Introduction
Dental implants have been widely used to treat patients with partial or complete edentulism and show long-term stability and a high survival rate [
1,
2]. Titanium is currently the most extensively applied biomedical implant material due to its strong anticorrosive characteristics, biocompatibility, and mechanical resistance [
3]. Although titanium is known to be a good osteoconductive material, previous research has shown that it always forms a passive and protective surface oxide layer once exposed to the atmosphere [
4], which further leads to poor osteointegration between bone and implant interfaces.
Currently, most commercially used titanium implants are provided in gas-permeable packaging with a combination of micro- and nanoscale surface roughness modified by sandblasting and acid etching (SLA) [
5,
6]. This topology has been reported to promote new bone formation and enhance osteointegration [
7]. However, because hydrocarbons continually accumulate on fresh titanium surfaces, the zeta potential on the surface changes from positive to negative and blocks the Arg-Gly-Asp (RGD) sequence, a cell adhesion terminal structure, which further leads to time-dependent degradation of titanium osteoconductivity [
8]. Several methods have been published to reactivate aged titanium surfaces, such as ultraviolet (UV) and nonthermal atmospheric pressure plasma (NTAPPJ) treatment [
9,
10]. Studies have shown that the effect of UV treatment could be maintained for only a short period, which could barely meet the criteria of dental clinical conservation [
10‐
13]. In addition, it would be difficult for dentists to apply UV treatment to a titanium surface immediately before use, and this method may result in an increased risk of infection.
Some authors reported that storing titanium implants in alendronate (ALN) or ddH
2O could prevent the accumulation of organic impurities and maintain a hydrophilic surface [
5,
14]. In addition, some authors believe that ALN can also directly stimulate osteoblast activity by increasing alkaline phosphatase (ALP) activity. However, ALN is mainly used as a bone antiresorptive agent by inhibiting osteoclastic activity, and its effect on osteoblasts remains unclear [
15,
16]. Its clinical application is also limited by potential complications [
17]. Our previous work showed that soaking in sodium bicarbonate, a mild alkaline compound with low biohazard potential, changed hydrophobic SLA titanium surfaces to hydrophilic and further improved osteoblast early adhesion and differentiation in vitro [
18,
19]. Moreover, sodium bicarbonate is a well-known pH buffer that regulates acid and base equilibrium by hydrolysis and ionization reactions. This process is influenced by the concentration of sodium bicarbonate and environmental temperature. The extracellular pH (pH
e) regulates the balance of resorption and formation/mineralization [
20], and a more alkaloid environment could promote mineralization by increasing osteoblast activation [
21]. Therefore, a proper concentration of sodium bicarbonate has the potential to avoid the aging and enhanced osseointegration of titanium implants. To the best of our knowledge, few studies have evaluated whether SLA titanium discs soaked in solutions with different pH values regulate seeded osteoblast fates.
In this study, we aimed to focus on the concentration of sodium bicarbonate used to soak SLA titanium discs. We investigated the characteristics of titanium surfaces after modification by sodium bicarbonate and evaluated the effect of varying concentrations of sodium bicarbonate on bone marrow mesenchymal stem cell (BMSC) adhesion, proliferation, and differentiation.
Discussion
In this study, we aimed to evaluate whether the treatment of SLA titanium discs with different concentrations of sodium bicarbonate would affect their biological activity and further regulate BMSC osteogenic differentiation. The results showed that 100 mM sodium bicarbonate could promote BMSC osteogenic differentiation. Due to its high biocompatibility and low cost, sodium bicarbonate has the potential to be applied in dentistry as a solution to conserve dental implants.
To date, most titanium implants have been stored under ambient conditions. Theoretically, titanium or the titanium oxide surface layer, which is produced on titanium contact with atmospheric oxygen, is a high-energy surface [
8]. However, the high surface energy enhances adsorption of hydrocarbons from the atmosphere and further decreases the surface energy. Given that this conversion occurs in a time-dependent way, this behavior has been defined as biological aging [
4,
22,
23]. Several previous studies investigated the biological aging of the titanium surface, which could impact the osteoconductive and osteoinductive abilities of implants [
7,
24,
25]. One important change in aging SLA titanium implants is the conversion of hydrophilic surfaces to hydrophobic surfaces due to carbon contamination [
26,
27]. Our results showed soaking in sodium bicarbonate with a concentration above 20 mM significantly enhanced the hydrophilicity of titanium surfaces. Tugulu et al. believed this change could be explained by the cavities on SLA titanium surfaces, which filled with liquid after immersion into any solution due to capillary forces [
28]. We also found that increased concentration of sodium bicarbonate improved the surface hydrophilicity, which was probably due to changes in the concentration of hydroxyl groups [
29]. Moreover, our previous studies have shown that the effect of sodium bicarbonate immersion on titanium discs was reduced by extensive rinsing [
18,
19]. In this study, we immersed the discs into sodium bicarbonate without rinsing shortly before BM seeding to simulate sodium bicarbonate preservation of dental implants. The results showed this experimental procedure had a predictable effect on the pH of cell culture media (Fig.
4b). The reproducibility of the results might come from the stable surface hydrophilicity and elemental components after sodium bicarbonate immersion, which limit the sodium bicarbonate affinity to titanium [
18]. Thus, this method may prevent the aging of titanium surfaces, and a suitable concentration of sodium bicarbonate for implant storage is above 20 mM.
In addition to these findings, once the concentration of sodium bicarbonate was within the range of 0–200 mM, it could obviously affect the pH value of the solution at room temperature. When the concentration was 100 mM, the pH value reached a maximum, approximately 9 (Fig.
4). Interestingly, we found that this variation further affected the pH value of cell culture media. Although this study did not clearly identify this phenomenon, our results suggested a correlation between the increased pH of the initial media after BMSC seeding and the pH of different concentrations of sodium bicarbonate solution for titanium disc immersion. Previous research demonstrated that the extracellular pH value is one of the significant aspects with respect to cells in contact with titanium surfaces. The extracellular acid-base equilibrium influences the function of bone cells and bone mineralization processes. There is a rapid decrease in the pH value of the surrounding areas of implants at the early phase of the bone healing process, which is due to the tissue inflammatory response and osteoclast activity [
30,
31]. Therefore, a higher extracellular pH value is thought to be beneficial for enhancing osteoblast differentiation and inhibiting osteoclast differentiation [
32,
33]. Liu et al. reported a biodegradable implant made of β-tricalcium phosphate (β-TCP), calcium silicate (CS), and 10% strontium-substituted calcium silicate (Sr-CS) powders by a chemical precipitation method, which could enhance the osteoblast differentiation and accelerate the repair of osteoporotic bone defects [
31]. Li et al. designed a Mg-Fe layered double hydroxide (LDH) film coating on commercial titanium surfaces via hydrothermal treatment, and by increasing microenvironment pH value, this new method showed good biocompatibility and osteogenic activity both in vitro and in vivo [
34]. Our results suggest that titanium discs soaked in sodium bicarbonate successfully promoted the pH value of cell culture media for 5–7 days (Fig.
4b) and further enhanced BMSC adhesion, proliferation, and differentiation (Figs.
5,
6,
7,
8,
9). This finding may be due to the activation of integrin receptors in this weakly alkaline environment, which stimulated the phosphorylation of FAK and further led to the upregulation of ALP and Runx2 expression (Figs.
10‐
11). Therefore, a suitable and continuous alkaline environment would promote early osteointegration between bone and titanium surfaces. With the rapid development of coating technology, several new strategies have been developed for modification of pure titanium surfaces, such as nanotubes, nanoparticles, and nanofibers [
35]. Interestingly, these structures have the ability to conserve small molecules, which made this facile sodium bicarbonate soaking design even more promising in implant conservation.
Several limitations of this study must be considered when interpreting the above data. The pH of cell culture media mentioned above cannot be precisely ascertained, because we did not find a feasible probe to detect the interaction between the cell membrane and the material surfaces. The current results could partially reflect the effect of sodium bicarbonate immersion, which is also affected by factors such as cell metabolism. Additionally, in vitro findings do not always translate to in vivo results. Here, we measured the variation in the extracellular pH value to evaluate the BMSC osteogenic ability, and therefore, we did not account for the competition of other cell lines or the effects of tissue fluid and blood pH balance, which may be affected by ventilation and respiration of the body. However, implants are always placed in a local area, and soaking in sodium bicarbonate could cause a cellular scale change, which could be recognized as a generalized gradient. This local gradient is expected to generate a broader tissue response, and further experiments are needed. In addition, in regard to the potential solutions for storing implants, an in vitro study is needed.
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