels in phosphate-buffered saline was performed up to 14 d. The DPSC response to hydrogels was characterized by viability, morphology, gene expression, and alkaline phosphatase (ALP) activity. In vivo investigation was conducted using an ectopic model of pulp exposure in human tooth fragments implanted in mice for 4 wk. Hematoxylin and eosin, trichrome staining, and immunohistochemistry were performed. Statistical analysis was performed using analysis of variance and Tukey post hoc (α = 0.05). Our in vitro studies showed that rWNT5B significantly upregulated ALP, RUNX2, and DMP1 after 7 d. rWNTs and CHIR significantly enhanced mineralization. rWNTs were almost completely released from the hydrogels in 14 d, with 2 release peaks at 6 h and 4 d. rWNT-loaded hydrogels did not affect cell viability and morphology. rWNT10B-loaded hydrogels significantly increased the expression of ALP and RUNX2. The rWNT-loaded hydrogels significantly increased ALP activity. Our in vivo histological data indicate the presence of gel in the exposure area, the formation of pulp-like tissue in contact with the hydrogels, new blood vessels, and polarized cells adjacent to dentin tubules. Canonical WNT10B and noncanonical WNT5B appear to play specific roles in DPSC differentiation. WNT-loaded hydrogels are cytocompatible, increase the expression of osteo-/odontogenic markers and ALP activity, and induce pulp-like tissue formation.