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ACS Appl Mater Interfaces
ACS Appl Mater Interfaces
am
aamick
ACS Applied Materials & Interfaces
1944-8244
1944-8252
American Chemical Society

39182190
10.1021/acsami.4c08047
Research Article
Carbonate Apatite Honeycomb Scaffold-Based Drug Delivery System for Repairing Osteoporotic Bone Defects
https://orcid.org/0000-0002-3147-5784
Hayashi Koichiro *
https://orcid.org/0000-0003-3299-6634
Zhang Cheng
Taleb Alashkar Ahmad Nazir
Ishikawa Kunio
Department of Biomaterials, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
* Email: khayashi@dent.kyushu-u.ac.jp.
25 08 2024
04 09 2024
16 35 4595645968
16 05 2024
29 07 2024
28 07 2024
© 2024 The Authors. Published by American Chemical Society
2024
The Authors
https://creativecommons.org/licenses/by-nc-nd/4.0/ Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/).

Osteoporotic bone defects are difficult to repair in elderly patients. This study aimed to repair osteoporotic bone defects using a combination of bone tissue engineering (BTE) and drug delivery systems (DDS). Herein, honeycomb granules (HCGs) composed of carbonate apatite microspheres were fabricated as BTE scaffolds. Each HCG possesses hexagonal macropores and abundant interconnected micropores between the microspheres. Owing to these multiscale interconnected pores, HCGs can readily contain antibodies against sclerostin (Scl), which causes imbalances in bone homeostasis. Anti-Scl antibody-loaded HCGs (Scl-Ab-HCGs) regulate the release of Scl-Abs in response to the pH of the osteoporotic environment. In ovariectomized rabbit osteoporotic femurs, HCG monotherapy forms new bone with less osteocyte damage (fewer empty bone lacunae) and fewer osteoclasts than osteoporotic bone; however, it is insufficient to prevent receptor activator of nuclear factor-kappa B ligand (RANKL) overexpression. Consequently, HCG monotherapy restores bone quantity better than no treatment but not to normal levels. In contrast, new bone tissue formed by Scl-Ab-HCG-based DDS predominantly expresses osteocalcin rather than RANKL, similar to normal bone, and shows a similar osteocyte apoptosis level, bone quantity, and osteoclast number as normal bone. Thus, Scl-Ab-HCG-based DDS is a promising approach for osteoporotic bone defect repair.

bioceramics
calcium phosphate
bone tissue engineering
drug delivery
osteoporosis
Japan Agency for Medical Research and Development 10.13039/100009619 JP24ym0126098h0003 Japan Society for the Promotion of Science 10.13039/501100001691 JP23K18593 Japan Society for the Promotion of Science 10.13039/501100001691 JP22H03954 Japan Agency for Medical Research and Development 10.13039/100009619 JP24ym0126811j0003 document-id-old-9am4c08047
document-id-new-14am4c08047
ccc-price
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pmc1 Introduction

Osteoporosis is a metabolic bone disease characterized by reduced bone mass, deteriorated bone microarchitecture, and poor bone regeneration, leading to a significantly increased risk of bone fracture.1−5 It is caused by an imbalance in bone homeostasis, i.e., rate of bone resorption exceeds that of bone formation.6 The balance between bone formation and resorption is disrupted in postmenopausal women and elderly individuals, causing osteoporosis. Globally, over 1 billion people suffer from severe pain, bone fractures, and physical inconvenience due to osteoporosis, and over 9 million osteoporotic fractures occur each year.7,8 Over half of the people over the age of 50 years in the United States have osteoporosis or low bone mass,9 and half of postmenopausal Asian or Caucasian women aged 65 years have osteoporosis.1,10 Due to the rapid aging of populations worldwide, the number of patients with osteoporosis is expected to continue to increase. Furthermore, the risk of secondary fractures in osteoporotic patients with fractures is extremely high, with an incidence rate of secondary fractures of 23 and 54% within 1 and 5 years, respectively.11 Therefore, treatments that rapidly increase bone density and reduce fracture risk are desirable.

Continuing treatment is difficult for several patients with osteoporosis, and half of them discontinue treatment within a year.12−14 Treatment discontinuation increases the risk of bone mineral density loss and fractures.15 Deng et al. reported that 54% of patients who discontinue treatment are self-discontinued, and 46.3% of those patients forget to visit the outpatient clinic.15 Furthermore, Rossini et al.19 reported that the main causes of treatment discontinuation were drug side effects, fear of side effects, lack of motivation for treatment, and inconvenient dosing.16 To mitigate barriers to treatment adherence, such as side effects and frequent dosing, a drug delivery system (DDS) is considered a promising strategy.17

Sclerostin (Scl), a glycoprotein secreted by osteocytes, is known to cause an imbalance in bone homeostasis. Scl suppresses the propagation of Wnt signals to β-catenin, which prevents bone formation by osteoblasts and promotes bone resorption by osteoclasts, eventually resulting in decreased bone mass.18−20 Therefore, the use of an antibody against Scl (Scl-Ab) is a rational strategy for neutralizing the inhibitory effects of Scl on Wnt signaling. Scl-Ab is reported to exert a dual effect of promoting bone formation and preventing bone resorption. Meta-analyses have demonstrated that Scl-Ab shows a higher antifracture efficacy than bisphosphonates and denosumab, which only inhibit bone resorption by osteoclasts, and teriparatide, which only promotes bone formation by osteoblasts.18−20

Furthermore, bone tissue engineering (BTE) is a prospective alternative for treatment of osteoporotic bone defects and fractures.21−23 Autografts are conventionally used as BTE scaffolds for repairing ordinary bone defects and fractures.21−23 However, unlike healthy bone, osteoporotic bone is low in volume and strength; therefore, autologous bone grafting is not adequate to repair bone defects and fractures in patients with osteoporosis.21−23 Although allografts and xenografts are also used as BTE scaffolds, they are often associated with risks of immunorejection and infection.21−23 To overcome these problems, synthetic BTE scaffolds are being developed.21−23 Typical synthetic BTE scaffolds used for repairing osteoporotic bone defects and fractures include osteoconductive bioceramics incorporated with therapeutic substances that enhance the metabolic activity of osteoblasts and inhibit the metabolic activity of osteoclasts.21,22,24 For example, the doping of strontium (Sr) ions that enhance bone formation into hydroxyapatite (HAp) and β-tricalcium phosphate (β-TCP) have been intensely studied.25−30 In these cases, Sr ions partially replace calcium (Ca) ions of HAp and β-TCP. Previous studies demonstrated that Sr-doped HAp increases bone volume in osteoporotic bone compared with pure HAp,25,26 whereas Sr-doped β-TCP prolongs the inflammation phase and forms a smaller volume of bone than pure β-TCP.28 Furthermore, excessively released Sr ions associated with fast scaffold degradation have been reported to prevent cell proliferation and bone formation.29,30 Based on the fact that β-TCP is degraded quickly in vivo, Sr-doped β-TCP may increase the Sr ion concentration excessively, which may result in slower bone defect restoration than that by pure β-TCP.28 In contrast, the Sr ion release rate from the Sr-doped HAp may be lower than the appropriate release rate because HAp degradation is extremely slow. Two recent studies demonstrated that Sr-doped HAp implantation requires a longer time to restore bone volume than the combination of HAp implantation and strontium ranelate drug administration.25,27 Thus, the above findings indicate that the therapeutic effects are not fully realized when the time points of Sr ion action are not appropriate. However, in the incorporation method, in which Sr ions are doped into the host material, the release of Sr ions depends solely on the host material degradation. Therefore, an incorporation method that can control the release of therapeutic substances in addition to the degradation of the host material is desirable. Furthermore, in case of doping, the amount of therapeutic substances (ions) is determined at the time of scaffold fabrication, and the amount of therapeutic substances cannot be controlled according to the patient’s symptoms. To control the amount of therapeutic substances according to the patient’s symptoms, it is desirable to incorporate any therapeutic substances into the scaffold at the time of implantation.

The incorporation of drugs into host materials by loading has been studied.31−36 In the case of drug incorporation by loading, in addition to drug release due to host material degradation, diffuse release of the drug due to concentration gradients can be utilized. Thus, the therapeutic substances on the surface of the host material can act early due to diffusion release, whereas the therapeutic substances inside the host material released due to the degradation of the host material can act in the medium to long-term. Furthermore, the drug content can be easily controlled because it can be incorporated after fabricating the host materials. Such a multistep drug release and precise control of drug content can enhance the efficacy of synthetic BTE scaffolds.

To efficiently load drugs onto the surface of the host material, the host material should be porous. Macropores that penetrate the host material and highly continuous micropores are required for loading drugs throughout the interior of the host material. Furthermore, a host material that is slightly degraded spontaneously, but is degraded by osteoclasts, is favorable. Host materials, i.e., scaffolds that exhibit such degradation behavior, can release drugs under osteoporotic conditions and stop releasing drugs when osteoporotic symptoms subside, thus enabling a DDS that spontaneously controls drug release according to the progression of osteoporosis.

In our previous studies, we found that carbonate apatite (CAp) scaffolds exhibited these aforementioned characteristics. Our previous studies demonstrated that CAp, a bone mineral, is osteoconductive and resorbed by osteoclasts37,38 and that the osteogenic and angiogenic abilities of CAp scaffolds can be controlled by controlling their shape,39,40 macroporous structure,41−51 and microporous properties.52,53 Macroporous structures with penetrating macropores (>100 μm in size) such as honeycomb37−39,44−50,52,53 allow the formation of new bone and blood vessels simultaneously on the scaffold surface and within the macropores. Notably, round honeycomb granules (HCGs) conform well to bone defect shapes and provide a suitably sized space for cell penetration and tissue ingrowth among granules; thus, they enable abundant formation of new bone tissue.39 Furthermore, the microporous properties control CAp scaffold degradation and bone formation.52,53 Micropores (<10 μm in size) are also expected to serve as spaces to load drugs. Previously, we established a method for introducing highly continuous micropores into CAp scaffolds by exploiting the fact that micropores inevitably form among microspheres when they are packed.50

In this study, we aimed to load antibodies against Scl (Scl-Abs) into HCGs containing CAp microspheres and evaluate the therapeutic effects of Scl-Ab-loaded HCGs (Scl-Ab-HCGs) in an ovariectomized (OVX) rabbit model of osteoporosis. To the best of our knowledge, this study is the first to investigate and clarify the therapeutic effects and bone formation behavior of a scaffold-based DDS that releases Scl-Abs from calcium phosphate scaffolds in osteoporotic bone defects.

2 Results

2.1 Material Characterization

HCGs comprising CAp microspheres were fabricated by extrusion, debinding, and phosphatization based on our previous study.39 The HCGs were approximately 1.5 mm in size and each HCG had seven penetrating hexagonal macropores, in which the length of each side was approximately 150 μm (Figure 1A,B). The walls surrounding the hexagonal macropores were approximately 130 μm in thickness (Figure 1A,B). The walls comprised microspheres with aggregated scale-like CAp crystals (Figure 1C). Micropores (<1 μm) were present among CAp microspheres in the interior of the walls (Figure 1C). Mercury intrusion porosimetry demonstrated that the HCGs had micropore sizes of 9.6, 18.0, and 628.0 nm (Figure 1D). Micropores with a size of 628.0 nm coincide with the micropores among the CAp microspheres within the walls. The micropores with sizes of 9.6 and 18.0 nm corresponded to spaces among CAp crystals comprising microspheres. The total volume of these micropores for each granule (1.5 ± 0.2 mg) was approximately 130 cm3. To load Scl-Abs onto HCGs, the HCGs were immersed in an Scl-Ab solution and subjected to vacuum deaeration. To evaluate the Scl-Ab distribution in HCGs, fluorescent dye (fluorescein isothiocyanate [FITC])-labeled antibodies were used. After loading Scl-Abs by vacuum deaeration, the HCGs maintained their original morphology and structure (Figure 1E). HCGs fluoresced from the entire granule (Figure 1F). Furthermore, fluorescence was observed uniformly across the entire cross-section of the HCGs (Figure 1G). Thus, the Scl-Abs penetrated the HCGs and were loaded throughout them.

Figure 1 (A) Scanning electron microscopic (SEM) image of honeycomb granules (HCGs). (B) High-magnification image of the center of an HCG. (C) High-magnification image of the wall surrounding the hexagonal macropore. Yellow arrowheads indicate micropores among carbonate apatite (CAp) microspheres. (D) Pore size distribution and cumulative pore volume measured by mercury intrusion porosimetry. (E) Optical and (F) fluorescent images of antisclerostin antibody-loaded HCGs (Scl-Ab-HCGs). (G) High-magnification fluorescent image of an Scl-Ab-HCG.

The X-ray diffraction (XRD) patterns of HCGs and Scl-Ab-HCGs coincided with those of a commercial CAp reference substance (Figure 2A). Thus, the inorganic phase of Scl-Ab-HCGs maintained apatitic crystals after being loaded with Scl-Abs. The absorption bands of the phosphate group were detected at 1020, 599, and 562 cm–1 in the Fourier transform infrared (FTIR) spectra of Scl-Ab-HCGs, HCGs, and HAp, respectively (Figure 2B). The carbonate group bands were present at 1473 and 1415 cm–1 in the spectra of Scl-Ab-HCGs and HCGs, respectively, but they were absent in the HAp spectrum (Figure 2B). In contrast, a band corresponding to the hydroxyl group was observed at 629 cm–1 in the HAp spectrum but was absent in the Scl-Ab-HCG and HCG spectra (Figure 2B). HCGs comprise CAp, in which the carbonate group replaces the phosphate and hydroxyl groups. Furthermore, the band of the amide group appeared at 1652 cm–1 in the Scl-Ab-HCG spectrum (Figure 2B), demonstrating that the Scl-Abs were loaded in the HCGs to form Scl-Ab-HCGs.

Figure 2 (A) X-ray diffraction (XRD) patterns of Scl-Ab-HCGs, HCGs, and CAp. (B) Fourier transform infrared (FTIR) spectra of Scl-Ab-HCGs, HCGs, and hydroxyapatite (HAp).

The content of Scl-Abs loaded in the Scl-Ab-HCGs was estimated from the absorbance of Scl-Abs in the supernatant solutions collected after immersing HCGs in Scl-Ab solutions of different concentrations (4.5, 45.0, and 90.0 mg/mL) for 1 min under vacuum. When the HCGs were immersed in 4.5, 45.0, and 90.0 mg/mL Scl-Ab solutions, the Scl-Ab-HCGs contained 66.8, 638.0, and 1,364.0 mg/g Scl-Abs, respectively (Figure 3A). The Scl-Ab-releasing behavior of Scl-Ab-HCGs was evaluated in acetate buffer at pH 5.5, which coincides with the weakly acidic environment created by osteoclasts, and in saline at pH 7.4 (Figure 3B). In saline at pH 7.4, 0.92 mg/mL of Scl-Abs was released from Scl-Ab-HCGs 1 h after immersion with a subsequent gradual release of Scl-Abs until 576 h (2.62 mg/mL of cumulative release at 576 h). In the buffer solution at pH 5.5, 1.48 mg/mL of Scl-Abs was released 1 h after immersion, and rapid release continued up to 48 h after immersion (3.74 mg/mL of cumulative release at 48 h). Subsequently, Scl-Abs were gradually released until 576 h (4.45 mg/mL of cumulative release at 576 h). Furthermore, to investigate the Scl-Ab release mechanism, the concentration of Ca ions released from the Scl-Ab-HCGs was analyzed (Figure 3C). The Scl-Ab-HCGs rapidly released Ca ions in a solution at pH 5.5 but released few Ca ions in saline (pH 7.4). Thus, although Scl-Ab-HCGs showed little spontaneous degradation in physiological environments, they degraded in weakly acidic environments created by osteoclasts. Based on the release behavior of Scl-Abs (Figure 3B) and Ca ions (Figure 3C), the initial release of Scl-Abs in saline (pH 7.4) depended on diffusion due to concentration gradients rather than Scl-Ab-HCG degradation. Furthermore, the amount of Scl-Abs released by diffusion was approximately 60% of that by Scl-Ab-HCG degradation.

Figure 3 (A) Correlation between the Scl-Ab solution concentration for loading Scl-Abs into HCGs and the final Scl-Ab concentration in the obtained Scl-Ab-HCGs. (B) Concentration of Scl-Abs released from Scl-Ab-HCGs in saline at pH 7.4 and acetate buffer at pH 5.5, which coincides with the weakly acidic environment created by osteoclasts. The released Scl-Abs concentrations were quantified from the absorbance of Scl-Abs in the supernatants. (C) The concentration of Ca ions released from Scl-Ab-HCGs in saline at pH 7.4 and acetate buffer at pH 5.5; ppm, parts per million. The released Ca ion concentrations were quantified by inductively coupled plasma atomic emission spectroscopy.

2.2 In Vitro Cell Morphology, Proliferation, and Mineralization

The effects of Scl-Ab-HCGs on vitro cell proliferation were evaluated in osteoblasts. In accordance with ISO 10993-12 (Biological evaluation of medical devices, Part 12: Sample preparation and reference materials),54 extracts were collected from HCGs and Scl-Ab-HCGs. The Scl-Ab-HCG extract contained 1.41 mg/mL of Scl-Abs. The extract was diluted 5-fold (5×) and 25-fold (25×) and contained 0.06 and 0.28 mg/mL of Scl-Abs, respectively. The 1×, 5× , and 25× diluted extracts were designated Scl-Ab-HCGs (1.41), Scl-Ab-HCGs (0.28), and Scl-Ab-HCGs (0.06), respectively. After incubation for 24 h, no significant differences in cell morphology were observed between the control and HCG groups (Figure 4A,B). In the Scl-Ab-HCG (0.06) and Scl-Ab-HCG (0.28) groups, cells were elongated and actin filaments were oriented in the cell elongation direction (Figure 4C,D). However, in the Scl-Ab-HCG (1.41) group, the cells were not elongated and their morphology was similar to cells of the control and HCG groups (Figure 4E). The cell densities in the Scl-Ab-HCG (0.06) and Scl-Ab-HCG (0.28) groups were significantly higher than those in the control, HCG, and Scl-Ab-HCG (1.41) groups (Figure 4F and Table S1). Furthermore, bone nodules were stained with alizarin red S (ARS) to evaluate osteoblast mineralization after incubation for 14 days (Figure 4G–K). No bone nodules were observed in the control group after staining with ARS (Figure 4G). A few bone nodules were observed in the HCG and Scl-Ab-HCG (1.41) groups (Figure 4H,K). In contrast, abundant bone nodules were observed in the Scl Ab-HCG (0.06) and Scl-Ab-HCG (0.28) groups (Figure 4I,J). The number of bone nodules was quantified by measuring the absorbance of ARS at 560 nm. The number of bone nodules in the Scl-Ab-HCG (0.06) and Scl-Ab-HCG (0.28) groups was significantly higher than that in the control, HCG, and Scl-Ab-HCG (1.41) groups (Figure 4L and Table S1).

Figure 4 Fluorescence images of cells after 24 h of culturing in (A) control, (B) HCG, (C) Scl-Ab-HCG (0.06 mg/mL; 25× dilution), (D) Scl-Ab-HCG (0.28 mg/mL; 5× dilution), (E) Scl-Ab-HCG (1.41 mg/mL; 1× dilution) groups. The cell nuclei and actin filaments were stained blue and red, respectively. Scale bars: 100 μm. (F) Cell number per square millimeter. A significant difference (p < 0.05) was found among the groups (shown as the letters a and b in the graph). Deposited minerals stained with alizarin red S (ARS) after 14 days of culturing in (G) control, (H) HCG, (I) Scl-Ab-HCG (0.06), (J) Scl-Ab-HCG (0.28), (K) Scl-Ab-HCG (1.41) groups. Scale bars: 1 mm. (L) Absorbance of ARS was measured at 560 nm to estimate the number of bone nodules. A significant difference (p < 0.05) was detected among the groups (shown as the letters a and b in the graph).

2.3 In Vivo Bone Formation in Osteoporotic Bone Defects

OVX rabbits were used as an osteoporotic model to investigate the efficacy of Scl-Ab-HCGs. Serum estrogen levels were significantly lower in normal rabbits than in OVX rabbits (Figure 5A). Furthermore, microcomputed tomography (μ-CT) confirmed that the normal rabbit femurs showed significantly higher bone volume fraction than the OVX rabbit femurs (Figure 5B). These results indicate that OVX rabbits can be used as an osteoporotic model. HCGs and Scl-Ab-HCGs were implanted into the osteoporotic bone defects of OVX rabbit femurs. Subsequently, μ-CT at postoperative week (POW) 4 demonstrated that new bone formed only in the vicinity of granules in the HCG-implanted group (Figure 5C), whereas abundant bone filled the space among granules in the Scl-Ab-HCG-implanted group (Figure 5D). In bone defects without implantation (empty group), little bone was formed (Figure 5E). At POW 12, in the HCG-implanted group, although a portion of the HCGs was resorbed, bone formation was limited to areas immediately nearby the granules, similar to bone formation at POW 4 (Figure 5F). In the Scl-Ab-HCG-treated group, a larger volume of granules was resorbed and replaced with new bone (Figure 5G). In the empty group, little bone was formed even on POW 12 (Figure 5H). The bone volume fractions (bone volume-to-total tissue volume ratio [BV/TV]) were analyzed using μ-CT (Figure 5I and Table S3). At POW 4, BV/TV in Scl-Ab-HCG-implanted bone defects (33.9 ± 5.1%) was comparable to that in normal femur (33.7 ± 8.3%). At POW 12, BV/TV in Scl-Ab-HCG-implanted bone defects (39.8 ± 3.3%) remained at the same level as that in normal femur. In contrast, the BV/TV in HCG-implanted bone defects at both POW 4 (27.1 ± 5.8%) and 12 (27.0 ± 4.0%) was significantly lower than that in Scl-Ab-HCG-implanted bone defects and normal femurs. Although the BV/TV tended to be higher in HCG-implanted bone defects than that in OVX femurs (20.9 ± 3.7%), this difference was not statistically significant. In the empty group, the BV/TV was almost zero at both POW 4 (0.5 ± 0.5%) and 12 (0.3 ± 0.3%).

Figure 5 (A) Serum estrogen levels in normal and ovariectomized (OVX) rabbits. *p < 0.05. (B) Microcomputed tomography (μ-CT) images of normal and OVX rabbit femurs. μ-CT images of OVX rabbit femurs at (C–E) 4 weeks and (F–H) 12 weeks after surgery: (C, F) HCG-implanted, (D, G) Scl-Ab-HCG-implanted, and (E, H) empty groups. Scale bars: 1 mm. Yellow arrowheads indicate the remaining materials. (I) Bone volume-to-total tissue volume ratio (BV/TV) in normal and OVX rabbit femurs and in newly formed bone in the osteoporotic bone defects of OVX rabbit femurs in HCG-implanted, Scl-Ab-HCG-implanted, and empty groups. Significant differences (p < 0.05) were found among the groups and are indicated using different letters (a, b, and c).

Hematoxylin and eosin (HE) staining demonstrated that osteocytes were present in almost all bone lacunae in normal femurs (Figure 6A) but were absent in several bone lacunae in the osteoporotic femurs of OVX rabbits (Figure 6B). Recent studies demonstrated that empty osteocyte lacunae appear as a result of osteocyte apoptosis and are observed in osteoporotic bone.55,56 In addition to the results on serum estrogen level and μ-CT, the results on empty lacunae also indicate that OVX rabbit femurs were osteoporotic. On POW 4 in the HCG-implanted group, new bone tissue comprising mature and immature bone formed on the surface of the granules (Figure 6C). In the Scl-Ab-HCG-implanted group, mature bone formed not only on the surface of granules but also in the spaces among granules (Figure 6D). In both the HCG-implanted and Scl-Ab-HCG-implanted groups, osteocytes were present in almost all bone lacunae in mature bone (Figure 6C,D). In the empty group, adipose tissue filled the bone defects (Figure 6E). At POW 12, in the HCG-implanted group, although mature bone formed on the surface of the granules, little bone formed in regions away from the granules (Figure 6F). Furthermore, bone did not always form in the regions where HCG was resorbed; however, adipose tissue formed in some regions where HCG was resorbed (Figure 6F). In the Scl-Ab-HCG-implanted group, abundant bone formed on the granular surfaces and in the intergranular spaces and filled the intragranular hexagonal macropores (Figure 6G). The Scl-Ab-HCGs were replaced with new bone rather than adipose tissue (Figure 6G). In the empty group, little bone was formed even on POW 12 (Figure 6H). The bone percentages in the normal and OVX rabbit femurs were 34.9 ± 4.7% and 26.2 ± 1.5%, respectively (Figure 6I and Table S4). The percentages of newly formed bone in the osteoporotic bone defects (i.e., new bone percentage) in Scl-Ab-HCG-implanted groups at POW 4 (31.2 ± 2.8%) were already equivalent to the bone percentage in normal femurs, and new bone percentage at POW 12 (36.7 ± 2.5%) was still equal to the bone percentage in normal femurs (Figure 6I). Furthermore, the new bone percentages in osteoporotic bone defects with Scl-Ab-HCG implantation at both POW 4 and 12 were significantly higher than the bone percentage in OVX rabbit femurs and the new bone percentages in osteoporotic bone defects with HCG implantation at POW 4 (26.4 ± 2.0%) and POW 12 (24.3 ± 1.6%). In contrast, the new bone percentage in osteoporotic bone defects with HCG implantation at both POW 4 and 12 was equivalent to the bone percentage in OVX rabbit femurs (no significant difference). New bone percentages in the empty group at POW 4 and 12 were 1.7 ± 1.0% and 1.1 ± 0.4%, respectively, which were significantly lower than all other groups. Furthermore, the percentage of empty bone lacunae in OVX rabbit femurs was significantly higher than that in normal rabbit femurs and osteoporotic bone defects with HCG and Scl-Ab-HCG implantation (Figure 6J and Table S5). No significant difference was detected in the percentage of empty bone lacunae between normal rabbit femurs and osteoporotic bone defects at 4 and 12 weeks after HCG and Scl-Ab-HCG implantation.

Figure 6 Hematoxylin and eosin (HE)-stained sections of (A) normal and (B) OVX rabbit femurs. HE-stained sections of bone defect sites in OVX rabbit femurs at (C–E) 4 weeks and (F–H) 12 weeks after surgery: (C, F) HCG-implanted, (D, G) Scl-Ab-HCG-implanted, and (E, H) empty groups. (A–E) The upper and lower rows show low-magnification and high-magnification images, respectively. The scale bars in the upper and lower rows indicate 500 and 20 μm, respectively. The black arrowheads indicate empty bone lacunae without osteocytes. MB, IB, AT, and # indicate mature bone, immature bone, adipose tissue, and remaining material, respectively. (I) Area percentages of bone in normal and OVX rabbit femurs and those of newly formed bone in the osteoporotic bone defects of OVX rabbit femurs in HCG-implanted, Scl-Ab-HCG-implanted, and empty groups. (J) Percentages of empty bone lacunae in the HCG-implanted, Scl-Ab-HCG-implanted, and empty groups.

Immunostaining for receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) and osteocalcin (OCN), which are bone metabolic markers, demonstrated that normal femurs mainly expressed OCN (Figure 7A), whereas OVX rabbit femurs predominantly expressed RANKL, rather than OCN (Figure 7B). At POW 4, the new bone in the osteoporotic bone defects mainly expressed RANKL in the HCG-implanted group, which was similar to that in OVX rabbit femurs (Figure 7C). In contrast, the new bone in osteoporotic bone defects in the Scl-Ab-HCG-implanted group mainly expressed OCN, similar to that in normal femurs (Figure 7D). In the HCG- and Scl-Ab-HCG-implanted groups, cells positive for both RANKL and OCN were present on the new bone surfaces (Figure 7C,D). In the empty group, RANKL-positive cells were observed in the adipose tissue formed in the defect at POW 4 (Figure 7E). New bone in osteoporotic bone defects in the HCG- (Figure 7F) and Scl-Ab-HCG-implanted groups (Figure 7G) mainly expressed RANKL and OCN, respectively, at POW 12 but not at POW 4. In the empty group at POW 12, RANKL-positive adipose cells were observed in the defects (Figure 7H). The area ratios of OCN-expressing tissues to RANKL-expressing tissues (OCN/RANKL) in the Scl-Ab-HCG-implanted group at both POW 4 and 12 were 5.2 ± 1.1 and 5.5 ± 2.7, respectively, which were equal to that in the normal femurs (5.2 ± 4.2) and were significantly higher than those in the OVX rabbit femurs (0.1 ± 0.1) and the HCG-implanted group (Figure 7I and Table S6). In contrast, the OCN/RANKL in the HCG-implanted group at POW 4 and 12 were 0.1 ± 0.1 and 0.3 ± 0.2, respectively, which showed no significant difference from that in OVX rabbit femurs (Figure 7I).

Figure 7 Double immunofluorescence staining of receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) and osteocalcin (OCN) in (A) normal and (B) OVX rabbit femur condyles and new bone in osteoporotic bone defects at (C–E) 4 weeks and (F–H) 12 weeks after surgery: (C, F) HCG-implanted, (D, G) Scl-Ab-HCG-implanted, and (E, H) empty groups. Scale bar: 20 μm. Damaged and healthy bone mainly expresses RANKL and OCN, respectively. White and pink arrowheads indicate osteoblasts and empty bone lacunae without osteocytes (damaged bone), respectively. The symbol # indicates remaining material. (I) Ratios of OCN to RANKL in normal and OVX rabbit femurs and new bone in osteoporotic bone defects in HCG-implanted, Scl-Ab-HCG-implanted, and empty groups. Significant differences (p < 0.05) are indicated using different letters (a and b).

Multinucleated cells positive for both tartrate-resistant acid phosphatase (TRAP) and receptor activator of NF-κB (RANK) (TRAP+ RANK+ multinucleated cells) were identified as osteoclasts.57 To determine the number of osteoclasts in each group, immunostaining for TRAP and RANK was performed. The number of TRAP+ RANK+ multinucleated cells in normal rabbit femurs (Figure 8A) was lower than that in OVX rabbit femurs (Figure 8B). At POW 4, the newly formed bone in the osteoporotic bone defects in the HCG-implanted group (Figure 8C) had a larger number of TRAP+ RANK+ multinucleated cells than that in the Scl-Ab-HCG-implanted group (Figure 8D). In the empty group at POW 4, TRAP+ RANK+ multinucleated cells were absent (Figure 8E). At POW 12, a greater number of TRAP+ RANK+ multinucleated cells was observed in the HCG-implanted group (Figure 8F) than in the Scl-Ab-HCG-implanted group (Figure 8G). In the empty group at POW 12, TRAP+ RANK+ multinucleated cells were absent, similar to that at POW 4 (Figure 8H). The number of TRAP+ RANK+ multinucleated cells in OVX rabbit femurs (122.2 ± 82.8 cells/mm2) was significantly larger than those in all other groups (Figure 8I and Table S7). The numbers of TRAP+ RANK+ multinucleated cells in the Scl-Ab-HCG-implanted group at both POW 4 (5.7 ± 4.3 cells/mm2) and 12 (6.1 ± 4.5 cells/mm2) were not significantly different from that in normal femur group (8.4 ± 5.2 cells/mm2). The numbers of TRAP+ RANK+ multinucleated cells in the HCG-implanted group at both POW 4 (23.7 ± 10.7 cells/mm2) and 12 (19.5 ± 8.9 cells/mm2) were significantly larger than those in normal femur group and the Scl-Ab-HCG-implanted group at both POW 4 and 12.

Figure 8 Double immunofluorescence staining of receptor activator of NF-κB (RANK) and tartrate-resistant acid phosphatase (TRAP) in (A) normal and (B) OVX rabbit femurs and new bone in osteoporotic bone defects at (C–E) 4 weeks and (F–H) 12 weeks after surgery: (C, F) HCG-implanted, (D, G) Scl-Ab-HCG-implanted, and (E, H) empty groups. Scale bar: 20 μm. The white arrowheads indicate TRAP+ RANK+ multinucleated cells, i.e., osteoclasts. The symbol # indicates remaining material. (I) Numbers of TRAP+ RANK+ multinucleated cells per square millimeter in normal and OVX rabbit femurs and new bone in osteoporotic bone defects in HCG-implanted, Scl-Ab-HCG-implanted, and empty groups. Significant differences (p < 0.05) were detected using different letters (a, b, and c).

3 Discussion

In this study, we achieved easy loading of controlled amounts of Scl-Abs throughout HCGs by taking advantage of the microspaces among the CAp microspheres that comprise HCGs. Scl-Ab-HCGs degraded gradually at physiologically normal pH and rapidly at osteoporotic weakly acidic pH. The Scl-Ab-HCGs released a certain amount of Scl-Abs by diffusion and released a large amount of Scl-Abs in response to weakly acidic pH. These results suggest that the Scl-Ab-HCGs released a large amount of Scl-Abs for a while after implantation into the osteoporotic bone defects, and subsequently, moderately released Scl-Abs as osteoporotic symptoms improved. Thus, Scl-Ab-HCGs may release Scl-Abs during osteoporosis progression. Furthermore, owing to the release of Ca ions associated with Scl-Abs, Scl-Ab-HCGs may allow for osteoporosis treatment while preventing hypocalcemia, which is a side effect of Scl-Abs.58,59 Moreover, our in vivo findings demonstrated that Scl-Ab-HCGs restored the bone quantity in osteoporotic bone defects to normal levels, whereas HCGs alone did not. Thus, the Scl-Ab-HCG-based DDS is an effective approach for repairing osteoporotic bone fractures and defects.

Importantly, in vivo immunohistological analyses demonstrated that scaffold-based DDS (Scl-Ab-HCG-based DDS) led to a different bone formation behavior from treatment with the scaffold alone (HCG monotherapy). The main in vivo immunohistological findings are as follows: Both the Scl-Ab-HCG-based DDS and HCG monotherapy led to the formation of new bone, where osteocyte apoptosis levels were almost equal to those in normal bone in osteoporotic bone defects. However, the new bone formed by HCG monotherapy expressed RANKL rather than OCN, similar to osteoporotic bone. In contrast, the new bone formed by the Scl-Ab-HCG-based DDS mainly expressed OCN, similar to normal bone. Furthermore, excess TRAP+ RANK+ multinucleated cells, identified as osteoclasts, were present in the new bone formed due to HCG monotherapy, whereas the number of TRAP+ RANK+ multinucleated cells in the new bone formed by Scl-Ab-HCG-based DDS was similar to that in normal bone. Previous studies have demonstrated that old or damaged bone mainly expresses RANKL, which binds to RANK to promote osteoclast maturation and bone resorption.60,61 These characteristics are consistent with those of new bone formed due to HCG monotherapy, in which RANKL is overexpressed and excess osteoclasts are present. Thus, HCG monotherapy leads to the formation of damaged bone, although less damage than osteoporotic bone. Consequently, excessive osteoclastic resorption occurs, and bone quantity is not restored to normal bone levels. In contrast, the Scl-Ab-HCG-based DDS can form OCN-expressing healthy bone, similar to normal bone, where remodeling-based bone formation occurs. Thus, in bone defects treated with the Scl-Ab-HCG-based DDS, bone quantity is restored to the same level as normal bone at an early stage and is maintained at the same level as normal bone thereafter.

Paradoxically, these findings indicate that HCGs can form healthier bones than osteoporotic bones without drugs for osteoporotic bone defects. Previously, Heng et al. reported that powdered calcium phosphates, particularly biphasic calcium phosphate (BCP) rather than HAp and TCP, prevent osteoclastic bone resorption and promote bone formation in osteoporotic bone defects without drugs.62 They indicated that although doping of ions such as Sr and Si into calcium phosphates does not inhibit osteoclastogenesis, powdered BCP inhibits osteoclastogenesis by promoting the extracellular Ca2+ and PO43– concentrations and inhibiting NF-κB activation.63 In this study, HCGs released Ca2+ and PO43–, which may increase the extracellular concentrations of these ions and relieve the damage to osteocytes. However, the HCG monotherapy did not prevent RANKL overexpression in the new bone, which may facilitate the binding of RANKL to RANK and NF-κB activation. Consequently, HCG monotherapy did not inhibit osteoclastogenesis and did not increase the bone quantity to normal levels. Thus, although HCG monotherapy results in the formation of new bone with less osteocyte damage (fewer empty bone lacunae) than in osteoporotic bone, it may be insufficient to prevent RANKL overexpression. Based on the above findings, the HCGs may regulate Ca2+ and PO43–, suggesting that the treatment of osteoporotic bone defects may be possible when the HCGs acquire the additional function of preventing NF-κB activation by some means. In this study, Scl-Abs released using the Scl-Ab-HCG-based DDS inhibited increased RANKL production, thereby preventing NF-κB activation and excessive bone resorption. Although powders are difficult to apply to osteoporotic bone defects because they cause inflammation, granular scaffold-based DDS developed in our study can be used with fewer inflammation concerns and a familiar operating feel.

In previous studies on scaffold-based DDS, BCP32,33 and composites of β-TCP with poly lactic-co-glycolic acid,36 polycaprolactone,31,35 and photocurable mesoporous bioactive glass34 have been used as scaffolds, and aspirin,36 vitamin C,35 berberine,33 lovastatin,31 parathyroid hormone,34 and icariin32 were released from the scaffolds. Among these studies, only Ren et al. actively attempted to utilize mesopores for drug loading; however, they did not report the size and interconnectivity of the mesopores,34 which are generally 2–50 nm in size.63 Although the size of an antibody drug is 9–16 nm, antibodies aggregate to a size >100 nm.64 To efficiently load the antibody drugs into the scaffolds, the scaffold should have pores of multiple sizes corresponding to the primary and secondary (aggregate) sizes of the antibody drugs, rather than mesopores only. Furthermore, the interconnectivity of the micropores is important for loading antibody drugs throughout the scaffold. In this study, mercury intrusion porosimetry demonstrated that each HCG had abundant micropores (130 cm3 per granule), indicating that the micropores within the HCGs were highly interconnected. As the HCGs were composed of CAp microspheres, high interconnectivity and a large volume of micropores were achieved. Furthermore, hexagonal macropores penetrating HCGs allow for more efficient loading of Scl-Abs. The multiscale pore formation in the present study may be the key to favorable in vivo outcomes.

Recently, BTE and DDS have been reported for osteoporosis using materials other than osteoconductive bioceramics, e.g., BTE and local DDS using injectable hydrogels.21 However, the mechanical properties of injectable hydrogels are generally insufficient to support body loads.21 Furthermore, injectable hydrogels with controlled degradation and drug release are lacking.21 In contrast, we previously reported that the mechanical properties and degradation of CAp honeycomb scaffolds can be regulated by controlling their micropores,52,53 which suggests that HCGs can function as mechanical supports with controlled drug release. Systemic DDS using nanomaterials have also been reported.21 However, nanomaterials have cytotoxicity (biosafety) concerns and cannot provide an extracellular environment that promotes cell adhesion and proliferation.21 Furthermore, the influence of structure, size, shape, and physicochemical properties of nanomaterials on their therapeutic effects is unknown.21 However, HCGs are composed of CAp, which has been approved by regulatory pharmaceutical bodies and whose safety is ensured, and can serve as the scaffold for promoting cell adhesion and proliferation. Furthermore, the influence of structure, size, shape, and physicochemical properties of HCGs on BTE has been revealed.37−53 Thus, in these aspects, HCGs have several advantages over nanomaterials.

In this study, we clarified the bone quantity and bone formation behavior in osteoporotic bone defects up to three months postoperation. However, this study has the following limitations: (1) We did not evaluate long-term treatment outcomes or the mechanical strength of the repaired bone. (2) Although the OVX animal model is commonly used as a typical osteoporosis model, this model does not fully represent the complexity of osteoporotic conditions. (3) Comparisons with various treatments lacked a more robust demonstration of the effectiveness of Scl-Ab-HCGs. (4) The mechanisms of the precise regulation of Scl-Ab release in the physiological environment have not been sufficiently elucidated. (5) The potential immunogenicity of scaffolds and loaded antibodies in the host environment remains to be elucidated. In future studies, these limitations should be overcome with long-term in vivo evaluations using a larger sample size, different animal models, and expanded comparisons, e.g., comparisons with other types of DDS or bone graft materials. Furthermore, the kinetics and factors influencing the release rate of Scl-Abs in the physiological environment should be clarified.

4 Conclusions

HCGs composed of CAp microspheres had a multiscale porous structure comprising abundant micropores and hexagonal penetrating macropores. The Scl-Abs were present throughout the HCGs, and the amount of loaded Scl-Abs was linearly controlled with respect to the preparation amount. Scl-Abs were released by both diffusion and scaffold degradation, and the release rate increased in a weakly acidic environment, which is present in osteoporotic bone. These results suggest that Scl-Ab-HCGs have the potential to release Scl-Abs as osteoporosis progresses. In vitro evaluation demonstrated that Scl-Ab-HCGs enhanced osteoblast proliferation and mineralization. In vivo evaluation showed that the Scl-Ab-HCG-based DDS restored bone quantity in osteoporotic bone defects of OVX rabbit femurs to normal levels, whereas HCG monotherapy did not. The empty bone lacuna percentage of bone newly formed by the Scl-Ab-HCG-based DDS was similar to that of normal femurs and was significantly lower than that of OVX rabbit femurs. New bone formed by Scl-Ab-HCG-based DDS expressed OCN, similar to normal rabbit femurs, whereas new bone formed by HCG monotherapy showed an overexpression of RANKL, similar to OVX rabbit femurs. Furthermore, the number of osteoclasts in the new bone formed by the Scl-Ab-HCG-based DDS was similar to that in normal rabbit femurs, which was significantly lower than that in the new bone formed by HCG monotherapy. Despite these results, HCG monotherapy was effective in reducing the percentage of empty bone lacunae and the osteoclast number. These results clarified that by adding the ability to release Scl-Abs to HCGs, which were originally superior for bone formation, new bone with properties and quantity similar to normal bone formed around 4 weeks post-treatment, and this bone was maintained for 12 weeks. Thus, HCG-based DDS represent a promising approach for osteoporotic bone defect repair.

5 Methods

5.1 Fabrication of HCGs Composed of CAp Microspheres

HCGs composed of CAp microspheres were fabricated using a modified version of our previously established protocol.39 First, long sticks of a mixture of CaCO3 microspheres (Sakai Chemical Industry, Osaka, Japan) and an organic binder (Nagamine Manufacturing, Kagawa, Japan) with a honeycomb structure were prepared by extrusion molding using an extruder (Lab Plastmill, Toyo Seiki, Tokyo, Japan). Second, the sticks were cut into 1.0–1.5 mm-long granules to yield HCGs composed of a mixture of CaCO3 microspheres and an organic binder. Subsequently, the HCGs composed of a mixture of CaCO3 microspheres and the organic binder were heated at 650 °C for 24 h to remove the organic binder. Consequently, HCGs composed of CaCO3 microspheres were obtained. Finally, the HCGs composed of CaCO3 microspheres were phosphatized at 80 °C for 7 days through dissolution–precipitation reactions in a 1 mol/L Na2HPO4 solution (Fujifilm Wako Pure Chemical, Osaka, Japan), resulting in the fabrication of HCGs composed of CAp microspheres.

5.2 Physicochemical and Structural Characterization of HCGs Composed of CAp Microspheres

Scanning electron microscopy (SEM; S3400N, Hitachi High-Technologies, Tokyo, Japan) was used to observe the microstructures of the HCGs composed of CAp microspheres. XRD (D8 Advance, Bruker AXS GmbH, Karlsruhe, Germany) was used to identify the crystal phases of HCGs composed of CAp microspheres. Commercial CAp (GC Corporation, Tokyo, Japan) was used as a reference for the XRD. FTIR spectrometry (FT-IR-6200, JASCO, Tokyo, Japan) was used to identify the functional groups of the HCGs composed of CAp microspheres and Scl-Ab-HCGs. Commercial HAp powder (Taihei Chemical Industrial, Osaka, Japan) was used as reference. The micropore properties of the HCGs composed of CAp microspheres were evaluated using a mercury intrusion porosimeter (AutoPore 9420, Shimadzu Corporation, Kyoto, Japan).

5.3 Loading Scl-Abs into HCGs Composed of CAp Microspheres

HCGs composed of CAp microspheres (130 mg) were immersed in 4.5, 44.9, and 89.7 mg/mL of Scl-Ab solutions (2 mL, Amgen, Tokyo, Japan) at 20 °C for 1 min under vacuum (6.65 kPa) using a diaphragm type dry vacuum pump (DAP-6D, ULVAC KIKO, Miyazaki, Japan). The distribution of Scl-Abs loaded in the HCGs composed of CAp microspheres was observed using a fluorescence microscope (BZ-X, Keyence, Osaka, Japan) after immersing the HCGs composed of CAp microspheres in a FITC-labeled Scl-Ab solution (bc-10200R-FITC, Bioss Antibodies, Woburn, MA, USA). The Scl-Ab concentrations in Scl-Ab-HCGs after immersion in Scl-Ab solutions of different concentrations were determined by measuring the absorbance of the supernatants using a UV–vis spectrophotometer (V-630, JASCO, Tokyo, Japan) based on the calibration curve method.

5.4 Release Behavior of Scl-Abs from HCGs Composed of CAp Microspheres

Scl-Ab-HCGs (130 mg) were immersed in saline at pH 7.4 (2 mL) and sodium acetate buffer solution at pH 5.5 (2 mL) at 37 °C for up to 576 h (24 days). Supernatants were collected at 1, 3, 24, 48, 72, 168, 240, 408, 504, and 576 h after immersion. The released Scl-Ab concentrations were quantified by measuring the absorbance of Scl-Abs in the supernatants using a UV–vis spectrophotometer (V-630, JASCO) based on the calibration curve method.

5.5 Release Behavior of Ca Ions from HCGs Composed of CAp Microspheres

Scl-Ab-HCGs (130 mg) were immersed in saline at pH 7.4 (2 mL) and sodium acetate buffer solution at pH 5.5 (2 mL) at 37 °C for up to 576 h (24 days). Supernatants were collected at 1, 3, 24, 48, 72, 168, 240, 408, 504, and 576 h after immersion. The released Ca ion concentrations were quantified using inductively coupled plasma atomic emission spectrometry (Optima 7300 DV, PerkinElmer, Waltham, MA, USA).

5.6 In Vitro Cell Morphology, Proliferation, and Mineralization

In accordance with ISO 10993-12 (Biological evaluation of medical devices – Part 12: Sample preparation and reference materials), extracts were collected from HCGs and Scl-Ab-HCGs after immersing them in a culture medium (Osteoblast Growth Medium [C-27001], PromoCell, Heidelberg, Germany) at 37 °C for 72 h.54 The Scl-Ab-HCG extract contained 1.41 mg/mL of Scl-Abs. The extract was diluted 5-fold (5×) and 25-fold (25×) with the culture medium (Osteoblast Growth Medium [C-27001], PromoCell), which contained 0.06 and 0.28 mg/mL of Scl-Abs, to prepare Scl-Ab-HCGs (1.41), Scl-Ab-HCGs (0.28), and Scl-Ab-HCGs (0.06). Osteoblasts (C-12720, PromoCell) were seeded in a 24-well plate (Falcon 24-well Clear Multiwell Plate, 353226, Corning, Corning, NY, USA) at a density of 5 × 104 cells/well and cultured in the extracts for 24 h to evaluate cell morphology and proliferation. As control, cells were cultured in the culture medium (Osteoblast Growth Medium [C-27001], PromoCell) instead of the extracts. After 24 h of culture, cells were fixed with 10% formalin solution (060-03845, Fujifilm Wako Pure Chemical) for 30 min. The cell morphology was observed using a fluorescence microscope (BZ-X, Keyence) after staining the cell nuclei and actin with Hoechst 33342 (H342, Dojindo Laboratories, Kumamoto, Japan) and red fluorescent dye-conjugated phalloidin (PHDH1-A, Cytoskeleton, Denver, CO, USA) solutions, respectively, according to the manufacturer’s instructions. Cell proliferation was evaluated by counting the number of cells per square millimeter using a fluorescence microscope (BZ-X, Keyence) and cell counting software (BZX Hybrid Cell Count Software, Keyence). Fourteen samples from each group were used to analyze cell morphology and proliferation. For assay mineralization, cells were fixed with 10% formalin solution (060–03845, Fujifilm Wako Pure Chemical) for 30 min after 14 days of cell culture and stained with 1% ARS solution (pH 4.2; 011-01192, Fujifilm Wako Pure Chemical) for 10 min at 20 °C. The cells were then rinsed eight times with phosphate-buffered saline. The optical images of the deposited minerals were acquired using the fluorescence microscope (BZ-X, Keyence). To quantify the deposited minerals, ARS was extracted using 10% (w/v) cetylpyridinium chloride (190177, Fujifilm Wako Pure Chemical) in 10 mmol/L Na2HPO4 solution (197-02865, Fujifilm Wako Pure Chemical).65 The extracts were collected after 24 h of incubation at 20 °C. The absorbance of the extracts was measured at 560 nm using a microplate reader (Multiskan FC Microplate Photometer, Thermo Fisher Scientific, Waltham, MA, USA). Five samples from each group were used to analyze the mineralization.

5.7 Ethics Statement

All animal experiments were conducted in compliance with the ARRIVE guidelines, Act on the Welfare and Management of Animals, and related laws and regulations. All the animal experiments were approved by the Animal Care and Use Committee of Kyushu University, Fukuoka, Japan (approval no. A22-299-0; issued June 7, 2022).

5.8 Preparation of the OVX Rabbit Osteoporotic Model

The OVX rabbit osteoporotic model was prepared as previously described.66−68 New Zealand white rabbits that had undergone bilateral ovariectomy were purchased from Japan SLC (female, 19-week-old, body weight of 3.0–3.5 kg, Shizuoka, Japan). The rabbits were acclimated to laboratory conditions for 1 week. To confirm the osteoporotic model preparation, serum estrogen levels were measured 10 months after ovariectomy using estrogen detection kits (Arbor Assays, Ann Arbor, MI, USA). Subsequently, OVX rabbits were euthanized by rapid intravenous administration of anesthesia (ketamine [10 mg/kg; Selactar, Elanco, Tokyo, Japan] and xylazine [3 mg/kg; Ketalar, DAIICHI SANKYO PROPHARMA, Tokyo, Japan]) through the auricular vein after intramuscular administration of anesthesia (ketamine [30 mg/kg; Selactar, Elanco] and xylazine [5 mg/kg; Ketalar, DAIICHI SANKYO PROPHARMA]); the femurs were then collected. The BV/TV of femur condyles was evaluated by μ-CT analysis (ScanXmate-L090T, Comscan, Kanagawa, Japan). Similarly, serum estrogen levels and BV/TV of rabbits that did not undergo ovariectomy (normal rabbits) were analyzed as controls. Five rabbits were used in each of the OVX and control groups.

5.9 Implantation of HCGs and Scl-Ab-HCGs into Osteoporotic Bone Defects in OVX Rabbit Femurs

OVX osteoporotic rabbits were anesthetized by intramuscular injection of ketamine (30 mg/kg) and xylazine (5.0 mg/kg) and their femoral sites were sheared. During the operation, anesthesia was maintained by intravenous administration of ketamine (10 mg/kg, Selactar, Elanco) and xylazine (3 mg/kg, Ketalar, DAIICHI SANKYO PROPHARMA) through the auricular vein, as needed. Local anesthesia (2% lidocaine, TAKATA Pharmaceutical, Saitama, Japan) was administered to the skin and muscle at the femoral condyle after disinfecting the femoral skin with a 10% w/v povidone-iodine solution (Meiji Seika Pharma, Tokyo, Japan). Incisions (approximately 1.5 cm) were made in the order of skin, muscle, and periosteum at the femoral condyle with a scalpel (SCALPELS No.10, Akiyama MEDICAL MFG., Tokyo, Japan). The periosteum was separated from the bone using a raspory (IA-08, YDM, Tokyo, Japan) to expose the femoral condyle. A cylindrical defect that is 6 mm in diameter and 5 mm in depth was created at the femoral condyle using a micromotor (VIVAMATE G5, Nakanishi, Tochigi, Japan) equipped with a trephine bar (08.910.07, HELMUT ZEPF Medizintechnik GmbH, Baden-Württemberg, Germany) while adding in a saline solution. HCGs and Scl-Ab-HCGs were randomly implanted into the femur defects, and the periosteum and incised skin were then sutured. Finally, to prevent infection, rabbits underwent disinfection at the surgical site using a 10% w/v povidone-iodine solution and intraperitoneal injection of a gentamicin sulfate solution (Gentacin, Takata Pharmaceutical, Saitama, Japan). At 4 and 12 weeks after implantation, the rabbits were euthanized by rapid intravenous administration of anesthesia (ketamine [10 mg/kg; Selactar, Elanco] and xylazine [3 mg/kg; Ketalar, DAIICHI SANKYO PROPHARMA]) through the auricular vein after intramuscular administration of anesthesia (ketamine [30 mg/kg; Selactar, Elanco] and xylazine [5 mg/kg; Ketalar, DAIICHI SANKYO PROPHARMA]); the femurs were then collected. The collected femurs were fixed in a 10% formalin solution (Fujifilm Wako Pure Chemical). Five samples were collected for each group.

5.10 In Vivo Analyses of Osteoporotic Bone Defect Restoration

The BV/TV of the defect site in the collected femur condyles was evaluated by μ-CT analysis (ScanXmate-L090T, Comscan). Subsequently, to evaluate the area percentage of new bone and percentage of empty bone lacunae, HE-stained tissue sections at the femur defect sites were prepared in the usual manner by demineralization, embedding in paraffin, thin sectioning at 5 μm thickness, and HE staining. To evaluate the osteoclast number and the expression of OCN and RANKL in the new bone, TRAP and RANK double immunofluorescence staining and RANKL and OCN double immunofluorescence staining were performed on the tissue sections with the cell nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI). In brief, for TRAP and RANK double immunofluorescence staining, the tissue sections were incubated with the primary antibody of mouse anti-RANK (ab13918, Abcam, Cambridge, UK) at 4 °C for 18 h, washed with tris-buffered saline (TBS, Fujifilm Wako Pure Chemical), treated with the secondary antibody (Alexa Fluor 488 Donkey Anti-Mouse IgG H&L, ab150105, Abcam) for 5 h, and washed with TBS. Subsequently, the tissue sections were incubated with the primary antibody against TRAP (PAA902Rb51, Cloud-Clone, Houston, TX, USA) at 4 °C for 18 h, washed with TBS, treated with the secondary antibody (Alexa Fluor 594 Goat Anti-guinea pig IgG H&L, ab150188, Abcam) for 5 h, and washed with TBS. Finally, the sections were sealed using a mounting agent containing DAPI (SlowFade Gold Antifade Mountant with DAPI, Thermo Fisher Scientific). RANKL and OCN double immunofluorescence staining was performed similarly. Primary anti-OCN and anti-RANKL antibodies were purchased from arigo Biolaboratories (ARG54608; Taiwan, China) and Bioss Antibodies (bs-0747R, Woburn, MA, USA), respectively. Alexa Fluor 594 Donkey Anti-Mouse IgG (ab150108, Abcam) and Alexa Fluor 488 Chicken Anti-Rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA) were used as secondary antibodies against OCN and RANKL, respectively.

5.11 Statistical Analysis

All results are presented as mean ± standard deviation. Comparisons among multiple groups were performed with one-way analysis of variance and subsequent Tukey–Kramer posthoc multiple tests using KaleidaGraph version 4.5 (Synergy Software, Reading, PA, USA). Student’s t-test was used to analyze the differences between the two groups. Statistical significance was set at p < 0.05.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsami.4c08047.Statistical analysis results for in vitro proliferation evaluation (cell density), statistical analysis results for in vitro mineralization evaluation (absorbance of alizarin red S), statistical analysis results for in vivo evaluation on bone volume-to-total tissue volume ratio (BV/TV), statistical analysis results for in vivo evaluation on bone area percentage, statistical analysis results for in vivo evaluation on empty bone lacuna percentage, statistical analysis results for in vivo evaluation on area ratios of OCN-expressing tissues to RANKL-expressing tissues (OCN/RANKL), and statistical analysis results for in vivo evaluation on numbers of TRAP+ RANK+ multinucleated cells per square millimeter (PDF)

Supplementary Material

am4c08047_si_001.pdf

The authors declare no competing financial interest.

Notes

All the animal experiments were approved by the Animal Care and Use Committee of Kyushu University (approval no. A22-299-0; issued June 7, 2022).

Acknowledgments

This research was supported by Japan Agency for Medical Research and Development under Grant Numbers JP24ym0126098h0003 and JP24ym0126811j0003 and Japan Society for the Promotion of Science under Grant Numbers JP23K18593 and JP22H03954.
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