Hydroxyapatite with High Carbonate Substitutions Promotes

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Hydroxyapatite with high carbonated substitutions promotes osteoclast resorption through osteocyte-like cells Miho Nakamura, Rumi Hiratai, Teuvo Hentunen, Jukka Salonen, and Kimihiro Yamashita ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.5b00509 • Publication Date (Web): 21 Dec 2015 Downloaded from http://pubs.acs.org on December 29, 2015

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ACS Biomaterials Science & Engineering

Hydroxyapatite with high carbonate substitutions promotes osteoclast resorption through osteocytelike cells AUTHOR NAMES Miho Nakamura1*, Rumi Hiratai1, Teuvo Hentunen2, Jukka Salonen2, Kimihiro Yamashita1

AUTHOR ADDRESS 1

Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University

2-3-10 Kanda-Surugadai, Chiyoda, Tokyo 1010062 Japan 2

Institute of Biomedicine/Cell Biology and Anatomy, University of Turku

Kiinamyllynkatu 10, 20520 Turku, Finland

KEYWORDS bone marrow, calcium phosphate, co-culture, osteoclast, surface energy, wettability

ACS Paragon Plus Environment

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ABSTRACT

The role of ceramic biomaterials in the repair of bone defects is changing from materials that purely fill the physical defects of the injured bone to scaffolds that control cellular behaviors. In this study, we investigated the osteoclast formation related to the osteoconductivity of ceramic biomaterials. We performed in vitro co-culture using osteocyte-like cells and bone marrow cells and in vivo implantation into rat femurs using hydroxyapatite with different amounts of carbonate substitutions. The co-culture evaluations and quantitative analyses revealed that bone marrow cells differentiated into osteoclasts and were activated to resorb the substrate when grown on hydroxyapatite with higher numbers of carbonate substitutions because of the expression of factors that induce osteoclast differentiation by osteocyte-like cells. The osteoclastogenesis in vivo showed osteoclast formation near the hydroxyapatite with more carbonate substitutions after implantation into the rat femurs. These results indicate that the content of carbonate ions in an apatite crystal lattice affects the induction of osteoclastogenesis in the vicinity of the implanted ceramic biomaterial. These results contribute to the design of biomaterials that would be resorbed by osteoclasts after fulfilling their primary function as scaffolds for cell growth and eventually bone regeneration.


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Introduction Several questions concerning the functions of ceramic biomaterials when applied as medical devices remain under discussion. The role of ceramic biomaterials in the repair of bone defects is changing from materials that purely fill the physical defects in injured bone to scaffolds that control cellular viability, adhesion, migration, proliferation, and differentiation. Commercial ceramic biomaterials, such as hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP), are recognized as scaffolds for adhesive cells that promote bone formation. Osteoconductivity is defined as the ability of an implanted material to support bone formation in its vicinity 1. For example, synthetic ceramic biomaterials such as HA and β-TCP, as well as biphasic calcium phosphate scaffolds made from a mixture of HA and β-TCP, have been shown to stimulate the osteogenic differentiation of mesenchymal stem cells derived from human bone marrow in vitro and to induce the formation of new mineralized bone in porous specimens in vivo 2. In another study, β-TCP granules induced the healing of bone tissue defects at 4 months after the implantation, whereas HA granules did not show significant new bone formation even after 12 months

3

. The reasons why ceramic biomaterials exhibit such differences in

osteoconductivity are not clearly understood. We focused on the natural mineral phase of bone tissue for the design of ceramic biomaterials. The inorganic constituent of bone tissue is actually not stoichiometric HA, but rather some partially substituted ions, such as 2-8 wt% of carbonate and small amounts of sodium, magnesium, chlorine, and potassium in its crystal structure 4. Such partial substitution feature of bone tissue has prompted numerous investigations of synthetic carbonate-substituted HA (CA) as an ideal clinical biomaterial because it is empirically hypothesized that artificial implants should closely resemble the natural mineral phase of bone tissue. As a bone mineral-resembling material, CA can be resorbed by osteoclasts and has the advantage of incorporating material dissolution into the remodeling of the bone metabolism process.


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Osteoclasts are thought to be derived from cells of the macrophage-monocyte system and are responsible for the resorption processes in bone. Calcium phosphate granules ectopically implanted into nude mice with human mesenchymal stem cells induced osteogenesis through the homing of macrophages and osteoclastogenesis 5. This induction was decreased by the blockage of osteoclast activity using an anti-RANKL treatment 5. Considering the importance of osteoclasts for bone remodeling and for responses triggered by trauma or functional demands, the understanding of the mutual interactions of osteoblasts, osteocytes, osteoclasts, and biomaterials is of great interest. An important function of osteocytes is to support osteoclastogenesis through the secretion of the receptor activator of the nuclear factor-kappa B ligand (RANKL) as a factor for osteoclast differentiation and activation

6-8

. The mouse osteocyte-like cell line, MLO-Y4, expressed

RANKL and induced osteoclast formation from bone marrow cells (BMCs) after co-culture 9. This co-culture of MLO-Y4 cells and BMCs could be used for the investigation of the interactions between osteogenic cells and biomaterials. Another important function of the osteocytes is their ability to sense mechanical stress applied to the extracellular matrix

10

. This

function suggests that these cells are able to perceive and respond to the physical and/or chemical properties of implanted biomaterials. We have recently demonstrated that CA enhanced osteoclast differentiation from human peripheral mononuclear blood cells 11. Although this result shows evidence for the bioresorption of CA by osteoclasts, osteocyte-mediated osteoclast formation would yield valuable information for the understanding of osteoconductivity. In view of the differences of monocytes derived from peripheral blood and bone marrow

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, osteoclast formation from BMCs is also important for

understanding biomaterial osteoconductivity. The aim of the present study was to assess the response of murine bone marrow osteoclast progenitor cells in an in vitro co-culture with osteocyte-like cells to apatite with various amounts


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of carbonate in its crystal lattice. A comparative study was performed by inserting corresponding compositions of synthesized apatite in rat femurs. We show here several novel insights into our understanding of ceramic biomaterial osteoconductivity. We hypothesized that ceramic biomaterials similar to bone promote osteoclastogenesis by stimulating osteocytes and thereby play a direct role in bone metabolism. Because the physiological bone minerals are not the same as those of stoichiometric HA, but rather contain a fraction of partially substituted ions, such as carbonate 4, the content of carbonate ions in an apatite crystal lattice alters its induction of osteoclastogenesis. The effects of apatite carbonate ion contents are discussed here in relation to the conventional understandings of the material properties of bone and osteoclastogenesis in vitro and in vivo.

Materials and Methods Sample preparation HA powder was synthesized from analytical grade calcium hydroxide and phosphoric acid reagents using the wet method 13. The HA powder was calcined at 850°C and pressed in a mold at 200 MPa. The HA compacts were sintered at 1250°C for 2 h in a saturated water vapor atmosphere. CA was synthesized using a modification of the procedure described by Doi et al

13, 14

. CA

powder was synthesized from analytical grade calcium nitrate tetrahydrate, disodium hydrogenphosphate, and sodium carbonate reagents with a CO3/PO4 molar ratio of 2 (CA2) and 5 (CA5) using the wet method. The CA2 and CA5 compacts were pressed in a mold at 200 MPa and sintered for 2 h in a carbon dioxide atmosphere to minimize carbonate loss from the CA surface at 830°C (CA2) and 780°C (CA5).


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After polishing with a 5-µm grain size diamond disk, the HA and CA specimens (φ 7 mm) were washed in ethanol with ultrasonication. The specimens were prepared at thicknesses of 0.8 mm. The roughness of the specimens was measured with a color laser microscope (VK-8500, Keyence, Osaka, Japan). Specimens with similar surface roughness (Ra = 0.8 µm) were used in the experiments. As a control, bone slices were prepared as previously described

15

. In brief, a frozen bovine

cortical femur was cut with a diamond saw (Buehler, Lake Bluff, IL, USA) into 130–180 µm thickness slices. The bone slices were rinsed by ultrasonication in distilled water.

Characterization The carbonate content present in the apatite structure of the specimens was quantitatively measured using Fourier transform infrared spectroscopy (FT-IR; Diamond-20 FT-IR spectra photometer, JEOL Ltd., Tokyo, Japan), thermogravimetric differential thermal analysis (TGDTA), and a CHN analyzer (Perkin Elmer, Waltham, MA, USA). The carbonate contents were estimated from the FT-IR spectra using the method of Cassella et al. 16. The contact angles of distilled and deionized water on the specimens were measured (Drop Master DM500, Kyowa Interface Science, Saitama, Japan) and calculated using Young’s equation: ߛௌ௏ = ߛ௅௏ cos ߠ + ߛௌ௅ where subscripts S, L, and V indicate solid, liquid, and vapor, respectively. To calculate the surface free energy of the specimens, contact angle measurements were performed using the two-liquid-phase method

17, 18

. The contact angle values of water on the

specimens were measured in hydrocarbon oils such as hexadecane, octane, heptane, decane, and hexane. The surface energy was then calculated using Jouany’s equation:


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௣ ௗ ߛௐ − ߛு + ߛுௐ cos ߠ = 2ටߛௌௗ ቆටߛௐ − ඥߛு ቇ + ‫ܫ‬ௌௐ

where subscripts W, H, and S indicate water, hydrocarbon, and solid, respectively. γsd represents the dispersive components and γsp represents the polar components 19.

Solubility The dense specimens were placed on the bottom of 48-well cell culture plate wells and immersed in 1 mL of cell culture medium and 1 M acetic acid solution (pH 5.6) for 0.5, 1, 2, 4, 8, 12, or 24 h. The calcium concentrations were measured using a calcium detection kit based on the methylene blue method (437-58201, Wako, Osaka, Japan). The phosphorus concentrations were measured using a phosphorus detection kit based on the p-methylamino phenol method (270-49801, Osaka, Wako).

Cell culture Mouse MLO-Y4 osteocyte-like cells

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were obtained from Prof. Bonewald (University of

Missouri-Kansas City, Kansas City, MO, USA). The cells were maintained in α-modified minimum essential medium (α-MEM, Gibco Life Technologies, USA) supplemented with 5% heat-inactivated fetal bovine serum (FBS, Gibco Life Technologies, USA), 5% heat-inactivated calf serum (CS, Gibco Life Technologies, USA), 100 units/mL penicillin, and 100 µg/mL streptomycin in a humidified atmosphere of 5% CO2 in air at 37°C. After reaching 70% confluence, the cells were detached with 0.05% trypsin/EDTA and then seeded onto the specimens at a density of 1 × 103 cells/cm2. Bone marrow cells (BMCs) were isolated from the tibiae and femurs of C57BI/6J mice (male, 8–10-week-old), as previously described in detail

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. In brief, the mice were euthanized and


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sterilized with 70% ethanol. The soft tissues were removed, and the tibiae and femurs were separated. The epiphyses were resected, and the bone marrow cells were aspirated with α-MEM supplemented with 10% FBS, 100 units/mL penicillin, and 100 µg/mL streptomycin using a syringe and a needle. The nucleated cells were counted using a hemocytometer. The MLO-Y4 cells were also co-cultured with BMCs on the specimens 9. The MLO-Y4 cells were cultured in α-MEM supplemented with 5% FBS, 5% CS, 100 units/mL penicillin, and 100 µg/mL streptomycin for 1 d and then washed with PBS. The BMCs were seeded onto the specimens at a density of 1 × 106 cells/cm2. The MLO-Y4 and BMC cell co-cultures were maintained in α-MEM supplemented with 10% FBS, 100 units/mL penicillin, and 100 µg/mL streptomycin, and the conditioned media (CM) were collected.

TRAP staining The cells were cultured on the specimens for 7 d, washed twice with PBS, and fixed with 4% paraformaldehyde for 20 min. To confirm the differentiation of the BMCs into osteoclasts, the cells adhered on the specimens were stained for tartrate-resistant acid phosphatase (TRAP; No. 387, Sigma–Aldrich, St. Louis, MO, USA) and observed using an optical microscope (IX71, Olympus, Tokyo, Japan). The number of TRAP-positive multinucleated cells per unit area was counted on each surface. A total of at least 30 fields on each specimen were assessed to obtain an average.

Fluorescence staining The cells cultured on the specimens for 7 d were washed with PBS and fixed with 4% paraformaldehyde for 20 min. After the PBS washes, the cells were incubated in blocking solution, 5% goat serum, and 0.1% Tween-20 in PBS. A mouse anti-RANKL antibody (Alexis


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Corporation, Switzerland) was added to the blocking solution for 1 h at room temperature (RT). Following extensive washing with PBS, the specimens were incubated in Alexa-conjugated goat anti-mouse immunoglobulin (Life Technologies, USA) in blocking solution containing rhodamine-phalloidin (Cytoskeleton, Inc., USA) for 1 h at RT. After nuclear staining with Hoechst (Dojindo, Tokyo, Japan), the fluorescent signals were observed using a fluorescence microscope (IX71, Olympus, Tokyo, Japan). The number of actin rings per unit area was counted on each surface. A total of at least 30 fields were assessed on each specimen to obtain an average.

ELISA for M-CSF and RANKL The concentration of soluble macrophage colony-stimulating factor (M-CSF) and RANKL in the CM samples at 7 d after beginning the co-cultivation of osteocyte-like cells and BMCs was detected using a commercial M-CSF kit (mouse M-CSF Quantikine® ELISA kit, MMC00, R&D, U.S.A.) and for RANKL (mouse TRANCE/RANKL/TNFSF11 Quantikine® ELISA kit, MTR00, R&D, U.S.A.).

Observation of resorption pits After fluorescence observations of each specimen were made, the osteoclasts were removed from the specimens by being scrubbed with a brush. All the specimens were then washed with distilled water and dried. The resorption activity of the osteoclasts was assessed by measuring the morphological parameters of the resorption pits. The dried specimens were examined with a color laser microscope (VK8500, Keyence, Osaka, Japan) equipped with an image analysis system. The depth of each resorption pit was measured to quantify the amount of osteoclast


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resorption. The measurements were performed for a minimum of 30 resorption pits on each specimen to obtain an average. Additionally, the specimens were observed with a scanning electron microscope (SEM; S-3400NX, Hitachi, Tokyo, Japan).

Implantation into rat femurs The HA and CA specimens were cut into pieces of 3 × 4 × 0.8 mm (thick) and sterilized with 70% ethanol. The following implantation experiments were carefully completed by veterinarians in accordance with the Guidelines for Animal Experimentation (Tokyo Medical and Dental University), as well as the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Pub. No. 85-23, Rev. 1985). The specimens were surgically implanted into the bilateral femoral diaphysis of Wistar rats (13-week-old males) under isoflurane anesthesia. An incision was made in the skin, intermuscular dissection was performed to expose the femoral periosteum, and rectangular defects were created from the cortex to the marrow using a dental engine bur (0.8-mm diameter). After the bone defects were washed with saline, the specimens were implanted into the holes and fixed in place with sutured muscle and skin tissues. After implantation surgery, 1 mg/kg butorphanol and 30 mg/kg trimethoprim sulfadiazine were injected. At 7 d after the implantation surgery, the rats were sacrificed under anesthesia and perfusion fixed with 4% paraformaldehyde. The femurs were extracted and immersed in 4% paraformaldehyde for 3 d at 4°C. After fixation, each sample was decalcified with 10% neutralized ethylenediamine tetraacetic acid (EDTA) for 5 weeks, dehydrated in an ascending gradient of ethanol concentrations, cleared in xylene, embedded in paraffin, and cut into 5-µm sections. Each section was deparaffinized with xylene, rehydrated with ethanol, and stained with hematoxylin and eosin (HE) and for TRAP using standard methods. The number of rat femurs was 5-6 for each specimen in one experiment. The same experiments were repeated four times.


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Statistical analysis Accurate quantifications of the different specimens were achieved by performing more than three independent experiments. A statistical analysis was performed using the Kruskal–Wallis analysis of variance (ANOVA). If that test indicated that the effects were significant, further pairwise comparisons of the image analysis data were then performed using Mann–Whitney U tests. A statistical significance level of p < 0.05 was used for all tests. All data are expressed as the mean ± standard deviation (SD).

Results The content of carbonate in the HA lattice was measured using FT-IR and TG-DTA methods and CHN analysis (Table 1). Examination of the CA specimens showed that the phosphate ions of the apatite were partially substituted by carbonate ions (B-type CA). The FT-IR spectra indicated that the content of carbonate ions in the HA lattice was 3.1 ± 0.1 wt.% for CA2 and 7.2 ± 0.1 wt.% for CA5. The content of carbonate ions in the HA lattice was calculated from the results of the CHN analysis as 3.5 ± 0.07 wt.% for CA2 at 8.9 ± 0.05 wt.% for CA5. The TGDTA spectra indicated that the content of carbonate ions was 3.1 ± 0.2 wt.% for CA2 and 7.7 ± 0.3 wt.% for CA5. Together, the results of the FT-IR, TG-DTA, and CHN analyses indicated that the resultant apatite was B-type CA containing approximately 3 wt.% for CA2 and 8 wt.% for CA5 of carbonate substituted for phosphate sites in the HA lattice. The surface roughness, surface energy, and solubility of the ceramic biomaterials were determined. The surface roughness (Ra) and wettability of the synthesized specimens are summarized in Table 2. No significant differences in surface roughness nor contact angle of water were found among the HA, CA2, and CA5 specimens. The dispersive components of the surface free energy of 11 mJ/m2 on HA, 5.3 mJ/m2 on CA2, and 4.7 mJ/m2 on CA5 were similar.


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The polar components of surface free energy were 34 mJ/m2 on HA, 28 mJ/m2 on CA2, and 27 mJ/m2 on CA5. The surface free energy calculated from the sum of the dispersive and polar components was 45 mJ/m2 for HA, 33 mJ/m2 for CA2, and 32 mJ/m2 for CA5. These results indicated that the substitution of carbonate ions into the HA lattice slightly decreased the surface free energy. The calcium and phosphorus concentrations were assessed after incubation of the HA, CA2, CA5, and bone slices for several hours in acetic acid solution (Fig. 1A and 1B) and in cell culture medium (Fig. 1C and 1D). The dissolution curves of the calcium and phosphorus concentrations into the acetic acid solution showed a significantly higher dissolution rate in the CA groups compared with the HA group. The concentrations of calcium and phosphorus were 0.0 mM during the control incubation, where no specimens were in the acetic acid solution. After 24 hours of immersion, the released calcium concentrations were 0.5 from HA, 5.9 from CA2, 8.0 from CA5, and 7.5 mM from the bone surfaces. After 24 hours of immersion, the released phosphorus concentrations were 0.4 from HA, 5.8 from CA2, 10.8 from CA5, and 12.0 mM from the bone surfaces. The CA2 and CA5 dissolution rates were approximately 4–5 times and 8–9 times, respectively, larger than that of HA. The culture medium calcium and phosphorus concentrations were 1.4 mM and 1.0 mM, respectively, and those levels were maintained throughout the solubility test incubation. The dissolution curves of calcium and phosphorus concentrations in cell culture medium showed that the HA, CA2, and CA5 specimens did not release calcium or phosphorus ions under nearly neutral conditions. To compare the osteoclast formation on the different types of apatite ceramic biomaterials, the osteocyte-like cells were co-cultured with BMCs for 7 d without any added differentiation factors. The osteoclasts derived from the BMCs were identified by staining for TRAP. The TRAP staining showed that a variable number of the BMCs that adhered to the specimens (HA, CA2, CA5, and bone slices) differentiated into osteoclast precursors or osteoclasts (Fig. 2Ba-d). Some TRAP-positive cells that had adhered to the HA and CA2 specimens were small and


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mononuclear, suggesting that they had not completely differentiated into osteoclasts. When the multinuclear TRAP-positive cells growing on each experimental substrate were counted (Fig. 2Be), it was found that the number of TRAP-positive cells was significantly larger on the CA5 substrates compared with those on the HA and CA2 substrates. The number of multinuclear TRAP-positive cells was largest on the bone slices. However, specimens cultured with BMCs but without osteocyte-like cells did not show TRAP-positive cells (Fig. 2A). The activation of osteoclasts derived from the BMCs through the expression of RANKL by the co-cultured osteocyte-like cells was investigated using immunohistochemical staining and ELISA. Images of the adhered cells double-stained for actin (red) and RANKL (green) are shown in Figure 3. Actin rings, which are specific markers for activated osteoclasts that resorb the substrate, were formed by osteoclasts on the CA5 specimens and the bone slices (Fig. 3c and 3d). On the contrary, no actin ring formation was observed on the HA and CA2 specimens (Fig. 3a and 3b), though some of the attached BMCs expressed stress fibers in their cytoplasm. To quantify the activation of osteoclasts on the four specimens, the number of actin rings per area was counted (Fig. 3e). Significantly more actin rings were found in cells on the CA5 specimens than in those on the HA and CA2 synthesized ceramic biomaterials. Compared with all the other specimens, the number of actin rings was largest on the bone slices. A positive immunoreaction for RANKL was found in the cytoplasm of the mononuclear cells that adhered to the HA, CA2, CA5, and bone slices (Fig. 3a-d). These RANKL-positive mononuclear cells were identified as osteocyte-like cells that proliferated and communicated with the BMCs on each specimen. The concentrations of soluble M-CSF (Fig. 3f) and RANKL (Fig. 3g) in the CM, measured using an ELISA kit, were significantly larger in the CA5 samples compared with those in the HA and CA2 synthesized ceramic biomaterial samples. The concentrations of soluble M-CSF and RANKL were the largest in the bone slice culture media. Osteoclast resorption pits on the HA, CA2, CA5, and bone slices were analyzed using SEM and laser microscopy. The sample surfaces were observed after culture with (Fig. 4e-l) or without


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cells (Fig. 4a-d). The most distinct resorption pits were formed on the CA5 specimens (Fig. 4g and 4k) and the bone slices (Fig. 4h and 4l), whereas the resorption pits that had formed on the CA2 specimens (Fig. 4f and 4j) were mostly shallow and less clear. The HA surface structural changes were small (Fig. 4e and 4i). As shown in the higher magnification SEM images (Fig. 4il), the areas resorbed by osteoclasts were rough and appeared granular compared with the intact surfaces of the specimens. The depth of the resorption pits was quantitatively analyzed using a laser microscope (Fig. 4m). The depth of the resorption pits formed by the osteoclasts on the CA5 specimens was approximately 4.9-fold greater than those on the HA specimens, followed by pits on the CA2 specimens, which were approximately 3.1-fold greater than those on the HA specimens (Fig. 4m). The H&E-stained histological images of rat femur bone marrow after sham surgery (i.e., no specimen implanted) showed new bone formation along the cortical bone surface facing the marrow (not shown). Histological examination of the rat femurs showed new bone formation caused by wound healing, also in the bone marrow, which was far from areas with immediate biomaterial contact, as shown in the H&E-stained sections after 7-d implantation of the HA (Fig. 5a-c), CA2 (Fig. 5d-f), and CA5 (Fig. 5g-i) specimens. In the case of the HA specimens, most of the surfaces were covered with fibrous tissue containing mononuclear fibroblast-like cells (Fig. 5a and 5b). Clusters of osteoid tissue were found only in the bone marrow. Newly formed bone was found in the regions approximately 100–200 µm away from the implanted HA. The trabeculae of the newly formed bone were covered with a single layer of osteoblasts (indicated by arrows) and included relatively few, small osteocyte lacunae. In the case of the CA2 and CA5 specimens, newly formed bone was similarly observed approximately 100–200 µm away from the implants (Fig. 5d-g and 5e-h). Giant and TRAP-positive multinuclear osteoclastic cells were found near the materials and on the newly formed bone in the bone marrow (indicated by arrows).


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The quantitative analysis of the TRAP-stained serial sections showed that more TRAP-positive osteoclastic cells were present near the CA2 (Fig. 5f) and in close proximity to the CA5 (Fig. 5i) than near the HA (Fig. 5c). The number of TRAP-positive, multinuclear cells per half of the bone marrow (within the 200 µm belt) was largest in the vicinity of the CA5, which was approximately 10-fold greater than that of the HA; the CA5 result was followed by the number of positive cells in the vicinity of the CA2, which was approximately 3-fold greater than that of the HA (Fig. 5j).

Discussion We have shown here that the content of carbonate ions in an apatite crystal lattice affects the induction of osteoclastogenesis in vitro and in vivo. This effect of the carbonate ion content in apatite will be discussed in relation to the conventional understanding of the material properties of bone and osteoclastogenesis. The in vitro co-cultures revealed that the BMCs differentiated into osteoclasts and were activated, resorbing the substrate when grown on the CA5 surfaces because of stimulation by the osteoclast differentiation factors expressed by osteocyte-like cells. Osteocytes have been reported to express RANKL both on their membrane and in soluble form

7-9

. Zhao S et al have

demonstrated that MLO-Y4 cells secrete M-CSF and RANKL into the CM, but that these secretions had no effect on BMC differentiation into osteoclasts 9. Juffer P et al demonstrated that the CM from unloaded MLO-Y4 cells increased osteoclast formation, while the CM from myotubes decreased osteoblast formation, and that the CM from cyclically strained myotubes increased osteoclast formation in comparison with the CM from unloaded myotubes

22

. Those

results indicate that MLO-Y4 cells need stimulation to secrete larger amounts of RANKL into the CM. Kurata K et al showed that damaged MLO-Y4 cells activate osteoclast precursors by soluble factors and thus can control the initial phase of targeted remodeling

23

. The in vitro


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results in the present study showed that the RANKL molecules expressed and secreted from the osteocyte-like cells stimulated the BMCs to differentiate into mature osteoclasts on CA5. We anticipated that the higher amount of carbonate in the apatite crystal structure would have effects on the osteoclast formation of osteocyte-like cells similar to those of osteocyte damage stimulation. The in vivo osteoclastogenesis results showed that osteoclasts were formed near the CA5 implanted into the rat femurs. One explanation for the observed difference in osteoclast differentiation is that the changes in the material properties caused by the substitution of carbonate ions into the HA lattice increase its similarity to bone tissue 4. Osteoclasts are formed by the fusion of monocytic precursors after stimulation by M-CSF and RANKL and are then activated, resorbing mineralized bone matrix

24

. This bone resorption

activity is essential for controlling bone development and turnover, as well as for calcium homeostasis. Osteoclast resorption depends on the organization of the actin cytoskeleton into an actin-rich structure—referred to as a sealing zone—that anchors the osteoclasts to the substrate. Therefore, the number of activated osteoclasts was estimated based on the morphology of the resorption stages, which have been categorized into three clearly distinct organizations of actin and five patterns of vinculin distribution 24. The podosome belt in mature osteoclasts is thought to evolve into the sealing zone of actively resorbing osteoclasts, forming a large circular band of actin that provides a tight attachment to the bone 25. The morphology of the actin ring is affected by surface roughness

26, 27

, crystallinity of the

solid surface 28, surface energy 29, and solubility 30. Based on these factors, we used synthesized specimens in the present study with approximately equal surface roughness (after polishing), crystallinity, and wettability. Therefore, the surface energy and solubility of the synthesized specimens were likely more influential on the formation of functional actin rings in the osteoclasts. The slightly decreased surface free energy (Table 2) and increased solubility (Fig. 1)


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of the CA5 compared with the HA may make it more suitable for the differentiation of osteoclast precursors into the osteoclastic phenotype. The surface free energy of biomaterials influences the initial attachment, spreading, and collagenous matrix deposition of human osteoblast-like cells 29. It has been shown that in A-type CA, the carbonate ions substituted for hydroxyl sites in the HA lattice decreased the polar component of the surface free energy compared with that in the HA. In addition, human osteoclasts cultured on A-type CA displayed a more spread cell morphology, as shown by actin staining 31. Although the lattice sites of the carbonate ion substitution were different, the B-type CA specimens used in the present study decreased the surface energy compared with that of stoichiometric HA. One hypothesis to explain this is that the conformation of the proteins for osteoclast adhesion may be different because of the decreased surface free energy on the CA. The specimens here were immersed in medium before being used for osteoclast culture and were presumably covered with a similar amount of adsorbed proteins as were in the medium. Based on the differences in surface free energy caused by ion substitution in the apatite crystal lattice

29

and the changes in protein conformation that can be influenced by surface chemistries 32, 33, it is possible that the conformation of the proteins adsorbed on the substrate varied between the specimens. Such conformational changes may induce differences in the exposed domains of the proteins adsorbed on the specimens. The CA2 and CA5 solubility rates in the acetic acid solution were approximately 4–5 times and 8–9 times larger, respectively, than that of the HA (Fig. 1). One possible contributing factor is that the extracellular calcium level affects the differentiation of osteoclast progenitors; another could be a difference in specimen solubility. The CA5 and bone slice specimen solubilities in acidic conditions, which mimic the resorption lacunae, were significantly higher than those of the HA and CA2, although solubility in neutral conditions was similar for each specimen. The higher solubility of the CA5 and bone slices is conducive to resorption at the sealing zone between the osteoclasts and the bone matrix. The calcium and phosphate ions released from the


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bone matrix by osteoclast resorption stimulate other osteoclast behaviors. The sensitivity of osteoclasts to increased extracellular calcium concentrations depends on their activation phase, which could be resting, migrating, or resorbing 34. In contrast with resting osteoclasts, resorbing osteoclasts are not sensitive to increased extracellular calcium concentrations. The inactivated osteoclasts surrounding the resorbing osteoclasts are stimulated by calcium ions released from the bone matrix to differentiate and resorb the intact areas of the bone matrix. The activated osteoclasts on CA5 resorbed the ceramic surface and released calcium

34

and phosphate

35

ions

into the cell culture medium. These released calcium ions stimulated the surrounding inactivated osteoclasts to differentiate, activate, and resorb the intact CA5 surface. One possible explanation for the osteoclast formation is acidification by the alteration of carbon dioxide or bicarbonate concentrations released from the CA5 surface; these might be released with calcium and phosphate ions from CA5 in acidic conditions at the surgical wound sites. Considering the acidity around the injured area, the acidic conditions could also induce osteoclast formation

36

.

The higher solubility of CA5 could be one of the important factors for osteoclast formation both in vitro and in vivo. Osteoconduction progresses in six stages: serum adsorption, recruitment of various cell types, osteoblast attachment and proliferation, osteoblast differentiation, matrix calcification, and bone remodeling 1. In the final stage of osteoconduction, the osteoclasts play reconstructive roles by resorbing the osteoid and bioresorbable materials and by interacting with the osteoblasts responsible for synthesis of bone matrix. One hypothesis suggested by the present study is that CA5 promotes osteoclastogenesis by stimulating osteocytes, thereby playing a direct role in bone metabolism. These results contribute to the design of biomaterials that would be resorbed by osteoclasts after fulfilling their primary function as a scaffold for cell growth and eventually bone regeneration.


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Conclusions The results of the present study provide new and important information on the effects of ceramic biomaterials on osteoclast formation. First, BMCs differentiated into osteoclasts and were activated to resorb the substrate when grown on CA5 because of the expression of osteoclast differentiation factor from osteocyte-like cells. Second, the in vivo osteoclastogenesis showed osteoclast formation near the CA5 after implantation into the rat femurs. These results indicate that the content of carbonate ions in an apatite crystal lattice affects the induction of osteoclastogenesis in the vicinity of the implanted ceramic biomaterial.

FIGURES

Fig. 1 The calcium and phosphorus concentrations after the incubation of HA (blue triangle), CA2 (pink circle), CA5 (red circle), bone slices (green diamond) and a blank control (open square) for 0.5, 1, 2, 4, 8, 12, or 24 h in 1 M acetic acid solution (pH 5.6) (Fig. 1A and 1B) and in cell culture medium (Fig. 1C and 1D).

Fig. 2 (A) Images of the specimens after co-culture of BMCs with or without osteocyte-like cells and TRAP staining. (B) TRAP staining of cells cultured on the (a) HA, (b) CA2, (c) CA5, and (d) bone slices. The BMCs cultured with osteocyte-like cells differentiated into osteoclast precursors or osteoclasts. Scale bar: 100 µm. (e) The number of multinuclear TRAP-positive cells was significantly increased on CA5 compared with on HA and CA2. *: p

Hydroxyapatite with High Carbonate Substitutions Promotes

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