Research ArticleRecruitment of dental pulp cells by dentine and pulp extracellular matrix components
Highlights
► Dentine & pulp extracellular matrix (ECM) molecules are chemoattractants for primary pulp cells. ► Chemoattractant effects of ECM were enhanced following physiologically relevant degradation. ► Pulp cell migration in response to dental ECMs was dependent on an active rho pathway. ► ECM recruited pulp cells exhibited increased stem cell marker expression. ► Release of dental ECM molecules in disease contributes to cell recruitment necessary for repair.
Introduction
Wound healing responses consist of a progressive series of events that occur with the purpose of restoring tissue integrity [1]. For dental tissue regeneration, this sequence of events occurs following severe or deep caries, in a process known as reparative dentinogenesis [2]. Localised dental tissue damage results in the death of primary odontoblasts and these cells are subsequently replaced by odontoblast-like cells which differentiate from precursor cell populations from within the pulp [3]. During reparative dentinogenesis, progenitors, such as dental pulp stem cells (DPSCs) or other pulpal stem/progenitor cell populations, migrate, proliferate and differentiate to give a new generation of odontoblast-like cells, which secrete new mineralised matrix for dental tissue repair and regeneration [4], [5], [6], [7]. The signalling pathways involved in the recruitment and differentiation of progenitor cells remain to be fully elucidated; however, several different factors have been postulated to influence their recruitment and differentiation [8], [9], [10].
Extracellular matrix molecules from dental tissues, including dentine (dECM) and pulp (pECM), are factors that contribute to signalling repair processes. Notably, dECM contains a range of bioactive molecules which have previously been shown to induce odontoblast differentiation and mineralisation both in vitro and in vivo [5], [11], [12], [13]. Indeed, small integrin-binding ligand, N-linked glycoprotein (SIBLING) family members which are present and released from dentine, such as bone sialoprotein, dentine sialoprotein, dentine phosphoprotein, and dentine matrix protein-1 have been implicated in signalling different aspects of reparative dentinogenesis [10], [14]. Another bioactive ECM derived SIBLING molecule, Dentonin which is a 23-amino-acid peptide derived from matrix extracellular phosphoglycoprotein (MEPE), has also been shown to enhance both DPSC proliferation and recruitment, processes vital for wound repair [15], [16], [17], [18].
Stem or progenitor cell recruitment is a key first step in the wound healing process and various molecules within ECMs from dental tissues (cementum, dentine and enamel) have been implicated in stimulating the migration of cells during dental disease [19]. Dentine phosphoprotein (DPP) and dentine sialoprotein (DSP) have been shown to induce the migration of neutrophils [20], while TGF-β1 induces the migration of mouse dental papilla derived cells [21]. Basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) alone, or in combination with nerve growth factor (NGF) and bone morphogenetic protein−7 (BMP-7) also reportedly induce pulp cell homing [22]. A recent study demonstrated that the ECM proteins laminin, fibronectin and collagen type-1, as well as growth factors contained within ECM, such as epidermal growth factor (EGF), fibroblast growth factor (FGF) and TGF-β1, and sphingosine−1–phosphate (S1P) also promoted the migration of a dental pulp stem cell line [23].
The mechanisms which underpin cell migration are divided into phases stages which include cell process extension, formation of focal adhesions, contraction of the body of the cell and tail detachment [24]. Three members of the rho GTPase family have been identified in regulating cell migration in general and include rho, rac and cdc42 [25], [26]. However, to date, the underlying mechanism of signalling of dental pulp cell migration has not been fully characterised.
Thus far, research into the effects of dental tissue derived ECM molecules has mainly focused on their bioactivity in an intact state. However, during caries, dental ECM molecules become degraded due to the action of acids and proteolytic enzymes derived from both infecting micro-organisms and host cells due to the innate immune defense response [27], [28], [29], [30], [31], [32], [33], [34]. Previous studies have demonstrated that degradation molecules and peptides derived from ECM are potent cell signalling molecules [35], [36] and studies in skin and bladder, have demonstrated that these molecules induce cell migration [37], [38], [39], although no data are available on partially degraded dental ECM molecules.
The signalling environment present within a diseased tooth is likely to be extremely complex and contains both intact and partially degraded dentine and pulp ECM alongside molecules derived from host cells. The present study therefore aimed to determine whether intact dentine tissue and preparations of ECM from pulp and dentine influence pulp cell migration. Pulp and dentine ECM preparations were also subjected to physiologically relevant degradative conditions to determine whether the relatively harsh conditions associated with carious disease modified the activity of these molecules. In addition, the intracellular signalling pathway involved in the migration of dental pulp cells was investigated together with the phenotypic characteristics of migratory and non-migratory pulpal cells.
Section snippets
Preparation of pulp and dentine ECM extracts
Pulps were dissected from 6 month old freshly extracted bovine mandibular incisor teeth and minced prior to homogenisation in 0.5 M NaCl (pH adjusted to 11.7) containing protease inhibitors (25 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 5 mM N-ethylmaleimide) and 1.5 mM sodium azide with gentle agitation for 24 h at 4 °C. The extraction was repeated three times with recovery of solubilized components by centrifugation followed by two further extractions with 1 ml 0.1 M tartaric acid solution (pH 2.0)
Chemoattractive activity of dentine
Primary dental pulp cells demonstrated preferential migration towards fragmented dentine particles (>60 μm dentine chips and >60 μm finely powdered) included within the agarose gels (Fig. 1A). The number of migrating pulp cells increased by 81% in the presence of dentine powder and by 37% in the presence of dentine chips within the agarose gel compared with the negative control agarose gels containing PBS solution (Fig. 1).
Chemoattractive activity of dECM and pECM
Using phase contrast microscopy, migration of pulp cells within the
Discussion
The present study demonstrated that intact dentine tissue (dentine chips/fragments) and ECM extracts from dentine and pulp could enhance primary pulp cell migration. Interestingly, powered dentine recruited higher numbers of pulp cells than dentine chips, possibly due to the increased surface area of the powered dentine which potentially provided greater exposure to the chemoattractant signals. Further study also demonstrated that acidic and enzymatic breakdown products of dental ECMs were also
Conclusions
ECM molecules released from the tooth during tissue injury promote pulp cell migration. The chemotactic effects of these molecules were enhanced when exposed to degradative conditions relevant to disease. The migration of pulp cells in response to dental ECMs was shown to be dependent on an active rho pathway and the recruited cells exhibited increased stem cell marker expression. ECM molecules and their breakdown products likely influence dental pulp stem cell migration in vivo during repair
Conflicts of interest
The authors deny any conflicts of interest.
Acknowledgments
This research was supported by the School of Dentistry, University of Birmingham.
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