Addition of Ca2+ to Titanium Plates by a Hydrothermal Method and the Effects on Human Gingival Fibroblasts


 Background: Human gingival fibroblasts (hGFs) have key roles in the formation of soft-tissue attachments around dental implants. We added calcium ions (Ca2+) to the surface of titanium plates (TPs) to make it more conducive to the early adhesion and proliferation of hGFs. Methods: Ca2+ was loaded onto the TP surface by a hydrothermal method. The morphology and composition of TP surfaces were determined by scanning electron microscopy and energy-dispersive spectroscopy. Proliferation of hGF-1 cells was measured by the CCK-8 assay. Immunofluorescence staining was done to detect adherent proteins on the TP surface. TPs were divided randomly into two groups: control and Ca.Results: In the Ca group, irregular lamellar crystals were found on the surface of TPs; The percentage of hGF-1 cells adhering to TPs in the Ca group was significantly higher than that in control group (P < 0.01); The fluorescence of integrin-β1 and F-actin in the Ca group was stronger than that in the control group. Conclusions: Our data suggest that Ca2+ can be added to TP surfaces by a hydrothermal method, and can enhance hGF adhesion. This property may be beneficial if Ca2+ is added to titanium surfaces before dental implantation.


Introduction
With the development of materials science, dental implants are being used increasingly in clinical treatment. Also, the long-term stability of dental implants is garnering attention [1,2].
In the past few decades, good osseointegration has been considered an important indicator to evaluate the success of dental implantation. In recent years, scholars have realized that the long-term stability of implants requires not only good osseointegration, but also good integration of soft tissue at the site of a transgingival implant [3,4]. The latter can separate the complex oral environment from alveolar bone, prevent the invasion of bacteria so as to protect the implant from bacteria and other in ammatory factors and, nally achieve long-term stability of the implant [5,6,7].
The soft tissue at the site of a transgingival implant is composed of epithelial tissue and its deep, dense connective tissue. A good combination of healthy connective tissue and implant abutment is particularly important [8]. Human gingival broblasts (hGFs) have a key role in the formation of soft-tissue attachments around an implant. They are the main cells in the connective tissue at the transgingival site, the main components that can synthesize the extracellular matrix (ECM), and participate in tissue repair/regeneration in the sealing of soft tissue [9,10]. Therefore, one must select suitable materials for the site of a transgingival implant and create them with suitable surfaces for the adhesion and proliferation of broblasts. This strategy facilitates early and rapid formation of good soft-tissue bonding around implants and reduces the risk of bacterial invasion so as to maintain the long-term stability of the implant.
Titanium is the main material for implant abutments due to its excellent biological properties. However, meeting the requirements of rapid integration with the surrounding soft tissue after repair to form an early soft-tissue seal using titanium is di cult. Therefore, several studies have treated the titanium surface to make it more conducive to the early adhesion and proliferation of broblasts so as to form good softtissue sealing faster [11,12,13,14].
The calcium ion (Ca 2+ ) plays a vital part in growth and development, osteogenesis, wound sealing, and antibacterial activities. Ca 2+ , as a signaling molecule, can play a part in the differentiation, proliferation, and apoptosis of cells by regulating different signaling pathways [15,16]. Seo  We added Ca 2+ to the surface of titanium metal by a hydrothermal method to make it more conducive to the early adhesion and proliferation of hGFs. We aimed to promote the formation of a good seal between a titanium-base platform and surrounding soft groups.

SEM of the TP surface
SEM results of the TPs in the two groups are shown in Fig.1 (all images were taken ×800 magni cation).
TPs in the control group had a smooth and at surface. In the Ca group, irregular lamellar crystals were found on the surface of TPs.

EDS of the TP surface
Surface elemental analysis of TPs in both groups as analyzed by EDS is shown in Fig.2. EDS of TPs in the control group showed them to contain oxygen (O) and titanium (Ti), whereas TPs in the Ca group contained O, Ti, and calcium (Ca).

Adhesion by hGF-1 cells
The CCK-8 assay was used to detect the adhesion of hGF-1 cells on the TP surface in the two groups (Fig.3). The percentage of hGF-1 cells adhering to TPs in the Ca group was signi cantly higher than that in control group at 1, 8, and 12 h (P < 0.01). After 1, 3, 5 days of culture, the proliferation trend of hGF-1 cells in the Ca group was signi cantly higher than that in the control group (P < 0.01).
2.4 Immuno uorescence staining for adherent proteins hGF-1 cells were cultured on the TP surface of both groups for 3 days. Then, we observed the distribution of F-actin (red) and integrin-β1 (green) by uorescence microscopy (Fig.4). hGF-1 cells on the TP in the control group had short spindles, were small, spread over a small area, F-actin morphology was discontinuous, the range of integrin-β1 distribution was small, and uorescence intensity was weak. In the Ca group, hGF-1 showed long spindles with a large area of spread, the uorescence intensity of Factin and integrin-β1 was wide, and the cell boundary was intact, continuous, and strong.

Discussion
The ECM-integrins-cytoskeleton is considered to be one of the most important signaling pathways [19]. Integrins act as protein receptors on cell membranes. Extracellular structures bind to proteins in the ECM. Intracellular structures bind to actin laments on the cytoskeleton through actin, thereby realizing bidirectional transduction of intracellular and intracellular signals. Cells have transmembrane adhesion molecules on their surface (including integrin) that facilitate adhesion to the ECM surface [20,21]. Most human cells proliferate, migrate, and differentiate after adhering to the ECM through their adhesion proteins. We hypothesized that Ca 2+ addition to a titanium surface using a hydrothermal method would provide a favorable surface for ECM proteins to adsorb onto the titanium matrix.
In vitro studies, SEM, and EDS showed that Ca 2+ could be added to the surface of TPs by a hydrothermal method. Immuno uorescence staining revealed that, compared with the control group, hGF-1 cells on TPs in the Ca group grew spindles and had a wider area of spread; also, more hGF-1 cells were on the TP surface and they had more intercellular connections. F-actin is a basic cytoskeletal component involved in the movement and proliferation of cells. Therefore, Ca 2+ seemed to promote the adhesion of hGF-1 cells to the surface of TPs by enhancing F-actin expression.
Palaiologou et al. demonstrated in an in vitro study that an integrin-β subgroup binding different integrinα subunits had the highest content on hGF membranes [22]. Hence, we selected integrin-β1 for observation. The cytoskeleton is a complex network comprising different protein bers and various regulatory proteins, including micro laments, microtubules, and intermediate laments. Among them, micro laments are the main purveyors of signal transduction in the cytoskeleton, and the latter is composed mainly of F-actin. Therefore, rhodamine was selected for F-actin-speci c staining in the present study. Staining showed that integrin-β1 and F-actin had stronger uorescence in the Ca group than that in the control group, which suggested that Ca 2+ adsorbed more ECM proteins, thereby promoting the formation of more adhesive plaques in hGF-1 cells. These results also suggest that Ca 2+ addition to TPs by a hydrothermal method can promote the adhesion and proliferation of hGF-1 cells on the TP surface.
The main limitation in our study was that we did not study the antibacterial properties of the TPs. In the mouth, implant abutments are surrounded by various types of bacteria.

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Ca 2+ can be added to the surface of TPs by a hydrothermal method, and can enhance hGF adhesion. This property may be bene cial if Ca 2+ is added to titanium surfaces before dental implantation.

Titanium plates (TPs)
Commercially available plates made of pure titanium (15 mm in diameter and 1.5 mm in thickness) were used (Fig.5). TPs were divided randomly into two groups: control (Cont) and Ca (hydrothermal treatment with CaCl2 solution (20 mmol/L) for 10 h at 160°C).

5.2
Scanning electron microscopy (SEM) of the TP surface After the two groups of TPs had dried naturally, the surface was sprayed with gold. We observed the morphology of each TP surface by SEM using an acceleration voltage of 15 kV and magni cation of ×800.

Energy-dispersive spectroscopy (EDS) of the TP surface
After TPs in the two groups had dried naturally, the surface was sprayed with gold. We analyzed the elemental composition on the surface of each TP by EDS.

Culture of hGF-1 cells
TPs in both groups were cleaned ultrasonically with pure acetone for 20 min, anhydrous ethanol for 10 min, deionized water for 10 min, and then sterilized with ultraviolet light. hGF-1 cells (2×10 4 cells/mL, CL-0356, Procell Life Science&Technology Co.,Ltd.) were inoculated on the surface of TPs and Dulbecco's modi ed Eagle's medium containing 10% fetal bovine serum was added. Culture was undertaken in a sterile environment at constant temperature (37°C) in an incubator containing 5%CO 2 .

Adhesion of hGF-1 cells
Cell Counting Kit (CCK)-8 diluent solution (500 μL) was added to each TP, and the latter placed in an incubator at 37°C for 1 h away from light. The optical density of each well at 450 nm was measured, and the reference wavelength was 450 nm. The adhesion and proliferation of hGF-1 cells on the TP surface were detected at 1, 8, and 12 h, as well as at 1, 3 and 5 d.

Declarations
Availability of data and material The datasets analyzed during the current study are available from the corresponding author on reasonable request.  Adhesion ratio of hGF-1 cells. (a) The adhesion ratio on the TP surface in the two groups at 1, 8, and 12 hours. Each bar represents the mean ± SD. T-test; * * p < 0.01 between the indicated groups. (b) The adhesion ratio on the TP surface in the two groups at 1, 3, and 5 days. Each bar represents the mean ± SD. T-test; * * p < 0.01 between the indicated groups.

Figure 4
Immuno uorescence staining for adherent proteins. hGF-1 cells were cultured on the TP surface in the two groups for 3 days. Observed the distribution of F-actin (red) and integrin-β1 (green) by uorescence microscopy.