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Journal of Periodontal & Implant Science logoLink to Journal of Periodontal & Implant Science
. 2024 Jan 10;54(5):336–348. doi: 10.5051/jpis.2303000150

Guided bone regeneration of calcium phosphate-coated and strontium ranelate-doped titanium mesh in a rat calvarial defect model

Seon Mi Byeon 1, Tae Sung Bae 1, Min Ho Lee 1, Seung Geun Ahn 2,3,✉
PMCID: PMC11543329  PMID: 38290999

Abstract

Purpose

When applied alone, titanium (Ti) mesh may not effectively block the penetration of soft tissues, resulting in insufficient new bone formation. This study aimed to confer bioactivity and improve bone regeneration by doping calcium phosphate (CaP) precipitation and strontium (Sr) ranelate onto a TiO2 nanotube (TNT) layer on the surface of a Ti mesh.

Methods

The TNT layer was obtained by anodizing on the Ti mesh, and CaP was formed by cyclic pre-calcification. The final specimens were produced by doping with Sr ranelate. The surface properties of the modified Ti mesh were investigated using high-resolution field emission scanning electron microscopy, energy-dispersive X-ray spectroscopy, and X-ray diffraction. To evaluate the effects of surface treatment on cell viability, osteoblasts were cultured for 1–3 days, and their absorbance was subsequently measured. In an in vivo experiment, critical-size defects were created in rat calvaria (Ф=8 mm). After 5 weeks, the rats were sacrificed (n=4 per group) and bone blocks were taken for micro-computed tomography and histological analysis.

Results

After immersing the Sr ranelate-doped Ti mesh in simulated body fluid, the protrusions observed in the initial stage of hydroxyapatite were precipitated as a dense structure. On day 3 of osteoblast culture, cell viability was significantly higher on the pre-calcified Sr ranelate-doped Ti mesh surface than on the untreated Ti mesh surface (P<0.05). In the in vivo experiment, a bony bridge formed between the surrounding basal bone and the new bone under the Sr ranelate-doped Ti mesh implanted in a rat calvarial defect, closing the defect. New bone mineral density (0.91±0.003 g/mm3) and bone volume (29.35±2.082 mm3) significantly increased compared to the other groups (P<0.05).

Conclusions

Cyclic pre-calcification of a Ti mesh with a uniform TNT layer increased bioactivity, and subsequent doping with Sr ranelate effectively improved bone regeneration in bone defects.

Keywords: Bone regeneration, Calcium phosphate, Rats, Strontium ranelate, Titanium

Graphical Abstract

graphic file with name jpis-54-336-abf001.jpg

INTRODUCTION

During dental implant treatment, patients with locally deficient alveolar bone are often treated with guided bone regeneration (GBR). This technique involves the application of a barrier membrane, which is necessary because a sufficient amount of bone mass is required for successful implantation [1]. Initially, the primary function of the barrier membrane was to prevent the infiltration of soft tissue by covering the area of missing alveolar bone. However, its use has evolved, and now it also serves to support the deficient alveolar bone and promote bone regeneration around the implant [2,3].

Barrier membranes are classified as either resorbable or non-resorbable depending on whether they are biodegradable [4]. Non-resorbable polytetrafluoroethylene (PTFE) barrier membranes and titanium (Ti) meshes are widely used, despite the requirement for secondary surgical removal, because they maintain the space for a sufficient period to allow bone regeneration [5,6]. Ti meshes offer greater strength than PTFE barriers, thereby preventing external deformation during the bone regeneration process [7]. Additionally, due to the inherent properties of the material, Ti meshes do not disrupt blood supply and exhibit excellent biocompatibility, leading to a less pronounced inflammatory response, even when exposed locally [8].

Ti has long been used as an implant material due to its excellent corrosion resistance, biocompatibility, and mechanical properties [9]. With advances in nanotechnology, numerous studies have focused on creating implant materials with nanostructured oxide layers on the surfaces of metals such as Ti, zirconium, and niobium [10,11]. A nanostructured surface is reported to offer a greater surface area than a micro-structured one, enhancing osteoblast attachment and proliferation [12]. One approach to modifying the Ti surface into a nanostructure is by forming a Ti oxide (TiO2) nanotube (TNT) layer through anodization, which significantly increases the surface area compared to machined-smooth surfaces [13]. The anodization process yields a TNT layer with uniform thickness, regardless of the underlying surface shape [14]. This TNT layer provides sufficient space to serve as a carrier for bioactive materials or drugs. When a drug is loaded onto the nanotube layer, it is released in a controlled manner to the targeted area, allowing for a substantial effect with a minimal dosage [15].

Studies investigating the promotion of the osseointegration of Ti implants have reported that osseointegration is facilitated by loading calcium phosphate (CaP)-based bioactive materials onto the nanotube layer [16,17]. The CaP coating elicits a favorable host response, chemically bonding the bone to the implant. Additionally, bone regeneration has been augmented by incorporating osteogenesis-inducing drugs into CaP-coated Ti implants [18,19].

Strontium (Sr) ranelate is utilized as a treatment for osteoporosis. It has a dual effect during in vivo bone remodeling, simultaneously promoting bone formation and inhibiting bone resorption [20,21]. Studies have shown that it can increase bone density and reduce the risk of fractures. The long-term sequelae of Sr ranelate use remain unclear, but to date, no serious adverse reactions have been documented. Diarrhea, the most frequently reported adverse reaction, usually subsides within three months of starting treatment. Some patients may also experience headaches and abdominal pain. Sr ranelate is contraindicated in individuals with severe renal impairment and those at risk for venous thrombosis [22,23].

Research is currently exploring how to generate new bone by applying a Ti mesh in GBR [18,19]. There is growing interest in methods that modify the surface of Ti mesh with various osteogenic materials to enhance new bone formation [18,24]. Among these, techniques that modify the Ti surface with Sr are being actively investigated [18,25]. Nanostructured Ti implants containing Sr have shown the ability to improve osseointegration by significantly increasing the expression of genes related to osteogenesis and the production of osteocalcin [26]. Xu et al. [27] evaluated the effect of Sr-doped Ti implant surfaces on osseointegration in diabetic rats. Their findings indicated reduced inflammation in the bone tissue surrounding the Sr-Ti implant during the early healing phase, which may contribute to faster osseointegration in diabetic rats. However, to date, no studies have explored the in vitro and in vivo effects of Ti meshes coated with CaP and doped with Sr following the formation of a TNT layer. In this study, we coated Ti mesh with CaP and doped the TNT layer with Sr ranelate, then evaluated the impact on bone regeneration through both in vitro and in vivo tests. The null hypothesis of the study was that cyclic pre-calcification of Ti mesh to precipitate CaP, along with Sr ranelate doping, would not influence bone regeneration.

MATERIALS AND METHODS

Sample preparation

This study used a thin Ti mesh (TMN355004, Neobiotech, Seoul, Korea; Figure 1A) that was 100 μm thick, 35 mm wide, and 50 mm long. Holes with a diameter of 0.429 mm were made in the specimen at regular 0.452 mm intervals, and the distance between the lines connecting the holes was 0.341 mm. Sixteen specimens (4 per group; Figure 1B) were prepared for animal testing by cutting the mesh into disks with a diameter of 10 mm.

Figure 1. HR FE-SEM image of Ti mesh and methods of experimental groups. (A) HR FE-SEM image of this study’s Ti mesh. Holes with a diameter of 0.429 mm were formed at regular intervals of 0.452 mm, and the distance between the lines connecting the holes was 0.341 mm. (B) Methods of surface treatment by group.

Figure 1

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HR FE-SEM: high-resolution field emission scanning electron microscopy.

Formation of the TNT layer

To remove the oxide layer that had formed on their surfaces, the specimens were immersed in a solution of HNO3, HF, and H2O mixed in a 12:7:81 ratio for 10 seconds. They were then rinsed with distilled water and dried. To create the TNT layer, a treated specimen was connected to the anode and a platinum plate to the cathode of a DC electrostatic power supply device (Inverter Tech Co., Ltd, Gwangju, Korea). Anodization took place over the course of 1 hour at a current density of 20 mA/cm2 and a voltage of 20 V, using an aqueous electrolyte solution. This solution was prepared by adding 20 wt% of H2O and 1 wt% of NH4F to glycerol. After anodization, all specimens were ultrasonically cleaned in distilled water for 20 seconds and subsequently dried in an oven at 50°C for 24 hours.

Cyclic pre-calcification and heat treatment

The specimens with the TNT layer were immersed in a 0.5 vol% silica aqueous solution for 5 minutes, followed by incubation in a 37°C oven (OF-12GW, Jeio Tech, Daejeon, Korea) for 1 hour. Subsequently, they underwent 20 cycles of pre-calcification in a 0.05 M NH4H2PO4 aqueous solution and a 0.01 M Ca(OH)2 aqueous solution, both maintained at 90°C. Finally, the specimens were placed in an electric furnace (Ajeon Industrial Co., Ltd, Namyangju, Korea) and heated to 500°C for 2 hours. This process aimed to stabilize the TNT structure, induce CaP crystallization, and remove impurities from the coating layer.

Sr ranelate treatment

The pre-calcified specimens were immersed in a physiological saline solution containing 3 mg/mL Sr ranelate (SML0596, Sigma Chemical Co., St. Louis, MO, USA) for 10 minutes, and then freeze-dried. This process was repeated 6 times.

Bioactivity testing

Specimens from each group were immersed in simulated body fluid (SBF) for 1 day to investigate the pattern of hydroxyapatite (HA) precipitation. Prior to incubation, all specimens were autoclaved at 120°C for 20 minutes and then maintained at 37°C in a 5% CO2 atmosphere for one day. The SBF was prepared by adding 0.185 g/L of calcium chloride dihydrate, 0.09767 g/L of magnesium sulfate, and 0.350 g/L of sodium hydrogen carbonate to Hank’s balanced salt solution (H2387, Sigma Chemical Co.). The pH was adjusted to 7.4 using a 1 M aqueous solution of HCL.

Surface analysis

The surface morphology of the treated specimens and the structural changes following immersion in SBF were examined using a high-resolution field emission scanning electron microscope (HR FE-SEM; SU8230, Hitachi, Tokyo, Japan). Elemental concentration changes were analyzed using energy-dispersive X-ray spectroscopy (EDS; Bruker, Billerica, MA, USA). The crystal structures of the elements in the coating were characterized by X-ray diffraction (XRD; Dmax III-A type, Rigaku, Japan).

In vitro tests

The effects of the surface treatment on cell viability were evaluated using osteoblasts (MC3T3-E1 cells, American Type Culture Collection, Manassas, VA, USA). The culture medium was prepared by adding 10% fetal bovine serum (Gibco Co., Waltham, MA, USA), 500 units/mL of penicillin (Gibco Co.), and 500 units/mL of streptomycin (Gibco Co.) to an α-minimum essential medium (Gibco Co.). The specimen surface was sterilized with ethylene oxide gas before the MC3T3-E1 cells were seeded onto it at a density of 2×104 cells/cm2 and cultured for 1 or 3 days. The specimens were transferred to a new 24-well plate and the medium was replaced with a fresh medium containing aqueous tetrazolium salt (WST)-8 reagent from Cell Counting Kit-8 (Enzo Life Sciences Inc., Farmingdale, NY, USA). After incubation for 1.5 hours, absorbance was measured at 450 nm using an ELISA microplate reader (SpectraMax Plus, Molecular Devices, San Jose, CA, USA).

In vivo testing

The animal tests were conducted in accordance with the Declaration of Helsinki and approved by the Animal Laboratory Management Committee (approval number: JBNU 2021-05) of the Experimental Animal Center at Jeonbuk National University (Jeonju, Korea). Twelve 8-week-old male Sprague-Dawley rats, weighing 230±20 g, were used as test animals (4 rats per group). For specimen placement, anesthesia was induced by intraperitoneal injection of zolazepam (Zoletil 50, Virbac, Carros, France) and xylazine hydrochloride (Rompun, Bayer, Seoul, Korea) at a 1:1 ratio. Additional local anesthesia was administered to the surgical site with 2% lidocaine supplemented with epinephrine (1:100,000). A vertical incision was made at the surgical site to separate the periosteum attached to the calvaria, and a circular defect, 8 mm in diameter, was formed by attaching a trephine bur to an endodontic motor (X-SMART, Dentsply, Tokyo, Japan). Then, the defect was covered with the prepared specimen, and the periosteum and skin were sutured with an absorbable suture. To prevent secondary infections, the antibiotic amikacin (300 μL/kg; Samu Media, Yesan, Korea) was subcutaneously administered for 3 days.

The rats were sacrificed by an overdose of thiopental sodium (Choongwae Pharma, Gwacheon, Korea) at 5 weeks. Bone blocks (15×15 mm) containing the mesh were obtained from each rat and stored in 10% formalin for micro-computed tomography (micro-CT) and histological analyses.

Micro-CT analysis

Five weeks after specimen implantation, micro-CT (Skyscan 1076, Skyscan, Kontich, Belgium) was used to examine the bone volume (BV) and bone mineral density (BMD) of the treated area. The X-ray tube’s voltage and current were 100 kV and 100 μA, respectively, and the exposure time was 240 ms. X-ray images were obtained using NRecon software (Skyscan) at 0.6° intervals within a 360°scanning rotation. Regions of interest were defined as a range including a mesh along the boundary of the critical defect with a diameter of 8 mm. In the scanned images, the response to the bone tissue appeared continuously at 80–200. A 3-dimensional (3D) image was constructed using the CTVol software (Skyscan). BV and BMD were measured per Hounsfield unit using CTAn software (Skyscan).

Histological analysis

After micro-CT analysis, the bone blocks obtained at 5 weeks were histologically analyzed. The blocks containing the mesh were fixed in a 10% neutral buffered formalin solution for 2 days. They were then stained with Villanueva solution (Polysciences Inc., Warrington, PA, USA) and dehydrated sequentially in an ethanol series (80%, 90%, 100%) and 100% acetone. The blocks were pre-infiltrated with methyl methacrylate (MMA monomer, Yaruki Pure Chemicals Co. Ltd., Kyoto, Japan) for 2 hours under a vacuum for resin embedding. Then, polymerization was performed at 35°C for 3 days and at 60°C for 1 day with a poly-MMA mixture to which 2 wt% benzoyl peroxide was added. The resin blocks were cut into 0.5-mm-thick slices vertical to the Ti mesh plane using a cutting machine (EXAKT 300 CP, EXAKT Technologies Inc., Oklahoma City, OK, USA). These slices were ground to a thickness of 40 μm using a fine grinder (EXAKT 400 CS, EXAKT Technologies Inc.). New bone formed under the Ti mesh was observed using an optical microscope (EZ4D, Leica, Teaneck, NJ, USA).

Statistical analysis

The data were statistically analyzed using SPSS 12.0 software (SPSS Inc., Chicago, IL, USA). The results’ significance was verified by one-way analysis of variance, and group differences were examined using the Tukey multi-range test. A P value <0.05 was considered statistically significant.

RESULTS

Figure 2 presents HR FE-SEM images of the surface-treated Ti mesh, and Table 1 shows the EDS analysis of Ti, Ca, P, and Sr precipitated on the surface. The TNT layer was even on the surface of the anodization + heating (AH) group, and small tubes had developed between large-diameter tubes, forming a dense structure (Figure 2A and D). On the surface of the anodization + cyclic pre-calcification (20 cycles) + heating (APH) group, CaP was densely precipitated as fine cluster-forming grains (Figure 2B and E), and high Ca and P concentrations were detected in the EDS analysis (Table 1). In the anodization + cyclic pre-calcification (20 cycles) + heating + Sr ranelate-doping (APHS) group, CaP precipitates in the form of fine grains, partially acicular Sr crystals were observed (Figure 2C and F), and Ca, P, and Sr were detected (Table 1).

Figure 2. HR FE-SEM images of the surface-treated Ti mesh. (A-C) The AH, APH, and APHS group, respectively. (D-F) Magnified images of the AH, APH, and APHS group, respectively.

Figure 2

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HR FE-SEM: high-resolution field emission scanning electron microscopy, AH: anodization + heating, APH: anodization + cyclic pre-calcification (20 cycles) + heating, APHS: anodization + cyclic pre-calcification (20 cycles) + heating + Sr ranelate-doping.

Table 1. Ca and P concentration (wt%) of surface-treated groups (n=3 per group).

Group Ti (wt%) Ca (wt%) P (wt%) Sr (wt%)
AH 64.13±1.58 - - -
APH 15.82±1.41 26.90±0.80 12.82±0.67 -
APHS 8.87±0.05 30.56±1.15 12.80±0.91 1.07±0.02
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Values are presented as mean±standard deviation.

AH: anodization + heating, APH: anodization + cyclic pre-calcification (20 cycles) + heating, APHS: anodization + cyclic pre-calcification (20 cycles) + heating + Sr-ranelate doping, Ti: titanium, Ca: calcium, P: phosphate, Sr: strontium.

Figure 3 shows the XRD analysis of the untreated (UT), AH, APH, and APHS groups. Only Ti peaks were observed in the UT group (Figure 3A). Ti and TiO2 peaks were observed in the AH group (Figure 3B). Ti, TiO2, HA, and octacalcium phosphate (OCP) peaks were observed in the APH group (Figure 3C). Sr peaks were observed in addition to Ti, TiO2, HA, and OCP peaks in the APHS group (Figure 3D).

Figure 3. XRD analysis of the surface-treated Ti mesh. (A) The UT group, (B) the AH group, (C) the APH group, and (D) the APHS group.

Figure 3

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UT: untreated, AH: anodization + heating, APH: anodization + cyclic pre-calcification (20 cycles) + heating, APHS: anodization + cyclic pre-calcification (20 cycles) + heating + Sr ranelate-doping.

Figure 4 shows the HR FE-SEM images of the surface-treated AH, APH, and APHS groups after immersion in SBF for 1 day, and Table 2 presents the results of the EDS analysis. The AH group did not significantly change after SBF immersion (Figure 4A and D), and Ca and P concentrations were low (Table 2). In the APH and APHS groups, the protrusions observed in the initial stage of HA precipitation appeared in a dense structure (Figure 4B, C, E, and F, respectively), and the Ca and P concentrations increased to a greater extent in the APHS group than in the APH group (Table 2).

Figure 4. HR FE-SEM surface images after immersion in SBF for 1 day. (A-C) The AH, APH, and APHS group, respectively. (D-F) Magnified images of the AH, APH, and APHS group, respectively.

Figure 4

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HR FE-SEM: high-resolution field emission scanning electron microscopy, SBF: simulated body fluid, AH: anodization + heating, APH: anodization + cyclic pre-calcification (20 cycles) + heating, APHS: anodization + cyclic pre-calcification (20 cycles) + heating + Sr ranelate-doping.

Table 2. The surface-treated groups’ EDS results after immersion in SBF for 1 day (n=3 per group).

Group Ti (wt%) Ca (wt%) P (wt%) Sr (wt%)
AH 64.12±1.89 0.19±0.01 0.12±0.02 -
APH 14.72±1.24 27.93±0.05 13.08±0.07 -
APHS 8.79±1.19 32.65±1.11 14.45±0.56 -
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Values are presented as mean±standard deviation.

EDS: energy-dispersive X-ray spectroscopy, SBF: simulated body fluid, AH: anodization + heating, APH: anodization + cyclic pre-calcification (20 cycles) + heating, APHS: anodization + cyclic pre-calcification (20 cycles) + heating + Sr-ranelate doping, Ti: titanium, Ca: calcium, P: phosphate, Sr: strontium.

Figure 5 shows the survival rates of osteoblasts measured by WST-8 assays after 1 and 3 days of cell culture. On the first day of culture, cell viability did not differ significantly between the AH and UT groups (P>0.05), but significantly decreased in the APH and APHS groups (P<0.05). On the third day of culture, cell viability did not differ significantly between the APH, APHS, and UT groups (P>0.05) but significantly decreased in the AH group (P<0.05).

Figure 5. MC3T3-E1 cell viability results from the WST-8 assay at days 1 and 3 post-incubation.

Figure 5

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UT: untreated, AH: anodization + heating, APH: anodization + cyclic pre-calcification (20 cycles) + heating, APHS: anodization + cyclic pre-calcification (20 cycles) + heating + Sr ranelate-doping.

a)Statistically significant difference (P<0.05).

Figure 6 displays 3D images of new bone formation (Figure 6A-D) and the results of the BMD (Figure 6E) and BV (Figure 6F) analyses when the mesh specimens were implanted in the rat calvarial defects (8 mm diameter dotted line area) for 5 weeks. The new BMD was significantly higher in the APH and APHS groups than in the UT group. In addition, BMD gradually but significantly increased from the AH to the APH to the APHS groups (P<0.05). The 3D micro-CT images illustrate that new bone had formed along the rats’ critical defects under the Ti mesh in all the groups (Figure 6A-D). The size of the new bone area increased from the UT group to the APHS group (Figure 6A-D), but the new BV was only significantly higher in the APHS group than in the UT group (P<0.05; Figure 6F).

Figure 6. Micro-computed tomography of new bone layers beneath the Ti mesh evaluated using the rat calvarial defect model. (A-D) 3D images. Green: Ti mesh, white circle: rat calvarial defect (8 mm in diameter). (E) BMD and (F) BV of new bone layers beneath the Ti mesh.

Figure 6

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UT: untreated, AH: anodization + heating, APH: anodization + cyclic pre-calcification (20 cycles) + heating, APHS: anodization + cyclic pre-calcification (20 cycles) + heating + Sr ranelate-doping, Ti: titanium, BMD: bone mineral density, BV: bone volume.

a)Statistically significant difference (P<0.05).

Figure 7 shows histological images of the bone formed inside the rats’ calvarial defects under the surface-treated Ti mesh at 5 weeks. Soft tissue covered the Ti mesh in the UT group, but in the APHS group, new bone had formed below the mesh and the defect had closed. In the AH and APH groups, new bone grew from the basal bone toward the center of the defect below the mesh.

Figure 7. Histological images after implantation of the surface-modified Ti mesh on the calvarial defect of a rat for 5 weeks. The black rectangles (left border), blue rectangles (center), and red rectangles (right border) show high-magnification images.

Figure 7

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UT: untreated, AH: anodization + heating, APH: anodization + cyclic pre-calcification (20 cycles) + heating, APHS: anodization + cyclic pre-calcification (20 cycles) + heating + Sr ranelate-doping.

DISCUSSION

Fixing and maintaining a dental implant can be challenging in the presence of significant alveolar bone loss [28]. When implant surgery is performed on compromised alveolar bone, GBR with a membrane is necessary to increase the bone both quantitatively and qualitatively, ensuring satisfactory aesthetic and functional results [29]. The recent development of Ti mesh for GBR is advantageous as it is rigid and prevents membrane collapse during the healing period, thereby providing stable support for the bone graft material [30]. Ti mesh is becoming more popular due to its resistance to bacterial contamination, superior mechanical properties, and biocompatibility [31]. There is a growing body of research focused on enhancing osseointegration by applying surface treatments that increase the bioactivity of Ti mesh surfaces, as well as optimizing the structure by adjusting factors such as pore size and thickness [32,33].

Nguyen et al. [34] reported that CaP particles penetrated the nanotube structure of Ti membranes treated with anodization and cyclic pre-calcification, enhancing HA precipitation and demonstrating bioactivity within two days using the SBF immersion test. Gu et al. [35] found that the CaP-deposited nanotubular Ti surface provided an effective area for cell attachment and proliferation, thereby accelerating early cell differentiation and extracellular matrix formation. In the current study, Ti mesh was pre-treated with TNT coating and cyclic pre-calcification, and the effects of additional doping with Sr ranelate on bone regeneration were investigated.

In the SBF immersion test, the initial protrusions observed at the onset of HA precipitation formed a dense structure on the surface of both the APH and APHS groups after one day (Figure 4). The presence of CaP was detected, indicating bioactivity, in line with previous research. The EDS analysis demonstrated a greater increase in CaP content in the APHS group compared to the APH group (Table 2). These findings imply that HA precipitation was augmented by the doping of Sr ranelate following pre-calcification, suggesting that the mesh's surface is likely to chemically bond with bone in vivo [36]. Lindahl et al. [37] reported that Sr2+ influenced the biological formation of HA in the early stages of SBF deposition.

It is crucial to investigate the effect of surface-treated Ti mesh on osteoblasts in vitro. In this study, the WST-8 assay analysis showed viable osteoblasts (MC3T3-E1 cells) and no toxicity on day 3 in the APH and APHS groups (Figure 5). Nguyen et al. [38] similarly reported that Sr-doped CaP Ti meshes exhibited the highest cell viability at 7 days. They also found that cell activity changed according to the Sr-to-HA ratio, suggesting that the Sr-doping concentration improved cell adhesion and proliferation, thereby promoting bone tissue growth. Avci et al. [39] observed that cells grown on Sr-doped HA coatings showed higher initial cell adhesion, with more cytoskeletal elongation.

At 5 weeks, new bone was observed under the Ti mesh in all groups, closing the rats’ calvarial defects. BV and BMD progressively improved from the AH group to the APH group to the APHS group. The pattern of new bone growth from under the mesh to the center of the defect in the APHS group showed consistent bone formation in micro-CT images (Figure 6) and histological examinations (Figure 7). This finding suggests that the mesh used in the APHS group promoted stable bone regeneration by increasing stability in the initially formed bone layer. This study’s null hypothesis was rejected because CaP was precipitated on the Ti mesh by cyclic pre-calcification, and the rats’ calvarial defects were restored through new bone formation by Sr ranelate-doping (in the APHS group).

Among Sr salts, Sr ranelate, which contains the organic compound ranelic acid, is known for its excellent physicochemical properties, bioavailability, and stability [40]. It promotes the differentiation of osteoblast progenitor cells into osteoblasts by stimulating calcium-sensing receptors. In the receptor activator of nuclear factor kappa-B ligand system, osteoblasts induce the secretion of osteoprotegerin, which inhibits osteoclasts and reduces bone resorption [41,42].

Sr enhances the adhesion and proliferation of human mesenchymal stem cells and induces superior osteogenic differentiation, as evidenced by the expression of specific genetic markers in Sr-doped HA [39]. Yan et al. [43] found that implants coated with Sr-HA significantly improved bone formation, as well as the quantity and quality of the bone tissue surrounding the implant.

Based on these results, GBR using CaP-coated, Sr ranelate-doped Ti mesh appears to effectively regenerate new bone in the bone defect area, in conjunction with bone graft material during implant surgery. To gain a deeper understanding of how surface-treated Ti mesh promotes new bone formation, further research involving the analysis of bone formation-related gene expression or protein levels is necessary.

Footnotes

Conflict of Interest: No potential conflict of interest relevant to this article was reported.

Author Contributions:
  • Conceptualization: Tae Sung Bae, Seung Geun Ahn.
  • Formal analysis: Seon Mi Byeon, Tae Sung Bae, Seung Geun Ahn.
  • Investigation: Seon Mi Byeon, Min Ho Lee.
  • Methodology: Seon Mi Byeon, Min Ho Lee, Seung Geun Ahn.
  • Project administration: Tae Sung Bae, Seung Geun Ahn.
  • Writing - original draft: Seon Mi Byeon, Tae Sung Bae.
  • Writing - review & editing: Tae Sung Bae, Min Ho Lee, Seung Geun Ahn.

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