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Polydopamine-templated hydroxyapatite reinforced polycaprolactone composite nanofibers with enhanced cytocompatibility and osteogenesis for bone tissue engineering Xiang Gao, Jinlin Song, Ping Ji, Xiaohong Zhang, Xiaoman Li, Xiao Xu, Mengke Wang, Siqi Zhang, Yi Deng, Feng Deng, and Shicheng Wei ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b12413 • Publication Date (Web): 12 Jan 2016 Downloaded from http://pubs.acs.org on January 16, 2016

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ACS Applied Materials & Interfaces

Polydopamine-templated hydroxyapatite reinforced polycaprolactone composite nanofibers with enhanced cytocompatibility and osteogenesis for bone tissue engineering Xiang Gao,#∆‡ Jinlin Song, #∆‡ Ping Ji, #∆‡ Xiaohong Zhang,§ Xiaoman Li,‖ Xiao Xu,† Mengke Wang,† Siqi Zhang,§ Yi Deng,§ Feng Deng,* #∆‡ and Shicheng Wei*∆§†

#

College of Stomatology, Chongqing Medical University, Chongqing 401147, China

∆

Chongqing Key Laboratory of Oral Diseases and Biomedical Sciences, Chongqing

Medical University, Chongqing 401147, China ‡

Chongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher

Education, Chongqing 401147, China §

Center for Biomedical Materials and Tissue Engineering, Academy for Advanced

Interdisciplinary Studies, Peking University, Beijing 100871, China †

Department of Oral and Maxillofacial Surgery, Laboratory of Interdisciplinary Studies,

Peking University School and Hospital of Stomatology, Beijing 100081, China ǁ

Department of Cariology and Endodontology, Peking University School and Hospital

of Stomatology, Beijing 100081, China KEYWORDS: polydopamine, nano-hydroxyapatite, nanofiber, human mesenchymal

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stem cells, bone tissue engineering

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ABSTRACT: Nano-hydroxyapatite (HA) synthesized by biomimetic strategy is a promising nanomaterial as bone substitute due to its physicochemical features similar to those of natural nanocrystal in bone tissue. Inspired by mussel adhesive chemistry, a novel nano-HA was synthesized in our work by employing polydopamine (pDA) as template under weak alkaline condition. Subsequently, the as-prepared pDA-templated HA (tHA) was introduced into polycaprolactone (PCL) matrix via co-electrospinning, and a bioactive tHA/PCL composite nanofiber scaffold was developed targeted at bone regeneration application. Our research showed that tHA reinforced PCL composite nanofibers exhibited favorable cytocompatibility at given concentration of tHA (0~10 w.t%). Compared to pure PCL and traditional nano-HA enriched PCL (HA/PCL) composite nanofibers, enhanced cell adhesion, spreading and proliferation of human mesenchymal stem cells (hMSCs) were observed on tHA/PCL composite nanofibers on account of the contribution of pDA present in tHA. More importantly, tHA nanoparticles exposed on the surface of composite nanofibers could further promote osteogenesis of hMSCs in vitro even in the absence of osteogenesis soluble inducing factors when compared to traditional HA/PCL scaffolds, which was supported by in vivo test as well according to the histological analysis. Overall, our study demonstrated that the developed tHA/PCL composite nanofibers with enhanced cytocompatibility and osteogenic capacity hold great potential as scaffolds for bone tissue engineering.

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1.

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Introduction Bone tissue engineering aims at the reconstruction and functional recovery of

seriously damaged bone tissue associated with trauma, osteoporosis, cancer, and congenital abnormalities.1-2 As a direct result, there is an urgent demand for developing advanced bioactive scaffolds that can facilitate the reorganization of functional tissue by guiding stem cell differentiation.3 A range of processing approaches like phase separation, extrusion, porogen leaching, computed tomography-guided fused deposition modeling and electrospinning have been employed to fabricate scaffolds with different compositions and three dimensional configurations.4-9 Among these scaffolds, electrospun nanofibers have gained tremendous interest in recent years owing to the structural similarity to the bone tissue extracellular matrix (ECM).5 It is well known that scaffold architecture plays a critical role in the regulation of stem cell behaviours such as attachment, proliferation and differentiation, and, thus, electrospun nanofibers represent a promising scaffold for bone tissue engineering.5 During past decades, a group of synthetic polymers like poly(glycolic acid), poly(lactic acid), poly(ε-caprolactone) and their copolymers have been extensively studied for bone tissue engineering applications due to their bioresorbability, high mechanical strength and low swelling ratio.3-4 Among them, PCL, a Food and Drug Administration (FDA) approved material, has attracted more attention, as it can be readily processed into scaffolds with specific shape due to its relative low melting point

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(55-60 ℃) and good blend-compatibility with other additives.4 However, PCL-based scaffolds have some shortcomings like slow in vivo degradation rate and lack of bioactivity,5, 10 limiting their further application in bone tissue engineering. To overcome these limitations, combining polymers with bioactive inorganic materials, such as hydroxyapatite,11 β-tricalcium phosphate12 and silica,3,

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via co-electrospinning is

considered a fascinating and advisable strategy to desigin scaffolds with appropriate properties for bone tissue engineering, because native bone ECM is essentially an organic–inorganic composite nanofibers organized on micro- and nanoscale.5 In view of the intrinsic osteoconduction and osseointegration potential,14 nano-hydroxyapatite (nano-HA; Ca10(PO4)6(OH)2), the major mineral constituent of bone matrix,15 is one of the most attractive inorganic materials for applications in bone regenerative medicine,6,

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and therefore, has been widely used to improve

physicochemical and biological properties of polymer nanofibers via blending approach.5 For example, Jyh-Ping Chen et al. developed PCL-HA composite nanofibers by incorporating nano-HA particles within PCL electrospun fibers.11 Compared to pure PCL nanofibers, the composite nanofibers containing nano-HA exhibited enhanced elastic modulus, as well as favorable adhesion, proliferation and osteogenic differentiation of hMSCs.11 Although some prospective results were achieved, the traditional methods for nano-HA synthesis often require harsh reaction conditions like elevated temperature and/or extreme pH value, giving rise to the concerns of researchers

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over the security of nano-HA for clinical applications.17 Moreover, the biological property of nano-HA largely depend on size, morphology, crystallinity and surface property that can be tailored in synthetic HA crystals to optimize their suitability for specific biomedical applications.15,

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However, the routine methods often yield HA

crystals with irregular size, morphology or crystallinity (unlike nanocrystals of natural HA),19-21 which may lead to uncontrolled biological behavior.18 Hence, there is a pressing demand to develop an approach which can produce nano-HA not only under environment-friendly reaction conditions but also in a controllable manner. Recently, intensive research efforts have focused on the synthesis of apatite crystals, which can resemble natural apatite in bones and dentals.18, 22-23 Biomimetic synthesis, a bottom-up strategy, has drawn extensive attention, since it can regulate the mineralization and growth direction of crystal nuclei, which simulates the process of HA nucleation and growth occurred in native bone tissue.24 Studies have demonstrated that nano-HA synthesized by biomimetic process exhibited improved chemical and physical properties, as well as biological property, compared to nano-HA produced by conventional

approaches,

such

as

chemical

precipitation

and

hydrothermal

synthesis.22-23 Many biomacromolecules like collagen, gelatin and polysaccharides have been reported to serve as biomimetic templates to synthesize nano-HA via functional groups present in the molecules,22-23 which can chelate Ca2+ ions and form hydrogen bonds with protonated PO43- and H2O on the surface of mineral. Inspired by the

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mechanism of biomimetic synthesis, a novel pDA-templated nano-HA (briefly named tHA) was developed in our study via catechol chemistry. pDA is a mimic of the specialized adhesive foot protein, Mefp-5 (Mytilus edulis foot protein-5), in which the catechol moiety could strongly bind to various metal ions.25-27 Jungki Ryu et al. previously reported an universal biomineralization route, which enable the directional c-axis growth of HA crystals upon the pDA film adhering to various scaffold materials.25 It is suggested that pDA is an intriguing candidate as template for biomimetic synthesis of nano-HA. Moreover, the surface modification of materials with pDA could also benefit cell adhesion and osteogenic differentiation, which has been well documented by several literatures.28-29 Therefore, it is reasonable to anticipate that the synthesized tHA-pDA nanocomposite would display improved cytocompatibility and osteogenic potential over traditional HA nanoparticles. Despite the attractive advantages and progress in preparation of various PCL composite nanofibers, the employment of tHA as nano reinforcement in PCL-based composite nanofibers for bone tissue engineering, to our knowledge, has not been reported. Hence, in the present study, a pilot and preliminary work was conducted on the fabrication of tHA/PCL composite nanofibers by co-electrospinning, and the biofunctionalities of composite nanofibers were then systematically investigated in vitro and in vivo (Figure 1). We believe the tHA-enriched electrospun PCL nanofibers with enhanced cytocompatibility and osteogenic activity hold a promising potential for bone

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regenerative medicine. 2. 2.1

Materials and methods Synthesis of pDA-templated nano-HA Ca (NO3)2 (Sino-pharm Chemical Reagent Co., Ltd) and Na2HPO4 (Sino-pharm

Chemical Reagent Co., Ltd) were dissolved in deionized water (DW) separately according to a Ca/P molar ratio of 1.67. Dopamine hydrochloride (Sigma) as template was added to Ca (NO3)2 solution at a concentration of 2 mg/ml. Then, Na2HPO4 solution was slowly dropped into Ca (NO3)2-dopamine mix solution with continuous stirring. Apatite growth occurred when kept at 60 ℃ for 12 h, and the pH of solution was maintained to approximate 8.5 via addition of Tris-HCl solution (10 mM, Aladdin). After reaction, the HA slurry was aged for 24 h at 37 ℃, and the precipitate was harvested after alternatively washing with DW and ethanol by centrifugation (6000×g), followed by dialysis in DW for 3 days to remove unattached dopamine molecules. Finally, the prepared precipitate was air dried overnight in an oven at 60 °C for future use. The traditional nano-HA was purchased from Sigma as control. 2.2

Preparation of electrospun tHA/PCL nanofibers PCL (MW=80000 g/mol, Sigma) was dissolved in Hexafluoro-2-propanol (HFIP,

Aladdin) at a concentration of 10% (w/v, PCL/HFIP). Then, a defined amount of tHA (0, 1 wt%, 5 wt%, 10 wt%, 20 wt%) was suspended in PCL solution by ultrasonic and vigorous stirring. The electrospinning of tHA/PCL composite nanofibers was carried out

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with a commercial electrospinning equipment (SS-2535, YongKang Technology Co., Ltd.) using a stainless-steel blunt needle (23 G). The electrospinning parameters were set at the applied voltage of 14 kv, the collection distance of 10 cm, and the feed rate of 1.0 ml/h. The as-prepared electrospun fibrous meshes were vacuum dried at room temperature (RT) to remove residual organic solvent for future use. For comparison, neat PCL and traditional nano-HA (10 wt%)-doped PCL nanofibers (10%HA/PCL) were also fabricated under same electrospinning conditions. 2.3

Morphological and chemical characterization The morphology of tHA was observed by transmission electron microscopy (TEM,

H-9000, Hitachi) with an operating voltage of 100 kV. Samples for TEM imaging were dispersed into DW by ultrasonic waves and the suspension was dropped onto carbon-coated copper grids, air-dried before observation. The incorporation of tHA within nanofibers was also confirmed by TEM at an acceleration voltage of 100 kV. Before observation, the nanofibers were electrospun directly onto holey carbon-coated copper grids. The surface morphology of tHA-incorporated nanofibers was observed using field-emission scanning electron microscopy (FE-SEM, S-4800, Hitachi). Before observation, the samples were sputtered with gold and examined at an accelerating voltage of 5 kV. 100 nanofibers from SEM images were randomly selected for measurement of fiber diameters using ImageJ software.

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The crystalline phases of tHA and traditional nano-HA were examined and compared by X-ray diffraction (XRD) using a Cu target as radiation source (λ= 1.540598 Å) at 40 KV. The diffraction angles (2θ) were set between 10° and 60° increments, with a step size of 4 min-1. Thermo-gravimetric analysis (TGA; Q600, TA Instruments) was employed to determine the actual yield of pDA in tHA. Samples (5 mg) were heated from 20 ℃ to 800 ℃ with a heating rate of 10 ℃/min under nitrogen atmosphere. Fourier transform infrared (FTIR) microspectroscopy (Nicolet 750, USA) was performed to identify the functional groups of tHA nanoparticles in a wavenumber range of 400 to 4000 cm-1. X-ray photoelectron spectroscopy (XPS; AXIS Ultra, Kratos Analytical Ltd.) was employed to identify the composition and elemental state of samples. The binding energies were calibrated by the C1s hydrocarbon peak at 284.8 eV. The quantitative analysis was conducted using CasaXPS software package. Water contact angle of the electrospun meshes was measured using a contact angle goniometer (SL200B, Kono, USA) under ambient temperature and humidity. The meshes were attached to coverslips and five droplets of DW were placed randomly at different locations on each sample for measurement. Mechanical property evaluation of the electrospun meshes was performed by universal testing machine (UTM, Instron 5900, Instron) at a crosshead speed of 10

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mm/min under a load of 10 N. Before measurement, five pieces of rectangular meshes were cut with dimensions of 20 mm × 60 mm. The elastic modulus of samples were obtained from stress–strain curves. Protein adsorption assay was performed by incubating FITC-labeled bovine serum albumin (FITC-BSA, 100 µg/ml, Sigma) with samples for 60 min at 37 ℃.30 To quantify the amount of absorbed proteins on nanofibers, the supernatant was collected after incubation and detected at 488 nm excitation wavelength and 525 nm emission wavelength using Multilabel Reader (2300, Perkin Eimer). The amount of absorbed BSA was calculated from a standard curve based on a series of FITC-BSA solutions with known concentrations. Afterwards, samples were rinsed with PBS and captured under laser scanning confocal microscope (LSCM, Carl Zeiss, Germany) to visualize the attached proteins. 2.4

Cell culture hMSCs were purchased from Sciencell Research Laboratories and maintained in

normal growth media containing α-MEM media (Gibco), 10% FBS (Gibco), 1% PS (Gibco) and 2 mM L-glutamine (Gibco) under standard conditions (humidified condition, 37 °C, 5% CO2). Prior to cell experiments, the prepared meshes were cut into round shape and sterilized with 70% ethanol for one hour, followed by thorough rinse with disinfected D-Hanks buffer. When reaching 70-75% confluence, cells were dissociated with trypsin-EDTA (Gibco) and seeded onto samples at a density of 2×104

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cell/cm2. After 24 h, the media was replaced with fresh normal growth media or osteoinductive media. Osteoinductive media was prepared by supplementing ascorbic acid (50 µg/ml, Sigma), β-glycerophosphate (10 mM, Sigma), and dexamethasone (100 nM, Sigma) to the normal growth medium. The media was changed every two days. 2.5

Cytocompatibility evaluation of tHA/PCL composite nanofibers

2.5.1 Early adhesion assay The early adhesion and spreading of hMSCs on samples were evaluated using SEM and LSCM. At 2 and 6 h after seeding, cells were fixed with glutaraldehyde (2.5%, Sigma) for 2 h at RT. Then, cells were dehydrated in gradient ethanol (30, 50, 70, 80, 90, 95, and 100%) for 10 min per step, and dried with hexamethyldisilazane. Afterwards, samples were sputtered gold and examined under SEM. For cytoskeleton observation, cells were fixed with paraformaldehyde (4%, Sigma) for 10 min, and permeabilized with Triton X-100 (0.1%, Sigma) for 5 min. Subsequently, cells were dyed with FITC-phalloidin (5 µg/ml, Sigma) for 20 min and captured under LSCM. Moreover, to quantify the number of early adherent cells on different samples, cells were detached using trypsin–EDTA and counted by haemocytometer at 2 h after seeding. 2.5.2 Cell proliferation assay Cell proliferation on samples was evaluated using cell counting assay kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan) following the manufacturer’s protocol. Briefly, at 1, 5, 7 days, the culture media was replaced with CCK-8 reagent solution (10%) and

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incubated at 37 ℃ for 4 h. After that, the media supernatant (100 µl) from each well was transferred to a fresh 96 well-plate for measurement of absorbance value at 450 nm using microplate reader (Model 680, Bio-Rad). 2.6

Cytotoxic mechanism of tHA/PCL composite nanofibers

2.6.1 ROS assay Intracellular production of reactive oxygen species (ROS) in hMSCs cultured on samples was evaluated using dihydroethidium (DHE) staining as previously described.31 After 24 h of seeding, cells were incubated with DHE (1 mM, Sigma) at 37 ℃ for 20 min. Then, cells were rinsed with PBS three times and viewed under LSCM (excitation at 488 nm and emission at 525 nm). To further measure cellular ROS, cells were harvested with trypsin after reaction. The fluorescence was immediately analyzed by flow cytometry (FACSCalibur, BD, USA). Untreated cells and cells pretreated with Rosup (50 µg/mL) for 60 min were used as negative and positive control, respectively. 2.6.2 Cell cycle assay For cell cycle assay, cells on samples were trypsinized and pooled by centrifugation at 1500 g for 5 min at 24 h after culture. Following washing twice with PBS, cells were fixed in cold ethanol (70 %) overnight at 4 ℃, and treated with Rnase A (0.1 mg/ml) for 15 min. Then, cells were dyed with PI solution (0.1 mg/ml) for 30 min. The DNA content in cells was detected by flow cytometry, and the percentage of cells in each phase of cell cycle was determined using ModFit software.

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2.6.3 LDH assay Lactate dehydrogenase (LDH) leakage assay was conducted to assess membrane integrity of cells cultured on samples. After incubation for 24 h, the media from each well was collected and detected using a LDH assay Kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s protocol. The absorbance was recorded at 450 nm using microplate reader. The lysed cells on tissue culture plate (TCP) were used as positive control. The LDH leakage was expressed as the percentage of the amount of LDH released from test group to the maximum amount of LDH released from positive control. 2.7 Osteogenic bioactivity evaluation of tHA/PCL composite nanofibers 2.7.1 Alkaline phosphatase (ALP) assay ALP activity of hMSCs on samples was assessed using an ALP assay reagent kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China). Briefly, after 14 days of culture, cells were lysed with Triton X-100 (1%) for 1 h at 4 ℃. Afterwards, cell lysates (30 µl) were mixed with ALP assay working solution and detected following the manufacturer’s instruction. The absorbance of reaction product was measured at 405 nm using a plate reader. The value of ALP activity was normalized by total protein content which was determined by a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific), and expressed as U/mg protein. Meanwhile, enzyme-histochemistry staining was conducted to visualize ALP distribution on sample surface using a BCIP/NBT ALP

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color development kit (Beyotime Biotechnology, Shanghai, China) following the manufacturer’s instruction. 2.7.2 Alizarin Red S (ARS) staining assay The calcium mineralization on samples were evaluated on day 21 by ARS staining assay. Briefly, cells were fixed in 95% ethanol for 1h at RT, followed by washing with DW. Then, the samples were immersed in ARS solution (2%, pH 4.2, Sigma) for 10 min at RT. After thorough washing with DW, the samples were captured using a scanner. To further quantify the extent of calcium mineralization, the stained samples were incubated with cetylpyridinium chloride solution (10%, Sigma) for 1h at RT, and the absorbance of eluate was recorded at 550 nm using a plate reader. The meshes (without seeding cells) in each group were also stained with ARS solution as control. 2.7.3 Real-time polymerase chain reaction (RT-PCR) After 14 days of culture, total RNA of cells on samples was isolated with Trizol reagent (Sigma) and reverse transcribed with RevertAid™ First Stand cDNA Synthesis Kit (Fermentas, Vilnius, Canada) according to the manufacturer’s instruction. Afterwards, RT-PCR was performed by ABI PRISM 7500 sequence detection system (Applied Biosystems, CA, USA) using SYBR Green PCR Master Mix. The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was employed as house keeping gene. The primer used in this study were: runt-related transcription factor 2 (Runx2),

(forward)

5´-AGGAATGCGCCCTAAATCACT-3´

and

(reverse)

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5´-ACCCAGAAGGCACAGACAGAAG-3´; type I collagen alpha 1 (Col1a1), (forward) 5´-AGACACTGGTGCTAAGGGAGAG-3´ 5´-GACCAGCAACACCATCTGCG-3´;

and osteocalcin

(reverse) (OCN),

(forward)

5´-CCTGAAAGCCGATGTGGT-3´ and (reverse) 5´-AGGGCAGCGAGGTAGTGA-3´; GAPDH,

(forward)

5´-CGACAGTCAGCCGCATCTT-3´

and

(reverse)

5´-CCAATACGACCAAATCCGTTG-3´. The cycle threshold values (Ct values) were applied to determine the fold differences by ∆∆Ct method. Untreated cells in normal growth media were assigned as control. 2.7.4 Immunocytochemistry After 14 days of culture, cells on samples were fixed with paraformaldehyde (4%) for 10 min at RT, permeabilized with Triton X-100 (0.1%) for 5 min. After washing three times with PBS, cells were incubated with BSA solution (1%, Sigma) for 2 h at 37 ℃ to block nonspecific binding. Then, cells were incubated with primary antibodies at 4 ℃ overnight. The following primary antibodies were used: mouse monoclonal anti-human OCN (1:100 dilution, Abcam), rabbit polyclonal anti-human osteopontin (OPN, 1:500 dilution, Abcam). After incubation with primary antibodies, cells were washed thrice with PBS and incubated for 1 h at RT with FITC-488 goat anti-rabbit (1:1000, CST) and TRITC-543 goat anti-mouse (1:1000, CST) secondary antibodies. Fluorescence signals were viewed under LSCM. 2.7.5 Western blot

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After 14 days of culture, cells on samples were lysed with RIPA buffer. Then, the total protein concentrations were determined using BCA protein assay kit. The proteins were separated on SDS-PAGE and transferred to PVDF membranes (Millipore, USA). After blocking with skim milk (5%) in TBST for 1h, the membranes were probed with Runx2 (1:3200, CST) and Col1a1 (1:1000, Santa Cruz) antibodies overnight at 4 ℃. Then, bands were visualized after incubation for 1 h with horseradish peroxidase (HRP)-conjugated secondary antibodies by chemiluminescence using an ECL detection kit (Amersham). GAPDH (1:1000, CST) antibody was used as an internal control. 2.8 In vivo experiment 2.8.1 Subcutaneous implantation Subcutaneous implantation experiment was performed on six healthy male BALB/c-nu mice aged 6 weeks and weighing 20±2 g for an observation period of 4 weeks after surgery, referring to Longwei Lv et al.’s study design.30 The protocol was approved by the Animal Ethics Committee of the Peking University (Approval no: LA2015149), and the animals were housed in compliance with the national guidelines for the care and use of laboratory animals. Before implantation, 10%HA/PCL and 10%tHA/PCL composite nanofibers were co-cultured with hMSCs in vitro for seven days. Afterwards, nude mice were anaesthetized via intraperitoneal injection of pentobarbital sodium (1%, 50 mg/kg, Sigma). After inducing general anesthesia, one longitudinal skin incisions was made on

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the dorsal surface of each mouse, and one subcutaneous pocket per mouse was created by blunt dissection. Each individual pocket held one cell-scaffold construct, and the incisions were closed with surgical sutures. Three samples for each group were implanted into three different animals, respectively. At four weeks post-implantation, the implants were retrieved and fixed in 4% paraformaldehyde overnight for further analysis. 2.8.2 Histology and immunohistochemistry (IHC) The samples from each group were decalcified in 10% EDTA at pH 7.4 for 2 weeks at 4 ℃ with a bi-weekly change of solution and subsequently embedded in paraffin. Serial sections (5 µm thick) from each sample were prepared for hematoxylin/eosin (HE) and Masson staining. For IHC staining, rehydrated paraffin-embedded sections were washed with PBS, and the endogenous peroxidase was blocked by incubation with 3% hydrogen peroxide for 15 min. Following thoroughly washing, the sections were incubated overnight with primary antibody against OPN (1:100; rabbit monoclonal) at 4 ℃. Afterwards, sections were rinsed in PBS and exposed to HRP-conjugated secondary antibody (1:100; goat anti-rabbit) for 60 min at RT. Finally, the 3-3’ diaminobenzidine (DAB) peroxidase substrate system was used for color development, followed by hematoxylin staining. The slices were examined under light microscope. 2.9 Statistical analysis All experiments were carried out in triplicate, and the data were expressed as mean

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± standard deviations and analyzed using SPSS software. One-way analysis of variance (ANOVA) and Tukey’s post hoc test were used to determine the significant differences among the groups. A value of P < 0.05 was considered statistically significant. 3

Results and discussion

3.1 Characterization of tHA nanoparticles Figure 2a illustrates the biomimetic synthesis mechanism of apatite crystals using pDA as template. Under weak alkaline (PH=8.5), dopamine molecules could initiate self-polymerization and form pDA structure.32 This polymer provides abundant catecholamine moieties as Ca2+ ion binder.25 With the formation of Ca-P nucleation centers, crystals began to precipitate and grow along the surface of pDA. The ultimate product is designated as tHA. To improve the nucleation and growth of crystals, the reaction temperature was maintained at 60 ℃ during synthesis and the resulting suspension was aged at 37 ℃ for 24 h. The morphology of tHA crystals was characterized by TEM (Figure 2b). Compared to rod-like traditional HA nanoparticles (Figure S1a), tHA crystals displayed a plate-like structure with a larger size (diameter: 31 ± 12 nm; length: 98 ± 22 nm), similar to the size and morphology of natural HA in bone tissue.18, 33 Partly as a result of strong interfacial adhesion effect of pDA, which accounted for about 9% of total weight based on the analysis of TG curves (Figure 2c), tHA nanoparticles were inclined to aggregate into clusters. XRD spectra (Figure 2d) showed that the Bragg diffraction

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peaks of tHA nanoparticles agreed quite well with those of traditional nano-HA at 2θ values of 25.9°, 31.8°, 32.8°, 39.7°, 46.6°, 49.4° and 53.1°, which were indexed to (002), (211), (300), (310), (222), (213) and (004) planes, respectively.22 In particular, the intense bands at around 2θ=26° and 2θ=33° proved that the mineralized phase was mainly HA.22 However, it is noted that three intense peaks corresponding to (211), (112) and (300) became broader in synthesized tHA compared to traditional nano-HA, indicating the low crystallinity of tHA nanoparticles as that of minerals present in human bone.34 Figure 2e presents the FTIR spectra of tHA nanoparticles. Similar to the spectra of traditional nano-HA, tHA samples displayed two broad absorption bands at 3435 cm-1 and 1632 cm-1, corresponding to the absorbed water. The typical peaks for CO32derived from atmosphere were recorded at approximate 1464, 1417, 875 cm-1, suggesting that trace amounts of PO43- were substituted by CO32-.25 Moreover, several characteristic absorption peaks of PO43- were also observed: the non-degenerate symmetric stretching mode ν1 at 960 cm-1, doubly degenerate bending mode ν2 at about 470 cm-1, the triply degenerate antisymmetric stretching vibration ν3 at 1105 and 1037 cm-1, the triply degenerate vibration ν4 at 605 and 567 cm-1.22 Particularly, the peak at 960 cm-1 is a representative indication of crystalline HA.25, 35 However, no additional peaks for pDA were detected in tHA samples, indicating that the vibration bands of pDA may be overlapped with those of nano-HA due to a relative low content of pDA in

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crystals. It is noteworthy that the stretching band of phosphate at 1037 cm-1 in traditional nano-HA shifted to 1031 cm-1 in tHA, implying the formation of strong interaction between the Ca-P phase and pDA constituent. To further verify the result obtained from FTIR, XPS was performed to provide direct evidence of chemical composition. As shown in Figure 2f, traditional nano-HA displayed carbon, oxygen, calcium, phosphorus peaks as the main atomic elements. On the other hand, a newly weak signal for nitrogen (N 1s) appeared at 399 eV in the wide spectra from tHA, which was clearly revealed in the high-resolution nitrogen spectra (Figure 2g). Moreover, the analysis of the elementary composition (Table S1) showed that tHA nanocrystals had a lower Ca/P ratio (1.37) than stoichiometric ration in HA, suggesting the calcium-deficient state on the tHA crystalline surface.22 But current calcium-deficient products are more beneficial for biological applications than stoichiometric ones, because the Ca/P atomic ratio in native bones is lower than 1.67.36 Calcium-deficient HA could elicit instant precipitation of biologically equivalent apatite on its surface when implanted in vivo, whereas precipitation on stoichiometric HA requires an induction time.37 In addition, An obvious change in carbon bond (C 1s) was also found in the high-resolution carbon spectra (Figure S1b, c). The C 1s spectrum of traditional nano-HA can be deconvoluted into three different curves: C-C, C-O and C=O, whereas a new curve for C-N bond was detected near 285 eV in tHA, proving the presence of pDA. Additionally, compared to traditional nano-HA, the quantitative

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results of funtional groups showed a significant increase in the content of hydroxyl and carbonyl groups from tHA samples(Figure S1d), which was ascribed to the profuse catechol and oxidized catechol (i.e. quinone) groups in tHA, suggesting that tHA nanoparticles may harbor similar bioactivity as pDA. 3.2 Characterization of electrospun tHA/PCL composite nanofibers The synthesized tHA nanoparticles displayed similar physicochemical features with nanocrystals in bone tissue, but their extremely brittle nature may limit their use to loading-bearing applications. Therefore, the tHA nanoparticles were then doped with PCL nanofibers via electrospinning to overcome their mechanical weaknesses for bone tissue engineering. Figure 3a displays the fabrication of tHA/PCL composite nanofiber scaffolds via electrospinning technology. The effect of tHA on the surface morphology and fiber diameter of the electrospun scaffolds was examined by SEM and TEM. As shown in Figure 3b and Figure S2, the pure PCL electrospun fibers showed uniform and smooth surface morphology. However, with the introduction of tHA, the nanoparticles made the fiber surface rough in a concentration dependent manner, and plenty of nodule-like structures were detected in fibers (Figure S2a), which was ascribed to the aggregation of tHA nanoparticles. Similar surface characteristic was also observed in 10%HA/PCL composite nanofibers (Figure S2g). Moreover, the distribution of fiber diameter was noted to gradually shift to smaller scale as the increase of tHA concentration in PCL nanofibers. The possible explanation for this

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change is that the electron charge density of the prepolymer solution was increased due to the addition of tHA.38 A similar phenomenon was also reported in earlier works. The incorporation of nano-HA into collagen type I,39 silk fibroin40 led to the decrease in fiber diameter and the formation of electrospun fibers with high surface roughness. However, there was no significant difference in fiber diameter between 10%HA/PCL and 10%tHA/PCL group (P>0.05). In view of the significant influence on hMSCs adhesion, proliferation, and differentiation,41-42 surface wettability and elastic modulus are two important parameters that should be considered, when designing scaffolds for bone tissue engineering. Table S2 presents the measurement of water contact angle and elastic modulus in our work. Numerous

superhydrophobic

materials,

like

polytetrafluoroethylene

and

poly

(vinylidene fluoride), were reported to show increased wettability when coated with pDA, owing to the hydrophilicity of catecholamine in dopamine molecules.32,

43

Moreover, the enhanced roughness was also reported to contribute to the surface wettability of hydrophobic substrates.44 Thereby, contact angle measurement showed that after co-electrospinning, the pDA-containing HA nanoparticles improved the hydrophilicity of PCL nanofibers to a certain extent, with increase in tHA concentration. Compared to 10%HA/PCL nanofibers, surface wettability of the 10%tHA/PCL was significantly enhanced (P0.05). Compared with PCL nanofibers, tHA/PCL composite nanofibers showed significant increase in elastic modulus as the nanoparticle concentration rose, indicating enhancement in stiffness of the scaffolds due to the presence of tHA, similar to the previous reports.38, 45 However, when the concentration of tHA was elevated to 20%, the elastic modulus of composite nanofibers was significantly reduced, which was probably ascribed to the obvious decrease in fiber diameter. For developing a scaffold for bone tissue engineering, the material should be able to withstand dynamic mechanical loading under in vivo conditions.46 From the mechanical testing results, all the composite nanofibers prepared in our study have elastic modulus in the range of 6 to 17 MPa, which endowed them with appropriate mechanical strength for bone tissue engineering.46 Moreover, the enhanced elastic modulus was also believed to have a favorable effect on the osteogenic differentiation of hMSCs.41 Protein adsorption is the first event taking place at the biomaterial-tissue interface when scaffolds are implanted into body, which plays a critical role in the subsequent

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cell behaviors like adhesion, proliferation and differentiation. Therefore, a nonspecific FITC-labeled BSA was employed to evaluate the bioaffinity of tHA/PCL composite nanofibers to proteins in vitro. Figure 3c displays the adhesion of albumin on the surface of samples within the initial 60 min. As a consequence of high specific surface area, pure PCL meshes induced uniform protein adsorption and showed weak green fluorescence on the fiber surface. With the addition of tHA particles, some fluorescence aggregates with higher intensity emerged in polymer matrix and increased with the augmentation of particle concentration. It is noted that more protein molecules were adsorbed to 10%tHA/PCL composite nanofibers when compared to 10%HA/PCL nanofibers according to the quantitative determination of adsorbed proteins (P

Polydopamine-Templated Hydroxyapatite ... - ACS Publications

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