Elsevier

Acta Biomaterialia

Volume 117, November 2020, Pages 283-293
Acta Biomaterialia

Full length article
Human neutrophils compromise the restoration-tooth interface

https://doi.org/10.1016/j.actbio.2020.09.025Get rights and content

Abstract

Neutrophils, cells of the innate immune system, enter the mouth and release factors that are hypothesized to contribute to the degradation of tooth dentin, methacrylate resin composites, and adhesives at the restoration-tooth-dentin interface. The objectives were to characterize neutrophils’ degradation towards resin composite, self-etch (SE) and total-etch (TE) adhesives, SE and TE resin-dentin interfaces and to identify proteins that could contribute to the degradation process. Neutrophils’ degradation of cured resin composite, and SE and TE adhesives, was quantified by measuring the specific resin degradation by-product, bishydroxy-propoxy-phenyl-propane (bisHPPP), released after 30 days incubation of the materials with the cells. Neutrophils’ degradative effect on resin-dentin interfaces was examined by recording the interfacial fracture toughness (FT), and surface analysis of the fracture mode following incubation of SE and TE miniature short-rod (mini-SR) specimens with the cells. Neutrophils increased degradation of polymerized resin composite, and TE adhesive, but not SE adhesive over 30 days (p < 0.05). Incubation of SE and TE resin-dentin interfaces with neutrophils led to a reduction in FT over time (p < 0.05). The effect was more pronounced for TE interfaces. Neutrophils also affected the fracture mode of SE and TE resin-dentin interfaces. Several proteins that could contribute to the degradative activity of neutrophils, including Neutrophil collagenase (MMP-8), Matrix metalloproteinase- 9 (MMP-9), Cathepsin G, Neutrophil- gelatinase associated lipocalin (NGAL) and Myeloperoxidase, were isolated. The ability of neutrophils to degrade resin, tooth dentin, and reduce the bond strength of resin-dentin interfaces suggest neutrophils’ potential role in primary and recurrent caries and dental restoration failure.

Introduction

Tooth decay (dental caries) is the most prevalent disease in the world, and is responsible for the loss of millions of working days due to dental visits or dental related sick-days [1], [2], [3]. Caries are mostly treated by surgical removal of decayed tooth which is followed by filling the lesion with a restoration [4]. Currently the most common restorative material is resin composite [5]. In the United States more than 166 million restorative procedures are done each year with resin composite restorations costing approximately $30 billion [6]. Around 40–70% of restorations placed are replacements for failed restorations due to caries reoccurring at the interface between the restoration and the tooth (recurrent caries), with bacterial acid production thought to be the major etiological cause [7], [8], [9], [10]. Repeated restorations could lead to more complex treatment modalities, such as endodontic treatment, more extensive restoration and even tooth loss, ultimately reducing people's quality of life [11].

Resin composites are bonded to the tooth structure by either self-etch (SE) or total-etch (TE) adhesives [12]. After the decayed tooth material is removed, the adhesive can be applied before filling the cavity with the restorative material [4]. Resin adhesives interweave with the dentinal collagen fibrils (exposed from acid demineralization) and enter the dentinal tubules [4,12], then it is photocured and hardened, forming the hybrid layer (HL). Resin composite restorative material is then placed over the adhesive and co-polymerized with it [4,12]. This adherence/sealing of the tooth-restoration margins is critical for longevity of the restoration [4,12]. SE systems consist of acidic resin that demineralizes the tooth dentin, as monomers penetrate into the collagen fibrils before they are cured. In TE systems, the dentin is conditioned with an acid etchant that is rinsed prior to the application and curing of a primer and an adhesive onto the exposed dentinal collagen [4,12].

2,2-Bis[4-(2-hydroxy-3-methacryloxypropoxy)phenyl]propane (bisGMA) is used as a universal monomer in methacrylate-based dental resins and adhesives. [13]. Polymerized bisGMA is hydrolized by esterases and cholesterol esterase (CE)-like activities from saliva, bacteria and neutrophils, leading to release of bishydroxy-propoxy-phenyl- propane (bisHPPP) [4,14]. Following demineralization by bacteria or the acidic conditioning during the bonding procedure of the restoration, tooth dentin can also be broken down by protease, matrix metalloproteinases (MMPs) or MMP-like activity from the saliva, dentin, bacteria and neutrophils [14], [15], [16]. It has been shown that esterase, CE, and matrix metalloproteinase (MMP) activities in the mouth can reduce interfacial fracture toughness (FT) of resin-tooth bonded specimens through the degradation of the restoration and tooth over time 17,18]. This allows for increased penetration of the restoration-tooth interface by oral fluids and bacteria [19,20]. As a result of the interfacial degradation, additional degradation occurs and can result in failure of the restoration due to detachment or recurrent caries [4,21].

Very little is known regarding the role of neutrophils in the formation and progression of recurrent caries, albeit these cells have catabolic activities and constantly populate the areas prone to this disease [3,9,15,16,22]. Neutrophils are white blood cells of the innate immune system and their primary function is to defend the body against microorganisms [23]. In a healthy individual, neutrophils constantly enter the oral cavity in response to immune system stimulation by the resident microorganisms [24], [25], [26]. In patients with periodontal disease, there is an increased recruitment and/or overacting of neutrophils, which can cause collateral damage to surrounding hard and soft tissues [27]. Neutrophils mostly enter the mouth from the gingival sulcus and are attracted to and form a layer over the oral bacterial biofilm (dental plaque) [28]. At the gingival pocket neutrophils exocytose their granules that are filled with catabolic and antibacterial factors towards the oral biofilm in order to combat it [24,28]. The degradative ability of neutrophils have been recently identified [14]; neutrophils were shown to have esterase-like activities, breaking down the universal resin monomer bisGMA, and polymerized resin composite. Neutrophils also have matrix metalloproteinase activities and can break down demineralized tooth dentin [14]. This suggests a potential contribution of neutrophils to the degradation of the tooth and/or tooth filling material within the gingival pocket; however, an analysis of neutrophils’ effects on resin-dentin interfaces have yet to be done. Recurrent caries are also more commonly found at the gingival margins, and are often accounted for by bacterial acid production, but the presence of neutrophils and their degradative activities supports the potential role of neutrophils involvement in the caries process [5,9].

The current study further explores neutrophils’ potential role in resin composite restoration failure. The objectives of the current study are to measure the degradative effect of neutrophils towards cured resin composite, TE adhesive, SE adhesive, and the resin-dentin interface over a period of up to 30 days, and to perform an initial identification of proteins from neutrophils that could be involved in the degradation of resin composites and human dentinal collagen. We hypothesize that human neutrophils degrade polymerised methacrylate resin composite and adhesives and the restoration-tooth interface in a material-dependent manner. This degradation may have clinical significance by contributing to resin-dentin interfacial breakdown, thus allowing for salivary and bacterial penetration, further interfacial degradation and formation of secondary caries [17], [18], [19], [20].

Section snippets

Neutrophil isolation

Healthy humans donated peripheral blood (University of Toronto Ethics Review Boards, Protocol # 38504). Inclusion criteria were 18 years of age or older, healthy, and not taking medication which could interfere with the normal function of white blood cells. Neutrophils were isolated by gradient separation, by layering blood onto 1-step polymorph (Accurate Chemical & Scientific Corp, Westbury, NY, USA) [29]. Samples were centrifuged at 500xG (24 °C) (Labnet Hermle Z400K centrifuge, Labnet

CE-like activities of neutrophils with stimulating factors

A statistically significant increase in average hourly CE-like activity was found only between the solution with neutrophils alone and the solution of neutrophils with 1 μg/mL LPS (p = 0.04) (Fig. 1). Based on this, 1 μg/mL LPS was chosen for biodegradation and fracture toughness studies described below in Sections 3.2, 3.3 and 3.4.

Biodegradation of polymerised resin composite and adhesives by neutrophils

Significantly more bisHPPP was released from resin composite when incubated with neutrophils compared to control after incubation period of 15 days or longer

Discussion

This study provides evidence that neutrophils can hydrolyze polymerized methacrylate resins (resin composite and total-etch adhesive, but not self-etch adhesive) and affect resin-dentin interfaces in a material-dependent manner, confirming our hypothesis. Initial analysis to identify the enzymes from neutrophils that could potentially be responsible for the breakdown of resins and restoration-tooth interfaces was performed, however the exact contribution of the factor(s) still remain unknown.

Conclusions

The results of this study provide knowledge on the potential contribution of neutrophils towards restoration failure via their ability to degrade the resin-dentin interface in vitro. Neutrophils showed a long-term degradative effect towards resin materials and resin-dentin interfaces made with self-etch and total-etch adhesives, reported on for the first time in this study. The above degradative impact on the restoration-tooth interface was much more rapid than previous reports on saliva and

Funding

This work was supported by the Canadian Institutes of Health Research [PJT-165 957]; Canada Foundation for Innovation John R. Evans Leaders Fund (CFI_JELF) [project #35378], and Ministry of Research and Innovation (MRI), Ontario Research Fund (ORF) [ORF-35 378].

Data availability

The raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study.

Statement of significance

This study provides evidence that neutrophils, the host's innate immune cells, could potentially impact dental restoration service life. Neutrophils enter the mouth at the gingiva as a defense mechanism against microorganisms. Neutrophils accumulate over the dental biofilm (plaque) that covers the tooth and restoration. They become activated in response to bacteria, releasing the cell's catabolic granule contents, comprising of degradative proteins. Recurrent caries frequently occurs at

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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