Use of Polyphenol Tannic Acid to Functionalize Titanium with Strontium for Enhancement of Osteoblast Differentiation and Reduction of Osteoclast Activity

Implant anchorage remains a challenge, especially in porous osteoporotic bone with high osteoclast activity. The implant surface is modified with osteogenic molecules to stimulate osseointegration. Strontium (Sr) is known for its osteogenic and anti-osteoclastogenic effects. In this study, Sr was immobilized on a titanium (Ti) surface using bioinspired polyphenol tannic acid (pTAN) coating as an ad-layer (Ti-pTAN). Two separate coating techniques were employed for comparative analysis. In the first technique, Ti was coated with a tannic acid solution containing Sr (Ti-pTAN-1Stp). In the second method, Ti was first coated with pTAN, before being immersed in a SrCl2 solution to immobilize Sr on Ti-pTAN (Ti-pTAN-2Stp). Ti-pTAN-1Stp and Ti-pTAN-2Stp augmented the alkaline phosphatase activity, collagen secretion, osteocalcin production and calcium deposition of MC3T3-E1 cells as compared to those of Ti and Ti-pTAN. However, osteoclast differentiation of RAW 264.7, as studied by TRAP activity, total DNA, and multinucleated cell formation, were decreased on Ti-pTAN, Ti-pTAN-1Stp and Ti-pTAN-2Stp as compared to Ti. Of all the substrates, osteoclast activity on Ti-pTAN-2Stp was the lowest. Hence, an economical and simple coating technique using pTAN as an adlayer preserved the dual biological effects of Sr. These results indicate a promising new approach to tailoring the cellular responses of implant surfaces.


Introduction
Low bone mineral density and altered microarchitecture are prominent characteristics of osteoporotic bone, especially in post-menopausal, elderly women [1]. Apart from being susceptible to fractures, the porous architecture of fragile osteoporotic bones affects the anchorage of screws, leading to post-surgical complications and implant failures [2]. Balanced regulation of osteoblast and osteoclast activity in the peri-implant region is crucial for successful osseointegration [3,4]. Thus, the surfaces of implants were functionalized with osteogenic molecules to promote bone-implant integration [5,6]. The physiochemical properties of the implant surface, such as roughness, topography and chemical modifications, play a crucial role in modulating the activity of bone cells at the bone-implant interface, and hence, influence osseointegration [7][8][9]. Surfaces were also functionalized with bone morphogenetic protein-2 (BMP-2), BMP-7, vascular endothelial growth factor and hydroxyapatite to regulate the cellular behavior on the implant surface and augment ossteointegration [6,[10][11][12]. Anti-osteoporotic drugs, such as bisphosphonates, have gained research interest in surface science by demonstrating an improvement of bone-implant integration in osteoporotic rats [13]. Yet, atypical fractures caused by bisphosphonates remain a concern [14].

Material Preparation
Sandpaper (600-and 1200-grit) was used to polish titanium alloy (Ti-6Al-4V foils, 10 mm × 10 mm, Goodfellow Cambridge Ltd. Huntingdon, England). The titanium (Ti) foils were then rinsed in water-bath ultrasonicator for 10 min. Sonication was further carried out in Kroll s reagent (4.0% HF, 7.2% HNO 3 , 88.8% water) to remove the accumulated carbide resulted from polishing [10]. Next, the reaction was stopped by 1 N sodium hydroxide addition, and subsequently, the substrates were sonicated 10 min each in dichloromethane, acetone, and water. The substrates were immersed for 30 min in 40% nitric acid for surface passivation and thoroughly washed with water to remove nitric acid.
The procedure for polyphenol tannic acid (pTAN) coating for Ti (named as Ti-pTAN) was followed as previously reported with slight modification [23]. In brief, Ti foils were immersed for 24 h in 2 mg/mL solution of pTAN prepared in bicine buffer (100 mM) with 0.6 M NaCl at 7.8 pH. The procedure was carried out in dark with gentle shaking. The substrates were washed thoroughly in water and further submerged in water for 2 days at 37 • C; the water was changed every 24 h.
Ti-pTAN substrates were functionalized with Sr by two different methods ( Figure 1). In the first one-step method, Ti foils were incubated in pTAN solution (2 mg/mL, 100 mM bicine buffer, pH = 7.8) containing SrCl 2 (0.3 M Sr) for 24 h with gentle shaking in dark. 0.3 M SrCl 2 was used to maintain the similar ionic stoichiometry as 0.6 M NaCl in bicine buffer. These substrates were named Ti-pTAN-1Stp. The second method was a 48 h-long, two-step process wherein pTAN coating and Sr coating were carried out separately. In the first 24 h, Ti was coated with pTAN, as discussed above. Subsequently, Ti-pTAN substrates were immersed in a SrCl 2 solution (0.1 M Sr in milli-Q water) for another 24 h in the dark. In this case, the Sr solution comprising minimum Sr concentration with maximum immobilisation efficiency was used. These substrates were denoted as Ti-pTAN-2Stp. After the Sr coating, Ti-pTAN-1Stp and Ti-pTAN-2Stp were sonicated for 5 min and washed thoroughly with milli-Q water to remove unbound Sr. The substrates were irradiated with UV for 30 min and transferred to a 24-well cell culture plate prior to cell seeding.

Substrate Characterization
Surface chemical composition of all the substrates were analyzed using X-ray photoelectron spectroscopy (XPS) (Kratos AXIS Ultra DLD spectrometer, Kratos Analytical Ltd, Manchester, UK). The binding energies of all the elements were referenced to C 1s signal at 284.6 eV. The surface roughness was studied using a contact based Nanomap-LS 3D profilometer (AEP Technologies, Santa Clara, CA, USA). Water contact angle (WCA) analysis for all the substrates were performed using contact angle analyzer Phoenix 300 touch (Surface Electro Optics, Gyeonggi-do, Korea). The measurements were carried out according to the sessile drop technique and a video camera. Ten microliters of sessile drop of distilled water was transferred onto the substrate using a micro-syringe. The contact angle values on both sides of the drop were measured using tangent rule to ensure symmetry and horizontal level. To estimate the amount of bound Sr ions, Ti-pTAN-1Stp and Ti-pTAN-2Stp substrates were soaked in a 2% nitric acid solution overnight with shaking. Sr ions dissolved in the nitric acid solution were quantified using inductively coupled plasma mass spectrometry (ICP-MS, CX 7700x Agilent, Santa Clara, CA, USA).

Strontium Ions Release Study
Ti-pTAN-1Stp and Ti-pTAN-2Stp substrates were immersed in 1 mL of phosphate buffer saline (PBS) at 37 • C for 14 days. PBS was collected at regular intervals and replenished with fresh PBS. Sr ions in released in PBS were measured using inductively coupled plasma mass spectrometry (ICP-MS, CX 7700x Agilent, CA, USA).
RAW 264.7 (ATCC, Manassa, VA, USA) murine pre-osteoclasts were cultured in complete Dulbecco s Modified Eagle Medium (10% heat inactivated FBS, 100 U/mL penicillin and 100 µg/mL streptomycin (Invitrogen, Waltham, MA, USA). For osteoclast differentiation studies, RAW 264.7 were cultured in presence of 50 ng/mL murine RANKL (R&D Systems, Minneapolis, MN, USA) in complete phenol red free MEM-α (10% heat inactivated FBS, 100 U/mL penicillin, 100 µg/mL streptomycin) at cell density of 2000/cm 2 on all the substrates. 100 µL of cell suspension was loaded on the substrates. After 6 h of incubation, an additional 400 µL of cell culture media was added to the wells. Cells were maintained in a humid environment at 37 • C with 5% CO 2 , and the spent media were changed every 2-3 days.

ALP Activity, Collagen-1 and Osteocalcin Production of MC3T3-E1 Cells
ALP activity of osteoblasts was estimated in the cell culture supernatant at various time points with QuantiChrom Alkaline Phosphatase Assay Kit (BioAssay Systems, Hayward, CA, USA). In brief, 50 µL of sample was incubated with 150 µL ALP working reagent for 4 min. Absorbance of 405 nm was measured at 0 and 4 min using microplate reader (Synergy H1). The ALP activity in terms of pNP produced was estimated as per the formula provided in the kit, and normalized with the total protein.
The unit for ALP was µM of pNP produced per minute per mg of protein. Collagen 1 and osteocalcin were also measured in cell culture supernatant on day 14 by using Pro-Collagen I alpha 1 SimpleStep ELISA kit (Abcam, Cambridge, UK) and Osteocalcin ELISA kit (Cloud-Clone Corp., Katy, TX, USA) respectively, and the values were normalized to total protein.

Collagen Staining of Osteoblasts
MC3T3-E1 was cultured on various substrates for 14 days. After washing with phosphate buffer saline (PBS), cells were fixed for 15 min in 4% paraformaldehyde which was followed by 5 min treatment with 0.1% triton X-100 to permeabilise cell membrane. Non-specific binding was blocked by overnight incubation of cells in 3% bovine serum albumin (BSA) at 4 • C. Cells were incubated with anti-mouse collage type I primary antibody (Merck, Darmstadt, Germany) for 90 min. Secondary antibody Alexa Fluor 546 tagged anti-rabbit IgG (H + L) (Invitrogen) and alexa Fluor 488 phalloidin (Invitrogen) for actin staining were added to cells for 30 min followed by nuclei labelling using DAPI. Cells images were acquired using Olympus FV1000 confocal laser scanning microscope (CSLM, Olympus, Tokyo, Japan).

Alizarin Staining and Calcium Estimation
After 14 days of culture MC3T3-E1 cells in osteogenic medium, the cells were washed with PBS and fixed with 70% ethanol at 4 • C for 1 h. Alizarin red solution (2% in water) was added to the cells for 1 h. The cells were washed with copious amount of water to remove unbound alizarin and observed under microscope (Leica Microsystems, Wetzlar, Germany).
Calcium deposited by MC3T3-E1 cells was also quantified after 14 days of culture. The substrates were submerged in 500 µL of 2% nitric acid overnight with gentle shaking, to dissolve the deposited calcium. The eluted calcium was then quantified using QuantiChrom Calcium Assay Kit (BioAssay Systems, Hayward, CA, USA) according to manufacturer s instructions. In brief, 5 µL of the sample was incubated with 200 µL of kit s working solution for 5 min. Absorbance was measured at 612 nm using a microplate reader (Synergy H1).

TRAP Activity
To trigger osteoclastogenesis, RAW 264.7 cells were exposed to 50 ng/mL of RANKL for 3 and 5 days. TRAP activity of the cells was estimated in cell culture supernatant at day 3 and day 5 of culture using TRAP activity assay kit (Takara Bio Inc, Shiga, Japan). Sample was mixed with acid phosphatase buffer (1:1 v/v) provided with the kit and incubated for 60 min at 37 • C. Stop solution, 0.5 N sodium hydroxide, was added and absorbance was taken at 405 nm by microplate reader (Synergy H1). The calculation of TRAP activity was based on pNP generated, which were normalized to total protein. Hence, the unit of TRAP activity was µM of pNP produced per minute per mg of protein.

DNA Quantification
RAW 264.7 cells were lysed by three freeze thaw cycles in milli-Q water following 5 days of culture with RANKL. After centrifugation for 20 min at 12,000 g (4 • C), the samples were mixed with picogreen reagent (Thermo Fisher Scientific, Waltham, MA, USA) and incubated for 5 min at RT. A microplate reader (Synergy H1) was used to excite the samples at 480 nm, and emission was measured at 520 nm.

Actin and TRAP Staining of Osteoclasts
RAW 264.7 cells were cultured on various substrates. After 5 days of RANKL exposure, the cells were fixed with 4% paraformaldehyde for 15 min and incubated for 5 min in triton 0.1% X-100 to increase cell membrane permeability. To prevent non-specific binding, the cells were immersed in 3% BSA (overnight, 4 • C). 90 min incubation in anti-TRAP primary antibody (Takara Bio Inc, Shiga, Japan) followed by 30 min incubation in premixed solution of secondary antibody (Alexa Fluor 546 tagged-Goat anti-Mouse IgG (H + L), Thermo Fisher Scientific) and phalloidin (Alexa fluor 488 tagged, Thermo Fisher Scientific) was carried out to stain TRAP and actin in the cells. After nuclei staining with DAPI, the cells were imaged with Olympus FV1000 CLSM (Olympus, Japan). Multinucleated cells with more than three nuclei (blue), and positive for TRAP (red) were classified as osteoclasts.

Statistics
All experiments were replicated more than three times and the data were presented as mean ± standard deviation (SD). The statistical analysis was carried out by one-way analysis-of-variance and Tukey post hoc test. p < 0.05 was considered as statistically significant.

Surface Characterization
In the current study, polyphenol tannic acid chemistry is utilized to immobilize Sr on a Ti surface. The elemental compositions of the surfaces were examined by XPS. Signal peaks for elements such as O 1s (530 eV), Ti 2p (460 eV) and C 1s (285 eV) were observed on pristine Ti ( Figure 2A and Table 1). C 1s signal on Ti was present due to inevitable hydrocarbon contamination. Therefore, C 1s signals are commonly used as reference for signal calibrations in XPS scans [27]. Ti-pTAN substrates displayed a decrease in Ti 2P signal signifying successful deposition of pTAN on Ti ( Figure 2B and Table 1). Ti-pTAN-1Stp and Ti-pTAN-2Stp also displayed a drop in Ti 2P signals. However, Ti 2P signals on Ti-pTAN-1Stp was higher than the Ti 2P signals on Ti-pTAN and Ti-pTAN-2Stp. This suggested that the substitution of Na with Sr in bicine buffer for 1 step coating method resulted in a comparatively thinner pTAN coating.  An increased C 1s signal was detected on Ti-pTAN substrate. The observed carbon to oxygen ratio (C/O, 2.17) was slightly higher than theoretical C/O ratio of pTAN (1.65), which may be the result of carbon contamination. Nonetheless, for Ti-pTAN-1Stp ( Figure 2C and Table 1) and Ti-pTAN-2Stp ( Figure 2D and Table 1), the C/O ratios were 1.52 and 1.64 respectively. Moreover, distinct Sr 3d signals were observed on Ti-pTAN-1Stp and Ti-pTAN-2Stp, suggesting the successful immobilization of Sr by pTAN coating.

Strontium Release
The release of Sr from Ti-pTAN-1Stp-Sr and Ti-pTAN-2Stp-Sr was estimated by ICP-MS. A burst release was observed after 24 h (Figure 4). However, subsequent time points revealed minimal release, with about 20% remaining on Ti-pTAN-1Stp-Sr and about 70% of the immobilized Sr remaining on Ti-pTAN-2Stp-Sr for 2 weeks. This shows that the binding of Sr 2+ ions on Ti-pTAN-2Stp-Sr were stronger, compared to Ti-pTAN-1Stp-Sr.
The effect of immobilized Sr on late differentiation markers such as osteocalcin [29] and matrix mineralization were investigated after 14 days of culture. Alizarin S stain and calcium estimation were performed to monitor the effects of Sr immobilized substrates on osteoblast mineralization. Stronger alizarin stain was observed on Ti-pTAN-1Stp-Sr (Figure 8Ac) and Ti-pTAN-2Stp-Sr (Figure 8Ad) as compared to Ti (Figure 7Aa) and Ti-pTAN (Figure 7Ab). Similarly, calcium estimated on Ti-pTAN-1Stp-Sr and Ti-pTAN-2Stp-Sr was higher than on Ti (p < 0.01) and Ti-pTAN (p < 0.01) ( Figure 8B). Moreover, calcium amounts on Ti-pTAN were elevated as compared to Ti (p < 0.05), suggesting that pTAN coatings have a positive influence on osteoblast mineralization. Osteocalcin production was also higher on Ti-pTAN-1Stp-Sr and Ti-pTAN-2Stp-Sr as compared to Ti (p < 0.01) and Ti-pTAN (p < 0.05 and p < 0.01, respectively).

Discussion
Since the imbalance in osteoclast and osteoblast activity in patients with bone metabolic disease can be an obstacle to bone implant success, coatings that actively aim at correcting this imbalance are required to recover bone turnover and improve the osseointegration of bone implants. In this study, a pTAN coating was used to immobilize Sr on a Ti surface. Two separate schemes were employed ( Figure 1); the first scheme was a one-step process, wherein Ti-pTAN-1Stp-Sr substrates were prepared by replacing sodium with Sr in tannic acid solution in bicine buffer. In the second scheme, Ti-pTAN-2Stp-Sr substrates were prepared by a two-step method, wherein a pTAN coating was carried out in a bicine buffer containing sodium, before coating the substrates with Sr. The Sr metal ions were immobilized on pTAN-coated Ti (Figure 2 and Table 1). The surface immobilization efficiency was higher for the two-step coating method. Hence, unlike the one-step coating method, the two-step coating method not only displayed higher surface density of immobilized Sr, but also showed stronger binding, as 70% of the Sr remained bound and was not released for two weeks (Figure 4). The thicker tannic acid coating conferred by the two-step method could contribute to more phenol groups increasing their interactions with Sr 2+ ions to increase immobilization efficiency. pTAN coating kinetics and mechanisms have been described previously [32]. It is proposed that the first layer of pTAN on the Ti surface is though the formation of TiO(OH)pTAN hydroxo complexes [25,32]. Subsequent layers are built up by the heterogeneous polymers formed by the autooxidation of pTAN [25]. pTAN is also known to form polymers through the formation of multivalent coordination bonding with metal ions [24,26]. Hence, it is assumed that the pTAN-Sr coatings on Ti-pTAN-1Stp-Sr and Ti-pTAN-2Stp-Sr are formed by auto-oxidation of pTAN and multivalent coordination bonding with Sr.
A decrease in osteoblast proliferation was observed for cells cultured on Ti-pTAN-1Stp-Sr and Ti-pTAN-2Stp-Sr ( Figure 5). Joseph Caverzasio observed that the addition of Sr to a cell culture medium enhanced osteoblast proliferation [3]. Almeida et al. reported that a Sr salt supplemented in media did not affect MC3T3-E1 proliferation at lower concentrations (0.05 and 1 mM), but at higher concentrations such as 0.5 mM, a decrease in proliferation was observed as compared to that of untreated controls [33,34]. Furthermore, Ti functionalized by Sr via magnetron co-sputtering technology also decreased proliferation of human dental pulp stem cells. [19] Yet, in both studies, osteoblast differentiation was augmented due to Sr introduction, which was consistent with our results, as described below. It is known that a decrease in osteoblast proliferation and growth arrest are associated with the elevated production of differentiation markers such as ALP, COLL 1 etc. [35,36] As such, the effects of Ti-pTAN-1Stp-Sr and Ti-pTAN-2Stp-Sr on osteoblast differentiation markers were investigated.
ALP and COLL 1 are early differentiation markers for osteoblasts, whereas osteocalcin is a late differentiation marker [29]. By secreting ALP and collagen 1 in the early phases of differentiation and mineralizing matrix in the later stages, MC3T3-E1 exhibits successive differentiation sequence which is comparable to osteoblast in bone [35]. Hence, this cell line is widely used as in vitro model for osteoblasts research. Generally, the ALP expression of differentiating osteoblasts augments in the early phases of differentiation, leading to matrix maturation, while the level drops before matrix mineralization [37]. As presented, ALP production was enhanced on day 5 for all the substrates with a decline in the activity on day 7 and day 14, suggesting the progression of the cells to the mineralization phase ( Figure 6A). Yet, compared to Ti, Sr-immobilized substrates such as Ti-pTAN-1Stp-Sr and Ti-pTAN-2Stp-Sr boosted ALP activity on day 5 and day 7. COLL 1 secretion, which is also an early differentiation marker of osteoblasts, was monitored after 14 days of culture ( Figures 6B and 7) [29]. Sr surface coated substrates displayed higher COLL 1 production. The majority of MC3T3-E1 cells on Sr immobilized substrates, in particular the cells on Ti-pTAN-1Stp-Sr, exhibited cuboidal morphology ( Figure 6), which is a characteristic feature of differentiating cells [38]. On the other hand, the majority of cells cultured on Ti and Ti-pTAN displayed a fusiform appearance (Figure 6), which is a predominant feature of proliferating cells [38]. This further corroborates the osteogenic potential of Sr immobilized substrates. Furthermore, the Sr-based surface coatings also augmented in the osteocalcin and matrix mineralization by the osteoblasts (Figure 8).
Studies had shown that strontium supplementation in media inhibited osteoblast mineralization in vitro [39,40]. Contrastingly, another report established that Sr enhanced ALP, osteocalcin secretion and the mineralization of mouse bone marrow stromal cells [41], and human primary osteoblasts [42]. Sr supplemented in cell culture media as a SrCl 2 triggered osteogenic differentiation of human mesenchymal stem cells (MSCs) and stimulated in vivo bone deposition when delivered locally as Sr-HA-collagen scaffold to treat rat calvarial defect via Wnt signal transduction [43]. In this study, Sr-immobilized substrates such as Ti-pTAN-1Stp-Sr and Ti-pTAN-2Stp-Sr augmented ALP activity, COLL 1 production, osteocalcin secretion and calcium deposition of MC3T3-E1 cells. However, Ti-pTAN-2Stp-Sr showed a higher surface density of Sr as compared to Ti-pTAN-1Stp-Sr, although the potential to augment osteogenesis was similar for both the substrates. This suggests that even if the two-step method of coating resulted in a higher efficiency of Sr binding properties, a low Sr density on the surface, such as 0.32 ± 0.02 µg/cm 2 , is sufficient in promoting osteoblast differentiation.
Osteoclasts are important cells for bone remodeling [44]; hence, the regulation of their activity on implant surfaces is crucial for osseointegration. The effects of osteoclasts on Sr-modified surfaces were also investigated. As a RANKL-sensitive pre-osteoclast population, RAW 264.7 can be easily procured and is easy to handle as compared to primary cultures, which makes is an attractive in vitro model for osteoclast research [45]. Osteoclastic differentiation of RAW 264.7 cells on Ti, Ti-pTAN, Ti-pTAN-1Stp-Sr and Ti-pTAN-2Stp-Sr were studied by estimating TRAP activity. TRAP activity was used as a marker for osteoclast differentiation and resorption [46][47][48]. pTAN coated substrates such as Ti-pTAN, Ti-pTAN-1Stp-Sr and Ti-pTAN-2Stp-Sr decreased TRAP activity and total DNA (Figure 9). However, on day 5, the decrease in TRAP production was greatest in Ti-pTAN-2Stp-Sr. Likewise, immunofluorescence labelling of intracellular TRAP production was also lowest on the Ti-pTAN-2Stp-Sr substrate (Figure 10d3). Strontium ranelate was shown to inhibit RANKL mediated osteoclastogenesis of RAW 264.7 and human peripheral blood monocyte cells [49]. As shown by Chung et al., Ti modified with Sr substituted HA by micro-arc treatment reduced osteoclast differentiation of RAW 264.7 cells [50]. Equally, this study demonstrated that Ti-pTAN-2Stp-Sr reduced osteoclast activity as compared to Ti and Ti-pTAN. Florencio-Silva et al. reported that osteoclast precursors fuse, polarize, and rearrange actin cytoskeleton to form actin rings and a sealing zone [51]. The formation of multinucleated cells was also reduced on all the pTAN coated substrates such as Ti-pTAN, Ti-pTAN-1Stp-Sr and Ti-pTAN-2Stp-Sr ( Figure 10). However, the inhibitory effects of Ti-pTAN-2Stp-Sr were superior to those of Ti, Ti-pTAN and Ti-pTAN-1Stp-Sr substrates.

Conclusions
In this study, a pTAN coating was used to immobilize Sr on a Ti surface. Although Ti-pTAN-2Stp-Sr showed a higher surface density of Sr as compared to Ti-pTAN-1Stp-Sr, the ability to augment osteogenesis was similar for both the substrates. Osteoclast differentiation studies revealed that the Ti-pTAN-2Stp-Sr substrates decreased the TRAP activity of osteoclast significantly as compared to Ti, Ti-pTAN and Ti-pTAN-1Stp-Sr. The pTAN coating also had an influence on the negative regulation of osteoclast differentiation, as reported [52]. Henceforth, this economical and simple coating of pTAN with inherent anti-osteoclastogenic properties could be exploited to immobilize osteogenic metal ions such as Sr 2+ on surfaces. The two-step method was more efficient to immobilize Sr, as well as to modulate osteoblast and osteoclast cells.
It is hoped that these findings will be beneficial to deliver Sr in order to tailor cellular responses at bone-implant interfaces, especially for osteoporotic bone. The imbalance in osteoclasts and osteoblasts activity in patients with bone metabolic disease such as osteoporosis can be an obstacle to bone implant success [53][54][55]. Coatings that actively aim at correcting this imbalance are required to recover bone turnover and improve the osseointegration of bone implants [56,57]. We believe that these surface coatings will be beneficial to regulate osteoclast and osteoblast development at implant surfaces. Nonetheless, we acknowledge that the in vitro results presented here are conducted in controlled laboratory settings and could not be extrapolated to in vivo or clinical conditions. The coupling of bone formation and bone resorption in a basic multicellular unit of a healthy bone is a multifaceted process which involves the interplay of various regulatory molecules and bone cells [58,59]. Further studies are needed to understand the viability of coatings and the potential to regulate the molecular and cellular interplay in in vivo settings.

Conflicts of Interest:
The authors declare no conflict of interest.