Role of connexin 43 in odontoblastic differentiation and structural maintenance in pulp damage repair

Abstract

Dental pulp can initiate its damage repair after an injury of the pulp-dentin complex by rearrangement of odontoblasts and formation of newly differentiated odontoblast-like cells. Connexin 43 (Cx43) is one of the gap junction proteins that participates in multiple tissue repair processes. However, the role of Cx43 in the repair of the dental pulp remains unclear. This study aimed to determine the function of Cx43 in the odontoblast arrangement patterns and odontoblastic differentiation. Human teeth for in vitro experiments were acquired, and a pulp injury model in Sprague-Dawley rats was used for in vivo analysis. The odontoblast arrangement pattern and the expression of Cx43 and dentin sialophosphoprotein (DSPP) were assessed. To investigate the function of Cx43 in odontoblastic differentiation, we overexpressed or inhibited Cx43. The results indicated that polarized odontoblasts were arranged along the pulp-dentin interface and had high levels of Cx43 expression in the healthy teeth; however, the odontoblast arrangement pattern was slightly changed concomitant to an increase in the Cx43 expression in the carious teeth. Regularly arranged odontoblast-like cells had high levels of the Cx43 expression during the formation of mature dentin, but the odontoblast-like cells were not regularly arranged beneath immature osteodentin in the pulp injury models. Subsequent in vitro experiments demonstrated that Cx43 is upregulated during odontoblastic differentiation of the dental pulp cells, and inhibition or overexpression of Cx43 influence the odontoblastic differentiation. Thus, Cx43 may be involved in the maintenance of odontoblast arrangement patterns, and influence the pulp repair outcomes by the regulation of odontoblastic differentiation.

Fig. 1

Odontoblast arrangement structure and Cx43 expression in the dental pulp. a Cx43 and α-tubulin immunofluorescence staining in rat, beagle, and human healthy dental pulp tissue. b Immunofluorescence staining of Cx43 and ZO-1 in dental pulp cells cultured in vitro. Od, odontoblasts

Fig. 2

Cx43 expression in the pulp tissue stimulated by caries. Cx43 (green) and α-tubulin (red) immunofluorescence staining of the odontoblast layer in teeth with moderate caries and healthy controls. The expression of Cx43 is slightly upregulated in caries teeth. Od, odontoblasts

Fig. 3

Cx43 expression and different dental pulp repair patterns in vivo. H&E and immunofluorescence staining in normal (a1–a3), inflammatory (b1–b3), necrotic (c1–c3), and reparative dentin pulp tissues (d1–f3) in rats. Regularly arranged odontoblast-like cells are present beneath acellular reparative dentin (d1–d3); however, these cells or structure are not detected beneath osteodentin (e1–e3)

Fig. 4

Cx43 expression and odontoblastic differentiation in vitro. a After differentiation induction, dental pulp cells were stained with Alizarin red. b Western blot was used to detect the protein expression level of DSPP and Cx43 after 7-day induction, and d 14-day induction. c Real-time PCR was used to assess the DSPP gene expression

Fig. 5

Odontoblastic differentiation of dental pulp cells with overexpressed or inhibited Cx43 in vitro. a Gene expression of GJA1 and DSPP, and b, c protein expression of DSPP and Cx43 at 7, 14, and 21 days were detected after inhibition or overexpression of the Cx43-encoding GJA1 gene. OE, over-expression group; control, OE null group; siRNA, gene silencing group; NC, siRNA null group; +, mineralization induction