Intra‐pulpal connective tissue formation and the advanced carious lesion: Is chondrogenesis and heterotopic ossification a response to pulpal inflammation?

Abstract Aims (a) The aim of this study was to investigate both the formation of dense connective tissue within the dental pulp, and its association with pulpal inflammation in teeth with advanced carious lesions; and (b) to investigate in vitro whether inflammation affects the expression of markers related to chondrogenesis/osteogenesis in pulp cells. Materials and methods Radiology and Histology: Forty‐six teeth with advanced carious lesions were radiographically investigated for intra‐pulpal radiodense structures. Specimens were processed for histology and stained with haematoxylin/eosin and proteoglycan‐specific stains. The intra‐pulpal connective tissue was scored as pulp stones or ectopic connective tissue. Cell culture: pulpal cells from human third molars ( n = 5) were cultured in chondrogenic medium +/− TLR2/4 agonists. Expression of the genes IL6 , TLR2/4 , SOX9 , COL1A1 , COL2A1 , TGFB1 , RUNX2 and ALPL was assessed by qPCR. Proteoglycan content within cultures was assessed spectrophotometrically. Results Radiodense structures were discovered in about half of all pulps. They were associated with ectopic connective tissue (χ 2 = 8.932, p = .004, OR = 6.80, 95% CI: [1.84, 25.19]) and with pulp stones (χ 2 = 12.274, df = 1, p < .001, OR = 22.167, 95% CI: [2.57, 200.00]). The morphology of the ectopic tissue resembled cartilage and was associated with inflammatory infiltration of the pulp (χ 2 = 10.148, p = .002, OR = 17.77, 95% CI: [2.05, 154.21]). After continuous stimulation of cultured cells with TLR2/4 agonists, the expression of two inflammatory markers increased: IL6 at Days 7 ( p = .020) and 14 ( p = .008); TLR2 at Days 7 ( p = .023) and 14 ( p = .009). Similarly, expression of chondrogenic markers decreased: SOX9 at Day 14 ( p = .035) and TGFB1 at Day 7 ( p = .004), and the osteogenic marker COL1A1 at Day 7 ( p = .007). Proteoglycan content did not differ between unstimulated and stimulated cells. Conclusions Ectopic connective tissue resembling cartilage can form in teeth affected by advanced carious lesions. This tissue type is radiographically visible and is associated with inflammatory infiltration of the pulp. Although TLR2/4 agonists led to an inflammatory response in cell culture of pulp cells, the effect on the expression of osteogenic/chondrogenic markers was limited, suggesting that immune cells are needed for connective tissue formation in vivo .