Bioinspired Mineralization Using Chondrocyte Membrane

Article Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Bioinspired Mineralization Using Chondrocyte Membrane Nanofragments Emilio Satoshi Hara,† Masahiro Okada,† Noriyuki Nagaoka,‡ Takako Hattori,§ Takuo Kuboki,∥ Takayoshi Nakano,⊥ and Takuya Matsumoto*,† †

Department of Biomaterials, ‡Advanced Research Center for Oral & Craniofacial Sciences, §Department of Oral Biochemistry and Molecular Dentistry, and ∥Department of Oral Rehabilitation and Regenerative Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama-shi, Okayama-ken 700-8525, Japan ⊥ Division of Materials and Manufacturing Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan S Supporting Information *

ABSTRACT: Biomineralization involves complex processes and interactions between organic and inorganic matters, which are controlled in part by the cells. The objectives of this study were, first, to perform a systematic and ultrastructural investigation of the initial mineral formation during secondary ossification center of mouse femur based on material science and biology viewpoint, and then develop novel biomaterials for mineralization based on the in vivo findings. First, we identified the very initial mineral deposition at postnatal day 5 (P5) at the medial side of femur epiphysis by nanocomputed tomography. Initial minerals were found in the surroundings of hypertrophic chondrocytes. Interestingly, histological and immunohistochemical analyses showed that initial mineralization until P6 was based on chondrocyte activity only, i.e., it occurred in the absence of osteoblasts. Moreover, electron microscopy-based ultrastructural analysis showed that cell-secreted matrix vesicles were absent in the early steps of osteoblast-independent endochondral ossification. Instead, chondrocyte membrane nanofragments were found in the fibrous matrix surrounding the hypertrophic chondrocytes. EDS analysis and electron diffraction study indicated that cell membrane nanofragments were not mineralized material, and could be the nucleation site for the newly formed calcospherites. The phospholipids in the cell membrane nanofragments could be a source of phosphate for subsequent calcium phosphate formation, which initially was amorphous, and later transformed into apatite crystals. Finally, artificial cell nanofragments were synthesized from ATDC5 chondrogenic cells, and in vitro assays showed that these nanofragments could promote mineral formation. Taken together, these results indicated that cell membrane nanofragments were the nucleation site for mineral formation, and could potentially be used as material for manipulation of biomineralization. KEYWORDS: biomineralization, endochondral ossification, cell membrane nanofragments, secondary ossification center, matrix vesicles

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as drug screening tools.1−4 Moreover, researchers have also attempted to manipulate mineral formation in vitro using a combination of scaffolds, growth factors, and cells.5 Nevertheless, apatite-related materials show brittle mechanical properties, and growth factors are still relatively expensive for application in clinical practice. Optimization of these techniques for synthetic bone tissue fabrication could allow further development of novel materials for biomedical application, as well as in vitro model systems to reduce animal experiments or costs for drug screening.

INTRODUCTION

Bone mineralization and formation involves complex and spatiotemporal dynamic physicochemical reactions and processes between organic and inorganic materials, which are coordinated in part by the cells. Bone formation takes place through two different processes, i.e., intramembranous and endochondral ossification, in which the space occupied by organic material composed of cells and extracellular matrix (ECM) components, is gradually replaced by inorganic carbonated apatite. Eventually, a hybrid material composed of approximately 70% of inorganic apatite and with extraordinary mechanical properties is accomplished. Recently, numerous studies have focused on designing and fabrication of bone-like materials, such as apatite-related inorganic materials, for regenerative medicine or to be used © XXXX American Chemical Society

Received: December 7, 2017 Accepted: December 23, 2017

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DOI: 10.1021/acsbiomaterials.7b00962 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

CTVol SkyScan softwares. Bone volume was analyzed under same parameters using CTAn SkyScan software. Nano-CT images were obtained with a Skycan 1272 scanner, with a resolution of 1 μm. Cross-sectional images were reconstructed to produce the final image using Nrecon. Cell Culture and in Vitro Mineralization Assay. ATDC5 prechondrogenic cells were maintained in Dulbecco’s Modified Eagle Medium and Ham’s F-12 medium (DMEM/F-12; Wako Pure Chemical Industries, Osaka, Japan) containing 10% fetal bovine serum (FBS; Life Technologies, Gaithersburg, MD, USA) and 1% penicillin and streptomycin (Sigma, St Louis, MO, USA). Cells were cultured until confluency, trypsinized and centrifuged. Cells were then suspended with phosphate buffer saline (PBS), and submitted to ultrasonication (VP-5S, Taitec, Saitama, Japan) for 30 s to induce cell lysis and fragmentation. Lysates were then submitted to a series of centrifugation steps at 10 000 g for 10 min, 20 000 g for 20 min and 150 000 g for 60 min for isolation of small cellular nanofragments, modified from a previously reported method.7 For TEM observation of nanofragments, fresh-isolated nanofragments were fixed with 2% PFA, 2% glutaraldehyde and 1% osmium tetroxide (TAAB Laborabories Equipment Ltd., Berkshire, UK) before sonication. Nanofragments were then dropped onto TEM grids before observation. For mineralization assays, cell nanofragments were incubated with alpha-Modified Eagle Medium (α-MEM) supplemented with βglycerophosphate in tissue culture plates for different time intervals. Mineralization was confirmed by alizarin red staining, EDS and electron diffraction. Field-Emission Scanning Electron Microscopy (FE-SEM) and Energy-Dispersive X-ray Spectroscopy (EDS) Analysis. For FESEM observation, samples were fixed and prepared with variations of the protocol as described previously.8 In brief, femur epiphysis samples were collected, fixed in 2% glutaraldehyde/2% PFA solution for 24 h, washed with phosphate buffered saline (PBS), processed with a solution containing 3% potassium ferrocyanide (Sigma-Aldrich, St. Louis, MO, USA) in PBS with an equal volume of 4% w/v aqueous solution of osmium tetroxide (TAAB Laborabories Equipment Ltd.). The tissues were incubated in this solution for 1 h on ice. Samples were then washed thoroughly and incubated with freshly prepared 1% thiocarbohydrazide (Sigma-Aldrich) solution for 20 min at room temperature. Tissues were washed thoroughly and incubated with 1% osmium for 30 min at room temperature. After washing, samples were dehydrated to a sequence of ethanol and acetone, and finally embedded in EPON 812 resin (TAAB Laborabories Equipment Ltd.). Specimen were then polished and cross-sectioned by an argon ion etching (SM-090101 Cross Section Polisher; JEOL, Tokyo, Japan). Specimens were then submitted to osmium coating (NeocSTB, Meiwafosis, Tokyo, Japan) before FE-SEM observation (FESEM; JSM-6701F, JEOL) with backscattered electrons, operated at 5 kV using an annular semiconductor detector, according to a previous report.9 For EDS analysis of the minerals, minerals were first washed by centrifugation, and then dropped onto a clean cover glass fixed onto an aluminum holder, and left to dry at room temperature. Minerals were then coated using an osmium coater (Neoc-Pro, Meiwafosis Co. Ltd., Tokyo, Japan) at an electrical discharge current of 10 mA and a degree of vacuum of 10 Pa for 10 s, before FE-SEM observation and EDS analysis using an electron microscope (S-4800, Hitachi, Tokyo, Japan) equipped with an EDS detector (EDAX, Mahwah, NJ, USA). Transmission Electron Microscopy (TEM), Scanning Transmission Electron Microscopy (STEM)-EDS Interfacial Analysis. P6 and P7 epiphysis samples, previously examined by FE-SEM, were further cut in 80 nm thick slices with diamond knife for observation in a transmission electron microscope (TEM; JEM-2100, JEOL) and a scanning transmission electron microscope (STEM; JEM-2100F, JEOL) equipped with a probe Cs-corrector (CEOS, Heidelberg, Germany) and a EDS spectrometer (JEM-2300T, JEOL) according to methods described previously.9 TEM observation of calcospherites was followed by electron diffraction performed in a TEM.

However, because all these engineering approaches are based on the knowledge of the biological understanding of bone tissue development, the still remaining controversies on the mechanism of bone formation could potentially be limiting the development of more efficient organic−inorganic hybrid structures related to bone tissue engineering methods. Therefore, it is crucial to understand the mechanisms behind bone formation process from both the Material Science and Biology point of view, to allow one’s to design new bioinspired or biomimetic materials and/or methods for bone tissue reconstruction and regeneration. In this study, we used the secondary ossification center as an in vivo model to investigate the initial bone formation process as it allows a unique and detailed spatiotemporal observation of site-specific changes in organic and inorganic matrix in the cartilage tissue (cells and extracellular matrix). The data showed that the initial stages of endochondral ossification are osteoblast-independent, and that chondrocyte membrane nanofragments, rather than matrix vesicles, were associated with initial amorphous calcium phosphate mineral formation. These evaluations were carried out based on a variety of material science-based and biology-based research methods including histological analysis, biochemical analysis, nanocomputer tomography, electron microscopy, crystallographical analysis, and elemental analysis. Finally, we fabricated artificial cell nanofragments from prechondrogenic cells, and demonstrate that these nanofragments in fact can promote mineralization in vitro.

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MATERIALS AND METHODS

Animals. New-born Balb/c mice from postnatal day 5 (P5) to P7 were used in the experiments according to the Guidelines for Animal Research of Okayama University. The Animal Care and Use Committee of Okayama University approved the research protocols (OKU-2014283 and OKU-2015542). Histological Analysis and Immunohistochemistry. Femur epiphysis were isolated and immediately embedded in cryosection medium (Section-lab, Hiroshima, Japan), with the posterior side of the joint toward the sectioning face. Serial cryosections were performed in a thickness of 10 μm using a cryostat (CM3050S, Leica Microsystems, Wetzlar, Germany). After sectioning, serial cryosections were fixed in 4% paraformaldehyde (PFA) for 2 min, and stained with toluidine blue for glycosaminoglycans for 30 s, or alizarin red S for mineralized material for 3 min. Samples were then washed to remove excess of staining solution, and observed under a microscope (Biozero BZX700, Keyence, Osaka, Japan). Chondrocyte size and number was quantified using ImageJ software (NIH, Bethesda, MD, USA) based on toluidine blue-stained serial sections. Average from at least 3 different sections from at least three different mice were used in the analysis. For immunohistochemical analysis, serial cryosections of epiphysis samples were fixed with 4% PFA, washed in PBS, blocked with 5% goat serum and then immunolabeled with primary antibody, or the isotype-matched IgG antibody at 4 °C overnight. Primary antibodies were against SOX-9 (Chemicon, Temecula, CA, USA), CD31 (Abcam, Cambridge, UK) and Runx2 (Calbiochem, San Diego, CA, USA). The target primary proteins were then visualized after incubation with secondary antibody conjugated with Alexa Fluor 488 or 647 (Life Technologies) under a fluorescence microscope (Biozero), as previously described.6 Cell nuclei was stained with Hoechst-3334 (Life Technologies). X-ray Micro-Computed Tomography (micro-CT) and NanoCT. Micro-CT images of the collected epiphysis were obtained using a SkyScan 1174 compact micro-CT (SkyScan, Aartselaar, Belgium). CT scans were performed at a resolution of 6.4 μm, and sections were reconstructed to produce the final 3D images using Nrecon and B


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Figure 1. Identification of the initial mineralized area in secondary ossification center and histomorphometric analysis of chondrocyte changes during initial mineralization. (A) Three-dimensional reconstruction of microCT scanned (6.4 μm resolution) knee joints from P5 to P7 new-born mice showing initial mineralization (arrows) in the medial side of P6 femur epiphysis. Images are representative of 4 different samples. L = lateral, M = medial. (B) Quantitative analysis of bone volume in P6 and P7 mice. N = 4. (C) Toluidine blue stained P5 epiphysis showing the presence of hypertrophic chondrocytes in the medial side. (D) Quantitative analysis of chondrocyte size (area) in the lateral and medial regions of P4 to P6 femur epiphyses. (E, F) Frequency of hypertrophic chondrocytes (>100 μm2) according to their size in the (E) lateral and (F) medial sides of P4 to P6 epiphysis. Toluidine blue stained samples were used for analysis. Quantitative analysis was performed with ImageJ software. N = 3.

the cartilage epiphysis of mouse femur, we first performed microCT analysis (6.4 μm resolution) of knee samples from newborn mice, and found initial bone formation at postnatal day 6 (P6) (Figure 1A, B). To obtain more detailed information on the spatial localization of initial mineral deposition in the cartilage epiphysis of mouse femur at a nanoscale, we used a nano-CT and identified low-contrast minerals outlining the chondrocytes in the center of the medial side of P5 epiphysis (Figure S1). Subsequently, from P6 to P7, there was a marked increase in the amount of mineralized bone, which expanded toward the lateral, anterior, and posterior regions (Figure 1A, B). Concomitant with these observations in the inorganic material formation, we could observe changes in chondrocytes in a time-, site-, and stage-specific manner. At P4, there was no significant differences in the average size and density of chondrocytes either in the medial or lateral side of the femur epiphysis (Figure 1C−F). At P5, there was a marked increase in

STEM-EDS was performed using a STEM, operated at 200 kV using a current density of 40 pA/cm2. STEM bright-field, annular darkfield detectors and EDS spectrometer attachments were used. The probe forming Cs corrector enabled sub-Å resolution STEM imaging (minimum probe size: 0.09 nm). For EDS, an electron spot with a 0.3 nm diameter was applied. A drift correction was performed each minute to avoid any possible drifts that might have occurred at nanoscale during the acquisition of STEM-EDS multielemental mapping. High-angle annular dark-field (HAADF) images were taken with a 167−228 mrad detector. Statistical Analysis. Analysis of the differences between groups were performed with unpaired Student’s t-test, or one-way ANOVA followed by a Tukey posthoc correction test when appropriate. Prism 5 software (GraphPad Software, La Jolla, CA, USA) was used for the analyses.

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RESULTS Systematic Identification of the Initial Mineralization Site and Analysis of Time and Stage-Specific Changes in Femur Epiphysis. To identify the initial mineral deposition in C


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Ultrastructural Analysis of Nucleation Site and Initial Minerals. Next, to investigate the process of mineral formation and maturation, we observed the initial minerals in P6 femur epiphysis embedded in resin by FE-SEM with a backscattered electron (Figure 3) and by STEM (Figure 4). Figure 3A shows

chondrocyte size in the medial side. From P5 to P6, there was a tendency for saturation in the cell size in medial side as shown by the average chondrocyte size (Figure 1D) and frequency of hypertrophic chondrocytes (>100 μm2) (Figure 1F). On the other hand, in the lateral side, the averaged cell size became marked increased, and the difference in the number of hypertrophic chondrocytes was higher only at P6 (Figure 1F). These data are in accordance with previous reports showing that chondrocyte hypertrophy is an important step preceding bone mineral formation, similar to the steps observed in the growth plate, despite the fact that chondrocytes were not arranged in cellular columns.10−12 Additional immunohistochemical analysis for the chondrocyte marker Sox-9, showed no staining or a week staining in the mineralized area (Figure 2A, B), indicating an absence of intact

Figure 3. Ultrastructural characterization of the initially formed minerals by SEM. (A−F) Initial mineralization occurred in the medial side of the femur epiphysis. (A) Photograph of femur epiphysis showing the medial, central and lateral sides. (B) Overview of the medial and central sides of P6 epiphysis, as highlighted in the square in A. BV = blood vessel. (C, E) Higher magnification images of initially formed calcospherites and cell membrane nanofragments in the central region of the epiphysis. Arrows indicate initial calcospherites. Arrowheads indicate membrane nanofragments. (D, F) Higher magnification images of mature and packed calcospherites (arrows) observed in the medial region of femur epiphysis. Images are representative of three different samples.

Figure 2. Histomorphological analysis of P5 to P7 femur epiphyses. (A) Alizarin red S staining of P5−P7 mouse femur epiphyses. Note that alkaline phosphatase activity preceded mineral formation in a spatiotemporal manner. (B−D) Immunofluorescence staining of epiphysis for Sox-9, CD31, and Runx2. (B) Sox-9 positive chondrocytes were observed throughout the intact cartilage, but were absent in the mineralized area. (C) Blood vessel invasion in the cartilage is indicated by immunostaining for CD31+ endothelial cells (arrows), as early as P5. (D) Osteoblasts (Runx2+ cells) were only detected at P7, in the mineralized area. Images are representative of at least three independent samples.

the medial, central and lateral regions of the epiphysis. Figure 3B shows the overview of the initially formed minerals in the medial and central region of the epiphysis. The region close to blood vessels, where the early minerals had been first formed at P5, corresponds to the medial region (higher magnification in Figure 3D, F); whereas the region where new minerals are being formed at P6 corresponds to the central region of the cartilage (higher magnification in Figure 3C, E). In the medial region, we could observe dense and compacted minerals surrounding chondrocytes (Figure 3D). These minerals achieved a maximum average size of 2−3 μm in diameter, and subsequently they become juxtaposed one to another, forming more densely packed mineral blocks (Figure 3F), which corresponded to those detected by microCT with a resolution of 6.4 μm (Figure 1A). Elemental mapping of the dense minerals in the medial region near the blood vessels,

chondrocytes after mineralization occurred. Additionally, immunostaining for endothelial cells (CD31, Figure 2C) showed initial vascular invasion at P5, confirming previous reports indicating the importance of vessel invasion in the cartilage for subsequent mineralization,13,14 possibly as a major supplier of calcium ions through diffusion. More importantly, no other cells (e.g., osteoblasts, endothelial cells) besides chondrocytes could be observed in the mineralized region at P5 and P6, whereas at P7, a substantial number of CD31+ endothelial cells and a few osteoblasts (Runx2+ cells) could be observed inside the cartilage, more specifically in the inner part of the initially formed minerals (Figure 2D). Taken together, these data indicate that the early steps of mineralization (at P5 and P6) during secondary ossification are not based on osteoblast activity, but mainly on that of hypertrophic chondrocytes. D


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the medial area (Figure 3D, F). Of note, we could not find round-shape matrix vesicles in the area surrounding the initial calcospherites in the femur epiphysis. In fact, close and together with the initial calcospherites, we could observe high contrast pleomorphic material of 50 to 200 nm in length surrounding the chondrocytes (arrowheads in Figure 3C, E, and Figure 4A, D, G). This high contrast pleomorphic material attached to the fibrous molecules (i.e., collagen) without showing round-shape vesicles. At first, we supposed this pleomorphic material to be initial minerals. However, elemental mapping could detect only low levels of calcium, but high levels of phosphorus (Figure 5A−D, Figure S3). Because of the affinity of osmium to stain carbon double bonds in phospholipids, the high amount of phosphorus in the phospholipids of the cellular membrane, we assumed that these structures would be cellular membrane nanofragments, which would probably mineralize in a later step. We additionally performed immunohistochemical analysis for integrin alpha 5, which is a receptor for collagens, and we found it to be present both in hypertrophic chondrocytes near the mineralized area, and in chondrocytes in the nonmineralized area (Figure S4). Chondrocytes in the nonmineralized area presented a clear and intact cell membrane. On the other hand, hypertrophic chondrocytes near the mineralized area presented defective cell membranes without clear boundaries. Remnants of integrin alpha 5-stained cell membrane nanofragments were observed in between the hypertrophic chondrocytes, similar to the nanofragments observed by SEM (Figure S5). Therefore, we concluded that the nanofragments in between adjacent hypertrophic chondrocytes near the mineralized area were cell membrane nanofragments. Of note, ultrastructural analysis of mouse calvaria, in which the mineralization process is based solely on the activity of osteoblasts, revealed the presence of matrix vesicles near collagen fibrils (Figure S6), as reported previously,10,15,16 and enabled a clear differentiation of the initial mineralization process between the intramembranous ossification in calvaria and endochondral ossification in the secondary ossification center. Next, we performed ultrastructural characterization and electron diffraction analysis of P6 initial calcospherites, and found that the initially formed minerals in the central region were noncrystallized amorphous mineral (Figure 6A−D). Minerals in the medial region, on the other hand, presented an electron diffraction pattern characteristic of hydroxyapatite (HAp) (Figure 6E−H). On the basis of these findings, the initial mineralization during secondary ossification did not involve either osteoblasts or matrix vesicles. We assumed that cell membrane nanofragments could be the nucleation site for nondense amorphous calcium phosphate, which in turn were

Figure 4. Ultrastructural characterization of the initially formed minerals by STEM. (A, D) STEM-HAADF images of cell membrane nanofragments (high contrast white pleomorphic material) surrounded by collagenous matrix. (B, E) Initial calcospherites and (C, F) mature calcospherites were observed together with membrane nanofragments as well as in the area close to the membrane nanofragments. D−F are high-magnification images of the squares shown in A−C, respectively. (G) Graph shows the average of the length (size) of cell membrane nanofragments, or the diameter of initial calcospherites and mature calcospherites, measured by ImageJ. N = 11, per group.

confirmed the high levels of calcium and phosphorus (Figure S2). On the other hand, in the central region of the epiphysis, we could observe small mineral aggregates, or the initial calcospherites. The size of these initial calcospherites was of approximately 150 to 400 nm in diameter (arrows in Figure 3C−E, and Figure 4G). These initial calcospherites would increase in size and become more dense, as those observed in

Figure 5. Elemental mapping of the cell membranes nanofragments for calcium, phosphorus and osmium. (A) STEM-HAADF images of cell membrane nanofragments. (B−D) Semiquantitative elemental maps of (B) calcium, (C) phosphorus, and (D) osmium, which were recorded simultaneously. Note the low calcium levels in the cell membrane nanofragments, indicating that the nanofragments are yet not mineralized. E


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Figure 6. TEM observation and electron diffraction analysis of initial calcospherites in P6 epiphysis. (A) Picture shows the front region where initial calcium phosphate minerals were formed in the ECM between the chondrocytes. (B, C) High-magnification images of the area shown in the square frame in A and B, respectively. Note the formation of initial calcium phosphate minerals (arrowheads) in the vicinity of cellular membrane nanofragments (in black). (D) Electron diffraction pattern showing diffuse rings characteristic of amorphous (noncrystalline) material. (E) Mature area where more dense and larger calcospherites are aggregated to each other. (F, G) High-magnification images of the area shown in the white square frame in E and F, respectively. Note the presence of nanofragments (in black) inside the mature and aggregated calcospherites. (H) Electron diffraction pattern showing a crystalline pattern characteristic of HAp.

Figure 7. In vitro synthesis of cell membrane nanofragments and mineralization assay. (A) Protocol for synthesis and isolation of cell membrane nanofragments, and for mineralization assay in vitro using cell nanofragments. (B) TEM images of the synthesized cell nanofragments. (C) Alizarin red staining of 2-day incubated cell nanofragments. Note that control (live cells) did not mineralize. (D) TEM observation and electron diffraction analysis, as well as (E) EDS analysis of minerals formed after cell nanofragments were incubated in α-MEM containing β-glycerophosphate (β-GP) for 7 days. Electron diffraction analysis indicated a pattern characteristic of hydroxyapatite. Note the high levels of calcium and phosphate peaks in E.

the precursors for more dense and packed poly crystal

be nucleation site for mineral formation, we expanded, collected, and submitted prechondrogenic ATDC5 cells to ultrasonication and ultracentrifugation to collect cell membrane nanofragments (Figure 7A, B). Subsequently, nanofragments

containing apatite blocks. In Vitro Mineralization Assay Using Cell Membrane Nanofragments. To demonstrate that cell nanofragments can F


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ACS Biomaterials Science & Engineering were incubated in α-MEM supplemented with 10 mM βglycerophosphate (Sigma) for 2 or 7 days. As shown in Figure 7C−E, membrane nanofragments promoted marked mineralization in vitro within just 2 days, as determined by alizarin red staining. Minerals with higher crystallinity were observed after 7 days of culture, as shown by the TEM images, electron diffraction and calcium and phosphate peaks in the EDS analysis (Figure 7E). On the other hand, when the cells were kept alive (intact cell membranes), there was no mineralization in the culture plates, suggesting that the cells could be synthesizing inhibitors of mineralization.

localized in the inner region of plasma membrane.18,19 Therefore, the enzymatic activity of TNAP in catalyzing the plasma membrane phospholipids could promote the release of phosphate ions, which in turn, could subsequently bind to calcium ions to form the initial amorphous calcium-phosphate minerals. The initially formed calcium phosphate clusters were amorphous, and these clusters subsequently turned into crystalline HAp (clusters), which further increased in size (radial growth) and then aggregated to form a more dense plaque. These findings are in accordance with previous reports showing that the initially formed bone mineral phase is amorphous.20,21 A recent study also suggested that under conditions in which apatite forms from an amorphous calcium phosphate precursor, prenucleation clusters of calcium triphosphate complexes are initially formed, and subsequently aggregate and take up an extra calcium ion to form amorphous calcium phosphate.22 On the other hand, amorphous calcium phosphate has been shown to transform into octacalcium phosphate (OCP) or into the thermodynamically more stable HAp, depending on factors such as pH, calcium, and phosphate ion concentration.10,23 Nevertheless, since the presence of OCP was not detected in the diffraction patterning, initial amorphous calcium phosphate would be a precursor phase and directly transform into the thermodynamically stable HAp through the phase transition, as demonstrated in previous reports.20,24 Cell membrane is mainly composed of a long natural carbon chain of phospholipids, containing numerous membranous proteins. Collagen is also a natural polymer, in which the positive net charge close to the C-terminal of collagen molecules promotes the infiltration of the fibrils with amorphous calcium phosphate.25 Synthetic polymers, including synthetic cell membrane-like phospholipids, can also be the backbone and the source for phosphorus in mineralization. Polymer-controlled mineralization has been used as a model for investigating biomineralization and for the synthesis of novel biomaterials.26 Several polymers, such as poly(methacrylic acid) and poly(2-dimethyl aminoethyl methacrylate, have been used for synthesis and control of mineral formation onto the polymer surface, and may have diverse applications.26,27 The role of noncollagenous proteins in mineral formation has also been largely investigated.28 Proteoglycans have been reported to inhibit HAp formation and growth. Hunter et al., also showed that osteopontin, a major protein driving mineralization in osteoblasts, inhibits the in vitro mineralization of HAp and other biominerals. On the other hand, phosphoproteins were reported to promote HAp formation, and could also be nucleation sites for mineralization.29 For instance, bone sialoprotein, which is an important protein in bone mineralization, can bind to type I collagen and is an efficient nucleator for HAp.30,31 Additionally, a number of studies reported that organic substances in the ECM adsorb onto HAp crystals and inhibit their growth.32−34 Previous models of in vitro HAp synthesis showed the formation of similar spherical minerals in hydrogels, but addition of specific amino acids induced changes in the growth and crystallinity of HAp crystals.33 Nevertheless, calcospherites in the femur epiphysis presented a similar size and structure in all mineralized area. Therefore, the direct and specific effect of organic molecules (e.g., proteins) on the initial crystal formation could be very limited in the early stages of mineral formation during secondary ossification. The role inhibitors of nucleation and the roles of ECM components of HAp crystal growth still requires further detailed investigation.

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DISCUSSION Bone formation is a complex process that has been target of debate for long years. A major concept on bone formation addresses that hypertrophic chondrocytes or osteoblasts secrete matrix vesicles (MVs), which are enriched in pyrophosphatase and tissue nonspecific alkaline phosphatase (TNAP). Upon calcium influx into MVs, which is hypothesized to be through the ability of annexin-V to bind to calcium, initial crystal deposition occur inside the MVs and subsequently, the crystals grow and expand beyond the MVs limit throughout the extracellular matrix (ECM).10,15,16 In this study, we focused on the secondary ossification of mouse femur epiphysis, which initiates during the first postnatal week, because it allows the observation of the dynamic changes in organic and inorganic material in a time- and stage-specific manner. On the basis of the histological analysis, we found that initial mineral formation in the medial side of femur epiphysis until P6 occurred in the absence of osteoblasts. Since osteoblasts and endothelial cells were observed inside the epiphysis onward P7, we were able to analyze the very initial steps of chondrocyte-based mineralization in femur epiphysis. Interestingly, in the absence of osteoblasts, matrix vesicles could not be detected in the cartilaginous type II collagen-based extracellular matrix, which is contradictory to what is observed in osteoblast-dependent intramembranous ossification in mouse calvaria. We here provide evidence that initial biomineralization of femur epiphysis is not based on matrix vesicles, and show that cellular membrane nanofragments are the nucleation site for mineral formation during the early stages of secondary ossification. Additionally, because these cellular membrane nanofragments initiate mineral formation in vitro, controlling the concentration and distribution of chondrocyte membrane nanofragments inside materials (e.g., hydrogels) could be alternative approaches for in vitro synthesis of boneinducing materials. Regarding the mechanisms of cell fragment formation in vivo, chondrocyte death (e.g., apoptosis, necroptosis) following hypertrophy could be a major phenomenon associated with the origin of cellular membrane nanofragments. We have also demonstrated that chondrocyte burst near the mineralized area.17 Taking in consideration that chondrocyte death (e.g., apoptosis, burst) leads to a decrease in cell volume, the cell membrane nanofragments, which were observed in the ECM in between the chondrocytes (Figure S5), could have remained attached in the ECM in the original place of the hypertrophic chondrocyte membrane boundaries before cell death (cell volume decrease). Cell membrane nanofragments are constituted of phospholipids and cell membrane proteins. Additionally, tissue nonspecific alkaline phosphatase (TNAP), the major phosphatase associated with bone formation, is known to be mainly G


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In summary, we identified and characterized the initially formed amorphous and HAp crystals during secondary ossification center in femur epiphysis. Additionally, the results indicated that cell membrane nanofragments are nucleation site for mineral formation in vivo, and can promote mineral formation in vitro. These data may enable more precise investigation from the crystallography viewpoint of the conditions and factors determining mineral formation, maturation (phase transition) and growth in a spatiotemporal manner. In the future, cell membrane fragment-based materials can also be developed and applied in bone tissue engineering and regeneration.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.7b00962. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +81 86 235 6665. Fax: +81 86 235 6669. ORCID

Emilio Satoshi Hara: 0000-0001-7374-3487 Author Contributions

E.S.H. designed and performed the experiments, collected and analyzed data, prepared figures, and wrote the manuscript. M.O. analyzed data. N.N. performed part of the experiments as well as the electron microscopic observations and analyzed data. T.H. performed part of the experiments and supplied materials. T.K. supplied materials. T.N. supplied materials. T.M. designed the experiments, analyzed data, and wrote part of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by JSPS Postdoctoral Fellowship for Foreign Researchers (to E.S.H.), as well as by JSPS KAKENHI Grant Number (JP16H06990, JP16H05533, JP25220912, JP25293402, and JP26106718).

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REFERENCES

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Bioinspired Mineralization Using Chondrocyte Membrane

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