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 bones^1,2). However, cytotoxic ions are released from Ti and its alloys, which decrease cell proliferation of osteoblasts^3,4). 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 periodontitis^1,5). 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 implant^6,9) 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 surfaces^1,3), 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 mechanisms^14,15). 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 activities^16-18). Recently, RA has been reported to increase alkaline phosphatase activity and bone mineralization for bone formation and prevent osteoporosis^16,19), 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.

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.

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 adhesion^2,6,8,20). 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 differentiation^2,21). 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

SLPI^1,2) and Tβ4^20,22), 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.

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 formation^3,16,24). The first stage after preosteoblasts come into contact with the implant material surface is cell adhesion and spreading^6,9). 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 FAK^20,25). 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 phosphorylation^8,20). FAs bind to F-actin bundles and recruit FAK and paxillin, which are signaling molecules that affect cell adhesion stability and multiple cellular processes^2,20). 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 ensured^2,28). Integrin-stimulated ERK2 activation induces cell proliferation, survival, migration, and differentiation through the FAK/Grab2/Ras/ERK1/2 signaling pathway^2,21). 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 response^2,29), 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 cytoskeleton^1,20,22). It also plays an important role in the mineralization of bone and dentin²²⁾. SLPI^2,29) and Tβ4^20,22) 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.

None.

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.

This article is not necessary for IRB screening.

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.

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).

Please contact the corresponding author for data availability.