Strontium Ranelate Incorporated Enzyme-Cross-Linked Gelatin

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Tissue Engineering and Regenerative Medicine

A Strontium Ranelate Incorporated Enzyme-crosslinked Gelatin Nanoparticle/ Silk Fibroin Aerogel for Osteogenesis in OVX-induced Osteoporosis Dize Li, Kaiwen Chen, Lian Duan, Tiwei Fu, Jiao Li, Zhixiang Mu, Si Wang, Qin Zou, Li Chen, Yangyingfan Feng, Yihan Li, Hongmei Zhang, Huanan Wang, Tao Chen, and Ping Ji ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b01298 • Publication Date (Web): 24 Jan 2019 Downloaded from http://pubs.acs.org on January 25, 2019

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ACS Biomaterials Science & Engineering

Chongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education

ACS Paragon Plus Environment

ACS Biomaterials Science & Engineering 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

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A Strontium Ranelate Incorporated Enzyme-crosslinked Gelatin Nanoparticle/Silk Fibroin Aerogel for Osteogenesis in OVXinduced Osteoporosis

Dize Li1, Kaiwen Chen2, Lian Duan3, Tiwei Fu1, Jiao Li1, Zhixiang Mu1, Si Wang1, Qin Zou4, Li Chen4, Yangyingfan Feng1, Yihan Li1, Hongmei Zhang1, Huanan Wang2, Tao Chen1, *, Ping Ji1, *

1Stomatological

Hospital of Chongqing Medical University, Chongqing Key Laboratory of Oral Diseases and

Biomedical Sciences, Chongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education, Chongqing Medical University, Chongqing, 401147, P. R. China 2School

of Life Science and Biotechnology, Dalian University of Technology, Dalian, 116023, P. R. China

3College

of Textiles and Garments, Southwest University, Chongqing, 400715, P. R. China

4Research

Center for Nano-Biomaterials, Analytical and Testing Center, Sichuan University, Chengdu,

610064, P. R. China

∗Corresponding authors： 426#Songshibei Road, Yubei District, Chongqing, 401147, P.R. China Tel.: +86-182-2502-9529 (Tao Chen); +86-139-0830-2957 (Ping Ji) Emails: [email protected] (Tao Chen); [email protected] (Ping Ji)

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Abstract Osteoporosis is a wide-range disease with negative impact on bone defects healing. Strontium ranelate (SR) has promising osteogenic potential for its dual function on stimulating osteoblasts and inhibiting osteoclasts activity. However, it has limitations for its dose-dependent effect and side effects on systemic application. Here, a sequentially crosslinking strategy including enzyme-crosslinking through tyrosinase from mushroom and physical folding is acquired to create SR loaded gelatin nanoparticle/silk fibroin aerogel (abbreviated as S/G-Sr-MT) with drug release controlling capacity. The results showed successful enzymecrosslinking, excellent spatial structure and enhanced mechanical of S/G-Sr-MT. Evenly Sr2+ loading and stable release with markedly inhibited initial burst release was detected. Biomineralization investigation showed rapid deposition of hydroxyapatite on the surface of S/G-Sr-MT. In vitro, spreading morphology and higher osteogenic gene expression of MC3T3-E1 seeded on S/G-Sr-MT were observed compared to other groups after 7-day culturing. In vivo, S/G-Sr-MT showed obvious osteogenic capacity in calvaria defects of ovariectomized rats in which high Runx2 expression and inhibited TRAP activity were observed. Such results suggested the S/G-Sr-MT scaffold could stimulate osteogenic differentiation of osteoblasts while inhibit osteoclast behaviors in vivo. These findings highlight the potential osteogenic ability and clinical application of SR incorporated enzyme-crosslinked scaffold in ovariectomized (OVX) bone healing.

Keywords: gelatin nanoparticle; tyrosinase; strontium ranelate; bone healing; enzyme-crosslinking.

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1.

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Introduction Low bone mass diseases like osteoporosis result in reducing of bone quantity and density1. Therefore,

the bone defect healing and quality of new bone was negatively affected. The difficulty of that is due to lower activity of osteoblasts, higher active bone resorption and active marrow adipogenesis2. Strontium ranelate (SR) rebalances bone remodeling by increasing bone formation and decrease bone resorption. Strontium ranelate (SR) not only boosts bone formation of osteoblasts, but also decreases bone resorption caused by osteoclasts3, therefore rebalances bone remodeling in osteoporosis. Notwithstanding, systemic administration of SR might raise the risk of angiocardiopathy4, 5. For dialyzed patients with renal insufficiency, using high doses of strontium may be associated with poor bone mineralization6. In order to solve these problems, long-term treatment with safe doses of SR is necessary. Thus, bone tissue engineering scaffolds are promising to be combined with anti-osteoporosis medicine as controlled release system and bone constructs. Gelatin nanoparticles (GNPs) can assemble into colloidal gel as vehicle for programmed SR release under the inducement of interparticle interactions7. However, this type of gel has inadequate strength and fast degradation behavior. Therefore, it is feasible and indispensable to integrate GNPs into other mechanically robust material to match the needs of bone tissue engineering with the prerequisite to assure its biocompatibility and biodegradability8. Bombyx mori silk fibroin (SF) exhibits remarkable biocompatibility, mechanical properties and mild immunological response in vivo9,

10.

High content of tyrosine residues make SF amenable to chemical

modifications11. SF also promotes the deposition of calcium phosphate, which is one of the main inorganic components of bone12. As for biomineralization, the carboxyl groups of SF can boost the nucleation of 3


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hydroxyapatite (HA) on the surface of scaffolds, which promote SF/HA matrix in the internal environment13. Based on the complementary characteristics of GNPs and SF, we presume that the combination of those two components above may provide surpassing bone tissue engineering scaffolds. In terms of composite scaffolds made of GNPs and SF, crosslinking methods are of great necessity for better physicochemical and biological properties. Chemical crosslinking may pose negative impact for its cytotoxicity from residues. Compared to that, enzymatic crosslinking is more preferable because it requires mild reaction conditions which prevent protein denaturalization14. Tyrosinases from mushroom (MT) are exceptionally selective on tyrosine residues which are highly contained in GNPs and SF (Figure1 A). The MT oxidize accessible tyrosine residues into o-quinone moieties which then undergo nonenzymatic reactions with amines of both GNPs and SF (Figure1 B)11. The accurate enzyme-crosslinking via MT therefore results in the sol-to-gel conversion of GNPs-SF without cellular toxicity. In addition, the GNPs-SF gel can change into aerogel with high porosity by extracting the liquid component through freeze-drying (Figure1 A). Moreover, alcohol-based agents was used to induce β-sheets crystallization of silk and thus improve mechanical characteristics15. Aerogels provide meshwork with larger specific surface area and lower density compared to other bone repairing scaffolds16 and can better maintain tissue healing space compared to hydrogels17. Up to now, few studies have focused on aerogel application on bone defect healing, and the existing aerogels had shortcomings like high cost, high brittleness and weak mechanical properties16. Besides, some studies had fabricated gelatin and silk scaffolds but did not involve osteogenic agents to enhance the bone formation promotion, meanwhile, few researches applied programmed local SR release systems in ovariectomized (OVX) model to evaluate their osteogenic effects18,

19.

Furthermore, some release controlling system for

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osteogenesis only target one specific type of bone related cells20. In order to solve these problems, based on a sequential enzyme-crosslinking strategy (MT followed by ethanol), we fabricated a composite aerogel scaffold assembled from SF and SR loaded GNPs (denoted as S/G-Sr-MT) for OVX bone regeneration. We used ninhydrin test and synchronous fluorescence analysis to examine the effect of sequentially-crosslinking. The morphology and properties of S/G-Sr-MT scaffold were evaluated and the release behavior of Sr2+ was explored. In vitro experiment, the mouse embryo osteoblast precursor cells linage (MC3T3-E1) were seeded on S/G-Sr-MT and the cell proliferation, morphology and osteogenic differentiation were detected. In vivo experiment, we implanted the composite scaffolds into calvaria defects of OVX rats to examine its bone healing capability.

Figure 1. Schematic representation. (A) Procedure of fabrication of aerogels in which two main steps are involved: firstly, the tyrosine residues of GNPs and SF was crosslinked by MT, then the β-sheets of SF was 5


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increased by treating with ethanol. (B) The mechanism of MT is to crosslink tyrosine residues and lysine from different chains of GNPs and SF.

2. Materials and methods 2.1 Animals and materials Female Sprague Dawley (SD) rats aged 14 to 16 weeks and weighing 200 to 300 g were obtained from the experimental animal center of Chongqing Medical University. Silkworm cocoons were kindly donated by Hongjuan Cui from Southwest University. Gelatin A (from porcine skin, 300 Bloom, isoelectric point (IEP) ≈ 9), strontium ranelate and the other reagents were obtained from Sigma-Aldrich (St.Louis, MO, USA). Runx2 polyclonal antibody was purchased from abcam (ab23981, Cambridge, UK). MEM Alpha Modification (αMEM), penicillin-streptomycin, phosphate buffered saline (PBS), trypsin and fetal bovine serum (FBS) were purchased from Hyclone (Logan, Utah, USA).

2.2 Preparation of SF/GNPs scaffolds GNPs were prepared according to Wang H et al.7 and SF solution was prepared according to the method of Chwalek et al.21 The brief description of GNPs and SF fabrication was added in the supporting information (S1). To make the final composite aerogels, GNPs and SF (6% w/v) solution was mixed at the concentration of 20% w/v and then freeze-dried for 24 h22,

23.

The samples in this step were denoted as S/G. For MT

crosslinking group, the GNPs/SF solution was mixed with MT (2.5 U/mg) and kept gently string at 30℃ for 2 h24, then freeze-dried for 24 h. The resulting samples were denoted as S/G-MT. For the drug-loaded aerogels, GNPs were mixed with SR solution (500 μl, 1 mM), then followed by the same process with S/G and S/G-MT groups as above-mentioned. The resulting aerogels were denoted as S/G-Sr and S/G-Sr-MT, respectively. 6



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Since SR is slightly soluble in ethanol, each scaffold was immersed into 100% ethanol for 30 min to create mechanically strengthened water-insoluble aerogels.

2.3 Surface characteristics The samples were sectioned into 1-mm-thick slices and the surface microstructure and morphology of S/G and S/G-MT were characterized by scanning electron microscopy (SEM, SU8010, HITACHI, Japan). The pore size and porosity of scaffolds were measured by SEM and ImageJ software (NIH, Bethesda, USA). The porosity of scaffolds were determined according to a method published by She et al18. Besides, the energy dispersive X-ray spectrometry (EDS) and X-ray photoelectron spectroscopy (XPS) was both used to detect the contained elements of scaffolds.

2.4 Fluorescence measurements and Ninhydrin assay To determine the crosslinking efficiency of MT, synchronous fluorescence spectra was used to verify the crosslinking reaction. The measurement was conducted with the wavelength shift (δ) as 15 nm, which is the characteristic wavelength of tyrosine25. Besides, the concentration of free amino groups was detected through ninhydrin assay. To verify this reaction involves both GNPs and SF, S/G, S/G+MT and (S+MT)/G (for this group, SF solution was treated with MT (2.5 U/mg) before mixed with GNPs) were measured. The detailed process of ninhydrin assay was described in Supporting Information (S2).

2.5 Mechanical properties The mechanical strength of S/G and S/G-MT were measured by using a universal testing machine (MTS 7


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Criterion, Model 43, USA). Cylindrical aerogel scaffolds at the height of 4 mm and diameter of 5 mm were used for the testing. The crosshead speed was 1 mm/min and the measuring range of strain was 0% - 50%. The compressive modulus of each group was calculated from elastic region of the stress-strain curve.

2.6 Swelling ratio, release and degradation behavior The detection of swelling ratio was performed according to Bindu et al26. We used the total immersion method to evaluate the in vitro release behavior of GNPs, S/G-Sr and S/G-Sr-MT (see details in Supporting Information S3). The measurement of in vitro degradation was performed according to Weili et al27, the materials at 1 mm of thickness and 5 mm of diameter were used and 3 same samples were evaluated in each group. For in vivo degradation, S/G and S/G-MT at the same size were implanted into intramuscular sacks at musculus vastus lateralis of each side and 6 rats (3 rats in each time point) were used in total for in vivo degradation. After surgery, SD rats were sacrificed at 4, 8 weeks. Implanted scaffolds and surrounding tissue were taken out, then paraffin-section method and H&E staining was used to evaluate the degradation behavior.

2.7 Biomimetic mineralization The simulated body liquid (SBF) was prepared according to Cuneyt Tas et al28. The samples were soaked in 10 × SBF for 6 h, then washed thoroughly with deionized water and dried in the air for 24 h. The surface morphologies of the mineralized scaffolds were characterized through SEM. Raman spectra of samples was measured by using Raman imaging microscope (DXR2xi, Thermo, USA) to confirm the existence of hydroxyapatite (HA). 8



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2.8 In vitro studies For cell spreading and proliferation assays, MC3T3-E1 cells (ATCC, Manassas, VA, USA) were cultured in α-MEM containing 10% FBS, 100 U/ml penicillin G and 100 mg/ml streptomycin at 37 °C with 5% CO2 (noted as basal growth medium). Cells were subcultrued to next passage by using 0.25% trypsin solution. Cells in passage 3 after thawed was used for in vitro testing. For cell differentiation and mineralization assays, cells were cultured in osteogenic medium (OM). The OM was the original α-MEM medium that consisted of β-glycerophosphate (10 mM), L-ascorbic acid (50 μg/ml) and dexamethasone (10 nM).

2.8.1 Cell seeding The cells were cultured on the surface of different aerogel scaffolds: S/G，S/G-MT， S/G-Sr， S/G-SrMT. For cell seeding, all scaffolds were sterilized with cobalt-60 for 24 h before used8. For each well, 100 μl of MC3T3-E1 suspension (2.5 × 105 cells/ml) was loaded onto scaffolds in 24 well plates and incubated at 37℃ for 2 h for cell attachment. Then 400 μl of basal growth medium or OM was added into each well to make the final concentration of cells 5 × 104 cells/ml. The medium was refreshed every 2 days.

2.8.2 Cell morphology As for cell morphology, the fixation and critical drying were performed according to Yulong et al29. Briefly, at set time point, MC3T3-E1 seeded materials were fixed in glutaraldehyde solution (2.5%) at 4℃ for 24 h. After dehydration and critical point drying, samples were measured by using SEM with an accelerating voltage of 1 kV. 9


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2.8.3 Cell proliferation Cytotoxicity of the aerogel scaffolds was evaluated by cell counting kit-8 (CCK-8) assay. Briefly, at 1, 4, 7 days cell loaded on scaffolds were incubated into medium containing 10% (v/v) CCK-8 agent for 3 h at 37 ℃. Then the colored medium was transferred into 96 well plates and measured at 450 nm by multimode plate reader (PerkinElmer, USA).

2.8.4 Cell differentiation Cell differentiation was evaluated through osteogenic related gene expression using quantitative realtime polymerase chain reaction (qRT-PCR). The specific steps of qRT-PCR and primer sequences are described in supporting information (see details in Supporting Information S4).

2.8.5 ALP staining and calcium content ALP staining of cells was performed according to Tao et al30. After 7 days of culture, the cells were stained using BCIP/NBT method. To evaluate mineralization of MC3T3-E1 on S/G-Sr-MT aerogels, Alizarin Red S (ARS) staining and quantification were performed referring to Xiaoshi et al31. Briefly, MC3T3-E1 cells were seeded on aerogels for 14 and 21 days. Then scaffolds were stained with 0.01% alizarin red dye for 1 h. Cetylpyridinium chloride was used to quantify ARS.

2.9 Animal and surgical protocols All the animal experiments complied with the ARRIVE guidelines. The calvaria bone defect model was 10



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used on OVX rats and they were divided into 3 groups: blank, S/G-MT and S/G-Sr-MT (Figure6 A). Detailed procedures of animals and surgeries are described in supporting information (see details in Supporting Information S5).

2.9.1 Microcomputed tomography After fixing in 4% paraformaldehyde overnight, the samples were measured by μCT imaging system (SANCO Medical AG, Switzerland). The scanning condition was 70 kV and 112 μA with a thickness of 0.048 mm per slice in medium-resolution mode 1024 reconstruction matrix and 200 ms integration time32, 33. The threshold values of bone were decided as 315 to 543 Hounsfield units32. The volume of interest (VOI) was determined as a cylinder (500 μm of thickness and 5 mm of diameter) at the center of each defect (Figure6 B). The bone volume faction (BV/TV), trabecular number (Tb.N), trabecular separation (Tb.Sp) and bone mineral density (BMD) were measured to evaluate the repair of critical bone defects. In addition, bonecovering rate (defined as the area ratio of new bone to whole circular defect) of each group was calculated by using ImageJ software.

2.9.2 Histological and histomorphometry analysis In order to evaluate the side effect of S/G-Sr-MT, at week 8, the vessel，heart and liver tissue were obtained and prepared using paraffin method followed by H&E staining. In addition, the H&E staining and aniline blue staining were used for the evaluation of new bone formation. Besides, to evaluate the activity of osteoclasts, tartrate-resistant acid phosphatase (TRAP) activity measurement was used. The aniline blue staining and TRAP staining were performed according to Chen et al30 (see details in supporting information 11


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S6 & S7).

2.9.3 Immunofluorescence (IF) analysis The expression of Runx2, which represents osteogenic activity in early stage of bone regeneration, was presented. The IF staining was performed by using process according to Mariana et al34 but slightly improved (see detail in supporting information S8).

2.10 Statistical analysis The statistical analysis was performed by SPSS 20.0 software (Chicago, IL, USA). One-way ANOVA and t-test were used to analyze the data which was expressed as mean ± standard deviation (SD). StudentNewman-Keuls method was chosen for post hoc test. The significance of difference was treated as ‘*’ for p≤0.05, ‘**’ for p≤0.01.

3. Results 3.1 Physicochemical characterizations 3.1.1 Crosslinking efficiency of MT After crosslinking, the synchronous fluorescence analysis (Figure2 B) showed lower peak at 303 nm of S/G-MT, which is recognized by typical mark of tyrosine, compared to S/G. The result of fluorescence highlighted markedly decreased tyrosine residues caused by MT crosslinking. Ninhydrin assay is characterized by specificity for amino acids and low susceptibility to interference, which based on its precise reaction35. For S/G, the concentration of free amino groups was 0.23 ± 0.01 μM. After SF was treated with 12



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MT, amino concentration decreased to 0.10 ± 0.01 μM (P

Strontium Ranelate Incorporated Enzyme-Cross-Linked Gelatin

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