Placement of a restoration to treat root caries disrupts many tissues. There is scope for the restorative material to interact with these to augment reductions in micro leakage afforded by an adhesive restorative material.
1) To investigate the effects of incorporating bioactive molecules into a glass polyalkenoate (GPA) 2) To quantify the changes in physical properties of the material.
Biocompatibility of the GPA cement (Chemfil Superior, Dentsply De Trey, Konstanz, Germany) in unmodified and modified forms was ascertained using cell culture techniques. The optimum concentration of bioactive components required to promote cell attachment was determined indirectly by quantification and localisation of the fibroblast marker vimentin. The properties of surface hardness, compressive strength and adhesive bond strength were also determined prior to and following addition of the bio-additives: collagen type I and a pentapeptide containing Arg-Gly-Asp (RGD).
Addition of Type I Collagen (100 μg/ml) and RGD (5 mg/ml) to ChemFil Superior had no statistically significant effect upon the compressive strength and bond strength to bovine enamel but significantly (P < 0.05) increased the materials shore hardness. The addition of RGD to ChemFil Superior increased most the expression of vimentin, indicating that the cells had become more fibroblastic. This may be indicative of increased synthesis of extracellular matrix macromolecules with the potential to foster adhesion of the modified glass polyalkenoate to distracted gingival tissues.
The results suggest that addition of bioactive molecules to GPA cement for subgingival restorations has potential clinical applications.
It is possible to envisage that the additions, as described in this paper, could foster the attachment of displaced gingival tissues to GPA restorative materials placed subgingivally where root caries has been treated. This would offer potential to form a seal around the restoration by the attached gingival tissues avoiding a periodontal pocket and depriving residual cariogenic bacteria of a nutrient supply. Further investigation of the effects upon other similar materials of such additions is warranted.
Root surface caries has been defined as “ a soft irregular shaped lesion either (a) totally confined to the root surface or (b) involving the undermining of enamel at the cemento-enamel junction clinically indicating that the lesion initiated on the root surface” . Root surface caries usually occurs supragingivally at or close to (within 2 mm) the cemento-enamel junction (CEJ) . Experts generally agree that root caries can occur anywhere on the root surface occlusal to the gingival margins, but there are contradictory views about root lesions involving the cemento-enamel junction (CEJ). These relate to the classification of such lesions for some think they should be classed as root surface caries extending onto the crown or indeed as coronal caries extending onto the root or even both. This however is more a measurement issue than a diagnostic one .
The occurrence and location of root surface caries are usually associated with age and gingival recession. This is consistent with the idea that root caries occurs in a location close to the crest of the gingiva, where dental plaque accumulates. Root surface caries most commonly occurs on the proximal surfaces followed by the facial surfaces of the tooth .
The prevalence of root surface caries in the general population increases with age . This increase in prevalence is related to the longer retention of teeth in older people, than in previous generations. Also, root surfaces at this age become exposed due to gingival recession putting the root surface at greater risk . The occurrences of root surface caries can be prevented using a variety of preventive methods e.g. water fluoridation and use of fluoridated dentifrices. Maximum efficiency of prevention could be achieved if high-risk individuals were identified earlier and appropriate preventive methods instituted .
The management of root surface caries should start by preventive and remineralisation therapies that will help inhibit or eliminate the lesion before further damage to dental tissues occurs. Restorative treatment is indicated where there is excessive distraction of the tooth tissues by active root surface caries . It is generally accepted that root surface caries can be prevented or arrested by plaque removal, diet modification and topical fluoride application .
When the active caries root lesion progresses, it causes destruction of the root tissues and so restorative treatment is indicated to remove the caries and replace the destroyed tissues. Sometimes such lesions extend subgingivally and the clinician is faced with many difficulties in removing the caries. These include impaired visibility, limited access, limited moisture control, pulpal proximity and the nature of the dental tissues themselves .
Many different restorative materials may be used to restore the root surface. Glass polyalkenoate (Conventional/Resin modified) cements are considered to be the materials of choice for restorative treatment of most root surface caries lesions . This is because such materials provide good adhesion to the hard tissues of the tooth and have anti-cariogenic effects due to sustained fluoride release. Resin composite materials, although possessing aesthetic qualities, undergo polymerisation shrinkage and have no or limited fluoride release . As these materials are resin based, they are intolerant of moisture. In the past dental amalgam was used, but its use is to be phased down , on environmental grounds, and its poor aesthetics do not endear it to an increasingly demanding public .
Placement of a restoration for a root caries lesion involves the disruption of many tissues namely the attached gingiva, periodontal ligament, enamel, dentin and cementum. Scope therefore exists for surface interactions of the restorative material with the cell populations of both the attached gingiva and periodontal ligament. The main component of the extracellular matrix of the periodontal ligament and gingiva is comprised of Type I collagen and its bioactive motif (RGD) . This offers the potential for cellular attachment to these tissues to augment the reductions in restoration microleakage afforded by the adhesive bonding of glass polyalkenoates to the hard tissues of the tooth.
This work sought to identify bioactive additions, potentially suitable for chairside incorporation at the time of mixing into glass polyalkenoate cement, to foster cellular attachment to subgingival restorations of root caries lesions. Although not tested in this study, the purpose of these additions was to provide a tissue seal for such restorations with a view to depriving residual cariogenic bacteria of their nutrient supply. As such additions could potentially affect the physical properties of the materials, those thought most likely to contribute to clinical success were determined for the material tested in both unmodified and modified form.
Materials and methods
Preliminary work, utilising cell culture and viability assays, identified that the glass polyalkenoate (GPA) Chemfil Superior was sufficiently biocompatible to advance this work. The compositional details of this material are summarised in Table 1 .
|Manufacturer||DENTSPLY D E T REY GmbH 78467 Konstanz
|Composition||Powder (1 g)||– Aluminium-sodium-calcium-fluoro-phosphoro-silicate (18:9:8:16:3:46) 0.84 g
– Polyacrylic acid (MW 30000–45000) 0.15g
The methods used in this study are best described under the subheadings of
Determination of optimal concentration of bioactive additions to promote cellular adhesion.
Effects of bioactive additions on the cements properties
Unless otherwise stated GraphPad PRISM software (version 5.0, GraphPad Software Inc., San Diego, California, USA) was used for all statistical analysis. Statistical significance was signified at P < 0.05.
Determination of optimal concentration of bioactive additions to promote cellular adhesion.
The effects of two different bioactive additions [1- Type I Collagen (Prepared in house ) and 2- Gly-Arg-Gly-Asp-Ser (RGD) (SIGMA-ALDRICH, St. Louis, MO, USA)] to ChemFil Superior GPA at two different concentrations were investigated.
In summary the additions investigated were;
Type I collagen was added to the mixing water of the GPA in two different weight ratios, 10 μg/ml and 100 μg/ml, to form aqueous solutions of 0.1% and 0.01% type I collagen.
RGD was added to the GPA mixing water at 1 mg/ml and 5 mg/ml.
These concentrations were chosen with reference to previously conducted cell attachment studies .
All bioactive additives were incorporated into the material and mixed according to the manufacturers’ instructions at a powder: liquid ratio of 1: 1.
To make the necessary material specimens for this aspect of the investigation, Polytetrafluoroethylene (PTFE) disc shaped moulds, with an inner diameter of 12 mm and thickness of 2 mm were used. Prior to use, they were washed thoroughly with water and detergent (Lipsol ® , Scilabware, Stoke-on-Trent, UK) for 5 min, and then sterilised by exposure to ultraviolet light for 24 h. All subsequent preparations of the glass polyalkenoate cement, in any form, were carried out under sterile conditions using sterilised instruments. To achieve this scoops, droppers and plastic spatulas provided by the manufacturers were rinsed thoroughly in 70% (v/v) ethanol and left to dry under ultraviolet light for 24 h before use. All specimens of the unmodified glass GPA cement were mixed according to the manufacturers’ instructions and condensed using plastic instruments into the PTFE mould. To achieve smooth specimen upper and lower surfaces, the mould, containing unset GPA cement, was sandwiched between two PTFE plates before being compressed between two metal plates. After setting the disks were removed from the mould by gentle hand pressure, following unscrewing of the metal plates. To ensure sterility, this work was undertaken under sterile conditions under a microbiological safety cabinet. The set discs were glued to the centre of the dishes using superglue (SHERAMEGA 200, Espohsrabe 53, Lemforde, Germany). Once all the specimens had adhered, the dishes were labelled and placed beneath a microbiological hood under UV light overnight (lid removed) for sterilisation. The specimens were washed over the next 24 h with Hanks balanced salt solution then left in serum free medium (SF-MEM) overnight prior to the biological testing.
Cell culture and viability scores
Normal oral mucosa fibroblast cells (MM1), Source – Dr M Macluskey University of Dundee (14) were cultured in 90 mm dishes with 6 ml growth medium each. These were incubated at 37 °C in a CO 2 incubator with 5% CO 2 until confluent. For all subsequent experiments, cells from these were seeded in 60 mm dishes around and over the material specimens at a concentration of 5 × 10 5 cells/dish for 21 days at 37 °C and 5% CO 2 . Control dishes contained cells and medium only. The medium was changed every 48 h. The cells were monitored under the light microscope (Olympus IX70-S8F2, Olympus, South-End-On-Sea, UK) Photographs were taken of the cells close (adjacent to the attached material) to and remote (at the perimeter of the dish) from the specimens every 3 days. Ten examiners, experienced in cell biology, were asked to rank cell viability using a visual observation method as detailed in Table 2 . The effects of the material and additives on the cells were compared to that of the control using the scoring values as indicators of cell viability seen over all observation times. This was undertaken by conducting a non-parametric one-way ANOVA (Kruskal-Wallis test) test of the data with localisation of significant differences using Dunn’s multiple comparison test. A note of the most common ranking scores for each experimental group was also made.
|VIABILITY SCORE||BRIEF DESCRIPTION|
|1||Normal cell morphology and cell density|
|2||Altered cell morphology and/or small gaps between cells|
|3||Altered cell morphology and/or large gaps between cells|
|4||Few (or no) visible cells|
Cell viability testing (MTT assay)
The MTT Assay was performed to determine the optimum concentration for each bio-additive to improve biocompatibility of the glass ionomer cement and promote cellular adhesion. The MTT assay is a colorimetric assay for measuring cellular viability .
Disks made from ChemFil Superior, without and with additives, were placed inside the wells of sterile plastic 48 well plates. These were then sterilised overnight under UV light. Specimens were then washed over the next 24 h with Hanks balanced salt solution, then left in SF-MEM overnight.
MM1 cells were seeded in two 48-well culture plates (50,000 cells per well) at a concentration of 5 × 10 4 cells/ml for 24 h and 72 h at 37 °C and 5% CO 2 . This concentration had previously been found to be optimal in pilot work for the purpose of the experiment.
After 24 h of incubation, cell attachment, morphology and confluence was checked on both plates and 250 μl of fresh growth medium was added to each well of the second plate, which was then returned to the incubator for another 48 h at 37 °C and 5% CO 2 . The medium was removed from the wells of the first plate by aspiration and the wells were then washed twice with SF-MEM. 250 μl MTT solution was added to each well and the plate was then incubated at 37 °C, 5% CO 2 for 3 h. The MTT solution was then removed from the wells by gently tapping the contents against paper tissues. Subsequently, 250 μl of DMSO was added to each well and the plate was placed on an orbital shaker for 20 min at room temperature. Optical density of the wells was then measured using a Fluorostar Optima plate reader (FLUOstar Optima, BMG Labtech, Aylesbury, Bucks, UK) at a wavelength of 540 nm.
The same procedure was repeated for the second plate after 72 h of incubation at 37 °C, 5% CO 2.
The Absorbance values obtained for each well represent the amount of MTT reduction, which is proportional to the number of viable cells. In order to assess the percentage of viable cells present in each well, the absorbance values were related to those of the control. This was achieved by setting the mean absorbance of the control to 100%. The percentage of viable cells was calculated using the equation:
P e r c e n t a g e o f v i a b l e c e l l s = Absorbance value Mean Absorbance of the control X 100 %
The effects of the material modification state on the cells were compared to each other using the percent viability values as indicators of cell numbers. An unpaired t -test was used to analyse the data to determine the effect of material state on cell viability.
Examination for the expression of vimentin
The expression of vimentin gives a measure of the fibroblastic activity level of the cells used in this work. A number of techniques were used to assess this namely: Immunocytochemistry and Protein Biochemistry.
Immunocytochemistry (ICC) to localise and quantify vimentin expression
The objective was to label MM1 fixed in situ with primary antibody against vimentin and visualise this by labelling with a fluorescent secondary antibody. The protocol used was the standard laboratory procedure as described in Islam et al. with vimentin (R28 rabbit monoclonal antibody # 3932S, Cell Signalling Technology) and fluorescent secondary antibody (anti-Rabbit IgG conjugated with Alexa Fluor 488 (#4412, Cell Signaling Technology)).
This involved the analysis of lysates of MM1 cells cultured alongside the glass polyalkenoate with and without additions of biopolymers.
The normal oral mucosa fibroblast cell line (MM1) was cultured along with glass polyalkenoate and bio-modified glass polyalkenoate cements at an initial density of 0.5 × 10 6 cells per 60 mm dish. After 3 weeks in culture, the medium was aspirated from the dishes and the cells were washed 3 times using phosphate-buffered saline (PBS). Total cell protein was then harvested using a RIPA (Radio immunoprecipitation Buffer) cell lysis buffer containing protease inhibitors (# 04693132001, Roche Diagnostics, Burgess Hill, UK). 500 μl of lysis buffer was added to each dish followed by incubation on ice for 10 min; finally, each dish was scraped and the lysates were then collected in Eppendorf tubes and stored at −20 °C.
SDS PAGE (polyacrylamide gel electrophoresis)
Frozen lysates were thawed and then spun at 13000 rpm for 5 min. Samples were combined with equal volume of Laemmli loading buffer (BioRad, Hemel Hempstead, Hertfordshire, UK) and were heated at 95 °C for 5 min, prior to loading onto the Any KD gel (Bio Rad). 5 μl cell lysate was loaded per well, and 3 μl of Magicmark XP (Invitrogen Ltd, Paisely UK) was also loaded onto the gel, for molecular weight estimation. Gels were run at a constant voltage of 110–150 V in TGS running buffer (BioRad) until the dye front reached the bottom of the gel. The gel was then removed from the cassette and placed into transfer buffer.
Proteins were transferred from the gel to nitrocellulose membrane using a semi-dry blotter (TransBlot Semi-Dry Transfer Cell, BioRad, Hemel Hempstead, Hertfordshire, UK) and Towbin transfer buffer for 42 min at 15 V. After blotting, the membranes were blocked in 1% milk TBST for 10 min and then exposed to a 1:2000 dilution of the anti-vimentin antibody (Cell Signalling #3932, Leiden, The Netherlands) overnight. The blot was then washed 3 times with TBS-T for 20 min for 20 min each wash. The membrane was then incubated with a 1:2000 dilution of the secondary antibody (Cell Signaling Anti-rabbit IgG – HRP labelled no. 7074) for an hour. Membranes were then washed again with TBST as previously described and then incubated with SuperSignal ® West Pico chemiluminescent substrate (Thermo Scientific, Life Technologies Ltd., Paisley, UK). Finally, the chemiluminescence was detected and documented by using a BioRad gel doc system. Protein expression was quantified using ImageLab software (BioRad 4.0.1. build 6).
In order to assess the percentage of Vimentin expression by cells cultured with materials, the band densities, following normalisation for gel loading, was related to those of the control (MM1 + ChemFil superior). This was achieved by setting the density of the control to 100%.
P e r c e n t a g e o f v i m e n t i n e x p r e s s e d b y c e l l s = B a n d d e n s i t y o f e a c h m a t e r i a l B a n d d e n s i t y o f t h e c o n t r o l × 100 %