One-Dimensional Hydroxyapatite Nanostructures with Tunable Length

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One Dimension Hydroxyapatite Nanostructures with Tunable Length for Efficient Stem Cell Differentiation Regulation Baojin Ma, Shan Zhang, Feng Liu, Jiazhi Duan, Shicai Wang, Jing Han, Yuanhua Sang, Xiaoqiang Yu, Dong Li, Wei Tang, Shaohua Ge, and Hong Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b13313 • Publication Date (Web): 14 Sep 2017 Downloaded from http://pubs.acs.org on September 17, 2017

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ACS Applied Materials & Interfaces

One Dimension Hydroxyapatite Nanostructures with Tunable Length for Efficient Stem Cell Differentiation Regulation

Baojin Ma,1 Shan Zhang,1 Feng Liu,1 Jiazhi Duan,1 Shicai Wang,1 Jing Han,3 Yuanhua Sang,1 Xiaoqiang Yu,1 Dong Li,5 Wei Tang,*4 Shaohua Ge*3 and Hong Liu*1,2

1 State Key Laboratory of Crystal Materials, Shandong University, Jinan, Shandong, 250100, China 2 Institute for Advanced Interdisciplinary Research, Jinan University, Jinan, Shandong, 250100, China 3 Shandong Provincial Key Laboratory of Oral Tissue Regeneration, School of Stomatology, Shandong University, Jinan, Shandong, 250100, China 4 School of Basic Medical Science, Shandong University, Jinan, Shandong, 250100, China 5 Department of Pediatrics, Qilu Hospital of Shandong University, Jinan, Shandong, 250100, China

* Corresponding Author. Hong Liu, [email protected]; Shaohua Ge, [email protected]; Wei Tang, [email protected]

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Abstract It is well-accepted that most osteogenic differentiation processes do need growth factors assistance to improve efficiency. As a material cue, hydroxyapatite (HAp) can promote osteogenic differentiation of stem cells only in a way. Up to now, rare work related to the relationship between HAp nanostructures and stem cells in osteogenic differentiation process without the assistance of growth factors has been reported. In this study, one dimension (1D) HAp nanostructures with tunable length were synthesized by an oleic acid assisted solvothermal method by adjusting the alcohol/water ratio (η). The morphology of 1D HAp nanostructures can be changed from long nanowires into nanorods with the η value change. Different substrates constructed by 1D HAp nanostructures were prepared to investigate the effect of morphology of nanostructured HAp on stem cell fate without any growth factors or differentiation induce media. Adipose-derived stem cells (hADSCs), a kind of promising stem cell for autologous stem cell tissue engineering, were used as stem cell model. The experiments prove that HAp morphology can determine the performance of hADSCs cultured on different substrates. Substrate constructed by HAp nanorods (100nm) is of little benefit to osteogenic differentiations. Substrate constructed by HAp long nanowires (50µm) causes growth and spread inhibition of hADSCs, which even causes most cells death after 7 d culture. However, substrate constructed by HAp short nanowires (5µm) can destine the hADSCs differentiation to osteoblasts efficiently in normal medium (after 3 weeks) without any growth factors. It is surprise that hADSCs have changed to polyhedral morphology and exhibited the tendency to osteogenic differentiation after only 24 h culture. Hydroxyapatite nanostructures mediated stem cell osteogenic differentiation excluding growth factors provides a powerful cue to design biomaterials with special nanostructures, and helps to elucidate the interaction of stem cell and biomaterials nanostructures. The results from this study are promising for application in bone tissue engineering.

Keywords: HAp nanostructures; tunable length; cell viability and spread; without


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growth factors; osteogenic differentiation

1. INTRODUCTION With the development of tissue engineering for regenerative medicine, the relationship between cells and materials has attracted many researchers’ interest and effort.1-3 It is well-known that stem cells have high sensitivity to their cell microenvironment.4,5 As an artificial stem cell niche, surface topography of biomaterials plays a highly important role in regulating stem cell fate from morphology to oriented differentiation.6,7 Several pivotal studies have demonstrated that well-designed biomaterials with special nanostructure can possess promotion and even act as growth factors8,9 in stem cells differentiation regulation, which is of great significance to tissue engineering. The progress of this field has encouraged researchers to search for proper nanostructures to regulate stem cell fates efficiently. To exclude the effect of materials’ nature on stem cell differentiation, nanostructure is often taken as the single variable to study its tuning ability to stem cells fates. Nanostructures usually were fabricated by template methods or photolithography methods10-12 with demonstrated technology. Unfortunately, most related materials for constructed nanostructures are silicon or polydimethylsiloxane, which are not suitable for tissue regeneration in human body. In most cases, the differentiation evaluation is performed in differentiation induce media with growth factors, which is far from the real in vivo environment for tissue medicine. Hydroxyapatite (HAp, Ca10(PO4)6(OH)2), a kind of homologous material present in humans, is a major inorganic component of many hard tissues (e.g., bone and teeth). As a result, it has been treated as one of the favorite materials in tissue engineering, especially in bone or teeth restoration/regeneration because of its high levels of biocompatibility, safety, and FDA approval.13-21 HAp has been widely studied for many different bio-applications, such as bio-coating, bone scaffold, cell imaging, and drug carrier.22-30 HAp-based materials with different morphology have been synthesized by various methods, such as hydrothermal, sol-gel, and solid phase methods.31-33 It is well-known that HAp can promote stem cells osteogenic



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differentiation to certain extent by releasing Ca2+ ions, which in itself is an advantage over other materials in bone reparation.34,35 Meanwhile, HAp nanoparticles are usually chosen as the major nanostructure used in stem cells regulation and coating design due to their accessibility of preparation.36 However, the efficiency of HAp nanoparticles to osteogenic differentiation is very low, and almost all the differentiation cases involved extra growth factors that are inconvenient to practical tissue engineering. Therefore, it is necessary to resort to nanostructure strength to promote osteogenic differentiation efficiently. However, the relationship between morphology of HAp nanostructures and differentiation tendency of stem cells has been rarely studied, rather than to find an efficient nanostructure for cell differentiation regulation. Normally, HAp nanostructures with different morphology can be synthesized with different methodologies, adjusting various conditions and surfactant selections. Simply using HAp nanostructures synthesized through different methods to investigate the effect of nanostructures on stem cells differentiation fails in study rigor because of surrounding surface differences. Therefore, there is a great opportunity to investigate the regulation of stem cell by morphological information of nanostructured HAp with the same surface property. In this study, we first synthesized 1D HAp nanostructures with tunable length and same surface property under the same synthesis system, changing only the solvent composition by a modified hydrothermal method. The modified hydrothermal not relies on the expensive templates or equipment, which will bring low cost in practical applications. Further, the length of prepared HAp nanostructures can be tuned only changing the ratio of alcohol/water. Therefore, the modified hydrothermal method has enormous advantages especially in HAp synthesis with tunable nanostructures for tissue engineering. As prepared, 1D HAp nanostructures with different lengths had the same crystal phase, growth direction and surface surrounding, which ensured that the nanostructure will be the single variable. Human adipose-derived stem cells (hADSCs), an abundant autogenetic stem cell with potential applications for tissue regeneration and can be easily extracted from adipose following almost no harm to human compared with other stem cells, were taken as a stem cell model to assess the


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effect of 1D HAp nanostructures on stem cell differentiation. It is noted that stem cells osteogenic differentiation can be promoted obviously by HAp short nanowires with ~5 µm in length without any growth factors, rather than short HAp nanorods and long HAp nanowires. The results have demonstrated that the fate regulation of hADSCs was determined by the length of 1D HAp nanostructure, which provides a reliable variable to direct the synthesis of HAp nanostructure if used properly in osteogenic differentiation engineering, and if chosen as an efficient coating modification.

2. MATERIALS AND METHODS 2.1. Materials All chemical reagents were of analytical grade and used without any further purification. Ca(NO3)2·4H2O (99.0%), Na3PO4·12H2O (98%), oleic acid and absolute ethanol (99.7%) were purchased from Sinopharm (Shanghai, China). For cell experiments, α-minimum essential medium (α-MEM), fetal bovine serum (FBS) were purchased from Gibco; penicillin−streptomycin (penicillin 10000 IU/ml and streptomycin 10 mg/ml) were purchased from Genview; CCK-8 (Cell Counting Kit-8) were purchased from Dojindo; Live/Dead staining kit was purchased from Sigma. hADSCs were obtained from liposuction waste fat in the Department of Orthopedic Surgery of Qilu Hospital. The informed consent of liposuction waste use was obtained from all patients. 2.2. Synthesis of 1D HAp with Different Nanostructures HAp with different nanostructures were synthesized by a modified hydrothermal method. In brief, 8 ml of oleic acid, a certain amount alcohol and a certain amount NaOH aqueous solution (0.5 g) were mixed in a 50 ml beaker under vigorously stirring. 176 mg CaCl2 aqueous solution and 230 mg NaH2PO4.2H2O aqueous solution were successively added to the solution. The total volume is 36 ml and the ratio of alcohol and water is from 0:14 to 8:6. The solution was stirred for 15 min. The mixture was moved into a 40 ml Teflon lined autoclave, and heated at 180℃ for 12 h. The resulting product was first dispersed in alcohol and then collected by centrifugation at 10000 rpm for 10 min. The collected HAp were then washed 3-6



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times with ethanol. 2.3. Characterization of 1D HAp Nanostructures with Different Length The morphology observation and elemental analysis were performed on a S-4800 (Hitachi, Japan) scanning electron microscope (SEM) and on a JEM-2100 (Jeol, Japan) transmission electron microscope (TEM). X-Ray diffractograms (XRD) were recorded on a Bruker D8 advance powder diffractometer equipped with a Cu Kα sealed tube. 2.4. Cell Culture hADSCs were cultured in α-MEM medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin. hADSCs were maintained in a humidified atmosphere of 5% CO2 at 37℃, and the culture medium was changed every 2 days. 2.5. Cytocompatibility Evaluation of 1D HAp nanostructures The HAp with different nanostructures were dropped on the surface of culture plate and dried to form a coating, then sterilized in 75% ethanol overnight. hADSCs were seeded in different substrate and incubated in α-MEM medium. After cultured for 24 h, live/dead cells staining was performed to observe the cell state visibly. For quantitatively measure the cell viability, the medium was replaced by the new medium containing 10% CCK-8 solution to quantitatively evaluate the cell viability. After 2 h of incubation at 37℃, the medium containing water-soluble formazan dye, which has an absorption at 450 nm wavelength, was assayed by a microplate reader (Multiscan MK3; Thermo, USA). Three parallel replicates were used for each sample. 2.6. Alkaline Phosphatase (ALP) Activity After culturing in normal media for 7,14 and 21 d, cells cultured on different substrate were lysed by 0.1% Triton X-100 for ALP activity assays. The ALP activity was analyzed using a Lab Assay ALP activity assay kit (Wako Pure Chemical, Osaka, Japan) according to the manufacturer’s instructions. The total intracellular protein content was measured by a micro-BCA protein assay kit (Thermo Pierce) according to the manufacturer’s instructions. 2.7. Quantitative PCR (q-PCR) After culturing in normal media for 7, 14 and 21 d, cells on different substrate were


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treated with TRIZOL Reagent (Invitrogen) to extract the total RNA. The RNA concentration, purity and integrity were performed using a Q-5000 spectrophotometer (Quwell) at 260/280 nm and agarose gel electrophoresis. q-PCR analysis was performed using the 7500 Real Time PCR system (Applied Biosystems) for one housekeeping gene, β-actin and the three genes of Runx2, osteocalcin (OCN) and osteopontin (OPN). The relative transcript levels of the target gene expressions were normalized to β-actin and expressed as the mean ±S.D. (n = 3/group). 2.8. Osteogenic Immunofluorescence Staining After culturing in normal media for 14 d, hADSCs on different samples were washed with PBS and fixed with 4% paraformaldehyde for 10 min. Then, they were permeabilized using 0.1% Triton X-100 for 10 min and blocked with 10% goat serum solution for 1 h at room temperature. After blocking, the cells were incubated overnight at 4°C with primary antibodies at 1:500 dilution against osteocalcin (mouse monoclonal anti-OCN, Abcam) and at 1:1000 dilution against osteopontin (rabbit polyclonal anti-OPN, Abcam). Goat antirabbit and goat antimouse secondary antibodies labeled by Cy3 and Alexa Fluor 488 at 1:200 dilutions in 5% goat serum solution were used for staining OPN and OCN for 1 h at room temperature, respectively. After rinsing off the second antibody with PBS, the cells were further stained by phalloidin conjugated to Alexa Fluor 568 for 60 min and Heochest 33342 for 5 min to stain the F-actin and nuclei respectively. Images of the stained samples were obtained using an inverted microscope (Olympus IX71, Japan).

3. RESULTS AND DISCUSSION 3.1. Characterization of 1D HAp Nanostructures. The morphology of 1D HAp nanostructures with tunable length synthesized in solvent with different η (alcohol/water) was characterized by SEM and TEM (Figure 1). As the ratio (η) value increased, the length of 1D HAp nanostructures correspondingly decreased (Figure 1 and S1). HAp nanostructure synthesized under η=4:10 shows long nanowires morphology with lengths over 50 µm (Figure 1a, d and g). By increasing the value of η, the morphology of HAp becomes shorter nanowires, with



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lengths approximately 5 µm (Figure 1b, e and h, η=5:9). When the η value is set at 8:6, HAp nanorods develop with lengths of about only 100 nm (Figure 1c, f and i). The transition process is documented clearly in Figure1 and S1, as η values process from 0:14 to 8:6. One can conclude that the adjusting η value decides the length of 1D HAp nanostructures in the same synthesis system. The nanostructures of η=4:10 (named as long nanowires), η=5:9 (named as short nanowires) and η=8:6 (named as nanorods) represent the three typical and uniform morphology, and thus are chosen as the focused objects of study. Although the diameters of the nanostructures with different length look different (due to the different degree of twists of the wire-like nanostructure), the real width of 1D HAp nanostructures with different length is very similar (Figure S2 and Figure 1i), which is approximately 15 nm. This indicates that the alcohol/water ratio of the solvent in the synthesis process controls length obviously, and almost has no effect on width. To observe crystal lattice and growth direction of as-prepared 1D HAp nanostructures, HRTEM characterization was performed, as shown in Figure 1k-l. The three samples possess same and typical lattice space at (002) (~0.344 nm) and at (100) (~0.817 nm) respectively. This means that all samples grow along c-axis which is consistent with previous reports.37,38 The insets in Figure 1k-l shows the typical Fast Fourier Transform (FFT) patterns of HAp,39 which provides evidence to support the growth direction.


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Figure 1. SEM characterization of HAp nanowires to nanorods at different η. a, d and g, SEM images at different magnification when η=4:10; b, e and h, SEM images at different magnification when η=5:9; c, f and i, SEM images at different magnification when η=8:6; k-l, HRTEM images and corresponding FFT patterns of HAp long nanowires short nanowires and nanorods respectively.

To confirm the crystal phase and surface properties, X-ray diffraction (XRD) patterns and FTIR spectra of as-prepared HAp were performed (Figure 2). As shown in

Figure

2a

and S3,

all samples

have

the typical diffraction

peaks

(PDF#74-0565).40,41 No peaks were found that belong to CaCO3 or Ca3(PO4)2 phase. These results confirm that all prepared samples are of single HAp phase. Furthermore, it is obvious that the intensity decrease of (300) peak and increase of (112) and (002) peaks correlate with the increased η value. These intensity changes of corresponding peaks are consistent with the SEM and HRTEM results. As the Table S1 shown, crystallite size (C) value of different 1D HAp nanostructures were calculated by Scherrer formula according the previous reports.42-44 The full width at half maximum



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(FWHM) value was obtained in JADE 6 software. When the value of η is below 4:10, HAp has the big C value (>60nm). When the value of η is over 5:9, HAp has the little C value (

One-Dimensional Hydroxyapatite Nanostructures with Tunable Length

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