Strontium-Doped Amorphous Calcium Phosphate Porous

Subscriber access provided by GAZI UNIV

Article

Strontium-Doped Amorphous Calcium Phosphate Porous Microspheres Synthesized through A Microwave-Hydrothermal Method Using Fructose 1,6-Bisphosphate as An Organic Phosphorus Source: Application in Drug Delivery and Enhanced Bone Regeneration Weilin Yu, Tuan-wei Sun, Chao Qi, Zhen-yu Ding, Hua-kun Zhao, Feng Chen, Dao-yun Chen, Ying-Jie Zhu, Zhongmin Shi, and Yaohua He ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b12325 • Publication Date (Web): 10 Jan 2017 Downloaded from http://pubs.acs.org on January 11, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Strontium-Doped

Amorphous

Calcium

Phosphate

Porous

Microspheres

Synthesized through A Microwave-Hydrothermal Method Using Fructose 1,6-Bisphosphate as An Organic Phosphorus Source: Application in Drug Delivery and Enhanced Bone Regeneration

Weilin Yu,a, 1 Tuan-Wei Sun,b, c, 1 Chao Qi,b, c Zhenyu Ding,a Huakun Zhao,a Feng Chen,b, c Daoyun Chen,a Ying-Jie Zhu,*, b, c, Zhongmin Shi *, a and Yaohua He *, a, d

a

Department of Orthopedics, Shanghai Jiao Tong University Affiliated Sixth People’s

Hospital, 600 Yishan Road, Shanghai 200233, China. b

State Key Laboratory of High Performance Ceramics and Superfine Microstructure，

Shanghai Institute of Ceramics, Chinese Academy of Sciences, 1295 Dingxi Road, Shanghai 200050, China. c

University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049, China.

d

Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, School of

Biomedical Engineering, 600 Yishan Road, Shanghai 200233, China.

ABSTRACT: Nanostructured calcium phosphate (CaP) porous microspheres are of great potential in drug delivery and bone regeneration due to their large specific surface area, biocompatibility and similarity to inorganic component of osseous tissue. In this work, strontium (Sr)-doped amorphous calcium phosphate porous microspheres (SrAPMs) were synthesized through a microwave-hydrothermal method

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 42

using fructose 1,6-bisphosphate trisodium salt (FBP) as the source of phosphate ions. The SrAPMs showed a mesoporous structure and a relatively high specific area. Compared

with

the

hydroxyapatite

(HAP)

nanorods

prepared

by

using

Na2HPO4˙12H2O as the phosphorus source, the SrAPMs with a higher specific surface area were more effective in drug loading using vancomycin as the antiobiotics of choice, and consequently having a higher antibacterial efficiency both on agar plates and in broths. Furthermore, to assess the potential application of SrAPMs in bone defect repair, a novel biomimetic bone tissue-engineering scaffold consisting of collagen (Coll) and SrAPMs was constructed using a freeze-drying fabrication process. Incorporation of the SrAPMs not only improved the mechanical properties, but also enhanced the osteogenesis of rat bone marrow mesenchymal stem cells (rBMSCs). The in vivo experiments demonstrated that the SrAPMs/coll scaffolds remarkably enhanced new bone formation compared with the Coll and APMs/coll scaffolds in a rat critical-sized calvarial defect model at 8 weeks post-implantation. In summary, SrAPMs developed in this work are promising as antibiotic carriers and may encourage bone formation when combined with collagen. KEYWORDS: calcium phosphate, drug delivery, biomimetic, scaffold, bone regeneration

1. INTRODUCTION Multifunctional bioactive materials for the repair of bone defects resulting from tumor resection, osteomyelitis, trauma or skeletal deformities have gained increasing interest


Page 3 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


in recent decades.1-4 Particularly, bone reconstruction in the defects caused by infections remains a great challenge because of the difficulty in eradication of pathogens. The patients with chronic bone infection usually need surgical debridement and long-term systemic antibiotics therapies.5 While long-term administration of antibiotics may cause adverse effects such as antibiotics resistance, and bone loss after debridement needs extraneous osseous grafts to maintain the bone integrity. Hence, development of biomaterials simultaneous with localized and sustained antibiotics delivery and the capacity to stimulate bone regeneration would be desirable for repairing bone defects caused by osteomyelitis. Calcium phosphates, such as HAP, as the main mineral composition of bone and tooth are widely used materials with good biocompatibility and osteoconductivity.6-7 Recently, nanostructured CaP porous microspheres with large specific surface area have attracted great attention because of their promising prospect in drug delivery, tissue engineering and bone regeneration.8-10 Until now, various methods, including solvothermal methods,11 polymer-assisted assembly from calcium phosphate nanorods,12-13 spray drying method,14 surfactant-assisted hydrothermal method,15 emulsion,16 ion-assisted assembly mineralization methods,17 etc., have been reported for the synthesis of nanostructured CaP microspheres. However, most of the synthesis methods involved the using of organic solvent, template, surfactant or chelating ligand, which may be harmful to environment and health.18 Moreover, despite the progress that has been achieved, the development of environment-friendly, harmless, facile and rapid synthesis methods of CaP nanostructured porous microspheres



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 42

remains a major challenge. Recently, microwave-assisted synthesis characterized with time saving, high efficiency and low energy consumption has become a hotspot of research.19-21 Generally, CaP microspheres are synthesized by using the inorganic phosphate such as Na2HPO4. In recent years, biocompatible organic phosphorus sources such as creatine phosphate and adenosine 5’-triphosphate disodium salt (ATP) have been used to synthesize nanostructured CaP porous microspheres through the microwave-hydrothermal method.19,

22

It was reported that the HAP mesoporous

microspheres synthesized using creatine phosphate biomolecules had a high drug-loading capacity and exhibited a sustained drug release profile due to their large specific surface area and hierarchically mesoporous nanostructure.19 Uskokovic et al. demonstrated that HAP nanospheres were more efficient in drug loading

and in

inhibiting bacterial growth because of their greater surface area than flaky, brick-like or elongated hydroxyapatite particles.8 Hence, it was hypothesized that the CaP porous microspheres synthesized using organic phosphorus sources would be more effective in antibiotics delivery than the HAP nanorods synthesized using Na2HPO4 as the phosphorus source. Although CaP based biomaterials such as β-tricalcium phosphate (β-TCP) and HAP, are extensivley applied for bone defect repair because of their good biocompatibility and osteoconductivity,23 the lack of the ability to stimulate new bone formation hampers their clinical application.24 Strontium is an essential trace element in human body, which can potentially substitute for calcium in calcium phosphate, calcium silicate or bioactive glasses to further enhance their biological performance due to


Page 5 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


their similarities in charge and ionic radius.25-26 More importantly, Sr has been demonstrated to stimulate bone regeneration and inhibit osteoclastic activity.27 Titanium implant surface modified with Sr element showed superior bone regeneration performance and better implant osseointegration.28 Sr-modified calcium phosphate cement possesses higher osteogenic activity and is particularly favorable for the repair of osteoporotic bone defects.29 As an essential trace element, Sr not only promotes the osteoblastic differentiation of mesenchymal stem cells, but also inhibits the osteoclast activity by suppressing the receptor activator of nuclear factor kappa-B (RANK) signaling pathway.30-31 In the present study, we reported a novel synthetic strategy for Sr-doped amorphous CaP porous microsphere (SrAPMs) through a microwave-hydrothermal synthesis process by using FBP biomolecules. On the one hand, the SrAPMs were loaded with vancomycin as the antibiotic of choice to investigate its drug delivery properties, and the antibacterial efficiency was evaluated both in broths and on agar plates. On the other hand, to explore the potential application in bone regeneration, the SrAPMs were incorporated into collagen that has been optimized for bone repair,32-33 with an intention to increase the bioactivity and mechanical properties, and more importantly, to enhance the osteogenic activity crucial for bone regeneration. Moreover, the SrAPMs/coll composite scaffolds mimic the structure and composition of the osteogenic niche in trabecular bone. The osteoinductive potential of the SrAPMs/coll scaffolds was evaluated in vitro by directly culturing with the rBMSCs, and the capacity to promote bone regeneration was assessed in vivo in a rat critical-sized



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

calvarial defect model.

2. MATERIALS AND METHODS 2.1. Synthesis of the SrAPMs. The SrAPMs with designed Sr/(Sr+Ca) molar ratio of 0.1 were synthesized through the microwave-hydrothermal process using FBP (Sangon Biotech, Shanghai) as the phosphorus source.34 The molar ratio of Sr/(Sr+Ca) was determined according to previous studies in which approximate 10 mol% Sr substitution for Ca was optimal for bone regeneration.35-36 For the synthesis of SrAPMs, 0.0999 g CaCl2 (Sinopharm) and 0.0267 g SrCl2·6H2O (Aladdin) were dissolved in 25 mL deionized water, then an aqueous solution of FBP (8.13 mg mL-1, 15 mL) was added dropwise under continuous stirring. Meanwhile, a 1 M NaOH aqueous solution was added to maintain the pH value of the above solution at 10. After continuous stirring for 10 min, the above mixture was transferred into a 60 mL autoclave and heated at 120 ˚C for 10 min in a microwave oven (MDS-6, Sineo, China). After cooling, the obtained products were collected by centrifugation, followed by rinse with deionized water and being dried. The control samples were synthesized under the same conditions using FBP or Na2HPO4˙12H2O (0.215 g, Sinopharm) as the phosphorus sources. 2.2. Vancomycin Loading and Release Assay. For the vancomycin loading, 100 mg of different samples (HAP nanorods, APMs and SrAPMs) were added separately into a 10 mL aqueous solution of vancomycin (2 mg mL-1). After 15 min of ultrasonic treatment, the suspensions were sealed and shaken (120 rpm) at 37 °C for 24 h, then


Page 6 of 42

Page 7 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the vancomycin-loaded samples (Van-HAP nanorods, Van-APMs and Van-SrAPM) were collected by centrifugation and dried. The vancomycin-loading capacities of different samples were determined by measuring the vancomycin concentrations in the adsorbed supernatants with a UV-vis spectrophotometer (UV-2300, Techcomp) at 280 nm wavelength. For the vancomycin release assay, 20 mg vancomycin-loaded samples were dispersed in 20 mL normal saline (NS). Then the suspensions were shaken at a constant speed of 120 rpm at 37 °C. At predetermined time points, 0.5 mL release medium was withdrawn to measure the concentration of released vancomycin, and replaced with 0.5 mL fresh NS. 2.3. Scaffold Fabrication. Three types of scaffolds corresponding to three groups were prepared: (1) Coll scaffold, (2) APMs/coll scaffold, and (3) SrAPMs/coll scaffold. For fabrication of the scaffolds, a homogeneous collagen suspension (40 mg g–1) was prepared by mixing the lyophilized collagen sponge (Kele Biological Technology Co. Ltd, China) with deionized water under continuous magnetic stirring. Then, APMs or SrAPMs were added to the collagen suspension, and the weight ratio of collagen to APMs or SrAPMs was 1:1. After vigorous stirring, the homogeneous mixture was loaded into a 48-well plate, frozen at −20 ˚C for 24 h and lyophilized at −20˚C for 48 h. Finally, the scaffolds were chemically cross-linked using 20 mM N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC, Sigma) and 8 mM N-hydroxysuccimide (NHS, Sigma) in 80/20 ethanol/deionized water for 8 h,37 followed by rinse with ethanol and deionized water for three times. The structures of



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

the as-prepared scaffolds were observed using scanning electron microscopy (SEM). The scaffolds were sterilized with 29 kGy of 60Co radiation before use. 2.4. Physical Characterization of Scaffolds. 2.4.1. Scaffold Mechanical Properties Measurement. The scaffolds (Φ10 mm × 10 mm) were first immersed in deionized water for 12 h. The SmartTest (Drick, China) universal testing machine was used to measure the mechanical properties of the scaffolds at a crosshead speed of 5 mm min−1 (n = 4). The compressive modulus was calculated in the strain range from 15 % to 25% of the stress-strain curve. 2.4.2. Ions Release from SrAPMs/coll Scaffolds. In a typical experiment, 25 mg of SrAPMs/coll scaffold was immersed in 15 mL NS38 under constant shaking. At predetermined time points, 0.5 mL of medium was collected for the measurement of the concentrations of Sr, Ca and P elements using an inductively coupled plasma (ICP) optical emission spectrometer (JY 2000-2, Horibia, France), and replaced with 0.5 mL fresh NS. 2.5. In Vitro Cellular Responses to Scaffolds. 2.5.1. Cell Viability on Scaffolds. The Cell Counting Kit-8 assay (CCK-8; Dojindo Molecular Technologies Inc., Japan) was used to evaluate the viability of rBMSCs on the scaffolds after 1, 3 and 7 days of culture. Briefly, the scaffolds (Φ10 mm × 2 mm) within a 48-well plate were seeded with 1×105 cells in 0.5 mL medium. At each time point, after removing the medium, 500 µL medium containing 10% CCK-8 solution was added to each well containing different scaffolds. After incubating for 2 h, aliquots (100 µL) were transferred to a


Page 8 of 42

Page 9 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


96-well plate, and the absorbance was measured using a microplate reader (Bio-Rad 680, USA) at 450 nm wavelength.

Seven days after seeding, the viability of rBMSCs was further assessed using a Live/Dead Cell Viability Assay kit (Invitrogen) according to the manufacturer’s instructions. The rBMSCs on the scaffolds were observed using a confocal laser scanning microscope (CLSM; LSM 510, Zeiss).

2.5.2. Cell Morphology on the Scaffolds. The cell morphology on the Coll, APMs/coll and SrAPMs/coll scaffolds was observed using SEM and CLSM. For SEM observation, 1×105 rBMSCs were seeded on each scaffold (Φ10 mm × 2 mm) and cultured for 7 days. Then the scaffolds were fixed with 2.5% glutaraldehyde for 4 h, followed by dehydration with different concentrations of ethanol. After freeze-drying, the scaffolds with cells were sputtered with platinum and observed using SEM.

As to the cytoskeleton staining, the samples prepared as described above were rinsed and subsequently fixed in 4% paraformaldehyde for 2 h, followed by a pretreatment with Triton X-100 (0.5% v/v) for 5 min. Subsequently, the samples were stained with rhodamine-phalloidin (Sigma) and DAPI (Sigma) for 30 min and 5 min, respectively, and the cells were imaged using CLSM. 2.5.3. Gene Expression Analysis. The effects of the scaffolds on the osteogenic differentiation of rBMSCs were assessed by real-time quantitative PCR (RT-qPCR). Briefly, approximately 2 × 105 rBMSCs were seeded on the scaffolds within 48-well



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

culture plates and cultured for 4 and 7 days. Total RNA was extracted by homogenizing the scaffolds with cells in 1 mL of Trizol reagent (Invitrogen). A PrimeScript 1st Strand cDNA Synthesis kit (Takara, Japan) was used to reverse transcribe the RNA into complementary DNA (cDNA). Quantification of cDNA was performed using a real-time PCR kit (SYBR Premix EX Taq, Takara) on an ABI7500 Thermal Cycler (Applied Biosystem, Australia). All assays were run in triplicate in three independent experiments. 2.6. Critical-Sized Rat Calvarial Defect Repair. 2.6.1. Animal Surgical Procedures. All experimental procedures concerning animals were approved by the Animal Research Committee of Sixth People’s Hospital, Shanghai Jiao Tong University School of Medicine. Eighteen SD rats were randomly divided into three groups: (1) Coll scaffold (n = 6), (2) APMs/coll scaffold (n = 6) and (3) SrAPMs/coll scaffold (n = 6). After anaesthesia, an approximately 2 cm sagittal incision was made on the scalp, followed by blunt dissection of periosteum to expose the calvarium. Two critical-sized defects were created using a 5-mm electric trephine (Nouvag AG, Goldach, Switzerland) under constant irrigation with NS. After implantation with the scaffolds (Φ5 mm × 2 mm), the incisions were closed layer by layer using absorbable suture. 2.6.2. Micro-Computed Tomography (Micro-CT) Measurement. Eight weeks following operation, the rats were sacrificed, and the calvaria were collected and fixed in 10% buffered neutralized formalin. Then, the calvaria were scanned using micro-CT (Skyscan 1176, Kontich, Belgium) at a resolution of 18 µm. The


Page 10 of 42

Page 11 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


reconstruction of three-dimensional (3D) images was completed using CTVox program (Skyscan Company). Both bone volume to total volume (BV/TV) and bone mineral density (BMD) in the defects were measured using the CTAn program (Skyscan Company). 2.6.3. Histological Observation. Following micro-CT scanning, the calvaria were decalcified, dehydrated and embedded in paraffin. Then, coronal sections (5 µm thickness) at the central region of the defects were prepared and stained with hematoxylin and eosin (HE). Osteogenic markers including OCN and OPN were examined with immunohistochemistry (IHC) staining. Briefly, the sections were incubated with primary antibodies against OCN (ab13420, 1:100; Abcam) or OPN (ab63856, 1:200, Abcam) at 4 ˚C overnight. Then the sections were incubated with goat anti-rat biotin-conjugated IgG (Beyotime, China) for 1 h. Color reaction was developed using the avidin-biotin-peroxidase complex (ABC) method. Finally, the sections were counterstained with hematoxylin and observed with an optical microscope. 2.7. Statistical Analysis. The data were presented as mean ± standard deviation and analyzed using SPSS software. One-way analysis of variance and Student-Newman-Keuls post hoc tests were used for multiple comparisons. Significant differences were accepted when p < 0.05. 3. RESULTS 3.1. Characterization of SrAPMs. The morphologies of the products and the EDS element mapping of the SrAPMs are shown in Figure 1. The control sample consisted



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

of HAP nanorods, whereas the APMs and SrAPMs exhibited a porous spherical structure with a relative uniform size. The distribution of Sr, Ca, P, C and O elements in SrAPMs by EDS element mapping indicates that Sr was homogenously distributed within the SrAPMs. As shown in Figure 2A, the crystal phase of the control sample consisted of a single phase of hydroxyapatite with a hexagonal structure (Ca10(PO4)6(OH)2, JCPDS No. 09-0432), whereas the XRD patterns of the APMs and SrAPMs showed the characteristic humps of the amorphous calcium phosphate phase at around 30˚, indicating that the APMs and SrAPMs consisted of amorphous calcium phosphate. Figure 2B shows the FTIR spectra of the products. From the FTIR spectrum of the control sample of HAP nanorods, one can see that the absorption peaks at around 1093 (ν3), 1038 (ν3), 961 (ν1), 602 (ν4) and 565 (ν4) cm−1 were assigned to the characteristic bands of PO43− in HAP. The FTIR spectra of APMs and SrAPMs exhibited intense absorption peaks at around 1093 and 565 cm−1, indicating the presence of amorphous calcium phosphate. Moreover, the FTIR spectra of APMs and SrAPMs showed the weak absorption peaks at around 1458 (δ−CH2−) and 798 cm−1 (=C−H), indicating the presence of FBP and/or fructose molecules within the APMs and SrAPMs. Figure 2C shows the TGA results of the products. A weight loss of 4.72% of HAP nanorods was observed after heating, whereas the weight loss for the APMs and SrAPMs were 25.10% and 27.34%, respectively. As shown in Figure 2D, the SBET of HAP nanorods, APMs and SrAPMs were 85.8, 106.7 and 110.1 m2 g-1, respectively, and the Barrett−Joyner−Halenda (BJH) desorption cumulative volume (VP) of HAP


Page 12 of 42

Page 13 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


nanorods, APMs and SrAPMs were 0.98, 1.02 and 0.69 cm3 g-1, respectively. The average BJH desorption pore sizes of HAP nanorods, APMs and SrAPMs were 32.3, 25.7 and 17.2 nm, respectively (Figure 2E). 3.2. In Vitro Vancomycin Release Properties. The vancomycin-loading capacities of APMs and SrAPMs were 28.8 ± 0.9 and 30.3 ± 0.6 mg g-1, respectively, which were higher than that of the HAP nanorods (22.9 ± 0.6 mg g-1). As shown in Figure 3A, the Van-APMs and Van-SrAPMs exhibited rapid vancomycin releases of approximately 63.8 ± 2.2% and 60.5 ± 2.4% during 12 h, respectively. With the increase of time, the drug release rates of the Van-APMs and Van-SrAPMs decreased gradually. The vancomycin that loaded in APMs and SrAPMs was not released completely and reached an equilibrium release of 86.9 ± 2.4% and 83.5 ± 2.0%, respectively, which may result from the strong interactions between the vancomycin molecules and the drug carriers. The Van-HAP nanorods showed a similar drug release performance with that of the Van-APMs or Van-SrAPMs, which reached an equilibrium release of 80.4 ± 4.3% in a period of about 36 h. As shown in Figure 3B, the relationships between the cumulative release amount of vancomycin and the square root of the release time for the HAP nanorods, APMs and SrAPMs drug-delivery systems showed good linear relationships with high regression factors of > 0.99, indicating that the drug release kinetics of the HAP nanorods, APMs and SrAPMs drug-delivery systems are governed by the diffusion of the Higuchi model.39 3.3. In Vitro Antibacterial Assays. The antibacterial assays were performed both



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

in broths and on agar plates to test the antibacterial efficiency of Van-HAP nanorods, Van-APMs and Van-SrAPMs. As shown in Figure 3C, an apparent difference in the antibacterial efficiency between Van-HAP nanorods and Van-APMs or Van-SrAPMs was observed in the particle concentration range of 1/32 to 1 mg mL-1. The minimum inhibitory concentration (MIC) values for Van-HAP nanorods, Van-APMs and Van-SrAPMs were 1/4, 1/8 and 1/8 mg mL-1, respectively. A similar result of the antibacterial efficiency was observed on contaminated surfaces (Figure 3D). The inhibition zone diameters on M-H agar plates for Van-APMs or Van-SrAPMs were significantly larger than those for the Van-HAP nanorods, and the inhibition zone diameters for the 3-mg group were significantly larger than those for the 1-mg group. However, no significant difference in antibacterial efficiency was observed between Van-APMs and Van-SrAPMs. No inhibitory effect was observed for the vancomycin-free samples. The images of visual appearance of the M-H broths and inhibition zones formed on the M-H plates are shown in Figure S1. 3.4. Scaffold Characterization. As shown in Figure 4A, the Coll, APMs/coll and SrAPMs/coll scaffolds were highly porous and exhibited a hierarchical structure. The pore structure was highly interconnected and uniformly distributed with pore sizes ranging from 100 to 300 µm. Higher magnification images showed that the surfaces of the APMs/coll and SrAPMs/coll scaffolds were rough and embedded with spherical particles, whereas the surface of the Coll scaffolds was smooth. As shown in Figure 4B and C, the compressive elastic moduli of APMs/coll and SrAPMs/coll scaffolds were 9.94 ± 1.09 kPa and 10.73 ± 0.93 kPa, respectively,


Page 14 of 42

Page 15 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


which were higher than that of the Coll scaffolds (4.22 ± 1.80 kPa). Figure 4D shows the dissolution performance of Sr, Ca and P elements from the SrAPMs/Coll scaffolds. The Sr, Ca and P elements showed a rapid release in the first day, followed by a much slower increase thereafter. The initial burst releases of Sr, Ca and P elements were about 34.7 ± 1.7%, 21.6 ± 0.8% and 25.3 ± 0.8% respectively within 24 h. The dissolution of Sr, Ca and P elements trended toward a release stage of equilibrium for 17 days and the cumulative releases of Sr, Ca and P elements were about 46.7 ± 1.1%, 28.4 ± 0.5% and 35.9 ± 0.1%, respectively. 3.5. Viability and Morphology of the rBMSCs on the Scaffolds. To assess the viability of the rBMSCs on the scaffolds, Live/Dead cell staining and CCK-8 assay were performed (Figure 5A and D). Live/Dead cell staining showed that cells remained alive for up to 7 days on each type of scaffold. The cell density on the Coll scaffolds was highest among the three types of scaffolds, and more cells attached on the SrAPMs/coll scaffolds than on the APMs/coll scaffolds. As determined by CCK-8 assay, there was no apparent cell proliferation for all the three types of scaffolds across the period examined, and the numbers of rBMSCs on the scaffolds followed the trend: Coll scaffold > SrAPMs/coll scaffold > APMs/coll scaffold. The cytoskeleton staining showed that the rBMSCs exhibited a well-spreading morphology in response to all the three types of collagen-based scaffolds (Figure 5B). The SEM micrographs further showed that the cells spread well and presented clear and prominent filopodia on the collagen-based scaffolds (Figure 5C). 3.6. Osteogenesis-Related Gene Expression of rBMSCs. To assess the osteogenic



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

response of rBMSCs on the Coll, APMs/coll and SrAPMs/coll scaffolds, RT-qPCR was performed. The gene expression of Runx2, ALP, OCN and OPN was analyzed at days 4 and 7 (Figure 6). Relative to the Coll scaffolds, the APMs/coll and SrAPMs/coll scaffolds enhanced the expression levels of Runx2, ALP and OPN at day 4, and SrAPMs/coll scaffolds stimulated the higher expression of Runx2 compared with the APMs/coll scaffolds. Prolonging the culture time to 7 days, the rBMSCs cultured on the APMs/coll and SrAPMs/coll scaffolds showed higher expression of Runx2, ALP, OCN and OPN compared with those cultured the Coll scaffolds, while the SrAPMs/coll scaffolds stimulated the highest expression levels of Runx2, ALP, OCN and OPN. 3.7. Evaluation of Osteogenesis In Vivo. 3.7.1. Micro-CT Assessment. To assess the new bone formation in the bone defects, micro-CT was first perfomed. A remarkable increase of calcified tissues was observed in the defects implanted with SrAPMs/coll or APMs/coll scaffolds compared to those implanted with Coll scaffolds, and the SrAPMs/coll scaffolds induced the most remarkable tissue calcification (Figure 7A). Moreover, quantitative analysis showed that the BMD and BV/TV for the SrAPMs/coll group (334.78 ± 41.08 mg cm-3 and 48.30 ± 11.75%) were higher than those for the APMs/coll group (213.12 ± 52.05 mg cm-3 and 20.64 ± 7.33%) and the Coll group (55.88 ± 14.89 mg cm-3 and 3.36 ± 0.76% ) (Figure 7B and C). 3.7.2. Histological Analysis of bone formation. The bone regeneration in the defects was further assessed by HE staining. HE staining of representative sections from each group is presented in Figure 8A and B. The defects implanted with Coll scaffolds


Page 16 of 42

Page 17 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


were mainly occupied by connective tissue and typical bone structure was scarcely observed. There was more new bone formed in the defects filled with SrAPMs/coll scaffolds compared with those implanted with APMs/coll scaffolds. The in vivo osteogenic activity of the scaffolds was further evaluated by IHC staining for osteogenic markers OCN and OPN (Figure 8C and D). The OCN and OPN were highly expressed in APMs/coll and SrAPMs/coll groups. Moreover, the defects implanted with SrAPMs/coll scaffolds showed more intense expression of OCN and OPN compared to those implanted with APMs/coll scaffolds.

4. DISCUSSION In the present study, Sr-doped amorphous calcium phosphate microspheres were successfully synthesized using FBP as the phosphorus source through a microwave-hydrothermal method. Compared with the HAP nanorods synthesized using Na2HPO4˙12H2O as the phosphorus source, the SrAPMs with a mesoporous structure and a higher specific surface area were more effective in vancomycin loading, consequently the Van-SrAPMs having superior antibacterial performance compared to Van-HAP nanorods. Furthermore, to explore the potential application of SrAPMs in bone regeneration, a biomimetic bone tissue-engineering scaffold was fabricated by incorporating the SrAPMs into collagen matrix that has been optimized for bone regeneration. The inclusion of SrAPMs not only improved the mechanical properties, but also enhanced the osteogenic responses in vitro and in vivo. The APMs and SrAPMs were mainly composed of amorphous calcium phosphate



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

and exhibited a uniform porous spherical morphology, while the control sample prepared using Na2HPO4˙12H2O consisted of HAP nanorods (Figure 1), indicating that biocompatible FBP can prevent the conversion of amorphous calcium phosphate into HAP crystal and plays an important role in the formation of CaP nanostructured microspheres. According to the experiment results and our previous studies,34, 40 a possible mechanism was proposed to explain the formation of SrAPMs. Under microwave-hydrothermal conditions, FBP molecules continuously hydrolyze to produce fructose and PO43- ions, followed by nuclei formation from a reaction among PO43-, Ca2+ and Sr2+ ions. Then the newly formed nuclei assemble into SrAPMs, driven by minimization of the surface free energy of the product. As shown in Figure 1, Sr was homogenously distributed within the SrAPMs, indicating that Sr was successfully doped into the APMs. Due to the chemical analogy to calcium, Sr could substitute the calcium positions in bone crystal.41 The TGA results showed that the weight losses of APMs and SrAPMs were larger than that of HAP nanorods (Figure 2C). The amorphous calcium phosphate generally contains 10-20 wt% of tight bound water, and the APMs and SrAPMs may also contain fructose or FBP molecules, which was confirmed by the FTIR spectra of the APMs and SrAPMs (Figure 2B). The APMs and SrAPMs had a higher specific surface area compared with the HAP nanorods. The average BJH desorption pore sizes of APMs and SrAPMs were 25.7 and 17.2 nm, respectively (Figure 2E), which were within the range of 2-50 nm. The mesoporous structure of APMs and SrAPMs may account for their higher specific surface area. Moreover, Lin et al. reported that the assembly of the HAP nanocrystals into porous


Page 18 of 42

Page 19 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


spherical structure leads to a larger specific surface area.18 The mesoporous structure and higher specific surface area of APMs and SrAPMs bode well for their applications as drug nanocarriers. Then, the drug delivery properties of the APMs and SrAPMs were investigated using vancomycin as the antibiotic of choice. The vancomycin-loading capacities of the APMs and SrAPMs were significantly higher than that of the HAP nanorods. The higher drug-loading capacities of APMs and SrAPMs may result from their mesoporous structure and higher specific surface area, which provides sufficient space for the adsorption of drug. The temporal drug release profiles for the HAP nanorods, APMs and SrAPMs vancomycin-delivery systems showed high similarity (Figure 3A). Since the drug was loaded by physisorption, all the vancomycin-delivery systems showed a rapid release in the first 24 h, followed by a zero-order release period with a slower release rate throughout the following 6 days. Although an initial burst release is not desirable for the delivery of many therapeutic drugs, this is not so for antibiotics where an initial rapid release is necessary to ensure that the released antibiotics can reach the therapeutic concentration within a short time and to prevent the bacterial strain becoming resistant to the antibiotics. For the HAP nanorods, APMs and SrAPMs drug-delivery systems, the relationships between the cumulative release amount of vancomycin and the square root of the release time showed a good linear relationship with a high regression factor of > 0.99 (Figure 3B), which is in agreement with the Higuchi model,39 indicating that the vancomycin release from Van-HAP nanorods, Van-APMs and Van-SrAPMs may be governed by a diffusion process. As



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

was to be expected, the antibacterial tests against S. aureus, a common pathogen of osteomyelitis, demonstrated that vancomycin-loaded APMs and SrAPMs showed superior antibacterial performance than the vancomycin-loaded HAP nanorods both on agar plates and in broths (Figure 3C and D). A direct correlation was observed between the specific surface area of the drug carriers and the antibacterial efficiency. Namely, the greater surface area of APMs and SrAPMs predisposed them to be more effective in adsorbing the vancomycin molecules and consequently, more effective in preventing the bacterial growth. The results of antibacterial tests are in agreement with a previous study by Uskokovic et al.8 It was reported that the hydrogen bonding interactions between the OH groups which both exist in the HAP crystals and drug molecules facilitated the adsorption of vancomycin molecules on the surface of HAP crystal.42 The APMs and SrAPMs with higher specific surface area may possess more OH groups, which promote the adsorption of vancomycin molecules. The satisfying drug loading and release properties of SrAPMs bode well for their potential application in treating osteomyelitis. To investigate the potential application of the SrAPMs in bone regeneration, a biomimetic composite SrAPMs/coll scaffold was synthesized by incorporating the SrAPMs into collagen matrix through a lyophilization fabrication process. Higher compressive moduli were achieved by incorporating the APMs or SrAPMs resulting from the reinforcing effect of the microspheres within the collagen matrix (Figure 4B and C). Quilan et al. demonstrated that the incorporation of bioactive glass particles improved the mechanical properties of the collagen-based scaffolds.43 The


Page 20 of 42

Page 21 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


submicron-sized APMs or SrAPMs have a relatively high specific surface area, and consequently a composite scaffold may exhibit improved mechanical properties resulting from strong interactions at interfaces. Moreover, the porosity of the collagen scaffolds was maintained after the incorporation of APMs and SrAPMs, which utilized the high surface area and strong interactions to reinforce the collagen scaffold while maintaining desirable porosity.44 The composite scaffolds fabricated in the present work presented interconnectivity with pore sizes ranging from 100 to 300 µm (Figure 4A), a size range that is suitable for the cell filtration and ingrowth of bone tissue.45 The rBMSCs spread well and maintained their phenotype on all the three types of scaffolds (Figure 5A-C ), which proved the biocompatibilty of these collagen-based scaffolds. However, on SrAPMs/coll scaffolds, cell viability was found lower than that on the Coll scaffolds but higher than that on the APMs/coll scaffolds (Figure 5D). Cell growth inhibition induced by excessive ions such as calcium and phosphate ions released form the scaffolds may account for the reduction of cell viability on the APMs/coll and SrAPMs/coll scaffolds, an effect that could be minimised under in vivo condition.43, 46 And the stimulative effects of Sr on cell proliferation 35, 47 may account for the higher cell viability on the SrAPMs/coll scaffolds than that on the APMs/coll scaffolds. The ability of the scaffolds to stimulate osteogenesis was carried out by culturing the rBMSCs in direct contact with the materials. Our results showed that the APMs/coll and SrAPMs/coll scaffolds improved the expression of Runx2, ALP, OCN



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

and OPN compared to the Coll scaffolds (Figure 6), indicating that the APMs/coll and SrAPMs/coll scaffolds enhaned the osteogenic differentiation of the rBMSCs. On the one hand, the composite scaffolds that resemble the structure and composition of trabecular bone may play as an osteogenic niche which is beneficial for the osteogenic differentiation of rBMSCs. The stem cell microenvironment significantly influences stem cell behavior and regulates its fate. The calcium and phosphate ions released from the composite scaffolds may also have a stimulatory effect on the osteogenic commitment of rBMSCs. Zhang et al. reported that CaP/silk scaffolds enhanced the osteogenic differentiation of BMSCs compared with the pure silk scaffolds.48 Rezwan et al. reported that the calcium and phosphate ions released from the calcium phosphate materials could induce osteoblast differention and mineralization.49 Minardi et al. reported that a biomimetic Mg-doped HAP/collagen composite scaffold mimicking the osteogenic niche could stimulate the osteogenesis of hBMSCs and augment ectopic bone formation.33 On the other hand, the incorporation of APMs or SrAPMs improved the mechanical properties of the Coll scaffolds, and the physical interactions with the extracellular matrix may affect the differentiation of stem cells. The commitment of cells to specific lineage is extreme sensitive to the tissue-level elasticity, and rigid matrices that resemble the texture of collageous bone have proven to be osteogenic.50 More importantly, the Sr ions released from the SrAPMs/coll scaffolds may enhance the osteogenic differentiation of rBMSCs. The SrAPMs/coll scaffold showed a sustained release of Sr ions (Figure 4), which may account for its higher osteoinductive potential compared with the APMs/coll scaffold. It is well


Page 22 of 42

Page 23 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


accepted that Sr is benefical for bone anabolism. Besides the positive effect on the proliferation of osteoblasts,47 Sr is also beneficial for the osteogenic differention of BMSCs. It was reported that the stimulative effect of SrCl2 on the osteogenesis of BMSCs might be mediated by the Wnt/β-catenin signaling pathway.51 Thus, the SrAPMs/coll scaffolds may potentially provide a more favorable environment for the osteogenesis of rBMSCs compared to the Coll and APMs/coll scaffolds both in terms of stimulating the osteogenic differentiation as well as being better mechanical properties. To further verify the osteoinductive potential of the SrAPMs/coll scaffolds, an in vivo study was performed by implanting the scaffolds into calvarial defects. Consistent with the in vitro results, the SrAPMs/coll scaffolds showed superior bone regeneration performance compared with the Coll and APMs/coll scaffolds. The in vivo results suggest that, following the incorporation of SrAPMs into the collagen matrix, it is possible to obtain biomimetic SrAPMs/coll composite scaffolds with excellent bone healing properties. It is worth noting that endogenous MSCs are crucial during bone generation, in which the MSCs could be motivated and participate in the repair process within the injury site.52 Therefore, the Sr-stimulated proliferation and osteogenesis of endogenous MSCs may contribute to the enhanced bone regeneration in the defects filled with SrAPMs/coll scaffolds. Moreover, Sr can also inhibit the bone resorption by suppressing the osteoclastic differentiation and activity.30 It was reported that Sr not only inhibited osteoclastic differentiation by suppressing the expression of receptor activator of nuclear factor kappa-B ligand



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(RANKL) but also stimulated the expression of osteoprotegerin (OPG), which can inhibit the differentiation and activity of osteoclast by blocking the interaction of RANK with its ligand.31, 53 Hence, the delivery of stimulative elements in the form of SrAPMs, combined with collagen that have been optimized for bone tissue engineering, provides great potential for enhanced bone regeneration. This study represents an important step on the development of a biomimetic biomaterial with a substantial commercial and clinical potential. Further studies remain to be carried out to test the regenerative capacities of SrAPMs/coll scaffolds in weight-bearing defects in conjunction with the conventionally calcium phosphate nanoparticles/collagen composite scaffolds. 5. CONCLUSIONS Herein, Sr-doped amorphous calcium phosphate porous microspheres were successfully synthesized through a microwave-hydrothermal method using FBP as the organic phosphorus source. Compared with the HAP nanorods synthesized using Na2HPO4˙12H2O as phosphorus source, the APMs and SrAPMs showed higher vancomycin-loading capacity and greater antibacterial efficiency due to their mesoporous structure and higher specific surface area, which bodes well for their potential application in treating bone infection. Furthermore, the SrAPMs were incorporated into collagen matrix to construct a biomimetic scaffold aiming to improve the bioactivity, mechanical properties and osteogenic activity. Our results demonstrated that the SrAPMs/coll scaffold could stimulate the osteogenesis of rBMSCs and promoted bone regeneration, which suggests that the SrAPMs/coll


Page 24 of 42

Page 25 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


scaffold may be a promising alternative to autogenous bone grafts for the repair of bone defects.

ASSOCIATED CONTENT Supporting Information Methods (characterization of the SrAPMs, bacteria preparation and culture of rBMSCs, antibacterial assay), images of visual appearance of the M-H broths and inhibition zones formed on the M-H plates. AUTHOR INFORMATION Corresponding Authors E-mail: [email protected]. (Y.-H.H.) E-mail: [email protected] (Z.-M.S.) E-mail: [email protected]. (Y.-J.Z) Author Contrubutions 1

These authors contributed equally to this work.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS Financial support from the National High-Tech Research and Development Program of China (863-Project, No. 2015AA020316), National Natural Science Foundation of China (81271961, 81572106, 81271998) and Shanghai Committee of Science and Technology, China (15ZR1431900, 15JC1491001) are acknowledged.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

REFERENCES (1) Vallet-Regi, M.; Ruiz-Hernandez, E., Bioceramics: from Bone Regeneration to Cancer Nanomedicine. Adv. Mater. 2011, 23, 5177-5218. (2) Habibovic, P.; Barralet, J. E., Bioinorganics and Biomaterials: Bone Repair. Acta Biomater. 2011, 7, 3013-3026. (3) Dimitriou, R.; Jones, E.; McGonagle, D.; Giannoudis, P. V., Bone Regeneration: Current Concepts and Future Directions. BMC Med. 2011, 9, 1-10. (4) Goldhahn, J.; Scheele, W. H.; Mitlak, B. H.; Abadie, E.; Aspenberg, P.; Augat, P.; Brandi, M. L.; Burlet, N.; Chines, A.; Delmas, P. D.; Dupin-Roger, I.; Ethgen, D.; Hanson, B.; Hartl, F.; Kanis, J. A.; Kewalramani, R.; Laslop, A.; Marsh, D.; Ormarsdottir, S.; Rizzoli, R.; Santora, A.; Schmidmaier, G.; Wagener, M.; Reginster, J. Y., Clinical Evaluation of Medicinal Products for Acceleration of Fracture Healing in Patients with Osteoporosis. Bone 2008, 43, 343-347. (5) Romano, C. L.; Logoluso, N.; Meani, E.; Romano, D.; De Vecchi, E.; Vassena, C.; Drago, L., A Comparative Study of the Use of Bioactive Glass S53P4 and Antibiotic-Loaded Calcium-Based Bone Substitutes in the Treatment of Chronic Osteomyelitis: A Retrospective Comparative Study. Bone Jt. J. 2014, 96-B, 845-850. (6) Li, H.; Yang, L.; Dong, X.; Gu, Y.; Lv, G.; Yan, Y., Composite Scaffolds of Nano Calcium Deficient Hydroxyapatite/Multi-(Amino Acid) Copolymer for Bone Tissue Regeneration. J. Mater. Sci.: Mater. Med. 2014, 25, 1257-1265. (7) Lee, J. S.; Baek, S. D.; Venkatesan, J.; Bhatnagar, I.; Chang, H. K.; Kim, H. T.; Kim, S. K., In Vivo Study of Chitosan-Natural Nano Hydroxyapatite Scaffolds for Bone Tissue Regeneration. Int. J. Biol. Macromol. 2014, 67, 360-366. (8) IUskokovic, V.; Batarni, S. S.; Schweicher, J.; King, A.; Desai, T. A., Effect of Calcium Phosphate Particle Shape and Size on Their Antibacterial and Osteogenic Activity in the Delivery of Antibiotics in Vitro. ACS Appl. Mater. Interfaces 2013, 5, 2422-2431. (9) Tang, Q. L.; Zhu, Y. J.; Wu, J.; Chen, F.; Cao, S. W., Calcium Phosphate Drug Nanocarriers with Ultrahigh and Adjustable Drug-Loading Capacity: One-Step Synthesis, in Situ Drug Loading and Prolonged Drug Release. Nanomedicine: NBM 2011, 7, 428-434. (10) Sun, F.; Zhou, H.; Lee, J., Various Preparation Methods of Highly Porous


Page 26 of 42

Page 27 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Hydroxyapatite/Polymer Nanoscale Biocomposites for Bone Regeneration. Acta Biomater. 2011, 7, 3813-3828. (11) Ma, M. G.; Zhu, J. F., Solvothermal Synthesis and Characterization of Hierarchically Nanostructured Hydroxyapatite Hollow Spheres. Eur. J. Inorg. Chem. 2009, 2009, 5522–5526. (12) Wang, K. W.; Zhu, Y. J.; Chen, X. Y.; Zhai, W. Y.; Wang, Q.; Chen, F.; Chang, P. J.; Duan, Y. R., Flower-Like Hierarchically Nanostructured Hydroxyapatite Hollow Spheres: Facile Preparation and Application in Anticancer Drug Cellular Delivery. Chem. -Asian J. 2010, 5, 2477–2482. (13) Qi, C.; Zhu, Y. J.; Lu, B. Q.; Zhao, X. Y.; Zhao, J.; Chen, F., Hydroxyapatite Nanosheet-Assembled Porous Hollow Microspheres: DNA-Templated Hydrothermal Synthesis, Drug Delivery and Protein Adsorption. J. Mater. Chem. 2012, 22, 22642-22650. (14) Sun, R.; Lu, Y.; Chen, K., Preparation and Characterization of Hollow Hydroxyapatite Microspheres by Spray Drying Method. Powder Technol. 2012, 29, 1088-1092. (15) Ma, M. G., Hierarchically Nanostructured Hydroxyapatite: Hydrothermal Synthesis, Morphology Control, Growth Mechanism, and Biological Activity. Int. J. Nanomed. 2011, 7, 1781-1791. (16) Shum, H. C.; Bandyopadhyay, A.; Bose, S.; Weitz, D. A., Double Emulsion Droplets as Microreactors for Synthesis of Mesoporous Hydroxyapatite. Chem. Mater. 2009, 21, 5548-5555. (17) Xia, W.; Grandfield, K.; Schwenke, A.; Engqvist, H., Synthesis and Release of Trace Elements from Hollow and Porous Hydroxyapatite Spheres. Nanotechnology 2011, 22, 305610-305619(10). (18) Lin, K.; Liu, P.; Wei, L.; Zou, Z.; Zhang, W.; Qian, Y.; Shen, Y.; Chang, J., Strontium Substituted Hydroxyapatite Porous Microspheres: Surfactant-Free Hydrothermal Synthesis, Enhanced Biological Response and Sustained Drug Release. Chem. Eng. J. 2013, 222, 49-59. (19) Qi, C.; Zhu, Y. J.; Lu, B. Q.; Zhao, X. Y.; Zhao, J.; Chen, F.; Wu, J., Hydroxyapatite Hierarchically Nanostructured Porous Hollow Microspheres: Rapid, Sustainable Microwave-Hydrothermal Synthesis by Using Creatine Phosphate as an Organic Phosphorus Source and Application in Drug Delivery and Protein Adsorption.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chem. -Eur. J. 2013, 19, 5332-5341. 20. Zhu, Y. J.; Chen, F., Microwave-Assisted Preparation of Inorganic Nanostructures in Liquid Phase. Chem. Rev. 2014, 114, 6462-6555. 21. Meng, L.-Y.; Wang, B.; Ma, M.-G.; Lin, K.-L., The Progress of Microwave-Assisted Hydrothermal Method in the Synthesis of Functional Nanomaterials. Mater. Today Chem. 2016, 1-2, 63-83. (22) Qi, C.; Zhu, Y. J.; Zhao, X. Y.; Lu, B. Q.; Tang, Q. L.; Zhao, J.; Chen, F., Highly Stable Amorphous Calcium Phosphate Porous Nanospheres: Microwave-Assisted Rapid Synthesis Using ATP as Phosphorus Source and Stabilizer, and Their Application in Anticancer Drug Delivery. Chem. -Eur. J. 2013, 19, 981-987. (23) Elliott, J. C., Structure and Chemistry of the Apatites and Other Calcium Orthophosphates. Elsevier: 1994. (24) Yuan, H.; Fernandes, H.; Habibovic, P.; Boer, J. D.; Barradas, A. M. C.; Ruiter, A. D.; Walsh, W. R.; Blitterswijk, C. A. V.; Bruijn, J. D. D., Osteoinductive Ceramics as Synthetic Alternative to Autologous Bone Grafting. P. Natl. Acad. Sci. U. S. A. 2010, 107, 13614-13619. (25) Zhang, W.; Shen, Y.; Pan, H.; Lin, K.; Liu, X.; Darvell, B. W.; Lu, W. W.; Jiang, C.; Deng, L.; Wang, D., Effects of Strontium in Modified Biomaterials. Acta Biomater. 2011, 7, 800-808. (26) Zreiqat, H.; Ramaswamy, Y.; Wu, C.; Paschalidis, A.; Lu, Z. F.; James, B.; Birke, O.; Mcdonald, M.; Little, D.; Dunstan, C. R., The Incorporation of Strontium and Zinc into a Calcium–Silicon Ceramic for Bone Tissue Engineering. Biomaterials 2010, 31, 3175-3184. (27)Zhao, L.; Wang, H.; Huo, K.; Zhang, X.; Wang, W.; Zhang, Y.; Wu, Z.; Chu, P. K., The Osteogenic Activity of Strontium Loaded Titania Nanotube Arrays on Titanium Substrates. Biomaterials 2012, 34, 19-29. (28) Park, J. W.; Kim, Y. J.; Jang, J. H.; Song, H., Positive Modulation of Osteogenesis- and Osteoclastogenesis-Related Gene Expression with Strontium-Containing Microstructured Ti Implants in Rabbit Cancellous Bone. J. Biomed. Mater. Res., Part A 2013, 101, 298-306. (29) Schumacher, M.; Lode, A.; Helth, A.; Gelinsky, M., A Novel Strontium(II)-Modified Calcium Phosphate Bone Cement Stimulates Human-Bone-Marrow-Derived Mesenchymal Stem Cell Proliferation and Osteogenic


Page 28 of 42

Page 29 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Differentiation in Vitro. Acta Biomater. 2013, 9, 9547-9557. (30) Saidak, Z.; Marie, P. J., Strontium Signaling: Molecular Mechanisms and Therapeutic Implications in Osteoporosis. Pharmacol. Ther. 2012, 136, 216-226. (31) Peng, S.; Zhou, G.; Luk, K. D.; Cheung, K. M.; Li, Z.; Lam, W. M.; Zhou, Z.; Lu, W. W., Strontium Promotes Osteogenic Differentiation of Mesenchymal Stem Cells through the Ras/MAPK Signaling Pathway. Cell. Physiol. Biochem. 2009, 23, 165-174. (32) Haugh, M. G.; Jaasma, M. J.; O'Brien, F. J., The Effect of Dehydrothermal Treatment on the Mechanical and Structural Properties of Collagen-GAG Scaffolds. J. Biomed. Mater. Res., Part A 2009, 89, 363-369. (33) Minardi, S.; Corradetti, B.; Taraballi, F.; Sandri, M.; Van Eps, J.; Cabrera, F. J.; Weiner, B. K.; Tampieri, A.; Tasciotti, E., Evaluation of the Osteoinductive Potential of a Bio-Inspired Scaffold Mimicking the Osteogenic Niche for Bone Augmentation. Biomaterials 2015, 62, 128-137. 34. Qi, C.; Zhu, Y. J.; Chen, F., Fructose 1,6-Bisphosphate Trisodium Salt as a New Phosphorus Source for the Rapid Microwave Synthesis of Porous Calcium-Phosphate Microspheres and Their Application in Drug Delivery. Chem. -Asian J. 2013, 8, 88-94. (35) Zhao, S.; Zhang, J.; Zhu, M.; Zhang, Y.; Liu, Z.; Tao, C.; Zhu, Y.; Zhang, C., Three-Dimensional Printed Strontium-Containing Mesoporous Bioactive Glass Scaffolds for Repairing Rat Critical-Sized Calvarial Defects. Acta Biomater. 2015, 12, 270-280. (36) Thormann, U.; Ray, S.; Sommer, U.; Elkhassawna, T.; Rehling, T.; Hundgeburth, M.; Henss, A.; Rohnke, M.; Janek, J.; Lips, K. S.; Heiss, C.; Schlewitz, G.; Szalay, G.; Schumacher, M.; Gelinsky, M.; Schnettler, R.; Alt, V., Bone Formation Induced by Strontium Modified Calcium Phosphate Cement in Critical-Size Metaphyseal Fracture Defects in Ovariectomized Rats. Biomaterials 2013, 34, 8589-8598. (37) Olde Damink, L. H.; Dijkstra, P. J.; van Luyn, M. J.; van Wachem, P. B.; Nieuwenhuis, P.; Feijen, J., Cross-Linking of Dermal Sheep Collagen Using a Water-Soluble Carbodiimide. Biomaterials 1996, 17, 765-773. (38) Jin, G.; Qin, H.; Cao, H.; Qiao, Y.; Zhao, Y.; Peng, X.; Zhang, X.; Liu, X.; Chu, P. K., Zn/Ag Micro-Galvanic Couples Formed on Titanium and Osseointegration Effects in the Presence of S. Aureus. Biomaterials 2015, 65, 22-31.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(39) Andersson, J.; Rosenholm, J.; Areva, S.; Linden, M., Influences of Material Characteristics on Ibuprofen Drug Loading and Release Profiles from Ordered Microand Mesoporous Silica Matrices. Chem. Mater. 2004, 16, 4160-4167. (40) Qi, C.; Chen, F.; Wu, J.; Zhu, Y.-J.; Hao, C.-N.; Duan, J.-L., Magnesium Whitlockite Hollow Microspheres: A Comparison of Microwave-Hydrothermal and Conventional Hydrothermal Syntheses Using Fructose 1,6-Bisphosphate, and Application in Protein Adsorption. RSC Adv. 2016, 6, 33393-33402. (41) Li, Z. Y.; Lam, W. M.; Yang, C.; Xu, B.; Ni, G. X.; Abbah, S. A.; Cheung, K. M.; Luk, K. D.; Lu, W. W., Chemical Composition, Crystal Size and Lattice Structural Changes after Incorporation of Strontium into Biomimetic Apatite. Biomaterials 2007, 28, 1452–1460. (42) Lin, K. L.; Zhou, Y. L.; Zhou, Y.; Qu, H. Y.; Chen, F.; Zhu, Y. J.; Chang, J., Biomimetic Hydroxyapatite Porous Microspheres with Co-Substituted Essential Trace Elements: Surfactant-Free Hydrothermal Synthesis, Enhanced Degradation and Drug Release. J. Mater. Chem. 2011, 21, 16558-16565. (43) Quinlan, E.; Partap, S.; Azevedo, M. M.; Jell, G.; Stevens, M. M.; O'Brien, F. J., Hypoxia-Mimicking Bioactive Glass/Collagen Glycosaminoglycan Composite Scaffolds to Enhance Angiogenesis and Bone Repair. Biomaterials 2015, 52, 358-366. (44) Cunniffe, G. M.; Curtin, C. M.; Thompson, E. M.; Dickson, G. R.; O'Brien, F. J., Content-Dependent Osteogenic Response of Nanohydroxyapatite: An in Vitro and in Vivo Assessment within Collagen-Based Scaffolds. ACS Appl. Mater. Interfaces 2016, 8, 23477-23488. (45) Yang, S.; Leong, K. F.; Du, Z.; Chua, C. K., The Design of Scaffolds for Use in Tissue Engineering. Part I. Traditional Factors. Tissue Eng. 2001, 7, 679-689. (46) Alcaide, M.; Portoles, P.; Lopez-Noriega, A.; Arcos, D.; Vallet-Regi, M.; Portoles, M. T., Interaction of an Ordered Mesoporous Bioactive Glass with Osteoblasts, Fibroblasts and Lymphocytes, Demonstrating Its Biocompatibility as a Potential Bone Graft Material. Acta Biomater. 2010, 6, 892-899. (47) Chattopadhyay, N.; Quinn, S. J.; Kifor, O.; Ye, C.; Brown, E. M., The Calcium-Sensing Receptor (Car) Is Involved in Strontium Ranelate-Induced Osteoblast Proliferation. Biochem. Pharmacol. 2007, 74, 438-447. (48) Zhang, Y.; Wu, C.; Friis, T.; Xiao, Y., The Osteogenic Properties of Cap/Silk Composite Scaffolds. Biomaterials 2010, 31, 2848-2856.


Page 30 of 42

Page 31 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(49) Rezwan, K.; Chen, Q. Z.; Blaker, J. J.; Boccaccini, A. R., Biodegradable and Bioactive Porous Polymer/Inorganic Composite Scaffolds for Bone Tissue Engineering. Biomaterials 2006, 27, 3413-3431. (50) Engler, A. J.; Sen, S.; Sweeney, H. L.; Discher, D. E., Matrix Elasticity Directs Stem Cell Lineage Specification. Cell 2006, 126, 677-689. (51) Yang, F.; Yang, D.; Tu, J.; Zheng, Q.; Cai, L.; Wang, L., Strontium Enhances Osteogenic Differentiation of Mesenchymal Stem Cells and in Vivo Bone Formation by Activating Wnt/Catenin Signaling. Stem Cells 2011, 29, 981-991. (52) Fong, E. L.; Chan, C. K.; Goodman, S. B., Stem Cell Homing in Musculoskeletal Injury. Biomaterials 2011, 32, 395-409. (53) Baron, R.; Tsouderos, Y., In Vitro Effects of S12911-2 on Osteoclast Function and Bone Marrow Macrophage Differentiation. Eur. J. Pharmacol. 2002, 450, 11-17.

Figureure Legends: Figure 1. SEM and TEM micrographs of HAP nanorods, APMs and SrAPMs. The EDS element mapping shows the distribution of Sr, Ca, P, C and O in SrAPMs. Figure 2. XRD patterns (A), FTIR spectra (B), TGA curves (C), Nitrogen adsorption-desorption isotherm (D) and BJH pore size distributions (E) of the HAP nanorods, APMs and SrAPMs. Figure 3. (A) Vancomycin-release curves of the vancomycin-loaded HAP nanorods, APMs and SrAPMs drug-delivery systems. (B) The cumulative release of vancomycin as a function of the square root of the release time. (C) Optical transparence at λ=600 nm of M-H broths as a function of the concentration of vancomycin-loade samples following 24 h of incubation (C- dashed line denotes the absorbance of negative control, incubated without bacteria, while the C+ dashed lines denotes the absorbance of the positive control, incubated with bacteria and no



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

vancomycin-loaded samples). (D) The average diameters of the inhibition zone on the M-H agar plates. (*Significant differences between Van-HAP nanorods and Van-APMs or Van-SrAPMs; #Significant differences between the 1-mg and 3-mg groups, p

Strontium-Doped Amorphous Calcium Phosphate Porous

Recommend Documents