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Article

In Vitro Coating Hydroxyapatite with 2-Heptylcyclopropane-1-Carboxylic Acid Prevents P. gingivalis Biofilm

by
Emily C. Montgomery
1,
Madelyn C. Wicker
1,
Tibirni Yusuf
1,
Elizabeth Matlock-Buchanan
1,
Tomoko Fujiwara
2,
Joel D. Bumgardner
1 and
J. Amber Jennings
1,*
1
Department of Biomedical Engineering, The University of Memphis, Memphis, TN 38152, USA
2
Department of Chemistry, The University of Memphis, Memphis, TN 38152, USA
*
Author to whom correspondence should be addressed.
Hygiene 2024, 4(4), 500-512; https://doi.org/10.3390/hygiene4040037
Submission received: 22 July 2024 / Revised: 22 October 2024 / Accepted: 31 October 2024 / Published: 7 November 2024
(This article belongs to the Section Oral and Dental Hygiene)
Browse Figures
Figure 1
<p>Schematic shows experimental workflow. Created in BioRender.com.</p> "> Figure 2
<p>Total 2CP loaded onto hydroxyapatite coupons (<span class="html-italic">n</span> = 3) determined by sum of three ethanol washes.</p> "> Figure 3
<p>Images depict contact angles for unloaded and 2CP-loaded HAp coupons (<span class="html-italic">n</span> = 5).</p> "> Figure 4
<p>(<b>A</b>) Individual and (<b>B</b>) cumulative amount of 2CP released as percentage of loaded amount from 2CP-loaded and 2CP+oral rinse-loaded hydroxyapatite samples (<span class="html-italic">n</span> = 6).</p> "> Figure 5
<p>Planktonic viability of <span class="html-italic">P. gingivalis</span> for HAp coupon groups (<span class="html-italic">n</span> = 3). “Before” and “after” refer to before and after 3-day elution. No significant differences were determined.</p> "> Figure 6
<p>Biofilm viability of <span class="html-italic">P. gingivalis</span> on HAp coupon surfaces (<span class="html-italic">n</span> = 3). “Before” and “after” refer to before and after 3-day elution. **** indicates significant difference (<span class="html-italic">p</span> &lt; 0.0001), and *** indicates significant difference (<span class="html-italic">p</span> &lt; 0.001).</p> "> Figure 7
<p>Photographs feature LIVE/DEAD images of biofilm growth on HAp coupon groups (<span class="html-italic">n</span> =1). “Before” and “after” refer to before and after 3-day elution.</p> "> Figure 8
<p>Viability of Saos-2 cells on HAp coupon surfaces (<span class="html-italic">n</span> = 3). ** indicates significant difference (<span class="html-italic">p</span> &lt; 0.01). Significant differences only shown for comparisons to control. Red line represents 70% threshold for viability outlined in ISO 10993-5 [<a href="#B34-hygiene-04-00037" class="html-bibr">34</a>].</p> "> Figure 9
<p>Images display LIVE/DEAD images of Saos-2 cell growth on HAp coupon groups (<span class="html-italic">n</span> = 1).</p> ">
Review Reports Versions Notes

Abstract

:
Infections are a common post-operative ailment for patients who have received a dental implant or device and can be attributed to biofilm formation in tissue or on the implant. Many current solutions for oral hygiene have side effects and affect the natural oral microbiome. 2-heptylcyclopropane-1-carboxylic acid (2CP) is a medium-chain fatty acid and synthetic diffusible signaling factor that can prevent and disperse biofilm. The purpose of this work was to evaluate an immersion strategy for coating hydroxyapatite (HAp) with 2CP to prevent biofilm attachment on and around natural teeth and dental implants. The release profile of 2CP-loaded and 2CP+oral rinse-loaded HAp coupons (n = 6) was assessed by a 3-day exposure to phosphate buffered saline (PBS). Antimicrobial properties against Porphyromonas gingivalis and cytocompatibility of 2CP-loaded HAp coupons (n = 4) were also assessed alone and in combination with 0.12% chlorhexidine gluconate oral rinse. The majority of 2CP is released by 12 h. 2CP, oral rinse, and 2CP+oral rinse significantly reduced P. gingivalis viability, though direct contact assay demonstrates a significant reduction in Saos-2 viability for oral rinse and 2CP+oral rinse coupons. Immersion or rinsing hydroxyapatite with 2CP could inhibit biofilm-associated dental infections and prevent further complications including caries, gingivitis, and peri-implantitis.

1. Introduction

Approximately ten million dental implants are implanted each year, with that number expected to increase by 2050 when the ratio of older adults (65 years and older) when compared to working age adults increases by 15%, from 25% up to 40% [1,2,3]. With over 700 different bacterial species populating the oral cavity, implanted dental biomaterials are often susceptible to bacterial contamination because they provide a substrate for bacterial attachment [4,5,6].
Porphyromonas gingivalis (P. gingivalis) is a significant contributor to periodontal disease and biofilm-associated infections among these pathogens [7]. P. gingivalis is a key pathogen implicated in periodontal disease and is commonly found in biofilms on dental implants and natural teeth [8]. If biofilm-associated infections are not addressed, there can be loss of gum tissue and bone, and bacteria can enter the bloodstream, traveling throughout the body and potentially causing cancers and chronic diseases including cardiovascular disease, diabetes, chronic kidney disease, Alzheimer’s disease, and rheumatoid arthritis [9,10]. Metallic implants are widely used due to high tensile strength, ductility, toughness, wear resistance, fatigue resistance, and creep resistance [11]. Still, metal implants also have limited biocompatibility and high corrosion in the biological environment, with failure occurring in 5–11% of dental implants [12,13,14,15,16]. Peri-implantitis is the primary reason for late-stage dental implant failure, accounting for 81.9% of late-stage failures [17]. On the contrary, ceramic coatings for implants have high biocompatibility, corrosion resistance, and inertness; however, these materials also have low impact resistance, reproducibility, and susceptibility to peri-implantitis [13]. Coatings for metal implants play an important role in promoting osseointegration and preventing infection. Chitosan-based coatings support bone growth and have reported antimicrobial properties [18]. Some of the other properties of chitosan-based coatings are biocompatibility, non-toxicity, regenerative properties, natural availability, and versatility as a drug carrier matrix, showing a natural antimicrobial activity against Streptococcus gordonii, one of the first bacteria to colonize surfaces in the oral cavity. Chitosan-based coatings have low stiffness and strength, and as such, the coating is usually modified as chitosan sol-gel preparations to improve their mechanical properties. It has been proven that chitosan/HA coatings protect the porous surfaces. [18] Hydroxyapatite (HAp) is a naturally occurring and non-toxic bioceramic making up 70% of human bone weight and constituting dental enamel and dentin [19]. HAp is also the most widely used calcium phosphate for metal implant coatings [13,20]. Coating titanium implant surfaces with HAp has shown enhanced integration with bone, improved corrosion and wear resistance, and decreased metallic ion release, compared to commercial titanium alloy implant surfaces [21,22]. Hydroxyapatite coatings support bacterial and fungal biofilm growth, however [23,24], and bacterial biofilm can destroy hydroxyapatite coatings and bone [25]. Crowns for dental implants are commonly made of ceramics like porcelain or zirconia, due to their durability and mechanical properties [26].
Previous research has suggested that unsaturated fatty acids like cis-2-decenoic acid (C2DA) and trans-2-decenoic acid (T2DA) disperse and inhibit biofilm formation [27,28]. Diffusible signaling factors such as these fatty acids also cause biofilm dispersal, increasing the effect of antibiotics through synergy and preventing implant removal, tissue debridement, and patient trauma [29]. Many DSF molecules become less effective when exposed to light, radiation, and other sterilization methods, but a synthetic DSF that is stable and resistant to isomerization by light, radiation, and sterilization has been developed; 2-heptylcyclopropane-1-carboxylic acid (2CP) is a medium-chain fatty acid and is useful for biomaterials due to its structural stability, cell compatibility, and antimicrobial activity [30]. Overall, the use of unsaturated cis isomers of fatty acids could be used to reduce oral biofilm formation. In studies by Harrison et al., an immersion method for loading 2CP on titanium resulted in bacterial and fungal inhibition for 72 h [31].
The overall goal of this work is to develop a simple 2CP loading strategy for teeth and HAp-coated dental implants to prevent dental plaque formation and subsequent occurrence of caries, gingivitis, and peri-implantitis. It was hypothesized that 2CP-loaded HAp would inhibit biofilm formation of dentally relevant bacteria while remaining cytocompatible with osteoblast cells.

2. Materials and Methods

To assess the qualities of 2CP-loaded HAp and the interaction of 2CP with commercial oral rinse, a 2CP coating for HAp was fabricated, the release profile of 2CP from HAp coupons with and without the presence of oral rinse was evaluated, the antimicrobial activity of 2CP released from HAp coupons, alone and in combination with oral rinse, against P. gingivalis was determined, and the cellular response of Saos-2 cells to 2CP released from Hap coupons, alone and in combination with oral rinse, was determined (Figure 1).

2.1. Loading and Characterization

2-heptylcyclopropane-1-carboxylic acid (2CP) was fabricated in a chemistry laboratory at the University of Memphis. Hydroxyapatite Disc Coupons were purchased from BioSurface Technologies Corporation (Bozeman, MT, USA). The manufacturer measures the composition of the coupons through X-ray diffraction to guarantee a minimum composition of 95% hydroxyapatite. Coupons are machine-pressed to achieve 0.5-inch (12.7 mm) diameter and 0.15-inch (3.8 mm) thickness. Based on methods from a study with alumina particles and the long-chain alcohols decanol, dodecanol, and octadecanol [32], an immersion loading method was developed for loading hydroxyapatite with fatty acids in methods similar to previous evaluations of titanium materials [31]. HAp coupons were immersed in 2CP in EtOH for 3 h at 40 °C and 50 rpm in capped vials. Loaded coupons were then dried overnight in a fume hood. Commercial dental rinse containing chlorhexidine was obtained to compare its effect on 2CP elution as well as to compare its antimicrobial properties to 2CP alone. The dental rinse chosen was 0.12% chlorhexidine gluconate oral rinse (Xtrrium, Madrid Dental Supply, Chatsworth, CA, USA), which is commonly prescribed for use after dental implant surgeries [33]. For loading HAp with 2CP+oral rinse, HAp coupons were immersed in 2.5 mg/mL 2CP in 0.12% chlorhexidine gluconate (CHG) oral rinse solution for 3 h at 40 °C in capped vials. Loaded coupons were then dried overnight in a fume hood.
To determine actual amount loaded, HAp coupons were soaked in four different concentrations of 2CP 100% ethanol solution: 30 mg 2CP/3 mL ethanol, 15 mg 2CP/3 mL ethanol, 7.5 mg 2CP/3 mL ethanol, and 3.75 mg 2CP/3 mL ethanol. Loaded coupons (n = 3) were washed with 100% ethanol and the wash-off solution was analyzed with HPLC-UV. This washing procedure was repeated until a minimal amount of fatty acid was present in the wash-off samples.
Contact angle measurements were collected for unloaded and 2CP-loaded HAp coupons (n = 3) using a VCA Optima measurement machine (Version 2.1.0.1, AST products, INC, Billerica, MA, USA) to determine the hydrophobicity of the coupon surface as a representation of whether 2CP was present. Water droplets (5 μL) were placed carefully onto the coupon surfaces, and after approximately one minute to allow any water to absorb, a digital camera recorded photographs of the droplets and the VCA OptimaXE goniometry software (AST Products Inc., Billerica, MA, USA) calculated the angle measurements.
Scanning electron microscopy (SEM) (Nova NANOSEM 650 FEI™, Hillsboro, OR, USA) was performed to characterize the HAp coupons and observe any differences in the surfaces of unloaded and 2CP-loaded coupons. Unloaded and 2CP-loaded HAp coupons (n = 1) were mounted on metal SEM stubs and a Au 80 Pt 20 sputter coating with 5 nm thickness was applied using a Q150T ES Plus turbomolecular pumped coater (EMS Quorum). SEM images were taken at multiple magnifications.
A fatty acid pre-column derivatization method was used to express 2CP in samples before HPLC analysis. After plating 100 µL of samples and standards in 50:50 PBS–methanol, 67 µL of 50 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in 10% pyridine and 24 µL of 100 mM indole-3-acetic acid hydrazide were added. To start the reaction, 10 µL of 100 mM hydrochloric acid was added before incubating at 50 °C for 3 h. After incubation, 10 µL of 200 mM sodium hydroxide was added to samples and standards to terminate the reaction. The concentration of 2CP in samples was measured with high-performance liquid chromatography–ultraviolet spectroscopy (HPLC–UV) using a reverse phase Hypersil GOLD Column (ThermoFisher Scientific, Waltham, MA, USA) with dimensions 150 × 4.6 mm. The mobile phase system was an 80:20 ratio of methanol–0.1% o-phosphoric acid at a flow rate of 0.800 mL/min for 8 min per sample, and the injection volume was 5 µL. The column was maintained at a temperature of 30 °C, and UV spectra were recorded at 220 and 280 nm.

2.2. Elution

Elution studies were performed to determine the release profile of 2CP from HAp coupons. Groups included 2.5 mg/mL 2CP-loaded (5 mg 2CP in 2 mL EtOH), 2.5 mg/mL 2CP+oral rinse-loaded, ethanol-loaded, and unloaded coupons (n = 6). HAp coupons were placed in 1 mL sterile 1X PBS in sterile well plates, and eluates were collected by complete solution change at 3, 6, 9, 12, 24, 36, 48, 60, and 72 h. The concentration of 2CP in samples was measured with HPLC–UV using the previously mentioned method. The following equation was used to calculate the percentage of 2CP released at each time point, where amount of 2CP lost during loading was determined by performing the loading procedure in plastic tubes without coupons (n = 3):
2 C P   R e l e a s e d % = 2 C P   i n   E l u t i o n   S a m p l e   ( µg ) 2 C P   i n   S o l u t i o n   b e f o r e   L o a d i n g µg − 2 C P   i n   S o l u t i o n   a f t e r   L o a d i n g µg − 2 C P   L o s t   d u r i n g   L o a d i n g µg × 100

2.3. Biofilm Prevention Direct Contact Assay

Biofilm prevention properties of 2CP-loaded, 2CP+oral rinse-loaded, oral rinse-loaded, and unloaded hydroxyapatite coupons (post-elution and fully loaded) were evaluated by direct contact assay (n = 4). Coupons were transferred to sterile well plates and inoculated with approximately 105 CFUs of P. gingivalis by the addition of bacteria solution in 100 µL increments to avoid significant loss of bacteria from the surface of the coupon. After 24 h, coupons were removed from wells, rinsed with sterile PBS to remove planktonic bacteria, and sonicated for 5 min at 40 kHz to remove biofilm-associated bacteria. Because the bacteria solution was added using a method to retain bacteria on the surface of the coupon, biofilm on the wells from which coupons were removed was considered negligible. Bacteria quantification (n = 4 planktonic; n = 3 biofilm) was determined using BacTiter-Glo® Microbial Cell Viability Assay (Promega, Madison, WI, USA). Groups were compared using ordinary one-way ANOVA with Tukey’s multiple comparisons test. Select coupons from each group (n = 1) were imaged with FilmTracer LIVE/DEAD assay using a Cytation 1 Imaging Reader (BioTek Instruments Inc., Winooski, VT, USA) and Gen5 software (Version 3.05, Agilent, Santa Clara, CA, USA).

2.4. Cytocompatibility Direct Contact Assay

2CP-loaded, 2CP+oral rinse-loaded, oral rinse-loaded, and unloaded hydroxyapatite coupons were assessed to identify cellular responses of Saos-2 osteoblast cells via direct contact assay. After loading, coupons were transferred to sterile 12-well plates, and Saos-2 Human Osteosarcoma Cell Line cells (ATCC, Manassas, VA, USA) were seeded at 1 × 104 cells/cm2 and grown in Dulbecco’s Modification of Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% GlutaMAX™ (Gibco®, Waltham, MA, USA) and without sodium pyruvate at 37 °C and 5% CO2. After 24 h, wells were imaged using a Cytation 1 Imaging Reader (BioTek Instruments Inc., Winooski, VT, USA), and cell viability was quantified using CellTiter-Glo® Viability Assay (n = 3; Promega, Madison, WI, USA).

2.5. Statistical Analysis

Results were normalized as percent viability versus cells grown on blank tissue culture plastic. Groups were compared using ordinary one-way ANOVA with Tukey’s multiple comparisons test. Statistical significance was determined at an alpha value of 0.05. Select coupons from each group (n = 1) were stained and imaged with LIVE/DEAD Viability/Cytotoxicity Kit using the Cytation 1 Imaging Reader (BioTek Instruments Inc., Winooski, VT, USA) and Gen5 software (Version 3.05, Agilent, Santa Clara, CA, USA).
There were no animal or human studies involved, and therefore, ethical approval was not needed.

3. Results

3.1. 2CP Is Retained on the Surface of HA

Based on the ethanol wash study, about 1.5% of 2CP from the loading solution is retained on the coupon surface after the loading procedure, which is about 100 µg for 2.5 mg 2CP in loading solution (Figure 2). Contact angles also indicate that loading HAp coupon with 2.5 mg/mL of 2CP reduces the wettability of the coupon surface (Figure 3).

3.2. 2CP Is Released in a Burst Profile over 12 Hours

Elution testing revealed that both 2CP-loaded and 2CP+oral rinse-loaded HAp coupons exhibited a burst release profile of 2CP, with nearly all of the 2CP being released within the initial 9–12 h (Figure 4). After the 12 h timepoint, there were small amounts under 50 ug at 24, 36, and 72 h with almost 100% of the 2CP + Oral Rinse being released, while only 75% or less of the 2CP + ETOH was released by the end of the 72 h.

3.3. 2CP Inhibits Biofilm Formation

2CP and oral rinse groups (before and after elution) reduced planktonic growth, compared to the unloaded control; however, controls have a high standard deviation due to one sample, and no statistical differences were detected between groups (Figure 5). 2CP and oral rinse groups reduced biofilm directly on coupons, compared to the unloaded control; however, post-elution 2CP-loaded coupons did not reduce biofilm significantly (Figure 6). Significant differences are only shown in figures for comparisons to unloaded HAp control and between the before and after elution groups. LIVE/DEAD staining produced comparable results, with oral rinse-loaded HAp eliminating the majority of biofilm growth on the coupon and slightly more growth for 2CP-loaded HAp groups (Figure 7).

3.4. 2CP Supports Osteoblast Viability

While oral rinse-containing groups significantly affected cell viability, 2CP-loaded coupons did not significantly affect Saos-2 viability compared to the TCP control (Figure 8 and Figure 9).

4. Discussion

Oral hygiene is a critical factor in maintaining healthy peri-implant tissue and preventing further tooth loss and periodontitis [35,36]. Patients are often given oral hygiene instructions, which include brushing, flossing, and use of antiseptic rinses. In this in vitro study, results suggest that a biofilm inhibitor may manage the oral microbiome and plaque formation on the implant and surrounding teeth. Combining 2CP with antimicrobials such as chlorhexidine may have longer-lasting effects on plaque removal and inhibition. Reducing peri-implantitis supports bone osseointegration and restoration of function.
Contact angles for unloaded and 2CP-loaded coupons indicate a hydrophobic surface after loading, suggesting that the majority of 2CP molecules are oriented with the hydrophobic portion exposed while the hydrophilic carboxylic acid portion is closest to the hydroxyapatite. If the majority of the 2CP molecules were oriented with their carboxylic acid functional group exposed, the wettability of the coupon would be greater, and the contact angle would be less than the reported contact angles in these studies. This finding is important, as less bacterial adhesion occurs on hydrophobic surfaces compared to hydrophilic oral surfaces [6]. As these studies were limited in the characterization of the 2CP-loaded HAp surface, other coating characterizations, including attenuated total reflectance Fourier-transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), and Raman spectroscopy, would be useful for understanding how 2CP is adsorbing and orienting on the HAp surface. ATR-FTIR was attempted with unloaded and 2CP-loaded HAp coupons; however, surface roughness of coupons caused signal noise.
Although elution testing suggests complete release of 2CP from the coupon surface in 12 h, this release profile would be acceptable for applying 2CP to teeth and dental implants, as mouthwashes are often prescribed for use twice daily. The elution results that the majority of the loaded 2CP is released by 12 h when sampling every 3 h support the bacterial result that previously eluted coupons were not able to significantly reduce P. gingivalis viability. Direct application to hydroxyapatite significantly reduces biofilm and planktonic bacteria in the short term but not after 72 h when the majority of 2CP is washed off, suggestive of weak adherence of 2CP to the ceramic material. Biomaterial matrices such as sponges and nanofibers can be used for localized long-term delivery, such as metronidazole. In studies of chitosan ESCMS, 2DA analogs like 2CP have been found to release over the course of 3 days [37]. Oral rinse-loaded and 2CP+oral rinse-loaded HAp coupons had the greatest reduction in P. gingivalis planktonic and biofilm growth compared to the control, which was expected due to the bactericidal action of chlorhexidine. Other medium-chain fatty acids, and in particular linoleic acid, have been found to inhibit oral bacteria [38]. Because of the need for healthy oral flora, removing opportunistic and disease-causing bacteria, or preventing their attachment, is of more importance than removing all oral bacteria. Further, titanium nanoparticles have been found to create gaps in bacterial cell walls. This causes increased cellular permeability and eventually cellular death while having a lower chance of developing resistance to the titanium. Cement with antibacterial agents have also been studied to determine changes in mechanical properties [39]. Nanoparticles can be utilized as antibacterial agents to coat brackets or as an additive to cement and other adhesives [40].
Moreover, findings from these studies suggest the potential of incorporating 2CP into chlorhexidine mouthwash to reduce plaque formation. In a 2015 study on the combination of low concentrations of C2DA and chlorhexidine to remove dental plaque formed by Streptococcus mutans and Candida albicans, biofilms were grown on saliva-coated hydroxyapatite discs for 48 h and then treated for one minute with chlorhexidine (0.08%, 0.06% and 0.04%) or combined chlorhexidine and C2DA two times a day for 3 days [41]. This study resulted in significant dispersal of biofilm by 310 nM C2DA with 0.04% chlorhexidine, suggesting synergy between C2DA and chlorhexidine, even at low concentrations [41]. These results, coupled with the current study’s findings on 2CP, support the possible inclusion of 2CP into chlorhexidine mouthwash for effective delivery to natural teeth and HAp-coated dental implants. Use of 2CP as a coating or additive with other common oral antiseptics, such as povidone iodine, hydrogen peroxide, or phenol, was not evaluated in this study.
Natural tooth enamel and dentin have trace amounts of ions such as sodium, magnesium, iron, and fluoride. A study investigating the combination of fluoride with HAp concluded that there is synergy between the two materials for preventing erosion and demineralization [42]. Studies have also reported that zinc-doped HAp specifically reduces the viability of S. mutans and prevents halitosis, or bad breath [43,44,45]. Other studies have suggested that silver ions released from HAp can inhibit E. coli and S. aureus growth [46]. Future work is needed to assess the combined effects of 2CP-loaded hydroxyapatite that is doped with ions. Another calcium phosphate, beta-tricalcium phosphate (β-TCP), appears as a transitional phase of HAp and is the most clinically used non-HAp calcium phosphate. In studies investigating the changes in β-TCP structure with the addition of carboxylic acids to produce functionalized β-TCP (fTCP), fTCP-containing toothpaste increased remineralization of enamel lesions compared to natural pH cycling and reduced white spot lesions compared to fluoride toothpaste [47,48,49]. A potential future study could assess 2CP-loaded TCP for antimicrobial and remineralization potential. Some studies have also developed Hap sheets that are ultra-thin to improve fusion with tooth surfaces as well as dental implant surfaces [28]. These sheets are advantageous because the structure mimics that of the natural teeth while having flexibility and strong adhesion with the tooth surface without bonding agents [50]. A future study could incorporate 2CP loading onto HAp sheets, instead of the Hap coupons used in this work.
The cytotoxicity results support the literature review, as oral rinse groups significantly reduced the viability of Saos-2 cells. It should also be noted that platelet-rich fibrin (PRF) and platelet-rich plasma have been used to augment soft tissue healing and to increase the rate of regeneration by enhancing the chemotaxis, angiogenesis, mitosis, and proliferation of potent stem cells [51,52,53], and the effects of 2CP on platelets have not been investigated. 2CP-loaded HAp, when fully loaded, reduces the viability of P. gingivalis biofilm while also not disrupting the viability of Saos-2 cells when using a loading concentration of 2.5 mg/mL, which deposits approximately 100 µg/mL of 2CP on the HAp surface. This is consistent with previous research showing that 125 μg/mL of 2CP disperses approximately 100% of S. aureus cells, and 2CP re. 2CP-loaded HAp coupon groups had a relative cell viability ≥70% of the control group, and therefore met the qualifications to be considered non-cytotoxic, as defined by the ISO 10993-5 Biological Evaluations of Medical Devices standard when evaluating medical devices for in vitro cytotoxicity [34]. Connecting the antimicrobial studies with the cytocompatibility studies, chlorhexidine-containing oral rinse is effective at reducing bacterial viability, but also reduces the viability of bone cells. The comparable qualities of 2CP to chlorhexidine may still lend credibility to the replacement of this “gold standard” with 2CP or 2CP in conjunction with lowered concentrations of antimicrobials in oral products due to the risk of toxicity with chlorhexidine. This cytocompatibility direct contact assay could be improved by including coupon groups after elution, similar to bacterial assays. Dental implants are often accompanied by growth promoting membranes and factors, such as platelet-rich fibrin. Chlorhexidine and other antimicrobials are likely toxic to cells and could damage materials, promoting inflammatory response. 2CP may be an alternative that supports better healing. Other studies have loaded fatty acids similar to 2CP onto tissue regeneration scaffolds for controlled delivery [54,55], so 2CP should not interfere with material properties of guided bone regeneration membranes.
It is recognized that this study has several limitations, particularly in using an in vitro model to investigate effects of immersion against a single strain of bacteria in simple buffered media. We chose P. gingivalis, as it is a representative g negative anaerobe that leads to periodontitis and has been identified as a contributor to peri-implantitis [56], although studies suggest that after dental implantation, P. gingivalis is not the predominant pathogen colonizing the oral microbiome [57]. In a study by Danser et al., patients with dental implants were found to be colonized with Peptostreptococcus spp., Fusobacterium spp., and other Prevotella species [57]. In studies by Harrison et al., activities of 2CP against g positive and g negative bacteria as well as fungi have been reported [30], so it would be valuable to investigate effects against these pathogens as well as mixed species biofilm. The methods used in these studies for loading HAp with 2CP were performed for 3 h at 40 °C and 50 rpm to represent coating of a hydroxyapatite-coated screw at the time of implantation. However, the percutaneous abutment and gingival tissue may be more likely to harbor biofilm. To meet more expected loading conditions when applying the 2CP loading strategy as an oral rinse, the time length should decrease to closer to 1 min and the rotational speed should increase to mimic the action of a mouthwash. While an oral rinse can reduce biofilm on teeth and the surfaces of implant exposed to the rinse, reaching the affected periodontal tissue may be limited by the alkaline pH and the slow rate of microliters/hour of gingival crevicular fluid flow [58,59] Other further studies should involve recharging of coated surfaces with 2CP as well as rechallenging coupons with bacteria. Additionally, future studies could be used to determine if lower concentrations of mouthwash would support the action of 2CP while supporting osteoblast growth. Lastly, future studies should evaluate the long-term cytotoxicity and biofilm inhibition properties of 2CP-loaded HAp and synthetic polymers representative of dental cement, as the current study duration was limited to 3 days and one type of ceramic material.

5. Conclusions

The purpose of this work was to determine if 2CP immersion loading could be applied to natural teeth and dental implants to help prevent and reduce dental plaque and development of caries and gingivitis. The studies presented in this thesis demonstrate successful loading of hydroxyapatite coupons with 2CP, elution of 2CP from the hydroxyapatite surface, and dual behavior of significantly reducing P. gingivalis biofilm viability while supporting Soas-2 cell viability. The developed 2CP loading strategy could potentially be applied to HAp-coated implants and natural teeth. In conclusion, the results of these studies support the hypothesis that 2CP-loaded hydroxyapatite coupons coated through immersion loading at a concentration of 2.5 mg/mL will release 2CP at levels sufficient for biofilm inhibition, demonstrating that 2CP-loaded hydroxyapatite coupons can have an antimicrobial effect on P. gingivalis. Further studies are needed to evaluate these claims in complex environments through preclinical and clinical investigations of peri-implantitis prevention.

Author Contributions

Conceptualization, J.A.J. and E.C.M.; methodology, J.A.J., E.C.M., T.F. and J.D.B.; formal analysis, E.C.M. and M.C.W.; investigation, E.C.M., M.C.W. and T.Y.; writing—original draft preparation, E.C.M.; writing—review and editing, M.C.W. and T.Y.; data curation: E.M.-B.; supervision, J.A.J.; project administration, J.A.J.; funding acquisition, J.A.J. All authors have read and agreed to the published version of the manuscript.

Funding

Study supported by the National Science Foundation, Award No. 1945094.

Institutional Review Board Statement

Not Applicable.

Data Availability Statement

The original contributions presented in the study are included in the article; further inquiries can be directed to the corresponding author/s.

Acknowledgments

The authors thank Jermiah Tate, Yogita Dintakurthi, and Hanna Jones for help with elution sample collection, Felio Perez and Rabeta Yeasmin for help with SEM, Jay Tippabattini for HPLC preparation, and Daniel Baker, Rachel Wiley, and Brian Hoffman for 2CP preparation.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Albino, J.; Dye, B.; Ricks, T. Surgeon General’s Report: Oral Health in America: Advances and Challenges; National Institutes of Health: Bethesda, MD, USA, 2020.
  2. Böheim, R.; Horvath, T.; Leoni, T.; Spielauer, M. The impact of health and education on labor force participation in aging societies: Projections for the United States and Germany from dynamic microsimulations. Popul. Res. Policy Rev. 2023, 42, 39. [Google Scholar] [CrossRef] [PubMed]
  3. Kasman, Ş.; Uçar, I.C.; Ozan, S. The effects of laser surface texturing parameters on the surface characteristics of biomedical-grade stainless steel. J. Mater. Eng. Perform. 2023, 33, 5793–5806. [Google Scholar] [CrossRef]
  4. Armellini, D.; Reynolds, M.A.; Harro, J.M.; Molly, L. Biofilm Formation on Natural Teeth and Dental Implants: What is the Difference? In The Role of Biofilms in Device-Related Infections; Springer: Berlin/Heidelberg, Germany, 2009; pp. 109–122. [Google Scholar]
  5. Padovani, G.C.; Fucio, S.B.; Ambrosano, G.M.; Correr-Sobrinho, L.; Puppin-Rontani, R.M. In situ bacterial accumulation on dental restorative materials. CLSM/COMSTAT analysis. Am. J. Dent. 2015, 28, 3–8. [Google Scholar] [PubMed]
  6. Busscher, H.; Rinastiti, M.; Siswomihardjo, W.; Van der Mei, H. Biofilm formation on dental restorative and implant materials. J. Dent. Res. 2010, 89, 657–665. [Google Scholar] [CrossRef] [PubMed]
  7. Fiorillo, L.; Cervino, G.; Laino, L.; D’Amico, C.; Mauceri, R.; Tozum, T.F.; Gaeta, M.; Cicciù, M. Porphyromonas gingivalis, periodontal and systemic implications: A systematic review. Dent. J. 2019, 7, 114. [Google Scholar] [CrossRef]
  8. Sahrmann, P.; Gilli, F.; Wiedemeier, D.B.; Attin, T.; Schmidlin, P.R.; Karygianni, L. The microbiome of peri-implantitis: A systematic review and meta-analysis. Microorganisms 2020, 8, 661. [Google Scholar] [CrossRef]
  9. Kurtzman, G.M.; Horowitz, R.A.; Johnson, R.; Prestiano, R.A.; Klein, B.I. The systemic oral health connection: Biofilms. Medicine 2022, 101, e30517. [Google Scholar] [CrossRef]
  10. Schaudinn, C.; Gorur, A.; Keller, D.; Sedghizadeh, P.P.; Costerton, J.W. Periodontitis: An archetypical biofilm disease. J. Am. Dent. Assoc. 2009, 140, 978–986. [Google Scholar] [CrossRef]
  11. Bandyopadhyay, A.; Mitra, I.; Goodman, S.B.; Kumar, M.; Bose, S. Improving biocompatibility for next generation of metallic implants. Prog. Mater. Sci. 2023, 133, 101053. [Google Scholar] [CrossRef]
  12. Klinge, B.; Hultin, M.; Berglundh, T. Peri-implantitis. Dent. Clin. 2005, 49, 661–676. [Google Scholar] [CrossRef]
  13. Bose, S.; Tarafder, S.; Bandyopadhyay, A. Hydroxyapatite coatings for metallic implants. In Hydroxyapatite (Hap) for Biomedical Applications; Elsevier: Amsterdam, The Netherlands, 2015; pp. 143–157. [Google Scholar]
  14. Berglundh, T.; Persson, L.; Klinge, B. A systematic review of the incidence of biological and technical complications in implant dentistry reported in prospective longitudinal studies of at least 5 years. J. Clin. Periodontol. 2002, 29, 197–212. [Google Scholar] [CrossRef] [PubMed]
  15. Snauwaert, K.; Duyck, J.; Van Steenberghe, D.; Quirynen, M.; Naert, I. Time dependent failure rate and marginal bone loss of implant supported prostheses: A 15-year follow-up study. Clin. Oral Investig. 2000, 4, 13–20. [Google Scholar] [CrossRef] [PubMed]
  16. Tonetti, M.S. Determination of the success and failure of root-form osseointegrated dental implants. Adv. Dent. Res. 1999, 13, 173–180. [Google Scholar] [CrossRef]
  17. Solderer, A.; Al-Jazrawi, A.; Sahrmann, P.; Jung, R.; Attin, T.; Schmidlin, P.R. Removal of failed dental implants revisited: Questions and answers. Clin. Exp. Dent. Res. 2019, 5, 712–724. [Google Scholar] [CrossRef]
  18. Paradowska-Stolarz, A.; Mikulewicz, M.; Laskowska, J.; Karolewicz, B.; Owczarek, A. The importance of chitosan coatings in dentistry. Marine Drugs 2023, 21, 613. [Google Scholar] [CrossRef]
  19. Gamagedara, T.; Rathnayake, U.; Rajapakse, R. Facile synthesis of hydroxyapatite nanoparticles by a polymer-assisted method: Morphology, mechanical properties and formation mechanism. J. Clin. Investig. 2018, 1, 1–5. [Google Scholar]
  20. Adamopoulos, O.; Papadopoulos, T. Nanostructured bioceramics for maxillofacial applications. J. Mater. Sci. Mater. Med. 2007, 18, 1587–1597. [Google Scholar] [CrossRef]
  21. Ben-Nissan, B.; Choi, A.; Roest, R.; Latella, B.; Bendavid, A. Adhesion of hydroxyapatite on titanium medical implants. In Hydroxyapatite (HAp) for Biomedical Applications; Elsevier: Amsterdam, The Netherlands, 2015; pp. 21–51. [Google Scholar]
  22. Zablotsky, M.H. Hydroxyapatite coatings in implant dentistry. Implant. Dent. 1992, 1, 253–257. [Google Scholar] [CrossRef]
  23. Westas, E.; Gillstedt, M.; Lönn-Stensrud, J.; Bruzell, E.; Andersson, M. Biofilm formation on nanostructured hydroxyapatite-coated titanium. J. Biomed. Mater. Res. Part A 2014, 102, 1063–1070. [Google Scholar] [CrossRef]
  24. Chevalier, M.; Ranque, S.; Prêcheur, I. Oral fungal-bacterial biofilm models in vitro: A review. Med. Mycol. 2018, 56, 653–667. [Google Scholar] [CrossRef]
  25. Junka, A.F.; Szymczyk, P.; Smutnicka, D.; Kos, M.; Smolina, I.; Bartoszewicz, M.; Chlebus, E.; Turniak, M.; Sedghizadeh, P.P. Microbial biofilms are able to destroy hydroxyapatite in the absence of host immunity in vitro. J. Oral Maxillofac. Surg. 2015, 73, 451–464. [Google Scholar] [CrossRef] [PubMed]
  26. Saeed, F.; Muhammad, N.; Khan, A.S.; Sharif, F.; Rahim, A.; Ahmad, P.; Irfan, M. Prosthodontics dental materials: From conventional to unconventional. Mater. Sci. Eng. C 2020, 106, 110167. [Google Scholar] [CrossRef] [PubMed]
  27. Davies, D.G.; Marques, C.N. A fatty acid messenger is responsible for inducing dispersion in microbial biofilms. J. Bacteriol. 2009, 191, 1393–1403. [Google Scholar] [CrossRef] [PubMed]
  28. Jennings, J.A.; Courtney, H.S.; Haggard, W.O. Cis-2-decenoic acid inhibits S. aureus growth and biofilm in vitro: A pilot study. Clin. Orthop. Relat. Res. 2012, 470, 2663–2670. [Google Scholar] [CrossRef]
  29. Roizman, D.; Vidaillac, C.; Givskov, M.; Yang, L. In vitro evaluation of biofilm dispersal as a therapeutic strategy to restore antimicrobial efficacy. Antimicrob. Agents Chemother. 2017, 61, 110–128. [Google Scholar] [CrossRef]
  30. Harrison, Z.L.; Awais, R.; Harris, M.; Raji, B.; Hoffman, B.C.; Baker, D.L.; Jennings, J.A. 2-Heptylcyclopropane-1-Carboxylic Acid Disperses and Inhibits Bacterial Biofilms. Front. Microbiol. 2021, 12, 645180. [Google Scholar] [CrossRef]
  31. Harrison, Z.L.; Montgomery, E.C.; Hoffman, B.; Perez, F.; Bush, J.R.; Bumgardner, J.D.; Fujiwara, T.; Baker, D.L.; Jennings, J.A. Titanium coated with 2-decenoic analogs reduces bacterial and fungal biofilms. J. Appl. Microbiol. 2023, 134, lxad155. [Google Scholar] [CrossRef]
  32. Bock, R.; Lange, F. Effects of CnTAB chain length and concentration on the rheological properties of aqueous suspensions of alkylated alumina powders. J. Am. Ceram. Soc. 2006, 89, 817–822. [Google Scholar] [CrossRef]
  33. Brookes, Z.L.; Bescos, R.; Belfield, L.A.; Ali, K.; Roberts, A. Current uses of chlorhexidine for management of oral disease: A narrative review. J. Dent. 2020, 103, 103497. [Google Scholar] [CrossRef]
  34. ISO 10993-5; Biological Evaluation of Medical Devices. Part 5: Tests for In Vitro Cytotoxicity. International Organization for Standardization: Geneva, Switzerland, 2009.
  35. Cheung, M.C.; Hopcraft, M.S.; Darby, I.B. Patient-reported oral hygiene and implant outcomes in general dental practice. Aust. Dent. J. 2021, 66, 49–60. [Google Scholar] [CrossRef]
  36. Remizova, A.A.; Sakaeva, Z.U.; Dzgoeva, Z.G.; Rayushkin, I.I.; Tingaeva, Y.I.; Povetkin, S.N.; Mishvelov, A.E. The role of oral hygiene in the effectiveness of prosthetics on dental implants. Ann. Dent. Spec. 2021, 9, 39–46. [Google Scholar] [CrossRef]
  37. Choi, L.R.; Harrison, Z.; Montgomery, E.C.; Bush, J.R.; Abuhussein, E.; Bumgardner, J.D.; Fujiwara, T.; Jennings, J.A. Chitosan Membranes Stabilized with Varying Acyl Lengths Release Cis-2-Decenoic Acid and Bupivacaine at Controlled Rates and Inhibit Pathogenic Biofilm. Front. Biosci. -Landmark 2024, 29, 108. [Google Scholar] [CrossRef] [PubMed]
  38. Masuda, M.; Era, M.; Kawahara, T.; Kanyama, T.; Morita, H. Antibacterial effect of fatty acid salts on oral bacteria. Biocontrol Sci. 2015, 20, 209–213. [Google Scholar] [CrossRef]
  39. Tüzüner, T.; Dimkov, A.; Nicholson, J.W. The effect of antimicrobial additives on the properties of dental glass-ionomer cements: A review. Acta Biomater. Odontol. Scand. 2019, 5, 9–21. [Google Scholar] [CrossRef]
  40. Tivanani, M.V.D.; Mulakala, V.; Keerthi, V.S. Antibacterial Properties and Shear Bond Strength of Titanium Dioxide Nanoparticles Incorporated into an Orthodontic Adhesive: A Systematic Review. Int. J. Clin. Pediatr. Dent. 2024, 17, 102. [Google Scholar]
  41. Rahmani-Badi, A.; Sepehr, S.; Babaie-Naiej, H. A combination of cis-2-decenoic acid and chlorhexidine removes dental plaque. Arch. Oral Biol. 2015, 60, 1655–1661. [Google Scholar] [CrossRef]
  42. Souza, B.M.; Comar, L.P.; Vertuan, M.; Fernandes Neto, C.; Buzalaf, M.A.R.; Magalhães, A.C. Effect of an experimental paste with hydroxyapatite nanoparticles and fluoride on dental demineralisation and remineralisation in situ. Caries Res. 2015, 49, 499–507. [Google Scholar] [CrossRef]
  43. Chen, L.; Al-Bayatee, S.; Khurshid, Z.; Shavandi, A.; Brunton, P.; Ratnayake, J. Hydroxyapatite in oral care products—A review. Materials 2021, 14, 4865. [Google Scholar] [CrossRef]
  44. Hannig, M.; Hannig, C. Nanomaterials in preventive dentistry. Nat. Nanotechnol. 2010, 5, 565–569. [Google Scholar] [CrossRef]
  45. Harks, I.; Jockel-Schneider, Y.; Schlagenhauf, U.; May, T.W.; Gravemeier, M.; Prior, K.; Petersilka, G.; Ehmke, B. Impact of the daily use of a microcrystal hydroxyapatite dentifrice on de novo plaque formation and clinical/microbiological parameters of periodontal health. A randomized trial. PLoS ONE 2016, 11, e0160142. [Google Scholar] [CrossRef]
  46. Díaz, M.; Barba, F.; Miranda, M.; Guitián, F.; Torrecillas, R.; Moya, J.S. Synthesis and antimicrobial activity of a silver-hydroxyapatite nanocomposite. J. Nanomater. 2009, 2009, 498505. [Google Scholar] [CrossRef]
  47. Hamba, H.; Nakamura, K.; Nikaido, T.; Tagami, J.; Muramatsu, T. Remineralization of enamel subsurface lesions using toothpaste containing tricalcium phosphate and fluoride: An in vitro µCT analysis. BMC Oral Health 2020, 20, 292. [Google Scholar] [CrossRef] [PubMed]
  48. Ionescu, A.C.; Cazzaniga, G.; Ottobelli, M.; Garcia-Godoy, F.; Brambilla, E. Substituted nano-hydroxyapatite toothpastes reduce biofilm formation on enamel and resin-based composite surfaces. J. Funct. Biomater. 2020, 11, 36. [Google Scholar] [CrossRef] [PubMed]
  49. Jo, S.-Y.; Chong, H.-J.; Lee, E.-H.; Chang, N.-Y.; Chae, J.-M.; Cho, J.-H.; Kim, S.-C.; Kang, K.-H. Effects of various toothpastes on remineralization of white spot lesions. Korean J. Orthod. 2014, 44, 113–118. [Google Scholar] [CrossRef]
  50. Hontsu, S.; Yoshikawa, K. Ultra-thin hydroxyapatite sheets for dental applications. In Hydroxyapatite (hap) for Biomedical Applications; Elsevier: Amsterdam, The Netherlands, 2015; pp. 129–142. [Google Scholar]
  51. Mijiritsky, E.; Assaf, H.D.; Kolerman, R.; Mangani, L.; Ivanova, V.; Zlatev, S. Autologous platelet concentrates (APCs) for hard tissue regeneration in oral implantology, sinus floor elevation, peri-implantitis, socket preservation, and medication-related osteonecrosis of the jaw (MRONJ): A literature review. Biology 2022, 11, 1254. [Google Scholar] [CrossRef]
  52. Albanese, A.; Licata, M.E.; Polizzi, B.; Campisi, G. Platelet-rich plasma (PRP) in dental and oral surgery: From the wound healing to bone regeneration. Immun. Ageing 2013, 10, 23. [Google Scholar] [CrossRef]
  53. Cheruvu, R.N.S.; Katuri, K.K.; Dhulipalla, R.; Kolaparthy, L.; Adurty, C.; Thota, K.M. Evaluation of soft tissue and crestal bone changes around non-submerged implants with and without a platelet-rich fibrin membrane: A randomized controlled clinical trial. Dent. Med. Probl. 2023, 60, 437–443. [Google Scholar] [CrossRef]
  54. Harrison, Z.; Montgomery, E.C.; Bush, J.R.; Gupta, N.; Bumgardner, J.D.; Fujiwara, T.; Baker, D.L.; Jennings, J.A. Cis-2-Decenoic Acid and Bupivacaine Delivered from Electrospun Chitosan Membranes Increase Cytokine Production in Dermal and Inflammatory Cell Lines. Pharmaceutics 2023, 15, 2476. [Google Scholar] [CrossRef]
  55. Harrison, Z.L.; Bumgardner, J.D.; Fujiwara, T.; Baker, D.L.; Jennings, J.A. In vitro evaluation of loaded chitosan membranes for pain relief and infection prevention. J. Biomed. Mater. Res. B Appl. Biomater. 2021, 109, 1735–1743. [Google Scholar] [CrossRef]
  56. Scarano, A.; Khater, A.G.; Gehrke, S.A.; Serra, P.; Francesco, I.; Di Carmine, M.; Tari, S.R.; Leo, L.; Lorusso, F. Current status of peri-implant diseases: A clinical review for evidence-based decision making. J. Funct. Biomater. 2023, 14, 210. [Google Scholar] [CrossRef]
  57. Danser, M.M.; van Winkelhoff, A.J.; van der Velden, U. Periodontal bacteria colonizing oral mucous membranes in edentulous patients wearing dental implants. J. Periodontol. 1997, 68, 209–216. [Google Scholar] [CrossRef] [PubMed]
  58. Subbarao, K.C.; Nattuthurai, G.S.; Sundararajan, S.K.; Sujith, I.; Joseph, J.; Syedshah, Y.P. Gingival Crevicular Fluid: An Overview. J. Pharm. Bioallied Sci. 2019, 11, S135–S139. [Google Scholar] [CrossRef] [PubMed]
  59. Bickel, M.; Munoz, J.; Giovannini, P. Acid-base properties of human gingival crevicular fluid. J. Dent. Res. 1985, 64, 1218–1220. [Google Scholar] [CrossRef] [PubMed]
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Figure 1. Schematic shows experimental workflow. Created in BioRender.com.
Figure 1. Schematic shows experimental workflow. Created in BioRender.com.
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Figure 2. Total 2CP loaded onto hydroxyapatite coupons (n = 3) determined by sum of three ethanol washes.
Figure 2. Total 2CP loaded onto hydroxyapatite coupons (n = 3) determined by sum of three ethanol washes.
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Figure 3. Images depict contact angles for unloaded and 2CP-loaded HAp coupons (n = 5).
Figure 3. Images depict contact angles for unloaded and 2CP-loaded HAp coupons (n = 5).
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Figure 4. (A) Individual and (B) cumulative amount of 2CP released as percentage of loaded amount from 2CP-loaded and 2CP+oral rinse-loaded hydroxyapatite samples (n = 6).
Figure 4. (A) Individual and (B) cumulative amount of 2CP released as percentage of loaded amount from 2CP-loaded and 2CP+oral rinse-loaded hydroxyapatite samples (n = 6).
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Figure 5. Planktonic viability of P. gingivalis for HAp coupon groups (n = 3). “Before” and “after” refer to before and after 3-day elution. No significant differences were determined.
Figure 5. Planktonic viability of P. gingivalis for HAp coupon groups (n = 3). “Before” and “after” refer to before and after 3-day elution. No significant differences were determined.
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Figure 6. Biofilm viability of P. gingivalis on HAp coupon surfaces (n = 3). “Before” and “after” refer to before and after 3-day elution. **** indicates significant difference (p < 0.0001), and *** indicates significant difference (p < 0.001).
Figure 6. Biofilm viability of P. gingivalis on HAp coupon surfaces (n = 3). “Before” and “after” refer to before and after 3-day elution. **** indicates significant difference (p < 0.0001), and *** indicates significant difference (p < 0.001).
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Figure 7. Photographs feature LIVE/DEAD images of biofilm growth on HAp coupon groups (n =1). “Before” and “after” refer to before and after 3-day elution.
Figure 7. Photographs feature LIVE/DEAD images of biofilm growth on HAp coupon groups (n =1). “Before” and “after” refer to before and after 3-day elution.
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Figure 8. Viability of Saos-2 cells on HAp coupon surfaces (n = 3). ** indicates significant difference (p < 0.01). Significant differences only shown for comparisons to control. Red line represents 70% threshold for viability outlined in ISO 10993-5 [34].
Figure 8. Viability of Saos-2 cells on HAp coupon surfaces (n = 3). ** indicates significant difference (p < 0.01). Significant differences only shown for comparisons to control. Red line represents 70% threshold for viability outlined in ISO 10993-5 [34].
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Figure 9. Images display LIVE/DEAD images of Saos-2 cell growth on HAp coupon groups (n = 1).
Figure 9. Images display LIVE/DEAD images of Saos-2 cell growth on HAp coupon groups (n = 1).
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MDPI and ACS Style

Montgomery, E.C.; Wicker, M.C.; Yusuf, T.; Matlock-Buchanan, E.; Fujiwara, T.; Bumgardner, J.D.; Jennings, J.A. In Vitro Coating Hydroxyapatite with 2-Heptylcyclopropane-1-Carboxylic Acid Prevents P. gingivalis Biofilm. Hygiene 2024, 4, 500-512. https://doi.org/10.3390/hygiene4040037

AMA Style

Montgomery EC, Wicker MC, Yusuf T, Matlock-Buchanan E, Fujiwara T, Bumgardner JD, Jennings JA. In Vitro Coating Hydroxyapatite with 2-Heptylcyclopropane-1-Carboxylic Acid Prevents P. gingivalis Biofilm. Hygiene. 2024; 4(4):500-512. https://doi.org/10.3390/hygiene4040037

Chicago/Turabian Style

Montgomery, Emily C., Madelyn C. Wicker, Tibirni Yusuf, Elizabeth Matlock-Buchanan, Tomoko Fujiwara, Joel D. Bumgardner, and J. Amber Jennings. 2024. "In Vitro Coating Hydroxyapatite with 2-Heptylcyclopropane-1-Carboxylic Acid Prevents P. gingivalis Biofilm" Hygiene 4, no. 4: 500-512. https://doi.org/10.3390/hygiene4040037

APA Style

Montgomery, E. C., Wicker, M. C., Yusuf, T., Matlock-Buchanan, E., Fujiwara, T., Bumgardner, J. D., & Jennings, J. A. (2024). In Vitro Coating Hydroxyapatite with 2-Heptylcyclopropane-1-Carboxylic Acid Prevents P. gingivalis Biofilm. Hygiene, 4(4), 500-512. https://doi.org/10.3390/hygiene4040037

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