search for




 

Effect of Rosmarinic Acid on the Focal Adhesions of MC3T3-E1 Preosteoblasts on Titanium Surface
J Dent Hyg Sci 2024;24:181-9
Published online September 30, 2024;  https://doi.org/10.17135/jdhs.2024.24.3.181
© 2024 Korean Society of Dental Hygiene Science.

Moon-Jin Jeong1 , Myoung-Hwa Lee1 , Do-Seon Lim2 , and Soon-Jeong Jeong3,4,†

1Department of Oral Histology and Developmental Biology, School of Dentistry, Chosun University, Gwangju 61452, 2Department of Dental Hygiene, Graduate School of Public Health Science, Eulji University, Seongnam 13135, 3Department of Dental Hygiene, College of Health Science, Youngsan University, Yangsan 50510, 4Institute of Basic Science for Well-Aging, Youngsan University, Yangsan 50510, Korea
Correspondence to: Soon-Jeong Jeong, https://orcid.org/0000-0002-8959-4663
Department of Dental Hygiene, College of Health Science, Youngsan University, 288 Junam-ro, Yangsan 50510, Korea
Tel: +82-55-380-9453, Fax: +82-55-380-9305, E-mail: jeongsj@ysu.ac.kr
Received September 2, 2024; Revised September 7, 2024; Accepted September 11, 2024.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: Focal adhesions (FAs) is the most important process in the first step of osseointegration between preosteoblasts and titanium (Ti). FAs improvement and pre-osteoblasts cell proliferation leads to successful Ti-based dental implants. This study aimed to confirm the applicability of rosmarinic acid (RA) as a functional substance for improving FAs and cell proliferation of MC3T3-E1 preosteoblasts on Ti surfaces during the first stage of osseointegration for successful Ti-based dental implants.
Methods: We used MC3T3-E1 preosteoblasts on Ti discs incubated in a medium supplemented with or without 14 μg/ml to decipher the effects of RA on FAs and cell proliferation. FAs and proliferation of MC3T3-E1 cells on Ti discs were assessed via MTT assay. Actin-labeled cells and paxillin contacts were observed and imaged by fluorescent microscopy, and the associated signaling pathways were revealed through western blot analysis.
Results: In RA-treated MC3T3-E1 cells on Ti discs, FAs between MC3T3-E1 preosteoblasts and Ti surfaces and the expression of focal adhesion kinase (FAK), phosphorylated FAK and paxillin proteins and filamentous-actin formation increased. RA increased the proliferation of MC3T3-E1 preosteoblasts on the Ti surface as well as the expression of Grab2, Ras, pERK1/2, and ERK1/2. In addition, the expression of secretory leukocyte protease inhibitor and thymosin b4, known as nanomolecules that enhance the interaction between implanted Ti materials and preosteoblasts in the RA-treated MC3T3-E1 preosteoblasts, increased. RA not only increased the FAs of MC3T3-E1 preosteoblasts on the Ti surface through the FAK/Paxillin signaling pathway, but also increased cell proliferation and mitosis through the FAK/Grab2/Ras/ERK1/2 signaling pathway.
Conclusion: RA can be applied as an effective functional substrate to improve the FAs and proliferation of MC3T3-E1 preosteoblats on Ti surfaces, which are essential in the first step of osseointegration between implanted Ti and bone tissue for the clinical success of Ti based dental implants.
Keywords : Focal adhesions, MC3T3-E1 preosteoblast,Osseointegration, Rosmarinic acid, Titanium
Introduction

1.Background

Titanium (Ti) and Ti alloys are widely used as implant materials in dentistry and orthopedics because of their light weight, high strength, and good biocompatibility with hard tissues such as teeth and bones1,2). However, cytotoxic ions are released from Ti and its alloys, which decrease cell proliferation of osteoblasts3,4). Although Ti-based dental implants have a high success rate in dentistry, dental implant failures still occur because of bone loss resulting from decreased cell proliferation of osteoblasts, delayed wound healing, peri-implant inflammation and periodontitis1,5). For successful Ti-based dental implants, preosteoblasts in contact with the Ti surfaces must survive and maintain cell viability and adhesion by activating extracellular matrix (ECM) protein complexes, integrins, cytoskeletal proteins and focal adhesion kinase (FAK)6,7). FAs are the most important process in the first step of osseointegration between preosteoblasts and Ti, and control subsequent spreading and cell proliferation8). The enhancement of FAs and proliferation of preosteoblasts are the initial steps towards a successful dental implant6,9) and greatly influence the integration of the Ti-based dental implant surface and host tissue, which is an important factor in determining the success and failure of dental implant6). Various studies have been conducted to improve the success of the first step in Ti-based dental implants, including cell adhesion, spreading, and proliferation of preosteoblasts1). Physical modification of Ti surfaces1,3), treatment and coating with fluoride10), transforming growth factor-b11), fibronectin11), and type I collagen12) increased the biocompatibility and osseointegration between osteoblasts and Ti-based implants through FAs and cell proliferation, resulting in successful dental implants13). Plants have long been used in traditional medicine and remain a source for developing new medicines to control and treat various diseases14). Herbal medicines, in particular, have a variety of biological activities and fewer side effects and are thought to control and treat various diseases via different mechanisms14,15). Rosmarinic acid (α-o-caffeoyl-3,4-dihydroxyphenyl-lactic acid; RA) is a natural phenolic substance contained in rosemary, basil, and mint, and has therapeutic potential with anti-oxidative, anti-bacterial, anti-viral, anti-inflammatory, and anti-mutagenic activities16-18). Recently, RA has been reported to increase alkaline phosphatase activity and bone mineralization for bone formation and prevent osteoporosis16,19), however, the cellular targets of RA in the bone are poorly understood.

2.Objectives

The purpose of this study was to identify the effects of RA on cell adhesion, including FAs and cell proliferation in MC3T3-E1 preosteoblasts on Ti surfaces and the related signaling pathways to confirm the applicability of RA as a functional and therapeutic substance for improving FAs and proliferation of MC3T3-E1 preosteoblasts on Ti surfaces during the first step of osseointegration for successful Ti-based dental implants.

Materials and Methods

1.Study design

1) Preparation of titanium discs and cell culture

Polished commercially pure Ti discs, 20 and 48 mm in diameter and 2 mm in thickness, were used in the experiment2). MC3T3-E1 preosteoblasts derived from murine calvaria2). The cells were planted on a Ti disc with alpha-modified eagle’s medium (α-MEM; WelGene Inc., Daegu, Korea) containing 1% antibiotic-antimycotic solution (WelGene Inc.) and 10% fetal bovine serum (WelGene Inc.), then incubated in a humidified 5% CO2 incubator at 37°C.

2) Cell adhesion and proliferation assay

MC3T3-E1 preosteoblasts, on Ti discs incubated in serum-free medium for 12 hours were incubated with or without 14 µg/ml RA (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) for 10, 30 and 60 minutes17). FAs and cell proliferation were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as previously described2).

3) Western blot analysis

Total protein was extracted from MC3T3-E1 preosteoblasts on Ti discs (1×106 cells/ml) using NP-40 lysis buffer and the protein concentration was determined using Bio-Rad quantitative reagent (Bio-Rad, Hercules, CA, USA). Proteins were electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gels. Separated proteins were transferred onto a polyvinylidene difluoride membrane (Merck Millipore, Darmstadt, Germany). The membrane was incubated for 16 hours at 4°C with primary antibodies, including anti-rabbit FAK (FAK; 1:1,000), anti-rabbit phosphorylated FAK (pFAK; Tyr397; 1:1,000), and anti-rabbit phosphorylated ERK1/2 (pERK1/2; 1:2,500) from Cell Signaling Technology Inc. (Danvers, MA, USA); anti-rabbit ERK1/2 (1:2,500), anti-mouse Grab2 (1:1,000), and anti-rabbit Ras (1:1,000) from Upstate Biotechnology Inc. (Lake Placid, NY, USA); anti-mouse paxillin (1:40,000) from BD Transduction Laboratories (San Jose, CA, USA); and anti-rabbit SLPI (1:1,000), anti-rabbit thymosin β4 (1:1,000), and anti-mouse β-actin (1:2,500) from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). After washing, the membrane was blotted with HRP-conjugated goat anti-rabbit or anti-mouse IgG secondary antibody (1:5,000; Enzo Life Sciences Inc., New York, NY, USA) for 1 hour at room temperature and developed using an X-ray film (Fuji Film Co., Tokyo, Japan) after detection using an enhanced luminescence solution (Merck Millipore, Burlington, MA, USA). The density of the expressed bands was measured using a Science Lab Image Gauge (Fuji Film Co.).

4) Immunofluorescence staining

MC3T3-E1 cells (1×105 cells/ml on the Ti discs, incubated in serum-free α-MEM with or without 14 µg/ml RA, were fixed with 4% paraformaldehyde for 10 min and then treated with 0.15% triton X-100 for 10 minutes. The cells were blocked with 1% bovine serum albumin for 1 hour and reacted with anti-mouse paxillin (1 µg/ml, BD Transduction Laboratories) for 16 hours at 4°C. After washing, cells were incubated with goat anti-mouse IgG conjugated to fluorescein isothiocyanate (FITC; Thermo Fisher Scientific, Waltham, MA, USA) for 1 hour at room temperature. For staining of F-actin, the cells were incubated with 50 µg/ml FITC-conjugated phalloidin (Sigma-Aldrich Chemical Co.) for 40 minutes at room temperature. The cells on the Ti discs were mounted using a mounting medium with DAPI (Vector Laboratories Inc., Burlingame, CA, USA). The actin-labeled cells and paxillin contacts were observed and imaged using a fluorescent microscope (Carl Zeiss, Oberkochen, Germany). The number of paxillin contacts per cell was counted at each time point. Data represent the mean of 40∼60 cells counted at each time point.

2.Statistical analysis

All experiments were independently performed in triplicate. All values were expressed as mean and standard deviation and were determined using SPSS (version 27.0; IBM Co., Armonk, NY, USA). Significant differences (p<0.05) were determined using independent t-tests.

Results

1.Effect of RA on focal adhesion of MC3T3-E1 preosteoblasts on Ti surface

Adhesion of MC3T3-E1 preosteoblasts on Ti discs treated with or without 14 µg/ml RA were confirmed by MTT assay. The cell adhesion rate of RA-treated MC3T3-E1 cells to Ti discs was higher than that of the untreated cells (p<0.05; Fig. 1).

Fig. 1. Effect of RA on cell adhesion in MC3T3-E1 preosteoblasts on Ti surface. Cell adhesion of MC3T3-E1 cells on Ti discs treated with or without 14 µg/ml RA were confirmed via MTT assay. The adhesion rate of MC3T3-E1 cells on Ti discs increased significantly in RA-treated pre-osteoblastic cells. RA: rosmarinic acid, Ti: titanium. *p<0.05, compared to control.

FAK and pFAK in preosteoblasts are predictable biomarkers of cell adhesion2,6,8,20). FAK also binds to filamentous-actin (F-actin) bundles to form cytoskeleton2). Paxillin is a FAs protein that interacts with FAK and directly affects cell adhesion1). FAK, pFAK and paxillin are associated with the FAK/Paxillin signaling pathway. To identify the effects of RA on FAs of MC3T3-E1 preosteoblasts on Ti surface and related signaling pathways, total protein was extracted from MC3T3-E1 preosteoblasts on a Ti disc treated with or without 14 µg/ml RA, and the expression of FAK, pFAK and paxillin proteins were confirmed via western blotting (Fig. 2A). In RA-treated MC3T3-E1 cells on Ti discs, pFAK expression increased at 10 and 30 minutes, but decreased at 60 minutes compared to that in untreated cells (p<0.05; Fig. 2A, 2B). Compared to untreated cells, paxillin expression increased in RA treated MC3T3-E1 cells on Ti discs (p<0.05; Fig. 2A, 2C).

Fig. 2. Effect of RA on focal adhesions in MC3T3-E1 preosteoblasts on Ti surface. After incubation of MC3T3-E1 cells on Ti discs for 12 hours in serum-free medium, the cells were incubated with or without 14 µg/ml RA, total proteins were extracted and analyzed via western blot using the indicated antibodies and β-actin was used as an internal control. (A) pFAK, FAK and paxillin expression. (B, C) Quantitative analysis of western blots. RA: rosmarinic acid, Ti: titanium, FAK: focal adhesion kinase, pFAK: phosphorylated FAK. *p<0.05, compared to control.

2.Effect of RA on F-actin formation and the number of paxillin contacts in MC3T3-E1 preosteoblasts on Ti surface

MC3T3-E1 cells on Ti discs treated with or without RA were fixed and immunofluorescently stained for microscopic observation of F-actin and paxillin contacts. The formation of F-actin in RA-treated MC3T3-E1 preosteoblasts on Ti discs was higher than that in untreated cells (Fig. 3A). The number of paxillin contacts in RA-treated MC3T3-E1 cells on Ti discs was higher than that in untreated cells (p<0.05; Fig. 3B, 3C).

Fig. 3. Effect of RA on F-actin and paxillin contacts formation in MC3T3-E1 preosteoblasts on Ti surface. MC3T3-E1 preosteoblasts on the Ti discs were incubated in serum-free α-MEM with or without 14 µg/ml RA. These cells were fixed and stained using immunofluoresce for fluorescence microscopy. (A) F-actin formation. (B, C) The number of paxillin contacts. All scale bars are 10 µm. RA: rosmarinic acid, Ti: titanium. *p<0.05, compared to control.

3.Effect of RA on cell proliferation of MC3T3-E1 preosteoblasts on Ti surface

Proliferation of MC3T3-E1 preosteoblasts on Ti discs treated with or without 14 µg/ml RA were assessed via MTT assay. Cell proliferation increased in RA treated MC3T3-E1 cells on Ti discs, compared to that in untreated cells (Fig. 4).

Fig. 4. Effect of RA on cell proliferation in MC3T3-E1 preosteoblasts on Ti surface. Proliferation of MC3T3-E1 cells on Ti discs treated with or without 14 µg/ml RA were confirmed via MTT assay. Cell proliferation in RA-treated preostoblasts on Ti discs increased (p<0.05). RA: rosmarinic acid, Ti: titanium. *p<0.05, compared to control.

The increase in cell proliferation after 24 hours of RA treatment was found to be significant when compared with that in untreated cells (p<0.05). pFAK binds directly to Grab2 during cell adhesion and cytoskeleton formation and induces Ras and ERK activity, which is involved in cell proliferation, survival and migration, and differentiation2,21). To identify the effects of RA and the related signaling pathways, protein expression of Grab2, Ras, ERK1/2 and pERK1/2 in MC3T3-E1 cells on Ti discs with or without RA was confirmed via western blotting (Fig. 5A). Grab2, Ras, ERK1/2, and pERK1/2 protein expression in RA-treated MC3T3-E1 cells on Ti discs was significantly higher than that in untreated cells (p<0.05; Fig. 5).

Fig. 5. Effect of RA on cell proliferation and mitosis in MC3T3-E1 preosteoblasts on Ti surface. MC3T3-E1 cells on Ti discs incubated for 12 hours in a medium without serum were treated with or without 14 µg/ml RA. Total protein was extracted from these preosteoblasts and western blot analysis was performed. (A) Protein expression of Grab2, Ras and pERK1/2. β-actin was used as an internal control. (B-D) Quantitative analysis of western blots. RA: rosmarinic acid, Ti: titanium. *p<0.05, compared to control.

4.Effect of RA on SLPI and Tβ4 expression in MC3T3-E1 preosteoblasts on Ti surface

SLPI1,2) and Tβ420,22), which increase cell adhesion and proliferation of MC3T3-E1 cells on the Ti surface, are also known as nanomolecules that enhance the interaction between implanted Ti materials and preosteoblasts. Western blot analysis was performed to identify the effects of RA on SLPI and Tβ4 protein expression in MC3T3-E1 cells on Ti discs (Fig. 6A). Secreted SLPI protein in RA-treated MC3T3-E1 preosteoblasts on Ti discs was significantly increased at 30 minutes, but was similar to that in untreated cells at 10 and 60 minutes (p<0.05; Fig. 6B). Cytoplasmic SLPI protein levels increased after 10 and 60 minutes of RA treatment, but decreased at 30 minutes (p<0.05; Fig. 6B). Tβ4 expression in RA-treated MC3T3-E1 cells on Ti discs significantly increased, compared to untreated cells (p<0.05; Fig. 6B).

Fig. 6. Effect of RA on SLPI and thymosin β4 protein expression in MC3T3-E1 preosteoblasts on Ti surface. Total protein was extracted from MC3T3-E1 cells on Ti discs treated with or without 14 µg/ml RA and western blot was performed. (A) Secreted SLPI (Sup), Cytoplasmic SLPI (Lys) and Tβ4 protein expression. β-actin was used as an internal control. Quantitative analysis of SLPI (B, C) and Tβ4 (D) protein. RA: rosmarinic acid, Ti: titanium. *p<0.05, compared to control.
Discussion

1.Key results, comparison with previous results, and interpretation

Osseointegration is the functional and structural interaction between an implant material and living bone tissue1). Bones are dynamic tissues that are continuously remodeled throughout life23). During bone remodeling, osteoblasts, formed by the differentiation of preosteoblasts originating from mesenchymal cells are responsible for the formation of an organic mixture in the bone matrix and mineralization for new bone formation3,16,24). The first stage after preosteoblasts come into contact with the implant material surface is cell adhesion and spreading6,9). Cell adhesion of preosteoblasts is essential for regulating proliferation, survival, migration, and differentiation into osteoblasts and is controlled by signal transduction through complexes of ECM proteins, integrins, cytoskeletal proteins, and FAK20,25). Stable adhesion of preosteoblasts induces signaling pathways associated with cell proliferation to increas cell viability and decreased cell apoptosis2). Adhered preosteoblasts interact with the ECM via integrin receptors, resulting in an integrin-dependent process involving FAs, FAK activation and phosphorylation8,20). FAs bind to F-actin bundles and recruit FAK and paxillin, which are signaling molecules that affect cell adhesion stability and multiple cellular processes2,20). Integrin, located in the cell membrane of preosteoblasts, directly binds to the cytoskeletal protein, α-actin to form the actin cytoskeleton and play an important role in osteoblast FAs, survival, differentiation, and bone formation by providing critical anchors facilitating cytoskeletal formation, mechanical forces and cell adhesion26). Paxillin, a scaffolding protein, is a structural FAs protein that directly interacts with FAK21). RA increased FAK phosphorylation, which is considered a biomarker for estimating cell adhesion quality (Fig. 1, 2), and increased paxillin expression and F-actin formation (Fig. 3). Consequently, RA enhanced the FAs between MC3T3-E1 preosteoblasts and Ti surfaces via the FAK/Paxillin signaling pathway (Fig. 7). pFAK directly binds to Grab2 during cell adhesion and cytoskeleton formation2). Grab2 is a small protein that is widely expressed many cellular functions and interacts with growth factor receptors for Ras27). The downstream region of Ras is ERK1/227). Growth factor receptors recruit Grab2 to the plasma membranes and Ras and ERK activity is ensured2,28). Integrin-stimulated ERK2 activation induces cell proliferation, survival, migration, and differentiation through the FAK/Grab2/Ras/ERK1/2 signaling pathway2,21). RA treatment increased the expression of Grab2, Ras and pERK1/2 in the preosteoblasts on Ti surface during cell adhesion and cytoskeletal formation (Fig. 5), resulting in increased cell proliferation (Fig. 4). Therefore, RA enhanced the proliferation of MC3T3-E1 preosteoblasts on the Ti surface through the integrin-initiated FAK/Grab2/Ras/ERK1/2 signaling pathway (Fig. 7).

Fig. 7. Simple schematic diagram of RA effect in the first stage of osseointegration between MC3T3-E1 preosteoblasts and Ti surface. RA enhanced FAs of preosteoblasts on Ti surface by increasing F-actin formation and paxillin contacts though the FAK/Paxillin signaling pathway. RA increased cell proliferation and mitosis of preosteoblasts on Ti surface through the FAK/Grab2/Ras/ERK1/2 signaling pathway via integrin-initiated signaling. RA: rosmarinic acid, Ti: titanium, FAK: focal adhesion kinase.

SLPI is an 11.7 kDa cysteine-rich protein found in saliva and seminal plasma produced by epithelial cells2). SLPI protects tissues from protease activity and increases cell proliferation and wound healing during inflammatory response2,29), acting as a modulator between immune response and the anti-inflammatory cytokines produced by macrophage30). Tβ4 is a small 4.9-kDa actin-sequestering peptide involved in cell proliferation, differentiation and motility through the regulation of dynamic modification of the actin cytoskeleton1,20,22). It also plays an important role in the mineralization of bone and dentin22). SLPI2,29) and Tβ420,22) increased FAs and proliferation of MC3T3-E1 preostoblasts on Ti surface. In RA-treated MC3T3-E1 preosteoblasts on Ti surface, SLPI and Tβ4 levels were increased during cell adhesion and cytoskeleto fromation (Fig. 6) and these results are consistent with previous studies on SLPI and Tβ4. Therefore, RA enhances FAs and the proliferation of MC3T3-E1 preosteoblasts on the Ti surface, which is essential during the first stage of osseointegration between the implanted Ti and bone.

2.Suggestion

RA not only enhances FAs between MC3T3-E1 preosteoblasts and Ti surfaces through the FAK/Paxillin signaling pathway but also increases cell proliferation through the FAK/Grab2/Ras/ERK1/2 signaling pathway. Therefore, RA should be applied as an effective functional substance to enhance the first stage of osseointegration by improving the FAs and cell proliferation of MC3T3-E1 preosteoblasts on the Ti surface.

3.Limitations

This invitro study was conducted using MC3T3-E1 preosteoblats. There are limitations in clarifying the effects of RA through this study alone. To confirm the efficacy and safety of RA treatment, preclinical and clinical studies using various cell lines and animal models are required.

Acknowledgements

None.

Conflict of interest

Prof. Soon-Jeong Jeong has been journal editor-in-chief of the Journal of Dental Hygiene Science since January 2023. She was not involved in the review process of this study. Otherwise, there is no potential conflict of interest relevant to this article was reported.

Ethical approval

This article is not necessary for IRB screening.

Author contributions

Conceptualization: Moon-Jin Jeong and Soon-Jeong Jeong. Data acquisition: Do-Seon Lim and Myoung-Hwa Lee. Formal analysis: Do-Seon Lim and Myoung-Hwa Lee. Funding acquisition: Soon-Jeong Jeong. Supervision: Soon-Jeong Jeong. Writing-original draft: Soon-Jeong Jeong. Writing-review & editing: Moon-Jin Jeong and Soon-Jeong Jeong.

Funding

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) Grant funded by the Korea government (grant number 2022R1F1A1069461).

Data availability

Please contact the corresponding author for data availability.

References
  1. Jeong SJ, Jeong MJ: Effect of thymosin beta4 on the differentiation and mineralization of MC3T3-E1 cell on a titanium surface. J Nanosci Nanotechnol 16: 1979-1983, 2016.
    https://doi.org/10.1166/jnn.2016.11928.
    Pubmed CrossRef
  2. Jeong SJ, Wang G, Choi BD, et al.: Secretory leukocyte protease inhibitor (SLPI) increases focal adhesion in MC3T3 osteoblast on titanium surface. J Nanosci Nanotechnol 15: 200-204, 2015.
    https://doi.org/10.1166/jnn.2015.8383.
    Pubmed CrossRef
  3. Choi BD, Lee SY, Jeong SJ, et al.: Secretory leukocyte protease inhibitor promotes differentiation and mineralization of MC3T3-E1 preosteoblasts on a titanium surface. Mol Med Rep 14: 1241-1246, 2016.
    https://doi.org/10.3892/mmr.2016.5381.
    Pubmed CrossRef
  4. Li Y, Wong C, Xiong J, Hodgson P, Wen C: Cytotoxicity of titanium and titanium alloying elements. J Dent Res 89: 493-497, 2010.
    https://doi.org/10.1177/0022034510363675.
    Pubmed CrossRef
  5. Alsaadi G, Quirynen M, Komárek A, van Steenberghe D: Impact of local and systemic factors on the incidence of oral implant failures, up to abutment connection. J Clin Periodontol 34: 610-617, 2007.
    https://doi.org/10.1111/j.1600-051X.2007.01077.x.
    Pubmed CrossRef
  6. Rossi MC, Bezerra FJB, Silva RA, et al.: Titanium-released from dental implant enhances pre-osteoblast adhesion by ROS modulating crucial intracellular pathways. J Biomed Mater Res A 105: 2968-2976, 2017.
    https://doi.org/10.1002/jbm.a.36150.
    Pubmed CrossRef
  7. Zambuzzi WF, Coelho PG, Alves GG, Granjeiro JM: Intracellular signal transduction as a factor in the development of "smart" biomaterials for bone tissue engineering. Biotechnol Bioeng 108: 1246-1250, 2011.
    https://doi.org/10.1002/bit.23117.
    Pubmed CrossRef
  8. Ma XY, Feng YF, Wang TS, et al.: Involvement of FAK-mediated BMP-2/Smad pathway in mediating osteoblast adhesion and differentiation on nano-HA/chitosan composite coated titanium implant under diabetic conditions. Biomater Sci 6: 225-238, 2017.
    https://doi.org/10.1039/c7bm00652g.
    Pubmed CrossRef
  9. Anselme K: Osteoblast adhesion on biomaterials. Biomaterials 21: 667-681, 2000.
    https://doi.org/10.1016/s0142-9612(99)00242-2.
    Pubmed CrossRef
  10. Mannherz HG, Hannappel E: The beta-thymosins: intracellular and extracellular activities of a versatile actin binding protein family. Cell Motil Cytoskeleton 66: 839-851, 2009.
    https://doi.org/10.1002/cm.20371.
    Pubmed CrossRef
  11. Lee SI, Kim DS, Lee HJ, Cha HJ, Kim EC: The role of thymosin beta 4 on odontogenic differentiation in human dental pulp cells. PLoS One 8: e61960, 2013.
    https://doi.org/10.1371/journal.pone.0061960.
    Pubmed KoreaMed CrossRef
  12. Zheng JX, Han YS, Wang JC, et al.: Strigolactones: a plant phytohormone as novel anti-inflammatory agents. Medchemcomm 9: 181-188, 2018.
    https://doi.org/10.1039/c7md00461c.
    Pubmed KoreaMed CrossRef
  13. Meyer U, Joos U, Mythili J, et al.: Ultrastructural characterization of the implant/bone interface of immediately loaded dental implants. Biomaterials 25: 1959-1967, 2004.
    https://doi.org/10.1016/j.biomaterials.2003.08.070.
    Pubmed CrossRef
  14. Kim MY, Bo HH, Choi EO, et al.: Induction of apoptosis by citrus unshiu peel in human breast cancer MCF-7 cells: involvement of ROS-dependent activation of AMPK. Biol Pharm Bull 41: 713-721, 2018.
    https://doi.org/10.1248/bpb.b17-00898.
    Pubmed CrossRef
  15. Na HS, Jeong SY, Park MH, Kim S, Chung J: Nuclear factor-κB dependent induction of TNF-α and IL-1β by the Aggregatibacter actinomycetemcomitans lipopolysaccharide in RAW 264.7 cells. Int J Oral Biol 39: 15-22, 2014.
    https://doi.org/10.11620/IJOB.2014.39.1.015.
    CrossRef
  16. Lee JW, Asai M, Jeon SK, et al.: Rosmarinic acid exerts an antiosteoporotic effect in the RANKL-induced mouse model of bone loss by promotion of osteoblastic differentiation and inhibition of osteoclastic differentiation. Mol Nutr Food Res 59: 386-400, 2015.
    https://doi.org/10.1002/mnfr.201400164.
    Pubmed CrossRef
  17. Jeong MJ, Lim DS, Kim SO, Park C, Choi YH, Jeong SJ: Effect of rosmarinic acid on differentiation and mineralization of MC3T3-E1 osteoblastic cells on titanium surface. Anim Cells Syst (Seoul) 25: 46-55, 2021.
    https://doi.org/10.1080/19768354.2021.1886987.
    Pubmed KoreaMed CrossRef
  18. Moon DO, Kim MO, Lee JD, Choi YH, Kim GY: Rosmarinic acid sensitizes cell death through suppression of TNF-alpha-induced NF-kappaB activation and ROS generation in human leukemia U937 cells. Cancer Lett 288: 183-191, 2010.
    https://doi.org/10.1016/j.canlet.2009.06.033.
    Pubmed CrossRef
  19. Xu Y, Jiang Z, Ji G, Liu J: Inhibition of bone metastasis from breast carcinoma by rosmarinic acid. Planta Med 76: 956-962, 2010.
    https://doi.org/10.1055/s-0029-1240893.
    Pubmed CrossRef
  20. Choi BD, Jeong SJ, Lee HY, et al.: The effect of thymosin β4 for osteoblast adhesion on titanium surface. J Nanosci Nanotechnol 15: 5663-5667, 2015.
    https://doi.org/10.1166/jnn.2015.10464.
    Pubmed CrossRef
  21. Schlaepfer DD, Jones KC, Hunter T: Multiple Grb2-mediated integrin-stimulated signaling pathways to ERK2/mitogen-activated protein kinase: summation of both c-Src- and focal adhesion kinase-initiated tyrosine phosphorylation events. Mol Cell Biol 18: 2571-2585, 1998.
    https://doi.org/10.1128/mcb.18.5.2571.
    Pubmed KoreaMed CrossRef
  22. Choi BD, Lim HJ, Lee SY, et al.: Thymosin β4 is associated with bone sialoprotein expression via ERK and Smad3 signaling pathways in MDPC-23 odontoblastic cells. Int J Mol Med 42: 2881-2890, 2018.
    https://doi.org/10.3892/ijmm.2018.3865.
    Pubmed CrossRef
  23. da Costa Fernandes CJ, Ferreira MR, Bezerra FJB, Zambuzzi WF: Zirconia stimulates ECM-remodeling as a prerequisite to pre-osteoblast adhesion/proliferation by possible interference with cellular anchorage. J Mater Sci Mater Med 29: 41, 2018.
    https://doi.org/10.1007/s10856-018-6041-9.
    Pubmed CrossRef
  24. Kang HJ, Jeong JS, Park NJ, et al.: An ethanol extract of Aster yomena (Kitam.) Honda inhibits lipopolysaccharide-induced inflammatory responses in murine RAW 264.7 macrophages. Biosci Trends 11: 85-94, 2017.
    https://doi.org/10.5582/bst.2016.01217.
    Pubmed CrossRef
  25. Burridge K, Chrzanowska-Wodnicka M: Focal adhesions, contractility, and signaling. Annu Rev Cell Dev Biol 12: 463-519, 1996.
    https://doi.org/10.1146/annurev.cellbio.12.1.463.
    Pubmed CrossRef
  26. Dejaeger M, Böhm AM, Dirckx N, et al.: Integrin-linked kinase regulates bone formation by controlling cytoskeletal organization and modulating BMP and Wnt signaling in osteoprogenitors. J Bone Miner Res 32: 2087-2102, 2017.
    https://doi.org/10.1002/jbmr.3190.
    Pubmed CrossRef
  27. Lowenstein EJ, Daly RJ, Batzer AG, et al.: The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling. Cell 70: 431-442, 1992.
    https://doi.org/10.1016/0092-8674(92)90167-b.
    Pubmed CrossRef
  28. Schwartz MA, Ginsberg MH: Networks and crosstalk: integrin signalling spreads. Nat Cell Biol 4: E65-E68, 2002.
    https://doi.org/10.1038/ncb0402-e65.
    Pubmed CrossRef
  29. Giancotti FG, Ruoslahti E: Integrin signaling. Science 285: 1028-1032, 1999.
    https://doi.org/10.1126/science.285.5430.1028.
    Pubmed KoreaMed CrossRef
  30. Jeong SJ, Choi BD, Lee HY, et al.: 660 nm red LED induces secretory leukocyte protease inhibitor (SLPI) in lipopolysaccharide-stimulated RAW264.7 cell. J Nanosci Nanotechnol 15: 5610-5616, 2015.
    https://doi.org/10.1166/jnn.2015.10465.
    Pubmed CrossRef


September 2024, 24 (3)
Full Text(PDF) Free

Social Network Service

Cited By Articles
  • CrossRef (0)
  • Download (61)

Author ORCID Information

Funding Information