Cells Recognize and Prefer Bone-like Hydroxyapatite: Biochemical

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Cells Recognize and Prefer Bone-like Hydroxyapatite: Biochemical Understanding of Ultrathin Mineral Platelets in Bone Cuilian Liu, Halei Zhai, Zhisen Zhang, Yaling Li, Xurong Xu, and Ruikang Tang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b10374 • Publication Date (Web): 18 Oct 2016 Downloaded from http://pubs.acs.org on October 22, 2016

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ACS Applied Materials & Interfaces

Cells

Recognize

Hydroxyapatite:

and

Biochemical

Prefer

Bone-like

Understanding

of

Ultrathin Mineral Platelets in Bone Cuilian Liu,† Halei Zhai, † Zhisen Zhang, † Yaling Li, † Xurong Xu‡ and Ruikang Tang. * †‡ †

Center for Biomaterials and Biopathways, Department of Chemistry, Zhejiang University,

Hangzhou, Zhejiang 310027, China ‡

Qiushi Academy for Advanced Studies, Zhejiang University, Hangzhou, China

KEYWORDS: Biomineralization; Crystallography; Fibronectin; Hydroxyapatite; Mesenchymal stem cells;

ABSTRACT: Hydroxyapatite (HAP) nanocrystallites in all types of bones are distinguished by their ultrathin characteristics, which are uniaxially oriented with fibrillar collagen to uniquely expose the (100) faces. We speculate that living organisms prefer the specific crystal morphology and orientation of HAP because of the interactions between cells and crystals at the mineral-cell interface. Here, bone-like platy HAP (p-HAP) and two different rod-like HAPs were synthesized to investigate the ultrathin mineral modulating effect on cell bioactivity and bone generation. Cell viability and osteogenic differentiation of mesenchymal stem cells (MSCs) were

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significantly promoted by the platy HAP with (100) faces compared to rod-like HAPs with (001) faces as the dominant crystal orientation, indicating that MSCs can recognize the crystal face and prefer the (100) HAP faces. This face-specific preference is dependent on the selective adsorption of fibronectin (FN), a plasma protein that plays a central role in cell adhesion, on the HAP surface. This selective adsorption is further confirmed by molecule dynamics (MD) simulation. Our results demonstrate that it is an intelligent choice for cells to use ultrathin HAP with a large (100) face as a basic building block in the hierarchical structure of bone, which is crucial to the promotion of MSCs osteoinductions during bone formation. INTRODUCTION Bone, the primary support for vertebrates, is a hierarchically structured organic-inorganic composite.1 At the nanoscale, an array of hydroxyapatite (HAP, P63/m) crystals embed into a collagen matrix to form biomineralized collagen fibrils, which are the basic building block for almost all bone.2 There is an intimate relationship between the self-assembled fibrillar collagen matrix and the uniaxially oriented, nanometer-sized, platy HAP crystals, which provide hard tissues with remarkable mechanical properties and remodeling capabilities.3 In bone, the HAP crystallites are extremely small, with lengths of 30–50 nm, widths of 15–30 nm, and thicknesses of 2–4 nm.2,4 These HAP platelets are ultrathin with a large (100) plane; the thickness of the platelets is most likely the smallest dimension for biologically formed crystals.2 HAP crystals are often illustrated as being coherently aligned, and all the platelets appear to be approximately parallel.5 As a result, the HAP platelets uniquely expose the (100) crystal faces in the biomineralized collagen fibrils. We postulate that HAP platelets with (100) face could optimize the bioactivity of osteogenic cells. This hypothesis may contribute to the understanding of biomineralization and biological hard tissues.


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Many studies have focused on the mechanical properties of HAP platelets in the protection of the brittle mineral phase in bone;3,6 however, little is known about the bioactive effects of ultrathin, platy HAP. Mimicking the formation of natural hard tissues significantly contributes to the determination of the biological functions of ultrathin HAP platelets in bone and of whether cells can recognize the crystallographic orientations of a biomineral. It has been documented that HAP can adsorb various proteins7 and that adsorptions are frequently anisotropic on different crystal faces. For example, amelogenin, a crucial protein in the generation of dental tissue, specifically binds to the (100) facet rather than the (001) face of HAP.8 Osteonectin, osteocalcin and phosphophoryn have been reported to bind specifically to the (100) face of HAP crystals.9 We note that the (100) face also dominates the ultrathin platelet crystals in bone. Because osteogenic cells play an important role in the generation of hard tissue,10 an emerging question is whether this biological choice of HAP orientation is related to the activities of these cells. To elucidate this interesting and important question, ultrathin, platy HAP with a large (100) face is required. Unfortunately, the apatite crystals typically synthesized in laboratories are rod-like with side faces of (100)/(010) and end faces of (001). Previously, a series of nano HAPs with welldefined sizes were synthesized, and biological assays revealed the effects of HAP size on cell proliferation and differentiation.11 However, these tiny crystallites were randomly aggregated; therefore, it was difficult to examine the bioactivity on each specific face. Despite the importance of studying face-specific mineral interactions to establish a fundamental understanding of the hierarchical structure of HAP in mineralized tissue, no direct measurements have yet been reported. We synthesized platy HAP with a large (100) surface and a thickness of approximately 2–4 nm. The ultrathin characteristic and crystal orientation of the prepared HAP are analogous to the biological HAP in bone.2,3 This bone-like HAP provided the


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opportunity to examine the HAP (100) facet exclusively; our results indicate that the ultrathin feature confers better biocompatibility and osteoinductivity onto platy HAP than the features of any other artificial HAP material. Thus, cells prefer ultrathin HAP and recognize the (100) face to achieve optimal biological function for bone generation; this result demonstrates an example of the intelligent selection of biominerals by living organisms. EXPERIMENTAL SECTION Synthesis of Hydroxyapatite (HAP). The chemicals, which were analytical-grade, were obtained from Aladdin Chemical Reagent Co., Ltd. (China). No additional purification was applied prior to use. All water in the experiments was triply distilled. A total of 0.2 g of CaCl2·2H2O was added to 200 mL of ethylene glycol (EG). A total of 2.27 mL of 0.3 M Na2HPO4 in aqueous solution and 0.52 mL of 1.0 M NaOH in aqueous solution were mixed with 40 mL of EG. The phosphate-containing solution was poured into the calcium-containing solution. The reaction solution was refluxed at 150 °C for 12 h. The precipitates were collected by centrifugation (8000 rpm) and were alternately washed with H2O and ethanol. The resulting HAP was named p-HAP. The solids were characterized by atomic force microscopy (AFM, Nano IVa, Veeco, USA; scan size, 5 µm; scan rate 0.7978 Hz), transmission electron microscopy (TEM, JEM-200CX, JEOL, Japan) and selected area electron diffraction (SAED). The details of the controllable syntheses of rod-like HAP were reported previously.11 Two kinds of rod-like HAP were used in this study: thick HAP (t-HAP) and narrow HAP (n-HAP). Briefly, 250 mL of 0.5 M CaCl2 was added to 500 mL water. The pH of the mixed solution was adjusted to 9.5. After the solution was stirred for 30 minutes, 250 mL of 0.3 M (NH4)2HPO4 was added to the reaction solution, and the pH was controlled at 9-10 during the reaction process. The mixed


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solution was kept at 100 °C for 24 h (t-HAP) and 75 °C for 48 h (n-HAP). The precipitates were collected by centrifugation (8000 rpm) and were alternately washed with H2O and ethanol. Preparation of HAP Films. Films of t-, n- and p-HAP were obtained by slowly coating coverslips (diameter = 15 mm) with 5 mg mL-1 solutions (150 µL) of HAP in ethanol at room temperature. Prior to the in vitro assays, all the films were sterilized overnight under ultraviolet germicidal lamps. The film samples were examined by field emission scanning electron microscopy (FESEM, Utral 55, CorlzeisD, Germany, operated at 5 KV), film X-ray diffraction (XRD, X'Pert PRO, PANalytical, Netherlands; Cu-Kα radiation, λ=1.5416 Å, scan step of 0.02 in 2θ from 10° to 60°) and AFM (Nano IVa, Veeco, USA; scan size, 5 µm; scan rate, 0.7978 Hz). Cell Culture. Animal experiments were approved by Zhejiang University and performed in strict accordance with the guidelines of the Laboratory Animal Centre of Zhejiang University. Mesenchymal stem cells (MSCs) were isolated from 4-week-old ICR mice following Kelly’s method.12 Briefly, the mice were sacrificed by vertebral dislocation. The femora and tibiae were harvested, and the entire bone marrow was washed with minimal essential medium α (α-MEM; HyClone) in a syringe. The cells were collected and were cultured in α-MEM supplemented with 10% fetal bovine serum (FBS; PAA), 100 U mL-1 of penicillin and 100 µg mL-1 of streptomycin (Life Technologies). After the cells were incubated at 37 °C in a humidified incubator with 5% CO2 for 4 days, the α-MEM was replaced with fresh medium. During the experiments, the culture medium was changed every 3 days. All MSCs used in our tests were primary MSCs (passage 0, p0). Cell Proliferation on HAP Films. Cell proliferation was measured by 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Different HAP films and uncoated


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coverslips were sterilized and placed into 24-well plates. The primary MSCs isolated from ICR mice needed 3-4 days for complete attachment. After 6 days, cell confluence appeared in some HAP films. Thus, we studied cell proliferation at days 5 and 6.The MSCs were cultured on the HAP films at a density of 8×106 cells per well and were incubated for 5 and 6 days; then, the MTT solution (50 µL, 5 mg mL-1) was added to each well, and the MSCs were incubated for another 4 h at 37 °C under 5% CO2. Subsequently, the medium was removed and dimethyl sulfoxide (DMSO, 400 µL) was added to dissolve the blue formazan crystals. Finally, the optical density of the solution was measured at 570 nm with a microplate reader (Eon, BioTek, USA). After the MSCs were incubated for 6 days on different substrates, they were rinsed twice in PBS and fixed with 4% paraformaldehyde in PBS overnight at 4 °C. These samples were washed with water, air-dried and coated with gold. The cell morphology was determined by FESEM. The film surfaces were washed with PBS after the cells were cultured for 6 days. The cells on the films were then incubated in 1 µM calcein acetoxymethyl ester (calcein AM, Invitrogen) at room temperature for 30 minutes. This assay identifies live cells through green fluorescence from calcein AM. The cell morphology and adhesion were investigated by fluorescence microscopy. Cell Differentiation on HAP Films. The osteogenic differentiation of MSCs was evaluated by measuring alkaline phosphatase (ALP) activity. The MSCs were seeded onto HAP films in 24-well plates at a density of 8×106 cells per well for 4 days. Then, the cells were cultured with an osteogenic induction supplement (OS) for 7, 12 and 14 days. MSCs cultured on glass without the OS were used as a negative control, while MSCs cultured on glass with the OS were used as a positive control. The ALP activity of MSCs was measured using an ALP measurement kit


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(Nanjing Jiancheng Bioengineering Institute, China). All the results were normalized with the total protein amount measured by a bicinchoninic acid (BCA) protein assay reagent kit (Beyotime Institute of Biotechnology, China). After 7 days of culture with the OS, total RNA from the MSCs was extracted with TRIzol reagent and reverse transcribed to single-stranded cDNA with Superscript TM II RTase (Invitrogen). PCR expression of the Runt-related transcription factor-2 (Runx2), ALP, collagen type I (Col I) and osteocalcin (OCN) genes was quantified using the iQTM5 Multicolor RealTime PCR Detection System (Bio-Rad) with SYBR Green detection reagents. The 18S rRNA gene was used as a housekeeping gene for normalization of the cDNA across samples. The relative amount of mRNA expression was expressed as a percentage relative to the positive control group. The primer sequences are listed in Table S1. After the MSCs were cultured with the OS for 7 days, the MSCs on the films were stained using a Cell Alkaline Phosphatase Assay Kit (azo-coupling method, Nanjing Jiancheng Bioengineering Institute, China). Fibronectin (FN) Adsorption. FN was selected as a typical model of an adsorbed protein and was used to evaluate the protein adsorption capability of different substrates. The FN used in our study was human plasma fibronectin (molecular weight = 440,000 Da, Gibco). The examined substrates were incubated in 100 µg mL-1 FN solution at 4 °C for 24 h. The remaining FN in the solution was measured with a BCA Kit. The amount of adsorbed FN was normalized by the surface area of the different HAP films. Then, the adsorption results were expressed as ng of FN per mm2.


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Molecular Dynamics Simulations. Adsorption of the 10th type III module of FN (FN-III10) on the HAP (001) and HAP (100) surfaces was systematically studied by molecular dynamics (MD). The Hauptmann HAP model13 was used. The creation and structures of the (100) and (001) faces were previously reported.14 TIP3P force fields15 were applied for water, and CHARMM27 force fields16 were applied for the protein. For both force fields, the dispersion terms were described by Lennard-Jones potentials. The MD simulations were performed with the Gromacs 4.5.2 software package17 in the NpT ensemble at a temperature of 310 K. Visual Molecular Dynamics (VMD)18 was used to visualize the output. Periodic boundary conditions were applied in all directions. Particle mesh Ewald (PME)19 summation was applied to treat the long-range Coulombic interactions. The cutoff distance was chosen to be 1.3 nm. The time step was 2 fs. Initial velocities according to the Maxwell distribution were used for the starting configuration. The interaction energies (EInter) between the protein and the HAP crystal were calculated from the sum of ECoul, the electrostatic interaction energy, and ELJ, the van der Waals interaction energy. Statistical Analysis. The data were collected from at least three separate experiments and are presented as the mean ± standard deviation (SD). Significant differences were analyzed by a paired Student’s t-test. P values less than 0.05 were considered to indicate significant differences. RESULTS AND DISCUSSION Three different HAP samples were used for in vitro biological assessments: thick nanorods (tHAP), narrow nanorods (n-HAP), and nanoplates (p-HAP). TEM images showed that the three HAP samples had similar lengths of 100 nm (Figure 1A-C) but varied greatly in their width/thickness. As shown, the width/thickness of t-HAP was ~50 nm but was only ~10 nm for


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n-HAP. The thickness of p-HAP was only 2–4 nm (Figure 1C), and this dimension was similar to that of HAP in bone (Figure S1).2,4 A high resolution transmission electron microscopy (HRTEM) image shows that the exposed crystal face of p-HAP was a (100) plane (Figure S2), indicating that the exposed crystal face of p-HAP is identical to the exposed crystal face of HAP platelets in bone.2,5 SAED results (insets of Figure 1A-C) confirmed that all the HAP materials were well crystallized. To determine the bioactivity and face-selectivity of cells on the mineral surface, different HAP films were prepared as the substrates for in vitro experiments. Although there were no obvious differences among the three HAP substrates at the optical macroscopic scale (Figure S3A-C), we found that these films had different textures under SEM (Figure 2A-C). The nanorods of t-HAP and n-HAP were randomly aggregated in the films, whereas the p-HAP plates tended to be aligned along their large planes to produce platy assemblies. Side-view SEM images confirmed this result (Figure S4). Although there was no collagen, the aggregation behavior of p-HAP was similar to that of bone HAP in hard tissue due to the ultrathin feature,2,20 and this behavior implied that the ultrathin crystals could self-assemble in a plane-parallel structure. Several studies have reported that the surface roughness (Ra) of the substrates can significantly affect cell adhesion and proliferation.21 Therefore, AFM was used to determine the Ra of the HAP films (Figure 2D-F). As expected, the surface of p-HAP was the smoothest of all the samples. The Ra values of the t-, n- and p-HAP films were 102.54 ± 13.92 nm, 97.64 ± 25.69 nm and 66.62 ± 14.84 nm, respectively. The crystallographic characteristics of the HAP films were studied by XRD (Figure 2G-I). The pattern of the p-HAP film was differed from those of the t- and n-HAP films. In comparison with the standard XRD pattern of HAP (JCPDS 9-432), the typical peak for (002) almost vanished in


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the p-HAP film, and only a broad band for 2θ at 30-35° was observed. The broad peak was caused by the overlap of the (211), (112), (300) and (202) diffraction peaks, and (300) became the strongest one (the broad peak of the p-HAP film, appearing between 2θ = 30-35°, was overlapped by (211), (112), (300) and (202)); Peakfit v4.12 software was used to separate the overlapped peaks (for details of the peak separation process, see SI). The weak (002) peak and the intense diffraction peaks of (300) in the p-HAP film implied that the (100) face dominated the substrate surface. In contrast, intense diffraction peaks for (002) and (300) were observed simultaneously for both the t- and n-HAP films; these peaks implied that (001) was one of the primary exposed crystal faces of the film in addition to (100). It should be noted that for (002), the n-HAP film had a lower intensity than the t-HAP film; this result is due to the lower exposure of the c planes. Thus, the ratio of (100):(001) on the surface of the film increased from t- to n- to p-HAP. The ratio of the diffraction intensities for (300) and (002) could be used to quantitatively estimate this change, and the values of the ratios were 1.25, 2.94 and 6.25 for the t-, n- and pHAP substrates, respectively. Higher ratios represented greater exposure of (100) in the HAP films. MSCs, adherent stromal cells of non-hematopoietic origin, can proliferate extensively and differentiate into several types of connective tissue cells, including bone, cartilage, tendon, fat, muscle, marrow, dermis and even nervous tissues.22 The osteogenic transformation of MSCs is particularly attractive in the context of bone engineering and reconstructive applications.23 The effects of different HAP substrates on the proliferation of MSCs were examined by MTT assay, with glass as a control (defined as 100%). Specifically, the bone-like p-HAP substrate significantly promoted cell proliferation and exhibited a better effect than the control group; the growth reached 111% and 114% at days 5 and 6, respectively (Figure 3A). However, both t- and


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n-HAP films significantly decreased cell proliferation by approximately 58% and 78% at day 5, respectively (Figure 3A). In our previous work, we systemically studied the biocompatibility of various artificial HAP particles and concluded that HAP particles with smaller dimensions could improve cell adhesion and proliferation.11 However, we noted that the control substrate, glass, was always better than any HAP material. For instance, the best HAP substrate in the previous study was 20 nm HAP, but the proliferation rate of MSCs on this substrate was only 93% of that on glass at day 6.11 In fact, rather than HAP, glass has been considered the best substrate for adhesion and proliferation of MSCs in vitro.24, 25 In the current study, we provide the first example of better MSCs bioactivity on bone-like p-HAP film than MSCs bioactivity on glass. This interesting result indicates the importance of controlling the morphology/facets of crystals to guarantee the optimal biocompatibility of mineral materials. The adhesion and viability of MSCs on different substrates were examined by SEM. Significantly, the cell density increased from the t-HAP film, to the n-HAP film, to glass and, finally, to the bone-like p-HAP film (Figure 3B-E); this result was consistent with the proliferation results. Cell morphology is closely related to cell viability and cell function,26 and a well-spread MSC should have a typically elongated and highly branched morphology.27 Cells on glass exhibited fibroblast-like morphology and were long and thin, characteristic of MSCs; however, some cells were still spherical or round and had a less branched morphology. Thus, not all the cells were well attached to the glass (Figure 3B). On the t-HAP surface, some individual spindle-shaped MSCs could be seen (Figure 3C). Cells with polygonal or fine filopodia were widespread on the n-HAP film, but no cell aggregates were found (Figure 3D). In contrast, cells on the bone-like p-HAP film were spindle-shaped and angular (Figure 3E). This phenomenon


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indicated the high cytoactivity of the cells, which were spread evenly over the bone-like p-HAP films, with cell-cell contact and communication. A similar result was observed using fluorescence staining (Figure 3F-I), which illustrated that the MSCs adhered to and spread over the bone-like p-HAP film better than on any other substrate. Both intramembranous and endochondral bone formation are dependent on osteoblasts that are differentiated from MSCs.28 Thus, the differentiation of pluripotent MSCs into bone-forming cells is an important step in bone generation. Additionally, this process is a complex, multistep pathway that depends on the presence of specific bioactive factors and is affected by environmental cues.29 ALP is an enzyme widely used to evaluate the osteogenic differentiation of MSCs. Upregulation of ALP has been considered an important indicator of osteogenic differentiation and bone formation.30 The potential effects of different HAP films on osteogenic differentiation of MSCs cultured for 7, 12 and 14 days were assessed by quantitatively studying the level of ALP activity normalized to the total protein content (Figure 4A). In the absence of an OS, MSCs did not display upregulation of ALP activity as expected. In the presence of an OS, MSCs displayed differential upregulation of ALP activity on each substrate type. The ALP activity values of MSCs cultured on the t-, n- and bone-like p-HAP films at day 14 were 5133.52 ± 870.40 U g-1, 5740.33 ± 720.39 U g-1 and 8891.55 ± 1881.41 U g-1, respectively. The data indicated that bone-like p-HAP had better osteogenic induction ability than t- and n-HAP. Runt-related transcription factor-2 (Runx2) belongs to the Runx family and is exclusively expressed in mineralized tissues.30 Runx2 is considered a key point for integration of a variety signals affecting osteogenesis because it stimulates osteo-related genes that encode collagen I (Col I), osteocalcin (OCN) and osteopontin (OPN).31 We noted that the bone-like p-HAP films significantly promoted Runx2 and ALP expression (Figure 4B). In addition, Col I is the most


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abundant protein found in the bone matrix. Upregulation of Col I is related to bone mineralization in the later stages of MSCs differentiation.32 OCN is the most abundant bonespecific non-collagenous protein synthesized by osteoblasts and is used as a marker to evaluate osteogenic differentiation and mineralization.33 The qRT-PCR results revealed that the expression levels of Col I and OCN induced by bone-like p-HAP films were approximately 247% and 351%, respectively, in comparison with the positive controls (Figure 4B). However, the OCN levels induced by t- and n-HAP films were only 128% and 135%, respectively. These results indicated that MSCs on the bone-like p-HAP films had significantly higher osteo-related gene expression than those on t- and n-HAP films, and this result was consistent with the ALP activity test. Faint ALP stain on the t-HAP film was detected, but the stain was much more extensive on the bone-like p-HAP films (Figure 4C). Again, the results confirmed that bone-like p-HAP films had the most positive influence on osteogenic differentiation of MSCs. Surface features have been shown to have an influence on cellular behavior.34 The most commonly used methods can be subdivided into microscopic, physical and chemical analysis. First, surface features on the microscopic level were observed. Using SEM and AFM, the topographical features of t-, n- and p-HAP films were found to be non-ordered, without regular patterns. It is well known that the most commonly used nanotopographies in studies are different grades of (semi-)ordered tubes, pits and pillars, and highly organized grooves. Ball et al. observed that substrates with an ordered topography could stimulate cell metabolic activity more than the disordered substrates,35 suggesting that ordered surface patterns might be a determining factor for specific effects on cellular behavior. Thus, in our study, topographical features did not act as a determining factor regulating the cellular activity of MSCs. Due to the non-ordered patterns of HAP films, the roughness (Ra) analysis of surface topographies was analyzed.


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Usually, it is accepted that bone marrow cell proliferation/differentiation can be enhanced by increasing the substrate Ra because cells prefer a rough surface.21 In our case, the bone-like pHAP film had the lowest Ra of the HAP samples while exhibiting the best MSC proliferation and differentiation. This atypical tendency implied that the Ra did not act as the key factor regulating MSC behavior on bone-like p-HAP films. In addition to roughness, we performed other important physical characterization examinations on the three HAP films, including surfacewettability and zeta potential; all films exhibited super-hydrophilicity and electro neutrality. Finally, the chemical characterizations of the surfaces were taken into consideration. SAED results show that all the HAP materials were well crystallized, indicating the same chemical compositions and crystal phases of HAP films. Here, the improved proliferation/differentiation of MSCs on p-HAP films can be understood by focusing on the crystal orientations. We noticed that the tendency for cell proliferation/differentiation coincided with the exposure ratio of (100) for the substrate, and this suggested a preference of the cells for this crystal facet. The surface properties of biomaterials can regulate protein adsorption, and the adsorbed proteins modulate subsequent cell attachment by interacting with receptors on the cell surface.36 Most mammalian cells are adherent and must spread over an extracellular matrix (ECM) to perform normal cellular functions such as proliferation and differentiation.37 The ECM is a fibrillar network of proteins,38 and FN39 is an ECM and plasma protein that plays a central role in cell adhesion at the cell-substrate interface. Extensive research on FN has made it a prototypical cell adhesion protein.39 It is widely accepted that FN not only has an essential role in anchorage and migration but also can affect cell proliferation and differentiation for various types of cells in the body.40


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We hypothesized that the increased proliferation and differentiation of MSCs on bone-like pHAP films over those on t- and n-HAP films might be because of enhanced adsorption of FN. The FN-cell interaction largely depends on the surface receptors of the cell.39 Here, the adsorption of FN, a model protein, was measured to evaluate the influence of HAP films on protein adsorption. Adsorption on the bone-like p-HAP film was 82.67 ± 1.92 ng mm-2, which was significantly higher than the adsorption on the t- and n-HAP films, 65.19 ± 2.22 ng mm-2 and 70.09 ± 1.99 ng mm-2, respectively (Figure 5A). As described above, the bone-like p-HAP film has higher biocompatibility and osteoinductivity than the t- and n-HAP films, and this tendency coincided with the proportion of the (100) crystal plane of the three film surfaces. We suggest that the different crystal facets exhibit different proliferation/differentiation for the cells. The adhesion of cells to the physical substrate requires an attachment factor.37,39 FN and its receptors provide this link.39 Thus, the experimental results imply that the increased adhesion, proliferation and differentiation of MSCs on the (100) HAP plane could be because of selective adsorption of adhesion proteins, such as FN, on the crystal facets. FN is a dimer composed of a number of repeating units, of which there are three types, FN-I, FN-II and FN-III.39 Cells primarily interact with FN at the Arg-Gly-Asp (RGD) cell attachment site.39, 41 The 10th type III module of FN (FN-III10) contains an RGD loop (Figure 5B). The adsorption of FN-III10 on the HAP (100) and HAP (001) surfaces was studied with molecular dynamics (MD). Quantitative analysis at the molecular level indicated the strength of the interaction energy between FN-III10 and the different HAP facets (Figure 5C-D). On the (100) HAP surface, the interaction energy was 1167.18 ± 4.1 kJ mol-1, whereas the value was only 760.20 ± 7.3 kJ mol-1 on (001) HAP. Clearly, (100) could provide better adsorption for FN than


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(001). Protein adsorption depends on the types and amounts of the adsorbed groups.41 On the (100) face, there were four adsorption sites for FN-III10 binding: two guanido groups (Figure 5C, binding sites 1 and 4) interacted with the HAP hydroxyl/phosphate anions, and two carboxyl residues (Figure 5C, binding sites 2 and 3) interacted with the HAP calcium ions. However, on the (001) surface, there were only three binding sites: two guanido groups (Figure 5D, binding sites 1’ and 3’) and a carboxyl residue (Figure 5D, binding site 2’). The difference between the FN-III10 binding energies on the (100) and (001) HAP faces, 406.98 kJ mol-1, was consistent with a typical carboxyl binding energy on HAP.41 On both HAP faces, the RGD loops exposed to water were in a conformation that supports the binding of cellular integrin receptors; this result indicates that the RGD sequence was preserved, and that the bioactivities were identical. Thus, in our test, the amount of FN adsorbed onto the substrates determines the adhesion ability of the cells, and the (100) HAP face has an advantage over the (001) face. It follows that the (100) crystal face adsorbs abundant FN from the ECM or serum with the RGD loop conformation exposed. Additionally, this special conformation promotes the binding of integrin receptors. FN can enhance the proliferation of MSCs through the α5β1 integrin receptor, and the osteogenic differentiation of MSCs is highly dependent on β1 integrin interactions.42 If adsorbed FN proteins bind to the integrin receptors on the MSC surface, this binding induces vigorous integrin-dependent signaling, which leads to strong cell adhesion, extensive cell spreading and enhanced cell survival. These events are crucial for the proliferation and differentiation of MSCs. The data suggest that the cells prefer the HAP ultrathin platelet structure because of the large (100) face; this preference may be attributed to the selective interaction between fibronectin and the HAP facet. The hierarchical construction of bone results in numerous exposed (100) HAP


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faces, which ensure cell adhesion, proliferation and differentiation. Therefore, biological organisms select the most reasonable crystal structure to achieve the optimal function. In bone, biomineralization refers to the deposition of HAP onto an organic matrix. During bone generation, the first crystals to appear are platelet-like crystals with c-axes aligned with the collagen fibers;43 this process results in an exposed (100) crystal face.44 In turn, the existing ultrathin HAP bone crystals generated by osteoblasts, which are formed by the differentiation of MSCs, can promote the adhesion, proliferation and osteogenic differentiation of MSCs through the selective adsorption of adhesion proteins. This intelligent mutual feedback mechanism may occur between bone-related cells and HAP to evolve the most suitable cycle for tissue generation; in this cycle, the (100) crystal face provides an optimized cell-mineral interface because of the superior biocompatibility and osteoinductive capacity. Meanwhile, the mineralized HAP deposited by the cells forms a large and unique (100) crystal face on the tissue interface. This discovery indicates that the ultrathin HAP is a biological preference in bone and provides a biochemical view of bone hierarchical structures in contrast to the well-established mechanical view.2, 45 The specific effect of the HAP (100) surface on the bioactivity of bone-related cells is inspired by natural process, and this effect can be used as an advanced strategy to improve material-induced bone repair and hard tissue engineering. CONCLUSIONS In our current study, we synthesized bone-like HAP and revealed that cells prefer plate HAP and can recognize crystal facets. These experimental cases demonstrate that the (100) HAP face can promote higher bioactivity of MSCs for adherence, proliferation and osteogenic differentiation than the (001) face and that this higher bioactivity facilitates bone generation.


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This benefit is highly dependent on the selective adsorption of functional proteins, such as fibronectin, on the HAP substrate. The specific bioactivity of the adhered cells enhanced by the crystal face may explain why biological bone tissue selects ultrathin HAP plates with a large (100) face to be the basic building block in the hierarchical structure of bone. This study provides an understanding of the optimization of the crystal morphology and orientation of the inorganic phase in biomaterials to ensure the best bioactivity during natural evolution. Accordingly, this feature also implies that bone-like HAP plates have great potential over the typically synthesized HAP rods in biomedical applications for bone engineering. FIGURES

Figure 1. Characteristics of different HAP powders. The TEM images reveal the different morphological characteristics of t-HAP (A), n-HAP (B) and p-HAP (C). The insets are the SAED of the corresponding samples under TEM; the inset AFM image indicates the thickness of p-HAP.


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Figure 2. Characteristics of different HAP films. The SEM images show different features of the t-HAP (A), n-HAP (B) and p-HAP (C) films. AFM images of the surfaces: t-HAP (D), n-HAP (E) and p-HAP (F); the calculated roughness values are also listed. XRD patterns: t-HAP film (G), n-HAP film (H) and p-HAP film (I). The insets resolve the overlapped spectra; the ratio of peak intensities for (300):(002) is given by R (300) /(002).


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Figure 3. Proliferation of MSCs on different HAP films and glass. The substrate effects of different HAP films on the proliferation of MSCs were examined with the MTT assay, with glass as a control (A). The results are represented by the mean ± SD (n = 3); *p

Cells Recognize and Prefer Bone-like Hydroxyapatite: Biochemical

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