Biological effects on tooth root surface topographies induced by various mechanical treatments

Colloids and Surfaces B: Biointerfaces 188 (2020) 110748

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Biological effects on tooth root surface topographies induced by various mechanical treatments

T

Xiaohui Qiua,b, Shuo Xua,b, Yuanping Haoc, Brandon Petersond, Baowei Lic, Kai Yanga,b, Xiaofang Lva,b, Qihui Zhoua,b,c,*, Qiuxia Jia,b,* a

Department of Periodontology, The Affiliated Hospital of Qingdao University, Qingdao, 266003, China School of Stomatology of Qingdao University, Qingdao, 266003, China c Institute for Translational Medicine, School of Basic Medicine, Qingdao University, Qingdao, 266021, China d W.J. Kolff Institute for Biomedical Engineering and Materials Science, Department of Biomedical Engineering, University Medical Center Groningen, University of Groningen, A. Deusinglaan 1, 9713 AV, Groningen, the Netherlands b

A R T I C LE I N FO

A B S T R A C T

Keywords: Dental calculus Mechanical treatments Tooth root biointerface Cell behaviors Inflammatory response

The cleaning and physicochemical properties on tooth root biointerfaces are pivotal for periodontal healing. Herein, this work investigated the impact of multi-treatment on the physicochemical features of tooth root surfaces and the responsive behavior of human gingival fibroblasts (hGFs). It was found that the combination of various mechanical treatments significantly affects the topographical pattern and size as well as wettability on tooth root surfaces. Furthermore, biological experiments revealed that hGF behaviors (i.e., cell adhesion, shape, spreading, arrangement, and viability) were regulated by the topography and wettability of tooth root surfaces. Also, there was no significant difference in the protein expression of NLRP3 inflammasome and IL-1β in hGFs among tooth root surfaces under various treatments. This study provides new insights to efficiently remove the dental calculus and to understand the interaction between the tooth root interface and cell, which could guide the clinical operation and thereby is more conducive to periodontal recovery.

1. Introduction Oral diseases are the most prevalent human diseases mainly caused by its open and complicated microenvironment extremely rich in bacteria [1,2]. Among them, dental calculus occurs in the majority of adults worldwide, caused by large amounts of oral bacteria, proteins, viruses, and food remnants as well as composed primarily of calcium phosphate mineral salts (calcified dental plaque) [3]. It can damage tooth enamel, result in tooth discoloration and resorption, gingivitis, periodontal disease, and even tooth loss [3]. Therefore, removal of dental calculus is of great importance and necessity. So far, several strategies, such as high-intensity laser, chemical, and mechanical treatments, are commonly used in dental calculus control. Lasers can selectively remove dental calculus with high precision and minimize damage to surrounding tissues [4]. However, this method is complicated and makes the tooth surface rougher than that induced by conventional removal. Chemical and mechanical methods are the earliest, most straightforward, and simplest way to remove the dental calculus [5]. However, chemical methods are less effective than mechanical treatments (e.g., ultrasonic scaling, hand scaling, and sandblasting) [6].

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The effect of individual mechanical treatment on the removal of dental calculus was studied extensively [5–10]. However, the effect of the multi-approach combination on the removal of dental calculus has not been widely reported. In addition, it is well-known that mechanical treatments can affect the structure of a tooth surface [9–11]. However, the effect of the posttreated root surface on periodontal recovery has not been investigated yet. In periodontal connective tissues, gingival fibroblasts (hGFs) are the predominant cell type interacting with the root and have distinct functional activities in the repair of periodontal tissues as well as in inflammatory periodontal diseases [12–15]. The gingival fibroblasts attachment to the root surface plays a critical role in the generation of an efficient mucosal seal. Therefore, human gingival fibroblasts (hGFs) were chosen for this research. Since cells adjacent to the biomaterials are significantly influenced by the altered physicochemical cues [16–18], herein we hypothesize that root surfaces with topographical structures would also be able to mediate the cellular responses of hGFs. To test the hypothesis, roots were treated by the mechanical approaches with different combinations (i.e., group U (ultrasonic scaling), group U+H (ultrasonic

Corresponding author at:Department of Periodontology, The Affiliated Hospital of Qingdao University, Qingdao, 266003, China E-mail addresses: [email protected] (Q. Zhou), [email protected] (Q. Ji).

https://doi.org/10.1016/j.colsurfb.2019.110748 Received 11 September 2019; Received in revised form 23 November 2019; Accepted 19 December 2019 Available online 20 December 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.

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Morphology of the root blocks was observed by scanning electron microscopy (SEM) (VEGA3, TESCAN, China). Prior to imaging, samples were sputter-coated with gold to increase conductivity. The false color in SEM images was obtained using Adobe Photoshop CS6 by opening the image, selecting RGB mode in image mode, creating a new layer (multiply), selecting the foreground color and coloring it with the brush tool.

scaling + hand scaling), group U + S (ultrasonic scaling + sandblasting), and group U+H+S (ultrasonic scaling + hand scaling + sandblasting)). The morphology, surface roughness, and wettability on root surfaces were systematically characterized by various techniques. Further, these roots with surface topographical features were seeded with hGFs to study the effect of root surface structures on cellular responses, including cell adhesion, morphology, proliferation, viability changes, expression of the inflammasome.

2.4. Saliva contact angle 2. Materials and methods To test wettability of the root blocks, the saliva contact angle (SCA) measurement was performed using the drop method by using a videoenabled goniometer (Theta, Biolin, Sweden). Saliva used here is naturally from a single person. The projected images of the droplets were automatically analyzed by the software. All measurements were repeated at least three times, and the results were averaged.

2.1. Preparation of human root cementum The present study was approved by the Ethics Committee of The Affiliated Hospital of Qingdao University, China (approval no. QYFYWZLL25562). Group B (blank) are healthy teeth without dental calculus as the control. Other teeth had dental calculus present on the cementum. Then, they were cleaned in distilled water and stored in phosphate-buffered saline (PBS) (pH 7.4) at 37 °C until the mechanical treatments were performed.

2.5. Cell culture and identification Primary human gingival fibroblasts (hGFs) used in this study were derived from adolescent gingival tissues obtained from the alveolar surgery at the department of periodontology, the Affiliated Hospital of Qingdao University. The present study with the patients’ informed consent has been approved by the Ethics Committee of the Affiliated Hospital of Qingdao University, China. The cells were cultured in αMEM (Biological Industries, Israel) supplemented with 10 % fetal bovine serum (Biological Industries, Israel) and 1 % penicillin/streptomycin (Biological Industries, Israel), in a humidified incubator of 5 % CO2 at 37 °C. The cells were harvested at approximately 80 − 90 % confluency from culture flasks by trypsin for 3 − 5 min at 37 °C for further subcultures. hGFs between passages 5 and 7 were used in the following analysis. HGFs were identified by immunofluorescence staining with anti-vimentin (proteintech, America), anti-FSP1 (fibroblast-specific protein 1) (abcam, Britain) and anti-cytokeratin antibodies (CST, China).

2.2. Mechanical treatments of specimens All specimens were rinsed for 1 min with 20 mL of saline solution and randomly treated by four methods, i.e., group U (ultrasonic scaling device from P5XS, ACTEON, France), group U+H (ultrasonic scaling + hand scaling device from Hu-Friedy, America), group U + S (ultrasonic scaling + sandblasting device from AIR-FLOW, classic, EMS, Switzerland with sodium bicarbonate powder), and group U+H+S (ultrasonic scaling + hand scaling + sandblasting)) shown in Fig. 1. In addition, treatment S was done with water. Then, the root blocks (5 × 5 mm size with 1 mm thickness) from the teeth above were cut using a Low speed precision cutting instrument (DTQ-5, Weiyi, China), soaked in PBS and dried overnight. All the instrumentations and treatments of teeth were carried out by a single operator. The criteria for adequate treatment were smooth, hard root surfaces, without clinical evidence of calculus.

2.6. Cell adhesion and morphology

2.3. Root surface roughness and morphology

All tooth roots were sterilized in an autoclave and placed in 48-wells microtiter plates. Afterward, hGFs were seeded onto the samples in 48well plates at a density of 4 × 104 cells/well for cell adhesion. All plates were stored in an incubator at 37 °C and 5 % CO2 for 1 day. The hGFs were fixated with 4 % glutaraldehyde (Tianjin beilian fine chemicals

The discs in each group (n = 3) were analyzed at 3 sites (scanning area = 400 μm) using 3D surface optical profilometer (ZETA-20, Zeta Instruments, USA) and an average for each group was calculated.

Fig. 1. Schematic diagram of different mechanical treatments. (A) Ultrasonic scaling, (B) hand scaling, (C) sand blasting. 2

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Fig. 2. 3D optical images of (A) group B: healthy root without any treatment, (B–E) root surfaces under U, U+H, U + S, and U+H+S, respectively. (F) The roughness of root surfaces determined from optical images (n = 3).

2.9. Statistical analysis

development co. LTD, China) in PBS for 2 h at room temperature and subsequently washed 3 times with PBS. Afterward, the samples were dehydrated in graded ethanol (30, 50, 70, 90, and 100 %), followed by critical-point-dried. Then, the morphology of cells was observed by SEM. Prior to imaging, samples were sputter-coated with gold to increase conductivity.

All data are presented as mean values ± standard deviation (mean ± SD). Three independent validity studies were conducted, and at least three samples per group were selected for statistical analysis. Statistical analysis between groups was analyzed via one-way analysis of variance (ANOVA) with Tukey’s test to determine differences. A value of p < 0.05 was considered statistically significant.

2.7. Cell viability 3. Results and discussion

The hGFs were seeded on the samples in 24-well plates at a density of 4 × 104 cells per well and cultured one and four days. Three samples per test group were used for the cell viability. The media was changed every two days during the cell culture. The samples were incubated in 400 μL of α-MEM with a supplement of 50 μL of CCK-8 solution (Absin Bioscience Inc., China) for 2 h. Culture medium served as a negative control. The collected solution was transferred to a 96-well plate with 100 μL per well. The CCK-8 assay was used, and optical density (OD values) at 450 nm were analyzed (SynergyH1/H1M, BioTek, China). The OD value of cells on each sample was calculated following the equation ODcell = OD(cell+medium) – ODmedium. The viability of hGFs was determined by the OD values.

3.1. Root surface morphology The root surface morphology and roughness were first characterized using 3D surface optical profilometer. As shown in Fig. 2A–E, the root surfaces under various mechanical treatments are relatively smooth. Further, the roughness of root surfaces was determined by quantitative analysis. As shown in Fig. 2F, a varied surface roughness for the group B, U, U+H, U + S, and U+H+S was 5.76 ± 0.93, 4.55 ± 0.51, 8.92 ± 2.89, 5.75 ± 1.29 and 6.11 ± 0.93 μm, respectively. However, there was no statistically significant difference among them (p＞ 0.05). Although 3D surface optical profilometer can select a larger scan area to give a better overall description of root morphology, this characterization technique has limited resolution. To further observe the detailed morphology on the root surfaces, root specimens were analyzed by SEM (Fig. 3). SEM images showed a notable change on the root surface morphology from different mechanical treatments. Interestingly, the root surface under U+H treatment is rough and has aligned topographical structure due to the directional hand scaling similar to the clinic treatment. Root surfaces without any treatment as the control (group B) as well as under U and U+H+S treatments were irregular and rough. Also, the root surface under the U treatment had lots of pits and cracks. Sand blasting further smoothed the root surface treated by hand scaling treatment, which could result in the loss of orientation structure. The root surface with U + S treatment was greatly smoother than the surface treated by U+H+S due to different initial roughness treated by U and U+H.

2.8. NAcht Leucine-rich repeat Protein 3 (NLRP3) inflammasome activation and interleukin-1β (IL-1β) production The hGFs were seeded on the samples in 24-well plates at a density of 4 × 104 cells per well and cultured seven days. Three samples per test group were used for the inflammasome activation test. The media was changed every two days during the cell culture. Total proteins from hGFs were lysed in (5×) Laemmli SDS buffer containing 0.01 % bromophenol blue, 1 % β-mercaptoethanol, and protease inhibitor cocktail (Roche). Then, equal amounts of protein were separated by SDS-PAGE gels and transferred onto PVDF membranes. After blocking with 5 % fat-free milk, the membranes were washed with TBST and incubated with primary and secondary antibodies according to the manufacturers’ instructions. Primary antibodies against NLRP3 (1:1000), apoptosisassociated speck-like protein containing CARD (ASC, 1:1000), caspase1 (1:1000), IL-1β (1:1000) are from Cell Signaling Technology. Secondary Horseradish Peroxidase (HRP)-linked antibodies from a rabbit-against human IgG (1:8000) and anti-GAPDH (1:5000) are from Elabscience. The protein was analyzed by an enhanced chemiluminescence kit (Beijing Biocoen Biotechnology CO.Ltd, China).

3.2. Root surface wettability Surface structures have a significant influence on material wettability [19,20]. Wettability is usually characterized by measuring the 3

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Fig. 3. SEM images of root surfaces under various mechanical treatments observed by different magnifications.

higher hydrophilicity or wettability of the root surface. As shown in Fig. 4B, the SCAs on all root surfaces initially decreased until the fourth second and then remained stable until the sixth second. There is no significant difference between group B and group U+H+S (p＞0.05) as well as between group U+H and group U + S (p＞0.05). However, the SCAs of group B and U+H+S were significantly higher than those on group U+H and U + S (p＜0.05). Also, the SCAs of group U+H and U + S were higher compared to that on the group U (p＜ 0.05). The SCA results indicated that the surface wettability of root samples depended on different treatments, but they retained a

water contact angle sample surfaces. Here, we used saliva instead of water, because teeth always contact with saliva in the mouth. Also, dynamic contact angle analysis was performed to investigate time-dependent wettability changes of root surfaces. As shown in Fig. 4A, although hydrophilicity of the root surfaces was examined by contact angle analysis with saliva, the SCAs were significantly different on the root surfaces under various treatments. The initial SCAs on the root surfaces under different treatments were 54.68° (group B), 33.09° (Group U), 40.71° (Group U+H), 43.12° (Group U + S), 55.76° (Group U+H+S), respectively. It is well-known that lower SCA corresponds to 4

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Fig. 4. (A) The initial SCA images on the root surfaces under different treatments. (B) Dynamic contact angles from 1−6 s on the root surfaces under different treatments.

Fig. 5. The staining of specific proteins for cell identification. (A) Vimentin. (B) Fspi. (C) Cytokeration.

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Fig. 6. SEM images of cell adhesion on the root surfaces under different treatments.

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Fig. 7. hGF viability cultured on TCP and root samples under different treatments for 1 and 4 days (n = 3). ** p < 0.01 vs group B. ## p < 0.01 vs group U+H & U + S.

treatments. On day 4, higher cell viability was observed in treated samples compared to the group B. Also, samples treated by U and U+H +S even displayed a significant effect of improvement compared to others. There was no statistical difference between the absorbance values of group U and group U+H+S. The NLRP3 inflammasome is an important regulator of inflammation and immunity [25,34,35]. To examine the inflammation response of hGFs, cells were cultured onto the root surfaces under various treatments and analyzed using the western blot. From Fig. 2S, it was found that NLRP3, ASC, Caspase-1, and IL-1β on root surfaces under different treatments had no significant difference as compared to the control (group B). This indicates that although there are various cell morphology and viability on root surfaces, this does not affect the protein expression of NLRP3 inflammasome and IL-1β. Probably, root surfaces treated by different mechanical methods could avoid causing periodontitis related to the host immune response. It is well-documented that cell responses (e.g., cell adhesion, viability, and inflammatory response) onto materials play a critical role on tissue repair and regeneration [16,36]. In our study, it was found that cell adhesion and morphology on root surfaces treated by U+H+S are closer to those on root surfaces of healthy teeth. After 4 days, cell viability on root surfaces treated by U+H+S was higher compared to that on samples from group B, U+H, and U + S. There was no inflammatory response on root surfaces treated by U+H+S. Taken together, the U+H+S treatment could be the optimal strategy for clinical operations.

hydrophilic character (< 90°). 3.3. Identification of hGFs and their adhesion on root surfaces The physicochemical properties on biointerfaces contribute significantly to regulating cell responses [16]. Therefore, it is necessary to investigate how root biointerfaces affect the gingival recovery. Here, hGFs were chosen for the following research. To verify whether the isolated cells were gingival fibroblasts, we observed cell morphology and detected the expression of specific protein markers. Fig. S1A shows that the cells still maintained a spindleshaped morphology after passage, which is a typical morphology for gingival fibroblasts [21,22]. Immunofluorescent results indicated that the expression of vimentin and FSP1 in cells was found (Fig. 5AB) while the expression of cytokeratin was undetected (Fig. 5C). The cytokeratin measurement can be further used to exclude epithelial cells. These results further suggest that the isolated cells from human gingival tissues were gingival fibroblasts [23–27]. Cells becoming adjacent to the biomaterials is the first and crucial step for cell responses, which precedes all other cellular events, such as spreading, migration, proliferation, and differentiation [20,28,29]. Fig. 6 displays that hGFs could adhere to the root surfaces, and their morphology was affected greatly by alterations to the root surface topographies. The morphology of hGFs on group B, U, U+H and U+H+S was similar and elongated. The elongation of cells on group B and U+H was more than those on the group U and U+H+S. Interestingly, the morphology of cells on the group U+H was aligned due to the contact guidance of oriented topography generated by the H treatment (Fig. 3). It has been reported that anisotropic topography (groove, wrinkle, aligned nanowire, etc.) that replicate the natural anisotropic ECM in vivo, can elicit cell alignment by way of directional elongation [30–32]. Also, the dimensions of aligned topography can greatly affect cell orientation [17]. Cells in the group U + S spread very well, probably because of the combined effects of better wettability and smoother surface compared to other samples. It was reported that surface roughness and/or pattern can affect ligand distribution and the formation of focal adhesions, which further regulates cell adhesion and morphology [33].

4. Conclusions In summary, various mechanical strategies greatly influenced the physical properties of root surfaces, including the topographical pattern, roughness, and hydrophilicity. Particularly, the aligned topography on the root surface was generated under the U+H treatment. The root surface under the U + S treatment is mostly smooth. Furthermore, biological results indicate that hGF behaviors (i.e., cell attachment, morphology, alignment, and viability) were affected by the topography and wettability of root surfaces. Specifically, cells spread better on the smoother surface with better wettability compared to other samples. Oriented cells were induced by aligned topographical surfaces. Higher cell viability was observed on treated samples compared to the control. In addition, the root biointerfaces under various treatments did not induce the protein expression of NLRP3 inflammasome and IL-1β. This study demonstrated that the combination of U+H +S multi-treatment could efficiently remove the dental calculus and regulate cell responses, which may promote periodontal healing.

3.4. hGF viability and their inflammatory response on the root surfaces Cell viability was measured using the CCK-8 assay and showed in Fig. 7. Polystyrene tissue culture plates (TCP) were used as positive control. On day 1, cells remained viable, and no significant difference was found on either surface (p > 0.05), which indicates that there were no cytotoxic effects from the root surfaces under different 7

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CRediT authorship contribution statement Xiaohui Qiu: Investigation, Visualization, Writing - original draft. Shuo Xu: Investigation. Yuanping Hao: Investigation. Brandon Peterson: Writing - review & editing. Baowei Li: Investigation. Kai Yang: Investigation. Xiaofang Lv: Investigation. Qihui Zhou: Visualization, Resources, Supervision, Writing - review & editing. Qiuxia Ji: Resources, Supervision, Writing - review & editing. Declaration of Competing Interest The authors declare no conflict of interests Acknowledgments The authors are very grateful for financial support of by the National Natural Science Foundation of China (Grant Nos. 81401526 and 31900957), the Major Research Plan of the National Natural Science Foundation of China (Grant No. 91849209), the Natural Science Foundation of Shandong Province, China (Grant No. ZR2019QC007 and ZR2019MH003), China Postdoctoral Science Foundation (Grant No. 2019M652326), Innovation program for the excellent youth scholars of higher education of Shandong province (Grant No. 2019KJE015), and the Scientific Research Foundation of Qingdao University (Grant No. DC1900009689). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfb.2019.110748. References [1] R.M. Benjamin, Oral health: the silent epidemic, Public Health Rep. 125 (2010) 158–159. [2] F.E. Dewhirst, T. Chen, J. Izard, B.J. Paster, A.C.R. Tanner, W.-H. Yu, A. Lakshmanan, W.G. Wade, The human oral microbiome, J. Bacteriol. 192 (2010) 5002–5017. [3] D.J. White, Dental calculus: recent insights into occurrence, formation, prevention, removal and oral health effects of supragingival and subgingival deposits, Eur. J. Oral Sci. 105 (1997) 508–522. [4] L.J. Walsh, The current status of laser applications in dentistry, Aust. Dent. J. (2003) 146–155. [5] B.M. Eschler, J.W. Rapley, Mechanical and chemical root preparation in vitro: efficiency of plaque and calculus removal, J. Periodontol. 62 (1991) 755–760. [6] E. Figuero, D.F. Nóbrega, M. García-Gargallo, L.M.A. Tenuta, D. Herrera, J.C. Carvalho, Mechanical and chemical plaque control in the simultaneous management of gingivitis and caries: a systematic review, J. Clin. Periodontol. 44 (2017) S116–S134. [7] E. Westfelt, Rationale of mechanical plaque control, J. Clin. Periodontol. 23 (1996) 263–267. [8] A. Mombelli, Maintenance therapy for teeth and implants, Periodontol. 2000 79 (2019) 190–199. [9] M. Maritato, L. Orazi, D. Laurito, G. Formisano, E. Serra, M. Lollobrigida, A. Molinari, A. De Biase, Root surface alterations following manual and mechanical scaling: a comparative study, Int. J. Dent. Hyg. 16 (2018) 553–558. [10] J.I. Rosales-Leal, A.B. Flores, T. Contreras, M. Bravo, M.A. Cabrerizo-Vílchez, F. Mesa, Effect of root planing on surface topography: an in-vivo randomized experimental trial, J. Periodontal Res. Suppl. 50 (2015) 205–210. [11] A. Dadwal, R. Kaur, V. Jindal, A. Jain, A. Mahajan, A. Goel, Comparative evaluation of manual scaling and root planing with or without magnification loupes using scanning electron microscope: a pilot study, J. Indian Soc. Periodontol. 22 (2018) 317–321. [12] T.D.K. Herath, R.P. Darveau, C.J. Seneviratne, C.Y. Wang, Y. Wang, L. Jin, Heterogeneous Porphyromonas gingivalis LPS modulates immuno-inflammatory response, antioxidant defense and cytoskeletal dynamics in human gingival fibroblasts, Sci. Rep. 6 (2016) 1–15.

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