Bone apatite anisotropic structure control via designing fibrous scaffolds

Bone tissue has an anisotropic structure, associated with the collagen fibrils' orientation and the c-axis direction of the bone apatite crystal. The bone regeneration process comprises two main phases: bone mineral density restoration (bone quantity), and subsequent recovery of bone apatite c-axis orientation (bone quality). Bone quality is the determinant factor for mechanical properties of bone. Control of osteoblast alignment is one of the strategies for reconstructing bone quality since the collagen/apatite matrix orientation in calcified tissues is dependent on the osteoblast orientation. In this work, fibrous scaffolds designed for reconstruction of bone quality via cell alignment control was investigated. The fibrous scaffolds were fabricated using the electrospinning method with poly(lactic acid) at various fiber collecting speeds. The degree of fiber alignment in the prepared fibrous scaffolds increased with increasing fiber collecting speed, indicating that the fibers were oriented in a single direction. The alignment of osteoblasts on the fibrous scaffolds as well as the subsequent apatite c-axis orientation increased with increasing fiber collecting speed. We successfully controlled cell alignment and apatite c-axis orientation using the designed morphology of fibrous scaffolds. To the best of our knowledge, this is the first report demonstrating that adjusting the degree of fiber orientation for fibrous scaffolds can manipulate the regeneration of bone quality.


Introduction
Bone has a multiscale structure with hierarchical levels from nano to microscale, which comprises collagen brils and biological apatite (BAp). 1 The hierarchical bone tissues exhibit anisotropic properties originating from the collagen bril orientation and the direction of the c-axis of BAp crystals. 2,3 Several researchers have focused on the distribution and orientation BAp in bone tissues, using X-ray, neutron and electron diffraction techniques. [4][5][6][7][8][9] Wenk et al. reported that preferred orientation of hydroxyapatite (HAp) crystallites in two mineralized tissue fragments was investigated using synchrotron X-rays. 5 The tissues exhibited signicant crystalline alignment with c-axis, which preferentially aligned parallel to the long axis of the bone. Ziv et al. showed the c-axis of HAp in bovine bone were aligned parallel to the collagen bril axes using electron diffraction methods. 4 In our previous review articles reported variety of the preferential alignment of the BAp c-axis of various type bones by the micro-beam X-ray diffractometer (mXRD) system equipped with two-dimensional detectors. 10,11 The orientation c-axis of BAp crystallites analyzed by mXRD offers a new index as a bone quality parameter; which can obtain relative intensity ratio of the 002 diffraction peak to the 310 peak in the X-ray prole. 3,12 Additionally, bone quality was dened by the National Institute of Health (NIH) in 2000 as parameters contributing to bone strength independent of bone mineral density (BMD). 13 The mechanical properties of bone tissue is strongly correlated with the degree of BAp c-axis orientation, which is one of the indices for bone quality. 14 Moreover, during bone regeneration, the recovery of c-axis orientation of BAp, which determines bone quality, is signicantly delayed compared to that of BMD, which determines bone quantity. Notably, bone quality dominates the mechanical properties of bone tissue rather than bone quantity. 12,14 Hence, bone quality reconstruction during bone regeneration is an important factor for designing scaffolds.
Fibrous scaffolds for bone regeneration via electrospinning methods could be applied to a biomimetic template for damaged tissue. 15,16 Oriented nanober scaffolds showed the ability of controlling cell alignment to the ber collecting direction. 17,18 Moreover, the cells producing collagen bril bundles were aligned in the direction of the cell orientation. Fee et al. reported that, broblasts on the oriented nanober scaffolds were aligned parallel to the bers, and their gene expression was upregulated through actin production, action polymerization, and focal adhesion formation. 19 Additionally, oriented nanober scaffolds were also found to upregulate the expression of osteogenic markers, such as runt-related transcription factor (Runx-2), type I collagen, alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OCN). 20 Kikuchi et al. reported that, collagen/HAp composites showed a self-organized nanostructure similar to bone, which HAp caxis of nanocrystals were parallel to the collagen brils. 21 In our previous work, we reported that osteoblasts orientation induced collagen/apatite matrix alignment in bone tissue. 22,23 Additionally, c-axis of BAp showed preferential alignment along the direction of osteoblast-produced collagen matrix. 22 Our previous work highlighted that anisotropic brous scaffolds with microbers exhibit controllability of osteoblasts alignment by designing their morphology. 24,25 Thus, controlling cell alignment is an invaluable strategy for reconstructing anisotropic bone matrices, such as the c-axis of BAp.
This work reports a fundamental investigation on designing brous scaffolds for reconstructing bone quality. In order to fabricate brous scaffolds, poly(lactic acid) (PLLA) was chosen, since it is the most widely used biodegradable polymer in biomedical elds. In this work, PLLA brous scaffolds were prepared using the electrospinning method, and their morphologies were controlled by ber collecting speed. The prepared brous scaffolds were evaluated for morphology, cell alignment, and bone apatite orientation.

Experimental
Fibrous scaffolds with various ber alignments were prepared using the electrospinning method. PLLA (LACEA, Mitsui Chemical, Japan) dissolved in dichloromethane (99.5%, Nacalai Tesque) at 14 wt% was the solution used for electrospinning. In our preliminary experiments, this ratio was found to be optimal for preparing the brous scaffolds with micrometer-sized diameter. The prepared solution was loaded into a syringe needle (18 gauge) set at 2.5 mL s À1 . A high-voltage supply (HARb-40P0.75, Matsusada Precision Inc., Japan) was used to apply 16 kV to the needle tip. The distance between the needle tip and drum collector was maintained at 200 mm. The drum collector (4 60 mm) was rotated at 0.1-10.0 m s À1 (30-3000 rpm). The obtained brous scaffolds were denoted as PLLA_x, where x is the ber collecting speed. The morphology of PLLA_x was observed by eld emission gun electron microscopy (SEM, JSM-6500, JEOL, Japan) aer coating with amorphous osmium layer using an osmium coater (Neoc CS, Meiwafosis, Japan). Fiber diameter, and angle (q) between the ber and collector rotation direction were measured using the ImageJ soware (NIH, USA).
Primary osteoblasts were isolated form newborn mouse calvariae as described in our previous reports. 26,27 Calvariae from newborn C57BL/6 mice were excised under aseptic conditions. The calvariae were placed in ice-cold alphaminimum essential medium (a-MEM, Invitrogen), and then brous tissues around the bone were gently removed. Subsequently, the calvariae were subjected to a series of collagenase (Wako Pure Chemical, Japan)/trypsin (Nacalai Tesque, Japan) digestions at 37 C for 15 min each. Since the broblasts were mixed, the supernatants of rst and 2nd digests were discarded. 28 The supernatants of 3rd-5th digests were neutralized with a-MEM and pooled. The pooled solution was ltered using a 100 mm mesh. The ltrate was centrifuged (1500 rpm, 5 min, 25 C), and the resulting pellet was resuspended in a-MEM. All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Osaka University and approved by the Animal Ethics Committee of Osaka University Committee for Animal Experimentation.
PLLA_x with 8 mm diameter was soaked in 70% ethanol for 30 seconds and subsequently dried under UV light for 30 min for sterilization. The cells were cultured in a-MEM containing 10% fetal bovine serum (FBS, Invitrogen). PLLA_x was then placed into 48 well plates, and primary osteoblasts were seeded by adding 0.5 mL of medium containing cells at a concentration of 3 Â 10 4 cells per mL. The culture medium was replaced aer day 1 and 3, and subsequently twice a week. Aer culturing for a week, the media was supplemented to achieve nal concentrations of 50 mg mL À1 ascorbic acid (Sigma-Aldrich), 10 mM bglycerophosphate (Sigma-Aldrich), and 50 nM dexamethasone (MP Bioscience). PLLA_x was analyzed for cell alignment and evaluation of calcied tissues aer 3 days and 4 weeks of culture, respectively.
The primary osteoblasts were cultivated for 3 days on PLLA_x (n ¼ 3). The cells were then xed with 4% formaldehyde in PBS for 20 min and washed 3 times with PBS-0.05% Triton X-100 (PBST). Subsequently, the cells were incubated in PBST containing 1% normal goat serum for 30 min to block nonspecic antibody binding sites, and then incubated with mouse monoclonal antibodies against vinculin (Sigma-Aldrich) at 4 C for 12 h. Aer washing 3 times with PBST, the cells were incubated with Alexa Fluor® 546-conjugated anti-mouse IgG (Invitrogen), followed by Alexa Fluor® 488-conjugated phalloidin (Invitrogen). Finally, the cells were washed 3 times, and mounted in Fluoro-KEEPER antifade reagent with DAPI (Nacalai Tesque). Fluorescent images were obtained using a uorescence microscope (BZ-X700, Keyence, Japan). Cell orientation angle (q) against the collector rotation direction was analyzed using Cell Proler soware (Broad Institute Cambridge). PLLA_x (n ¼ 7) cultivated for 4 weeks were xed with 4% formaldehyde in PBS for 20 min. Bone apatite crystals produced by primary osteoblasts were analyzed by mXRD system (R-Axis BQ, Rigaku, Japan) equipped with a transmission optical system (Mo-Ka radiation, 50 kV, 90 mA) and an imaging plate (storage phosphors) (Fuji Film, Tokyo, Japan) place behind the specimen. Detailed conditions for measurement have been described in the previous paper. 29,30 In this work, the incident beam focused into a diameter of 800 mm was used and diffraction data were collected for 1200 s. The preferred orientation of apatite c-axis was evaluated as the relative intensity ratio of the 002 diffraction peak to the 310 peak, which was measured in parallel to the collector rotation direction of the scaffolds. The intensity of 002 and 310 peaks in XRD prole were obtained from patterns reconstructed using multipeak tting package (Igor Pro, WaveMetrics).
The orientation order parameter FD and CD was calculated to evaluate the degrees of ber and cell alignment. 31 This system was derived by using a distribution function n(q), which is dened as the number of measured bers or cells at the angle q. The expected value of the mean square of cosine hcos 2 qi and FD and CD is calculated as follows: FD or CD ¼ 2(hcos 2 qi À 0.5) The degree of ber or cell alignment, FD or CD takes a value ranging from À1 (ber or cell were completely aligned perpendicular to the collector rotation direction), 0 (ber or cell were oriented randomly), to 1 (ber or cell were completely aligned parallel to the collector rotation direction).
Statistical comparisons between the two means were performed using a two-tailed unpaired Student's t-test followed by a F-test for homoscedasticity. p < 0.05 was considered signicant. PLLA_0.1 was selected for comparison group, which the bers randomly oriented.

Results and discussion
SEM images of PLLA_x are shown in Fig. 1(a-h), and their ber orientation angle histograms are presented in Fig. 1(i-p). The ber orientation angle was distributed at a center of 0 degree, and the breadth decreased with increasing ber collecting speed. Fiber diameter of PLLA_x is shown in Fig. 2(a), and the calculated FD value of PLLA_x is shown in Fig. 2(b). During the electrospinning process, bers were formed by the creation and elongation of an electric eld uid jet. 32 The velocities of the jets were measured in the range of 0.5 to 5.0 m s À1 via high framerate video camera. 32 In this work, ber collecting speed was set between 0.1 and 10.0 m s À1 , and the jet was single and stable throughout the fabrication. Fiber diameter of PLLA_x with the collecting speed x ¼ 0.1-3.0 showed no signicant difference, and that with x ¼ 4.0-10.0 decreased with increasing collecting speed. In case of the collecting speed x > 3.0, the uid jets were stretched during the fabrication of scaffolds, since velocities of ber collecting speed were larger than the uid jets formed in the present electrospinning conditions. Specically, the ber diameters of PLLA_x with x > 3.0 showed negative linear correlation with the collecting speed (p < 0.01, R 2 ¼ 0.94), and the diameter exhibited signicant smaller than that of PLLA_0.1. In contrast, the ber collecting speed of x # 3.0 is smaller than the velocity of the uid jet in this work; thus, the ber diameters showed no signicant difference for PLLA_x with x # 3.0. FD of PLLA_0.1 was À0.05 AE 0.09, which indicates that the bers were randomly oriented. FD of PLLA_10 was 0.96 AE 0.05, which shows that almost all bers were aligned parallel to the collector rotation direction. FD of PLLA_x with x > 0.1 showed signicant larger values compare with PLLA_0.1, which the bers randomly oriented. Moreover, FD of PLLA_x showed good correlation with the ber collecting speed using negative exponential decay function (R 2 ¼ 0.99), indicating that the Fig. 4 (a) Cell aspect ratio, (b) cell orientation degree (CD) on PLLA_x, and dashed lines represent correlation between the fiber collecting speed and cell aspect ratio and CD, respectively. Error bars represents standard deviation. morphology of brous scaffolds, such as ber diameter and alignment, can be controlled by the condition of electrospinning, e.g., ber collecting speed.
Cell uorescence images on PLLA_x are shown in Fig. 3(a-h), and their cell orientation angle histograms are shown in Fig. 3(i-p). The breadth of distribution for cell orientation angle decreased with increasing ber collecting speed, similar to the ber orientation angle distribution. Sun et al. reported that cells on the brous scaffolds showed different adhering behavior depending on the diameter of ber: a single ber for diameters larger than 10 mm, and several bers with spreading for diameters smaller than 10 mm. 33 Our previous work also showed similar tendency in the brous scaffolds with diameters > 6 mm, indicating that cells adhered on a single ber. 24,25 In this work, ber diameter of PLLA_10, which is the smallest ber diameter in PLLA_x, was approximately 6 mm; the cells on PLLA_x can adhere to a single ber surface. Cell aspect ratio on PLLA_x is shown in Fig. 4(a), and the ratio showed a linear correlation with the ber collecting speed (p < 0.01, R 2 ¼ 0.82). In case of PLLA_x with decreasing ber collecting speed, the bers showed larger number of cross points and the angles between the bers were larger, too. The cells on PLLA_0.1, where the bers were randomly arranged, were spread and adhered on several bers. However, those on PLLA_10 were adhered to single ber surfaces, elongated in the longitudinal direction of the ber. The cell aspect ratios on PLLA_x with x $ 5.0 exhibit signicant larger values compare with PLLA_0.1, due to the cells adhered on single ber. This is caused by the bers in the scaffolds were elongated and aligned during the electrospinning process, and followed decrease number of cross points. Thus, the aspect ratio of cells on PLLA_x increased with increasing ber collecting speed. The calculated CD on PLLA_x is shown in Fig. 4(b). CD of PLLA_0.1 was 0.02 AE 0.10, while that of PLLA_10 was 0.96 AE 0.01, indicating that the cells were random and parallel to the collector rotation direction, respectively. CD of PLLA_x with x > 1.0 showed signicant larger values compare with PLLA_0.1, which the bers randomly oriented. Additionally, CD and ber collecting speed showed a good correlation by negative exponential decay function (R 2 ¼ 0.98). Moreover, CD and FD showed linear correlation (p < 0.01, R 2 ¼ 0.95), as shown in Fig. 5. That is, cell alignment was successfully controlled by the morphology of the brous scaffolds, such as ber alignment.
Preferential orientation of the c-axis of apatite crystals was analyzed by mXRD system, and schematic illustration of analysis shown in Fig. 6(a). X-ray proles of apatite produced by primary osteoblasts on PLLA_x cultured for 4 weeks were showed in Fig. 6(b). PLLA_x showed the peaks corresponding to hydroxyapatite (ICCD card: 74-0566). The obtained X-ray proles were tted with Lorentzian functions; the dotted lines were reconstructed peaks of PLLA_0.1, which showed representative example of PLLA_x. The degree of preferential orientation of the c-axis in the apatite crystals was determined as the relative intensity ratio of the 002 diffraction peak to the 310 peak in the X-ray prole. This was previously reported as a suitable index for evaluating apatite orientation. 3,9,12,29,30 The degree of apatite caxis orientation (I 002 /I 310 ) of PLLA_x is shown in Fig. 6(c), with a linear correlation with the ber collecting speed (p < 0.01, R 2 ¼ 0.96). I 002 /I 310 values of PLLA_x with x > 5.0 showed signicant larger values compare with PLLA_0.1, which the bers randomly oriented. Moreover, FD and CD showed good correlation with the degree of apatite c-axis orientation by negative exponential decay function (vs. FD: R 2 ¼ 0.98, vs. CD: R 2 ¼ 0.99), as shown in Fig. 6(d). In our previous work, collagen matrix produced by aligning primary osteoblasts were oriented in the direction of cellular alignment, and the c-axis of the deposited apatite crystals indicated preferential alignment along the direction of the collagen matrix. 22 Consequently, c-axis orientation of bone apatite produced by primary osteoblasts on PLLA_x could be controlled by the morphology of ber alignment, i.e., ber collecting speed, which is similar to those of FD and CD. Therefore, morphology for the designed brous scaffolds in this work has successfully controlled cell alignment, as well as the direction of calcication, i.e., bone quality.

Conclusion
Designing brous scaffolds for reconstruction of bone quality was investigated. FD of PLLA_x increased with increasing ber collecting speed. Similarly, CD on PLLA_x increased with Fig. 6 (a) Schematic illustration of the analysis of apatite orientation using transmission mXRD system. Preferential orientation of the c-axis of apatite crystals was analyzed with integrated intensity ratio of 002/310 in X-ray profile. (b) X-ray profiles of apatite produced by primary osteoblasts on PLLA_x, which profiles were obtained parallel to the collector rotation direction of the scaffolds. Dotted line represent the Lorentzian curves of each peaks, which represents reconstructed pattern of PLLA_0.1. (c) Correlation between integrated intensity ratio of I 002 /I 310 , i.e. degree of apatite c-axis orientation along the collector rotation direction, and the fiber collecting speed. (d) Correlation between degree of apatite c-axis orientation and FD, and CD. Error bars represents standard deviation.
increasing ber collecting speed. Thus, cell alignment on the brous scaffolds can be controlled by their morphology, such as ber alignment. Furthermore, the apatite c-axis orientation degree, which is produced by primary osteoblasts, also increased with increasing ber collecting speed. Therefore, designing ber alignment of brous scaffolds with larger FD is more effective for bone quality reconstruction. These fundamental investigations are crucial to achieve further breakthrough in the research on regeneration of bone quality.

Conflicts of interest
Authors have no conict of interests to declare.