The role of osteoclasts in osteoinduction triggered by calcium phosphate biomaterials in mice

Abstract

OBJECTIVE:

Bone remodeling is mediated by the interaction between osteoblasts and osteoclasts, so does osteoinduction triggered by calcium phosphate (CaP) biomaterials. This study aims to investigate the role and function of osteoclasts in ectopic bone formation induced by CaP biomaterials.

METHODS:

Four kinds of mice, two outbred mouse strains (KM and ICR) and two inbred mouse strains (C57BL/6 and BALB/c), were chosen for the experiments. The hydroxyapatite/𝛽-tricalcium phosphate (HA/𝛽-TCP) biomaterials were implanted into the bilateral thigh muscle of each mouse, and then all mice ran on the treadmill to accelerate the ectopic bone formation. Five and ten weeks later, five mice in each group were euthanized and the samples were harvested for electron microscope scanning or histological identification: hematoxylin and eosin (HE), Masson-trichrome and tartrate-resistant acid phosphatase (TRAP) staining, respectively. The inflammation indexes, angiogenesis, and osteogenic ability were compared among the four kinds of mice, and the role of osteoclasts was analyzed based on this evidence.

RESULTS:

The number of multinucleated cells, the number of new blood vessels, and the area percentage of new bone tissues were higher in outbred mouse strains than in inbred mouse strains; and there were more TRAP-positive cells in the outbred mouse strains group. We believe that the monocytes from the peripheral blood could migrate into new bone tissues to form osteoclasts.

CONCLUSION:

Bone induction could be triggered by CaP biomaterials in mice, and osteoclasts could maintain the dynamic balance between bone resorption and remodeling, and induce the production of new bone marrow tissues.

Keywords

Calcium phosphate biomaterials osteoinduction osteoblasts osteoclasts

1. Introduction

Osteoinduction is that only the biomaterial itself induces ectopic bone formation at non-bone sites of organism, which was proposed in the 1990s [1]. Common biomedical materials included inorganic non-metallic materials (such as calcium phosphate, calcium sulfate, etc.) and metallic materials (such as titanium, etc.), which could be used as bone graft substitutes; however, the amount of new bone formation induced by different biomaterials varied [2–4]. Nowadays, calcium phosphate (CaP) ceramic, also called biphasic calcium phosphate (BCP) ceramic, was one of the most popular osteoinductive biomaterials, which has been widely studied and applied in clinic [5]. BCP ceramic was composed of stable hydroxyapatite (HA) and degradable tricalcium phosphate (TCP), which have the similar chemical composition with bone tissue itself and could release Ca and P ions in the microenvironment to induce ectopic bone formation [6]. However, the mechanism of osteoinduction was fairly complicated, in addition to involving physical and chemical characteristics of materials, it might be associated with lots of immunological and cytobiological events, such as inflammation, cell migration, cell proliferation and differentiation [7,8].

Although the immunological process of osteoinduction triggered by CaP biomaterials was still unclear, we could reference the mechanism of bone growth and development. The dynamic balance of bone development depended on the interaction between osteoblasts and osteoclasts, more than 10% bone tissues were reconstructed through bone formation and bone resorption each year; if osteoblasts and osteoclasts were disequilibrated, it would cause bone diseases, such as osteoporosis and bone sclerosis [9,10]. Therefore, we speculated that the ectopic bone formation induced by biomaterials in mice also kept the dynamic balance of osteoblasts and osteoclasts. In our previous studies, it has been proved that the source of osteoblasts was bone marrow mesenchymal stem cells (MSCs), then what's the source of osteoclasts? Osteoclast was one kind of microphage, which was derived from peripheral blood monocytes in normal development process [11]. Therefore, we have reason to believe that the osteoclast also originated from the peripheral blood monocytes, and maintained the balance of bone resorption and remodeling.

In this study, we will explore the role of osteoclasts in osteoinduction triggered by CaP biomaterials, and further analyze the process of inflammation, angiogenesis, chondrogenesis, osteogenesis, bone resorption and myelopoiesis in osteoinduction. This study will reveal the immunological mechanism of bone induction triggered by CaP biomaterials in mice, and lay the foundation for the clinical application of CaP artificial bone.

2. Materials and methods

2.1. Implantation

The (𝛷3 × 5) mm HA/𝛽-TCP biomaterials with 50% porosity and 300–500 μm pore size were purchased from GongChuang Biofunctional Materials Company (Hunan, China). The microstructure of HA/𝛽-TCP biomaterials was captured under scanning electron microscope (Fig. 1A). Eight-week-old male KM, ICR, C57BL/6 and BALB/c mice (n = 10) were purchased from Dossy Biological Technology Company (Chengdu, China), and were maintained in a temperature and light controlled environment ventilated with filtered air. During the surgery, all animals were intraperitoneally anesthetized and their bilateral thighs were disinfected, then the hair was removed and an approximately 8-mm muscle pouch was prepared, next, the HA/𝛽-TCP biomaterials were implanted into the bilateral muscle pockets. At last, the wounds were closed by single interrupted suturing, and penicillin was injected intramuscularly to prevent infection. To accelerate the ectopic bone formation, three days after implantation, all mice ran as a speed of 6 m/h in a treadmill, 30 min per day for 10 weeks. Five and ten weeks later, five mice of each group were killed to harvest the material samples, respectively.

Fig. 1.

(A) The SEM micrographs show the micropore of HA/𝛽-TCP biomaterials; (B) HE staining shows the microscopic characteristics inside the HA/𝛽-TCP biomaterials 5 weeks after implantation in four kinds of mice: KM, ICR, C57BL/6 and BALB/c, respectively. Red circle: cartilage; short arrow: blood vessels; long arrow: mesenchymal tissues; bar: 200 μm.

2.2. Hematoxylin and eosin staining

After harvesting, the samples were immediately fixed in 10% neutral formalin buffer solution for 24 h at room temperature, decalcified in 10% ethylenediaminetetraacetic acid (EDTA, pH 7.0) for about 20 days at room temperature, dehydrated with gradient of ethanol solutions at 70%, 80%, 90%, 95% and 100% concentration and then embedded in paraffin. The embedded samples were cut into 5-μm thick histological sections. The sections were performed with hematoxylin and eosin (HE) staining. The steps are as follows: (1) the sections were wiped off paraffin with xylene; (2) washed with gradient of ethanol solutions at 100%, 95%, 90% and 85% concentration, and then rinsed with water; (3) stained with hematoxylin for 3 min; (4) differentiated with 1% hydrochloric alcohol for 20 s after wash; (5) back to blue with 1% dilute ammonia for 30 s after wash; (6) stained with eosin for 2 min; (7) dehydrated with gradient of ethanol solutions at 85%, 90%, 95%, 100% concentration; (8) transparent with xylene, dried, and mounted.

2.3. Masson’s trichrome staining

The sections were dewaxed to water the same as the steps of HE staining, then performed as the following steps: (1) the sections were soaked in the Bouni solutions: 75 ml saturated picric acid, 25 ml formaldehyde, 5 ml glacial acetic acid, overnight; (2) preparing the Weigert A solutions: 1 g hematoxylin, 100 ml 90% ethanol; Weigert B solutions: 4 ml 30% ferric chloride, 100 ml distilled water, 1 ml hydrochloric acid; then the nuclei were stained with the mixture of Weigert A and B solutions (1:1) for 10 min; (3) differentiated with 1% hydrochloric alcohol for 20 s, then washed with running water; (4) stained with I solution (90 ml 1% scarlet water solution, 10 ml 1% acidic red water solution) for 6 min; (5) stained with II solution (2.5 g phosphomolybdic acid, 2.5 g phosphotungstic acid, 100 ml distilled water) for 3 min; (6) stained with III solution (2.5 g aniline blue, 2 ml glacial acetic acid, 100 ml distilled water) for 8 min; (7) soaked with 1% glacial acetic acid for 2 min; (8) dehydrated with gradient of ethanol solutions, transparent, dried, and mounted.

2.4. Tartrate-resistant acid phosphatase staining

The sections were dewaxed to water as the previous steps, then the sections were placed in tartrate-resistant acid phosphatase (TRAP) incubated buffer (1 ml naphthol AS-BI phosphoric acid solution, 0.1 ml fast garnet GBC base solution, 9 ml TRAP buffer) at 37 °C for 1 h; after the sections were washed, the nucleus were stained with hematoxylin for 3 min; lastly, the sections were dehydrated with gradient of ethanol solutions, transparent, dried, and mounted.

2.5. Transmission electron microscope

To discriminate the morphology of multinucleated cells, the implanted samples of week 5 were also performed transmission electron microscope (TEM), tissue blocks were rinsed in 0.1 mol/L phosphate buffer for 4 h at 4 °C, next, fixed in 1% osmic acid for 2 h, then rinsed with buffer for another 5 min, after that, dehydrated in gradient alcohol and acetone, soaked, embedded and polymerized, at last, 80 nm ultrathin sections were observed under TEM (Hitachi, Japan).

2.6. Scanning electron microscope

To identify the erythrocytes in new blood vessels, scanning electron microscope (SEM) was used to observe the morphology of red blood cells. Briefly, implanted samples of week 5 were fixed in 3% glutaraldehyde for 2 h at room temperature; then the specimens were dehydrated in alcohol gradient, and critical point drying for 1 h; lastly, specimens were intersected, sprayed with gold-palladium coat and observed under SEM (Hitachi S-4800, Japan).

2.7. Morphological analysis

The number of multinucleated cells, including macrophages, granulocytes and multinucleated giant cells, was counted based on five scanned HE sections of four groups at week 5; at the same time, the number of new blood vessels was also counted with the above-mentioned sections. Moreover, the areas of the new bone and total tissues were measured based on five scanned HE sections of each group at week 10, and the area percentage of new bone in four groups was calculated as: new bone area/total tissue area (%).

2.8. Statistical analysis

Data are expressed as mean ± standard deviation (X ± s) and were analyzed by student T test (SPSS 22.0, USA). P < 0.05 was considered statistically significant.

3. Results

3.1. Early stage of osteoinduction

Inflammation and angiogenesis were the main events in the early stage of osteoinduction. Therefore, we focused on the number of multinucleated cells and new blood vessels 5 weeks after implantation. In all groups, there were lots of inflammatory cells, capillaries, mesenchymal tissues and connective tissues in the pores of materials; however, the inflammation indexes were difficult to distinguish with the naked eye among the four groups: KM, ICR, C57BL/6 and BALB/c (Fig. 1B), it needs further statistical analysis. There was no new bone tissue formation in all groups, but some cartilage tissues were observed in group KM (Fig. 1B).

3.2. Multinucleated cells in inflammation

Lots of inflammatory cells appeared after the wound occurrence caused by implantation, we speculated that the level of inflammation was also related to the later osteogenesis. The multinucleated cells were circled in HE sections (Fig. 2A), and the typical macrophage was shown in TEM micrograph (Fig. 2B). To analyze the relationship between inflammation and multinucleated cells, the number of multinucleated cells was counted in all groups and the results showed that both the number of multinucleated cells in groups KM and ICR was higher than in groups C57BL/6 and BALB/c (Fig. 2C); suggesting that the inflammation was more intense in outbred mice.

Fig. 2.

(A) HE micrograph shows the inflammatory cells at week 5, circle: multinucleated cells; bar: 50 μm; (B) TEM micrograph shows the macrophage embedded in the HA/𝛽-TCP biomaterials, bar: 5 μm; (C) The number of multinucleated cells compared among groups KM, ICR, C57BL/6 and BALB/c. ^∗ P < 0.05.

3.3. New blood vessel formation

The bone formation was closely associated with angiogenesis, which could provide nutrients for new bone tissues. To analyze the precondition of new bone formation, the prominent vascularity was chosen (Fig. 3A), and the typical red blood cells were detected by SEM (Fig. 3B). To further compare the number of blood vessels and analyze the relationship between blood vessels and bone tissues in different kinds of mice, the number of blood vessels was counted and the results showed that the number of new blood vessels in groups KM and ICR were significantly higher than in groups C57BL/6 and BALB/c (P < 0.05, Fig. 3C), suggesting that there is more capillary formation in groups of outbred mice.

Fig. 3.

(A) HE micrograph shows the new blood vessel formation at week 5, circle: one blood vessel; bar: 50 μm; (B) SEM micrograph shows the red blood cells embedded in the HA/𝛽-TCP biomaterials, bar: 5 μm; (C) The number of new blood vessels compared among groups KM, ICR, C57BL/6 and BALB/c. ^∗ P < 0.05.

3.4. Late stage of osteoinduction

The osteoinduction triggered by CaP biomaterials has been verified in mice in our previous studies, and this study also showed that lots of cartilage tissues, bone tissues and bone marrow tissues formation induced by HA/𝛽-TCP biomaterials in mice 10 weeks after implantation. However, the amount of new bone tissues was higher in groups KM and ICR than in groups C57BL/6 and BALB/c, since only a small amount of bone tissues were observed in the latter (Fig. 4A); to identify the new bone tissues, the serial section of HE and Masson’s trichrome staining was performed to locate the osteoblasts, osteocytes and osteoclasts (Fig. 4B); the area of new bone tissues were calculated and statistical analyzed with image-pro plus (IPP) and SPSS software, the results showed that the area percentage of new bone was significantly higher in groups KM and ICR than in groups C57BL/6 and BALB/c, ^∗ P < 0.05 (Fig. 4C).

Fig. 4.

(A) HE micrograph shows the new bone and bone marrow tissues formation10 weeks after implantation in KM, ICR, C57BL/6 and BALB/c mice; (B) The bone tissues and cartilage tissue were identified with the serial sections of HE and Masson’s trichrome staining; (C) The area percentage of new bone tissues in four groups, ^∗ P < 0.05. BM: bone marrow; arrow: bone tissues; bar: 200 μm.

3.5. The role of osteoclasts in osteoinduction

To identify osteoclasts, TRAP staining was performed as the osteoclasts expressed the specific enzyme: TRAP. The results showed that the osteoclasts were stained fuchsia, while other cells displayed green; in the outbred mouse strains, there were lots of TRAP-positive cells, while there were almost TRAP-negative cells in the inbred mouse strains, suggesting that if there are more bone tissues, there are more osteoclasts as well. Therefore, the osteoclasts did appear in mature bone tissues and played an important regulatory function in osteoinduction, and it seemed bone marrow tissues could generate from osteoclasts in the HE micrograph (Fig. 5).

Fig. 5.

The process of osteoinduction mediated by endochondral ossification. Dots: osteoblasts; circle: osteoclasts; bar: 200 μm.

3.6. The formative process of osteoinduction

From a large amount of microscopic images, we speculated that the way of osteogenesis was intramembranous ossification combined with endochondral ossification in bone induction with CaP biomaterials. For example, the process of typical endochondral ossification was as follows: first, cartilage was induced by mesenchymal tissues in the pores of biomaterials under the circumstance of Ca²⁺, (PO₄)³⁻ and new blood vessels, etc.; second, some cartilage tissues were replaced by bone tissues, and osteoclast appearance to induce bone marrow tissue formation; third, all cartilage tissues were completely replaced by bone tissues which turned into mature lamellar bone, and the osteoclasts maintained the balance of bone resorption and remodeling (Fig. 6).

Fig. 6.

Tartrate-resistant acid phosphatase staining shows positive cells in outbred mouse strains, not in inbred mouse strains.

4. Discussion

The osteogenic ability of HA/𝛽-TCP biomaterials was different in muscle of different strains of mice, our results showed that amount of new bone tissues was significantly higher in outbred mouse strains (KM and ICR) than in inbred mouse strains (C57/B6 and BALB/c), which might be related to the levels of cytokines in body fluid, such as BMP-2 and TGF-𝛽 [12,13]. Tsukanaka et al. implanted purified porous 𝛽-TCP with 60% porosity into subcutaneous pockets of inbred mice FVB and C57BL/6, and found that more vessels and TRAP-positive cells were detected in FVB mice than in C57BL/6 mice, and osteoinduction was observed in half of the materials implanted into FVB mice [14]. Barradas et al. subcutaneously implanted osteoinductive TCP in 11 different inbred mouse strains and found that new bone formation was only observed in FVB and 129S2 mice [15]. Although the outbred mouse strains were rarely used for experiments due to its complicated genetic background, it still could reveal that the immunological factors, such as leukocytes and cytokines, could impact the ectopic bone formation of osteoinductive biomaterials in different strains of mice.

The immunological events were complicated during ectopic bone formation induced by CaP biomaterials, which included inflammation, angiogenesis, chondrogenesis, osteogenesis, bone resorption and myelopoiesis. Firstly, inflammation was caused by operative wound and foreign matter (CaP biomaterials) implantation, and along with inflammation came leukocytes infiltration including lymphocytes, granulocytes, mononuclear macrophages and dendritic cells [16]. On the one hand, the lymphocytes migrated from the peripheral lymphoid organs, granulocytes and monocytes came from peripheral blood and the monocytes would differentiate into macrophages in inflammatory sites [17]; on the other hand, the leukocytes might originate from the hematopoietic stem cells (HSCs) of bone marrow [18]. In this study, the macrophages, granulocytes and multinucleated giant cells were multinucleated cells, which could reflex the inflammation indexes and related to the angiogenesis and osteogenesis. Secondly, angiogenesis was always accompanied by inflammation, and provided the nutrition for new bone formation [19]. Our results showed lots of blood vessel formation in outbred mouse strains, and there was a correlation between the amount of blood vessels and bone tissues. Thirdly, chondrogenesis had a certain randomness, we didn’t observed cartilage in all groups; therefore, we speculated that in addition to endochondral ossification, there was also intramembranous ossification in osteoinduction of CaP biomaterials. Fourthly, osteogenesis was the most important step in osteoinduction, however, osteogenesis wouldn’t happen without inflammation and angiogenesis; our results also revealed that the inflammation indexes, the number of new blood vessels, the area percentage of new bone tissues were higher in outbred mouse strains than in inbred mouse strains. Lastly, the TRAP-positive cells, that is, osteoclasts played an important role in bone resorption and myelopoiesis, bone tissues couldn’t uncontrolled grow even during ectopic bone formation, otherwise, it might grow into a bone tumor [20]; therefore, osteoclast appearance maintained the balance between bone resorption and remodeling [21,22]. What we were interested in is where did osteoclasts come from? It might come from the peripheral blood monocytes according to the available evidence. We were also curious about the function of osteoclasts. They could contribute to bone resorption and remodeling and induce the production of bone marrow tissues. The new bone marrow tissues were very important in the bone transplantation, which could establish perfectly functional bones and reserve HSCs [23,24].

5. Conclusion

CaP biomaterials could induce more new bone formation in outbred mouse strains, the bone tissues turned into mature lamellar bone undergoing 10 weeks’ induction in the niche with immunological cells and cytokines; the osteoclasts were also induced to maintain the balance between bone resorption and remodeled and promoted the production of new bone marrow tissues. Our study revealed the immunological mechanism of bone induction triggered by CaP biomaterials in mice, and laid the foundation for the clinical application of CaP artificial bone.

Footnotes

Acknowledgements

This work was supported by the Department of Science and Technology of Sichuan Province, China (No. 2018JY0348), the Scientific Research Innovation Group Foundation of Educational Committee of Sichuan Province, China (No. 17TD0010) and the Sichuan Funds for Distinguished Young Scientists (2017JQ0060).

Conflict of interest

There is no conflict of interest regarding the submission of this manuscript.

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