
==== Front
Regen Ther
Regen Ther
Regenerative Therapy
2352-3204
Japanese Society for Regenerative Medicine

S2352-3204(24)00157-3
10.1016/j.reth.2024.08.021
Original Article
Superior bone regenerative properties of carbonate apatite with locational bone-active factors through an inorganic process
Sugiura Yuki yuki-sugiura@aist.go.jp
ab⁎
Ono Fumiko c
Nohara Masakatsu c
Funabiki Mai c
Kutara Kenji c
Kanda Teppei c
Yamada Etsuko a
Horie Masanori a
a Health and Medical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14, Hayashi-cho, Takamatsu, Kagawa, 761-0395, Japan
b Health and Medical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1, Higashi, Tsukuba, Ibaragi, 305-3095, Japan
c Department of Veterinary Associated Science, Faculty of Veterinary Medicine, Okayama University of Science (OUS), 1–3 Ikoi-no-oka, Imabari, Ehime, 794-8555, Japan
⁎ Corresponding author. Health and Medical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14, Hayashi-cho, Takamatsu, Kagawa, 761-0395, Japan. yuki-sugiura@aist.go.jp
09 9 2024
6 2024
09 9 2024
26 760766
19 8 2024
29 8 2024
© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Rapid bone regeneration is crucial for restoring alveolar bone and oral functions following periodontal diseases. However, the development of effective biomedical materials for this purpose remains insufficient. While bone autografts can enhance bone regeneration, they are invasive to healthy areas. Specifically, for alveolar bone regeneration, the implanted material must possess adequate mechanical strength. Moreover, local administration is preferred for older adults, who are a primary target population, to maintain their quality of life. We developed a silica-substituted carbonate apatite (CO3Ap–silica) block as newly bone substitute with a bone growth factor, featuring the major inorganic component of mature bone to enhance bone regeneration. CO3Ap–silica block stimulated the bone remodeling process at the implantation site and demonstrated significantly better bone regeneration compared to currently used carbonate apatite substitutes. Therefore, this new material is expected to advance technologies for restoring occlusal function after periodontal disease.

Graphical abstract

Image 1

Keywords

Periodontic
Bioceramics
Bone regeneration
Silica
Carbonate apatite
==== Body
pmc1 Introduction

For dental periodontal diseases, rapid bone regeneration is a crucial technology for recovery in the global elderly population [1,2]. Historically, bone autografts have been regarded as the gold standard for treating bone defects. However, limitations such as the limited amount of harvestable bone, the invasiveness of the procedure, and the increased risk of infection present significant challenges [[3], [4], [5]]. The clinical field demands bone regeneration substitutes produced through chemical processes that offer unlimited availability and eliminate infection risks, unlike autografts or allografts with unknown etiological risks.

In response to this demand, bioceramic-based bone substitutes have been introduced [6]. Carbonate apatite [CO3Ap: Ca10-a(PO4)6(CO3)c(OH)2-d], the primary inorganic component of aged bone mineral, has been proposed as a bone substitute [[7], [8], [9]]. CO3Ap bone substitutes are expected to exhibit high osteoconductivity and effectively reconstruct and regenerate defects [10]. However, bone regeneration using these materials remains slow, prolonging the recovery of skeletal functions [11]. For older patients, prolonged rest can deteriorate their overall condition [12,13]. Therefore, rapid regeneration of the treated area is essential.

Silica shows promise as a support material to promote skeletal growth when administered [[14], [15], [16]]. However, organic materials such as tetraethyl orthosilicate, used for silica loading, have risks owing to residual organic molecules [[17], [18], [19]].

Our previous work introduced a wet synthesis method for silica-supported calcium phosphate [[20], [21], [22], [23]]. We discovered that the mechanical strength of octacalcium phosphate [OCP: Ca8(PO4)4(HPO4)2·5H2O] improves when loaded with silica and dicarboxylic acid molecules simultaneously [24]. In this process, dicarboxylic acid molecules leach out during the phase transition to apatite, while silica remains in the interlayer [20,25]. Thus, OCP loaded with both silica and dicarboxylic acid molecules can transition to silica-substituted apatite with enhanced strength. In this study, we aimed to prepare silica-loaded apatite via phase transition from OCP and evaluate its bone regeneration ability.

2 Result and discussion

As an initial trial in this study, we investigated the preparation of silica-substituted apatite blocks with sufficient mechanical strength from both dicarboxylic acid (succinate) and silica-substituted OCP blocks through a hydrolysis reaction. Preprepared succinate–silica composite-supported OCP blocks were immersed in (NH4)2CO3 solution. The immersed samples maintained the shape of the OCP blocks (Fig. 1a). After immersion, XRD patterns indicated the disappearance of the OCP peak and the appearance of the apatite peak (Fig. 1b). In samples treated with (NH4)2CO3 solutions concentrations above 2 mol/L, a calcite peak was also observed. Fourier transform infrared (FT–IR) spectra showed several bands around 1420 and 1460 cm−1, indicating B-type CO3Ap carbonate adsorption (Fig. 1c). The intensities of these bands increased with higher (NH4)2CO3 concentrations [26,27]. Notably, no dicarboxylic acid bands were observed after immersion. Additionally, silanol bands were clearly present, indicating that silica remained substituted in the apatite blocks. CHN analysis supported the FT–IR results, showing a monotonic increase in CO3 content with increasing (NH4)2CO3 concentration (Fig. 1d). The silica content slightly decreased with higher (NH4)2CO3 concentrations (Fig. 1e). The pH of the solutions after impregnation decreased slightly in both cases (Fig. 1f).Fig. 1 Material characterization of apatite-silica blocks with different CO3 amounts from OCP-silica:Succinate blocks. (a) Photographs of the blocks before and after (NH4)2CO3 solution immersion. (b) Bulk XRD patterns of the blocks before and after (NH4)2CO3 solution immersion. (c) FT-IR spectra of the blocks before and after (NH4)2CO3 solution immersion. (c’) Expanded around 1200-1800 cm−1 of (c). (d) CO3 contents of the blocks after (NH4)2CO3 solution immersion. (e) Si contents of the blocks after (NH4)2CO3 solution immersion. (f) The pH values of (NH4)2CO3 solutions before (●) and after immersion (◆).

Fig. 1

Having prepared the silica-substituted apatite in bulk, we proceeded to evaluate its mechanical strength, which is crucial for its use as a bone regeneration material, requiring at least 0.2 MPa. Impregnation in solution initially reduced the mechanical strength (Fig. 2a). However, the mechanical strength of the blocks increased with higher (NH4)2CO3 concentrations (Fig. 2b) [28,29]. The system with the highest carbonate content and single-phase apatite, treated with 1.0 mol/L (NH4)2CO3, showed a DTS strength of approximately 0.7 MPa. This strength was significantly higher than that of the initially prepared CO3Ap–silica from only silica-substituted OCP (<0.1 MPa) and was sufficient for implantation in animals. The stress–strain curves exhibited similar behavior. Fig. 2c shows SEM micrographs of the samples. The cross-sectional microstructure of the specimens was examined to understand the reason for their high mechanical strength. A structure of closely packed, plate-like crystals was observed regardless of the carbonate content. This suggests that the samples exhibited high mechanical strength because they were not scabbed. Additionally, the density was significantly higher than that of the initially prepared silica-loaded CO3Ap.Fig. 2 Mechanical properties and fine structures of apatite-silica blocks with different CO3 amounts from OCP-silica:Succinate blocks. (a) Typical force curves of the samples. Red: OCP-silica:Succinate block. Purple: H2O immersion. Green: 0.1 mol/L (NH4)2CO3 immersion. Black: 0.5 mol/L (NH4)2CO3 immersion. Blue: 1.0 mol/L (NH4)2CO3 immersion. (b) DTS values of the OCP-silica:Succinate blocks before and after immersion. (c) SEM micrographs of cross sections of the OCP-silica:Succinate blocks and after immersion into H2O and 1.0 mol/L (NH4)2CO3.

Fig. 2

We obtained silica-loaded carbonate apatite with sufficient strength and evaluated its bone regenerative capacity in vivo. As a reference, CO3Ap blocks without silica were used, which are analogous to a commercially available bone regeneration material [6]. The prepared blocks were implanted into rabbit femur defects (Fig. 3a). One month after implantation, bone tissue formed firmly around both samples, demonstrating high osteoconductivity (Fig. 3b). However, the new bone surrounding the CO3Ap–silica material was notably thicker. Significant resorption of CO3Ap–silica was observed three months after implantation, along with partial bone regeneration at the implantation site. In all cases, a clear bond was evident between the bone tissue and the specimen (Fig. 3c). The ratio of new bone formed at the implantation site tended to be higher for CO3Ap-silica than for CO3Ap. The percentage of new bone formation at the implantation site tended to be higher for CO3Ap-silica than for CO3Ap at the 3 month follow-up (Fig. 3d). The ratio of the remaining specimen and bone contact were as follows (Fig. 3e). Both the bone contact rate and bone replacement rate were significantly higher for CO3Ap–silica compared to CO3Ap (Fig. 3f). The bone in contact with CO3Ap–silica was more substantial than that with carbonate apatite. Additionally, bone marrow tissue surrounding the bone, rich in reticulocytes and low in fatty structure, was observed (Fig. 3g).Fig. 3 In vivo evaluation results of feasibility studies for CO3Ap-silica bone regeneration ability using rabbit femur bone defect model. (a) Schematic illustration of implanted site. (b) Histological images of CO3Ap (reference) and CO3Ap-silica blocks 1 and 3 months after implantation. (c) Magnified images of the boundary of samples and tissues 1 and 3 months after implantation. ∗: samples. Blue broken lines: the boundary of samples and bone tissues. (d) Statical analysis results of sample area. (e) Statical analysis results of newly bone formed ratio. (f) Statical analysis results of bone contact onto samples. (g) Statical analysis results of red bone mallow contact onto samples. ∗: p < 0.05.

Fig. 3

This material's high bone contact rate and bone replacement properties were evaluated in greater detail to confirm its impact on the bone remodeling process. Tartaric acid-resistant acid phosphatase (TRAP) staining, which highlights osteoclasts, was used to assess bone remodeling activity (Fig. 4a). CO3Ap–silica exhibited a significantly higher density of osteoclasts at both implantation periods (Fig. 4b), despite silica's known inhibitory effect on osteoclast differentiation [16,30]. Both osteoblast and osteoclast density tended to be significantly higher in CO3Ap-silica than in CO3Ap. Osteoblast density was observed to decrease over time in both samples. This was also consistent with the rate of new bone formation (Fig. 4c and d). This relationship between osteoclast activity and new bone formation suggests that in silica-loaded carbonate apatite, osteoblast activity and bone remodeling are more active, leading to enhanced bone regeneration at the implantation site.Fig. 4 TRAP-stained histological images of CO3Ap and CO3Ap-silica blocks 1 and 3 months after implantation. (a) Low magnified images of CO3Ap (reference) and CO3Ap-silica blocks 1 and 3 months after implantation. (b) Magnified TRAP stained histological images of CO3Ap and CO3Ap-silica blocks 1 and 3 months after implantation. ∗: materials. Red arrow: TRAP-positive multinuclear giant cells. (c) Statical analysis result of the density of osteoblast. (d) Statical analysis result of the density of osteoclast. ∗: p < 0.05.

Fig. 4

CO3Ap–silica has demonstrated promising results as a bone regeneration material. One of its advantages over silica-loaded OCP is its high osteoconductivity from the early implantation stage, which may prevent strength loss at the bone implantation site. The higher bone conductivity of CO3Ap compared to OCP can be attributed to differences in their solubility [31,32]. Specifically, the higher acid resistance of CO3Ap to osteoclast-produced acid may harmonize the bone remodeling process with the material's regenerative capacity.

CO3Ap–silica is expected to continuously supply silica in the immediate vicinity in response to osteoclastic activity. The dissolved silica reduces osteoclastic activity while significantly enhancing osteoblastic activity [33]. This increased osteoblast activity likely drives bone regeneration and an active bone remodeling process primarily mediated by osteoblasts.

Historically, bone replacement materials have been designed to promote bone regeneration in harmony with the bone remodeling process [6,34]. However, in elderly patients whose bone remodeling processes have declined, current bone replacement materials exhibit low regenerative capacity [34]. Additionally, autologous bone grafting is often not an option. Bone loss is typically a local issue. Thus, it is desirable to induce rapid bone regeneration, specifically in the defect area. Moreover, there is a need for processes that enhance bone remodeling, the metabolic aspect of bone health, rather than drugs that inhibit bone metabolism, such as bisphosphonates [[35], [36], [37]]. CO3Ap–silica, with its sufficient mechanical strength, biocompatibility of carbonate apatite, and ability to activate osteoblasts and the bone remodeling process, shows great potential. Evidence of this includes the formation of red marrow-like tissue with high bone regeneration capacity around CO3Ap–silica. In this study, we evaluated CO3Ap–silica in the form of a dense block, which is suitable for such assessments. Future developments are expected to demonstrate even higher bone regenerative capacity by incorporating mechanical structures, such as foam and cementing processes, which further induce bone formation [[38], [39], [40]].

Currently, the primary cause of tooth loss is not dental caries but the dissolution and loss of the alveolar bone that supports the teeth owing to periodontal disease [[41], [42], [43]]. The resulting loss of occlusal function and deterioration of the oral environment can contribute to the expansion of oral flora and lead to serious conditions such as aspiration pneumonia and cardiac disease [44,45]. Reconstruction and regeneration of the alveolar bone are essential to restore occlusal function. Although treatments involving heat-generating nanoparticles, fungal breakdown factor particles, and growth factors have been proposed for periodontal disease, they do not address cases where the alveolar bone itself has been lost [[46], [47], [48], [49]]. This material can serve as a base for nanoparticles and tissue growth factors, potentially enabling early recovery of bone volume and formation. Combining it with these materials may prevent tooth loss and maintain occlusal function in patients with periodontal disease.

3 Conclusion

In this study, we fabricated silica-containing CO3Ap blocks with sufficient strength (DTS: ∼1 MPa) and evaluated their bone regeneration potential using a rabbit femur defect model to assess their usefulness for CO3Ap. This silica substitution enhanced periosteal bone production. Despite the silica substitution, CO3Ap's high biocompatibility and osteoconductivity remained unchanged. The development of this new material will address the non-adaptability of bone replacement materials, particularly for older adults, owing to the low bone regeneration capacity of current materials.

4 Materials and methods

4.1 Fabrication of silica-substituted apatite blocks with different CO3 contents

All reagents were purchased from FUJI Film Wako Pure Inc., Japan. We prepared stock solutions of 4.0 mol/L H3PO4 and 2.0 mol/L (NH4)2CO3 by diluting and dissolving them in distilled water.

As an initial step, we fabricated OCP blocks containing succinate and silica (OCP–silica) as intermediate materials. The details of the OCP–silica blocks are described in a published study [24]. Briefly, the fabrication process of the OCP component is as follows. First, 2.0 mL of H3PO4 and 0.60 g of succinate were placed into an agate mortar and dissolved. Then, 1.2 g of CaCO3 powder was gradually mixed into the solution. Subsequently, 1.08 mL of 38 wt% Na2SiO3 was added while stirring with a pestle. To promote the interlocking of the formed OCP–silica crystals, the mixture was placed in a silicone rubber mold (φ6 × 3 mm) and tightly sealed with a 0.3 mm thick polypropylene sheet to prevent evaporation. The reaction process was performed at 60 °C for 1 day. Finally, the treated materials were thoroughly washed with distilled water three times and then dried in a 40 °C oven for 1 day.

After fabricating and evaluating the OCP–silica blocks, 10 pieces of these blocks were immersed in 20 mL of 0.0–2.0 mol/L (NH4)2CO3 at 80 °C for 5 days. The treated blocks were washed with distilled water thrice and then dried in an 80 °C oven for 1 day. The pH of the solutions before and after immersion was measured using a pH electrode (ThoupH 9615S-10D, Horiba Co., Kyoto, Japan) connected to a multimeter (LAQUA F–2000PC, Horiba Co., Kyoto, Japan).

4.2 Fabricated material characterization and evaluation

Crystallographic information about the samples was obtained via X-ray diffraction (XRD: MiniFlex600, Rigaku Co., Japan) at an acceleration voltage of 40 kV and a current of 15 mA. All samples were finely crushed using an agate mortar and pestle, and the resulting powder was mounted onto a φ10 mm × 0.2 mm Si nonreflective plate. The diffraction angle was continuously scanned over 2θ values ranging from 3° to 70° at a scanning rate of 5°/min for characterization and from 2° to 12° and 20°–40° at a scanning rate of 1°/min for crystallographic parameter analysis.

The chemical bonding structure of the samples was analyzed via Fourier transform infrared spectroscopy (FT–IR: IRTracer-100, Shimadzu Co., Japan) using a triglycine sulfate detector (30 scans, resolution 2 cm−1) with a diamond attenuated total reflection prism. The atmospheric background was used for the measurements.

The fine structure of the samples was examined using field emission scanning electron microscopy (FE–SEM: JSM-6700F, JEOL Co., Japan) at an acceleration voltage of 3 kV after Os sputtering.

The Ca and P (PO4) (and Na) contents of the samples were measured by inductively coupled plasma atomic emission spectroscopy (ICP–AES: 5110VDV, Agilent Technology Co., Japan) after the samples were completely dissolved in a 2 wt% HNO3 solution.

The Si content of the samples was measured by energy-dispersive X-ray fluorescence spectroscopy (EDX-8100, Shimadzu Co., Japan) at an acceleration voltage of 15 kV under vacuum conditions. The Si/Ca ratio was determined using disk-shaped sintered blocks with serial mixture ratios of hydroxyapatite and SiO2.

The carbonate content in each sample was estimated from the at% of C in the CO3Ap-silica block, as measured with a carbon–hydrogen–nitrogen (CHN) coder (MT-6; Yanako Analytical Instruments, Kyoto, Japan) under O2 and Ar gas carrier.

The mechanical strengths of cylindrical-shaped specimens were evaluated in terms of the diametral tensile strength (DTS). The diameter and thickness of each specimen were measured using a micrometer (MDC-25MU, Mitutoyo Co. Ltd., Kawasaki, Japan). The specimens were then tested under compression by crushing at a constant crosshead speed of 1 mm/min using a universal testing machine (AGS-J, Shimadzu, Kyoto, Japan). For each specimen, the mean DTS values of five specimens were estimated and reported as the mean ± standard deviation using Student's t-test analysis (p < 0.05) because all factors were independent.

4.3 In vivo evaluation for bone regeneration properties

All animal experiments were conducted with the approval of the ethics committee of animal experimentation of AIST (approval numbers: A2018–327, A2020–376) and Okayama University of Science (approval number: E2020–096).

Eight male Japanese white rabbits (19 weeks old, each weighing 3.0–3.5 kg) were used in this study. The femurs of both legs of the rabbits, under systemic anesthesia, were carefully exposed by exfoliation. After the dissection of the periosteum, bone defects with dimensions of φ6.0 × 3.0 mm3 were artificially created in the cancellous bones of both femurs using a trephine bur attached to a dental handpiece. For comparison, the artificial bone defects were reconstructed to the level of the bone surface using CO3Ap–silica and reference CO3Ap blocks (φ6.0 × 3.0 mm). The fabrication method for CO3Ap blocks is detailed in a published study [28].

The specimens were sterilized at 80 °C for 8 h before implantation. Finally, the skin flaps were closed using suture thread. Buprenorphine hydrochloride (LepetanVR, Otsuka Pharmaceutical Co., Japan) and gentamicin sulfate (GENTACINVR, MSD, Tokyo, Japan) were injected intramuscularly to prevent postoperative pain and bacterial infection, respectively.

Four rabbits were euthanized at 1-month post-implantation and the remaining four at 3-months post-implantation by KCl overdose injection. The extracted femurs were fixed in PO4-buffered 10 % formaldehyde for 7 days. The fixed tissues were then decalcified using ethylenediaminetetraacetic acid (EDTA) or K-CX solution, dehydrated with graded ethanol solutions, and embedded in paraffin. The embedded tissues were sliced into 5–10-μm-thick sections using a microtome. HE and tartrate-resistant acid phosphatase (TRAP) stains were applied to the specimens. An all-in-one microscope (BZ-X710, KEYENCE, Osaka, Japan) was used to observe the stained specimens. The ratios of new bone formation in each sample were measured using ImageJ software (NIH, USA). The total length and area of the samples and tissues were measured using HE-stained histological samples. The results for each sample were statistically analyzed (N = 4).

Statistical analysis was performed using the Student's t-test method. A p-value of less than 0.05 was considered statistically significant.

4.4 Calculation for tissue response of samples

The data measured using ImageJ software, NIH, USA, were used for the analysis (N = 4 for each sample).

The areas of new bone and reminded samples were calculated as follows. The embedding site of the sample is a 6 × 3 mm rectangle. Then, the surrounded area of the sample including sample (8 × 4 mm rectangle) defined as area of interest; S. The area of newly formed bone; Ab and reminded sample area; As were measured. The ratio of newly formed bone; Rb was calculated as:(1) Rb = Ab / (S – As)

The area of bone tissue and the area of the sample in this area were each measured, and the quotient of the areas was calculated for each sample.

The density of osteoclasts was calculated as follows. The circumferential length of the samples was measured beforehand. The number of TRAP-positive, multinucleated giant cells in contact with the embedded sample was then measured and divided by the circumferential length of the sample to obtain the density of osteoclasts. The density of osteoblasts was determined by measuring the number of osteoblasts distributed either directly above the sample or on bone beams in direct contact with the sample. As in the case of osteoclasts, the density was determined by dividing the number of osteoblasts by the circumferential length of the sample.

Declaration of competing interest

Authors declare no financial interest and no conflict of interest.

Acknowledgement

We thank Dr. T. Nakanishi, RIST, Kagawa, Japan, for performing the FT-IR measurements. This study was financially supported by the 10.13039/501100005691 AMED Seeds H program (10.13039/501100001697 Keio University ; grant number H424TS ), AMED Seeds A program (Keio University; grant number A424TR), 10.13039/501100012013 Kazuchika Okura Memorial Foundation 2022FY Grant-in-Aid, and JST-CREST (grant number: JPMJCR22L5 ).

Peer review under responsibility of the Japanese Society for Regenerative Medicine.
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