Biodegradable Multifunctional Bioactive Glass-based

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Biological and Medical Applications of Materials and Interfaces

Biodegradable Multifunctional Bioactive Glass-based Nanocomposites Elastomers with Controlled Biomineralization Activity, Realtime Bioimaging Tracking and Decreased Inflammatory Response Yannan Li, Yi Guo, Wen Niu, Mi Chen, Yumeng Xue, Juan Ge, Peter X Ma, and Bo Lei ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b04856 • Publication Date (Web): 08 May 2018 Downloaded from http://pubs.acs.org on May 8, 2018

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ACS Applied Materials & Interfaces

Biodegradable Multifunctional Bioactive Glass-based Nanocomposites Elastomers with Controlled Biomineralization Activity, Real-time Bioimaging Tracking and Decreased Inflammatory Response Yannan Li a, Yi Guo a, Wen Niu a, Mi Chen a, Yumeng Xue a, Juan Ge a, Peter X Ma e, Bo Lei a, b, c, d* a

Frontier Institute of Science and Technology, Xi’an Jiaotong University, Xi’an 710054, China

b

State Key Laboratory for Manufacturing Systems Engineering, Xi’an Jiaotong University, Xi’an 710054,

China c

State Key Laboratory for Mechanical Behavior of Materials, Xi’an Jiaotong University, Xi’an 710054,

China d

Instrument Analysis Center, Xi'an Jiaotong University, Xi’an 710054, China

e

Department of Biologic and Materials Sciences, Department of Biomedical Engineering, Macromolecular

Science and Engineering Center, Department of Materials Science and Engineering, University of Michigan, Ann Arbor 48109, USA * Corresponding author: Bo Lei, [email protected] Abstract: Controlled biomineralization activity of biomaterials is rather important in bone regeneration and osseointegration avoiding the formation of fibrous capsule. However, most of conventional biodegradable elastomeric biomaterials for bone regeneration do not possess biomineralization ability and inherent multifunctional properties. Herein, we report a multifunctional bioactive glass (BG)-based hybrid poly(citrate-siloxane) (PCS) elastomer with intrinsical biomineralization activity and photoluminescent properties for potential bone tissue regeneration. Monodispersed bioactive glass nanoparticles (BGNs) were used to control the elastomeric behavior, biomineralization activity, photoluminescent ability and osteogenic cellular response of PCS elastomers. BGNs significantly enhanced the elastomeric modulus of

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PCS from 20 MPa to 200 MPa (10 times improvement) and the hydrophilicity (from 82°to 28°in water contact angle). The photoluminescent properties of PCS elastomers were also tailored through the incorporation of BGNs. The in vivo degradation of PCS-BGN nanocomposites could be efficiently tracked through non-invasively monitoring their fluorescent change. PCS-BGN nanocomposites enhanced the proliferation and osteoblastic differentiation of osteoblasts (MC3T3-E1), and decreased the in vivo inflammatory response. This study provided a novel tactics for designing the bioactive elastomeric biomaterials with multifunctional properties for bone regeneration medicine. Keywords: Multifunctional biomaterials; Bioactive glass; Elastomeric nanocomposites; Biomineralization; Bone regeneration; Introduction Due to the increased bone-related disease and accident, the bone tissue repair and regeneration has been one of the important issues in regenerative medicine.1 Although bone and joint replacement has received success in orthopedic clinic, the regeneration of native bone architecture is still not ideal.2 It is a reasonable way to design biomaterials to integrate with bone tissue, via imitating the structure of natural bone tissue.3 Native bone shows representative nanocomposites formed by biomineralized apatite nanocrystals and polymer nanofibers.4 Additionally, the extracellular matrix for bone tissue also shows a hierarchical structure with the sizes from nanoscale to macroscale.5 As a consequence, bioactive nanocomposites were becoming important in enhanced bone regeneration. 6 In the past decades, bioactive inorganic materials have achieved clinical success in bone and teeth restoration.7-9 Among the bioactive materials, silica-based bioactive glasses are showing increased interests in the field of bone regeneration, owing to their good biocompatibility, osteoconductivity, osteoinductivity,


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gene-activation bioactivity and controlled degradation.

10-13

A lot of studies showed that the good

biocompatibility of bioactive glasses (BGs) was due to the forming capacity of bone-like apatite phase (biominerialization) which can directly bond with bone or soft tissues.14 15 Compared with traditional BGs, sol-gel derived BG nanoparticles (BGNs) showed the enhanced biomineralization and osteoblastic cells response.16-18 Thus, BGNs have been employed to fabricate the bioactive nanocomposites for enhanced bone tissue regeneration. Up to now, BGNs-based poly(ε-caprolactone), poly (hydroxybutyrate-2-co-2-hydroxyvalerate), poly(L-lacic acid), chitosan, collagen and gelatin nanocomposites have shown their potential bone regeneration application.19-24 Although some positive results have been received in previous studies, most of BGN-based polymer nanocomposites lack biomimetic elastomeric mechanical behavior and inherent multifunctional properties such as bioimaging. In recent years, due to the biocompatible monomer, biomimetic elastomeric behavior, biodegradability, and good biocompatibility, polycitrate-based (PC) materials have been paid more attention in regenerative medicine including bone regeneration.25-27 However, the low mechanical modulus, poor osteogenic activity and biomineralization ability has limited their efficient application in bone tissue regeneration.28 Recently, our group developed a series of poly (citrate-siloxane) (PCS) elastomers using direct thermal-induced polymerization of siloxane.29-31 Compared with PC, PCS showed the significantly improved mechanical property and osteogenic differentiation bioactivity. In addition, PCS also presented stable photoluminescent property which endows the potential bioimaging application. Even so, there is still no significant biomineralization activity and apatite-forming ability for PCS. It is necessary and reasonable to develop bioactive multifunctional PCS-based biomaterials with controlled biomineralization ability for bone regeneration.



Herein, we employ monodispersed BGNs to regulate the mechanical behavior, photoluminescent properties, biomineralization activity, and osteogenic capacity of PCS elastomers. The surface morphology, elastomeric mechanical and photoluminescent properties, biodegradability, biomineralization in vitro, cytotoxicity, osteogenic differentiation, inflammatory response and bioimaging in vivo of PCS-BGN nanocomposites were investigated. 2. Experimental section 2.1. Materials Citric acid (CA, 99%), 1,8-octanediol (OD, 99%), (3-aminopropyl) triethoxysilane (AS, 98%), tetraethyl orthosilicate (TEOS, 99%), dodecylamine (DDA, 99%), triethylphosphate (TEP, 99%), ethyl alcohol (ETOH, 99%), calcium nitrate tetrahydrate (CN, 99%)，1，6-hexamethyl diisocyanate (HDI, 99%)， stannous octoate (100.0%), phosphate-buffered saline (PBS, 99%) and dimethylsulfoxide (DMSO, 99%) were purchased from Sigma-Aldrich. 2.2. Fabrication and cauterizations of PCS and PCS-BGN nanocomposites The PCS prepolymers were synthesized according to our previous report.31 Monodispersed BGN was synthesized with a simple sol-gel template method in accordance with previous report.32 PCS-BGN nanocomposites were fabricated by a chemical crosslinked method in the mild condition. In short, the PCS prepolymer was dissolved in DMSO to get a 10 wt% solution. Then, BGNs were mixed with PCS solutions at different mass ratios (from 5 wt% to 20 wt%) and stirred constantly for 2 h at 25 °C. The PCS/BGN mixture slurries reacted with molar ratio (1:0.3) of HDI and residual hydroxyls in PCS with 0.1 wt % catalyst (stannous octoate) for 60 min at 55 °C under stirring. At last, the solvents were evaporated completely at 55°C. After postpolycondensation at 80 °C for 48 h, the PCS-BGN nanocomposites were


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obtained. The obtained nanocomposites were named after PCS-BGN X where X represents the weight percent of BGNs. The structure characterizations of samples were shown in supporting information. 2.3. Mechanical properties of PCS-BGN nanocomposites The mechanical behavior of samples was analyzed by a mechanical testing machine (Criterion 43, Instron). The speed of tensile testing was set as 50 mm/min. The size of tested specimens was 50 mm×6 mm). Fatigue curves were obtained by continuously pulling and loosening for 6 cycles in 30% strain (MTS， CMT4104). The tensile stress, elongation and Young's modulus of the samples was calculated according to the following formulas： Stress σ= P/(a*b);

(1)

Strain ε = (L-L0) / L0;

(2)

Elongation (%) ε = (l-l0) / L;

(3)

Young's modulus E =σ0 /ε0;

(4)

In the formulas, P, a and b respectively represents the payload, the width and the thickness of the sample; L0 and L is on behalf of the original length and the length after stretch of the sample; σ0 and ε0 are the stress and strain of the sample for linear deformation. 2.4. Degradation behavior analysis of PCS-BGN nanocomposites in vitro For evaluating the degradation behavior of the PCS-BGN in vitro, the weight loss (WL %) were measured by soaking nanocomposites (6 mm×20 mm) in phosphate saline (PBS) at 37 °C (pH=7.4). Briefly, all materials were weighed (W0) and put into test tubes containing PBS at 10 mg/mL. The centrifuge tube with the materials was transferred to a 37 °C shaker and incubated for different periods (1, 3, 7, and 14 days). Material weight degradation was evaluated according to the following equation:



Weight Loss (WL %) =（W0-Wt）×100% /W0;

(5)

Where W0 and Wt is the initial weight and the weight after the degradation of the sample which was washed and dried at 60 °C to the constant weight. 2.5. Hydrophilicity analysis of PCS-BGN nanocomposites The hydrophilicity of PCS-BGN was performed by measuring the contact angles of water at 25 °C, using a goniometer (SL200KB-Kino). A drop of deionized water was put on the surface of samples and the picture was taken concurrently. The contact angles were calculated from five different positions on samples. 2.6. In vitro biomineralization bioactivity of PCS-BGN nanocomposites The biomineralized bioactivity of samples in vitro was confirmed by assessing the formation of apatite on the surface of the PCS-BGN nanocomposites after immersion in a simulated buffer fluid (SBF). SBF were made up according to the reference.32,

33

Briefly, the nanocomposites with a rectangle of 10 mm×6

mm were immersed in SBF with a 10 mg/mL and placed in an air shaker at 37 °C for 1 d, 3 d and 7 d. The solution was replaced every two days. At every time point, the soaked samples were took out, washed, and dried at 25 °C. The apatite formation of the surface of the material was evaluated through scanning electronic microscope (SEM, Quanta 250), powder diffractometer (D/MAX-RB, Rigaku) and infrared spectroscopy (Nicolet 6700). 2.7. In vivo degradation and real-time bioimaging of PCS-BGN nanocomposites To assess the relationship between material degradation and fluorescence imaging in vivo, at the same time point, the samples were removed, washed with absolute ethanol and deionized water and dried in the oven. Finally, all the samples were weighed and fluorescent behavior was tested. Prior to bioimaging, the


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circular PCS-BGN 15% and PCS-BGN 0% (diameter= 8 mm) and sterilized. After anesthesia, PCS-BGN 15% (the left side) and PCS-BGN 0% (the right side) were implanted subcutaneously on the mouse's back. Finally, the implanted mice were subjected to fluorescence bioimaging with different time points (15 d, 35 d, 60 d) by the animal imaging system (Xenogen, IVIS spectrum). The relative fluorescent intensity analysis was performed. To assess the relationship between material degradation and fluorescence imaging in vivo, at the same time point, the nanocomposites were removed, washed with absolute ethanol and deionized water and dried in the oven. Finally, all the samples were weighed and fluorescent behavior was tested. 2.8. Biocompatibility analysis in vitro of PCS-BGN nanocomposites The biocompatibility in vitro of samples was assessed by the osteoblast (MC3T3-E1). The cell activity and proliferation were determined using the Live-Dead kit and AlamarBlue assay kit (Thermo Fisher). Before cell culture, nanocomposites were sterilized by 75% ethanol (2 h) and exposed to UV (30 min). Cells were seeded (4000 cells/well) on the surface of the sample in a 24-well plate. At different time points, the cell activity and proliferation was measured according to the instructions of kits. The tissue culture plate (TCP) as a positive control was used. The live-dead cells on the surface of the samples were tested by the fluorescence microscopy (IX53, Olympus). To evaluate the osteogenic bioactivity of PCS-BGN nanocomposites, the early osteogenic markers bone sialoprotein (BSP) and collagen-1 (COL-1) from the cells after cultured for 7 d and 14 d was detected using a real-time polymerase chain reaction. The detailed method was as follows: Extraction of total RNA from MC3T3 was performed by TRIzol assay kit (Thermo Fisher) and 0.5 µg RNA single-stranded cDNA was prepared using RevertAid kit (Roche). The detailed procedure was in accordance with our previous



study.31 2.9. Inflammatory response evaluation in vivo The male rats (6 weeks, 200 g, Xi’an experimental animal company) employed to test the inflammatory response of samples, according to a protocol approval by Animal Care and Use Committee (Animal Ethics Approval #1002016019) from Xi’an Jiaotong University. Subcutaneous implantation was performed as described previously in our previous report

31

with a slight modification. Briefly, films were

cut into a 5 mm×3 mm area and sterilized for 30 min under UV. Four experimental groups per sample were implanted into the subcutaneous tissue of rat. After 2 and 4 weeks, nanocomposites were collected for subsequent histological analysis. The rats without any implants but with the same surgery were used as normal controls (NC). Sections of subcutaneous implants were embedded in paraffin and stained with Hematoxylin-Eosin (H&E) for a microscopic (BX53，Olympus) observation and histological analysis. 2.10. Statistical analysis The statistical analysis was carried out by GraphPad Prism 6.0. The t-test analysis was used to evaluate the significant difference between different groups and *p

Biodegradable Multifunctional Bioactive Glass-based

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