Hydroxyapatite Scaffold Promotes

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Biological and Medical Applications of Materials and Interfaces

Quercetin Inlaid Silk Fibroin/Hydroxyapatite Scaffold Promoting Enhanced Osteogenesis Jeong Eun Song, Nirmalya Tripathy, Dae Hoon Lee, Jong Ho Park, and Gilson Khang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b08119 • Publication Date (Web): 06 Sep 2018 Downloaded from http://pubs.acs.org on September 9, 2018

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ACS Applied Materials & Interfaces

Quercetin Inlaid Silk Fibroin/Hydroxyapatite Scaffold Promoting Enhanced Osteogenesis Jeong Eun Song§,†, Nirmalya Tripathy¶,†, Dae Hoon Lee§, Jong Ho Park§, Gilson Khang§,* §

Department of BIN Convergence Technology, Department of Polymer Nano Science & Technology and Polymer Materials Fusion Research Center, Chonbuk National University, 567 Baekje-daero, Deokjin-gu, Jeonju-si, Jeollabuk-do, 54896 Republic of Korea ¶

Department of Bioengineering, University of Washington, 3720 15th Avene Northeast, Box 355061, Seattle, Washington 98195 *

Corresponding author: [email protected] (G. Khang), Tel: +82-63-270-2336; Fax: +8263-270-2341 † Both authors equally contribute to this work. ABSTRACT There is a significant rise in the bone grafts demand worldwide in order to treat bone defects owing to continuous increasing conditions such as injury, trauma, diseases or infections. Therefore, development of three-dimensional (3D) scaffolds has evolved as a reliable technology to address the current limitations for bone tissue regeneration. Mimicking the natural bone, in this study, we have designed silk fibroin/hydroxyapatite scaffold inlaid with a bioactive phytochemical (quercetin) at different concentration for promoting osteogenesis, especially focusing on quercetin ability for enhancing bone health. Characterization of the quercetin/silk fibroin/hydroxyapatite (Qtn/SF/HAp) scaffolds showed an increased pore size and irregular porous microstructure with good mechanical strength. The Qtn (lowcontent)/SF/HAp scaffold was found to be an efficient cell carrier facilitating cellular growth, osteogenic differentiation and proliferation as compared to SF/HAp and Qtn (highcontent)/SF/HAp scaffolds. However Qtn (high-content)/SF/HAp was observed to inhibit cell proliferation without any effects on cell viability. In vitro and in vivo outcomes studied using bone marrow-derived mesenchymal stem cells (rBMSCs) confirm the cytocompatibility, osteogenic differentiation ability and prominent up-regulation of the bone-specific gene expressions for the rBMSCs seeded Qtn/SF/HAp scaffolds. In particular, the implanted Qtn (low-content)/SF/HAp scaffolds at the bone defect site were found to be well-attached and amalgamated with surrounding tissues with approximately 80% bone volume recovery at 6 weeks post-surgery as compared with other groups. Based on the aforementioned observations highlighting quercetin efficiency for bone regeneration, the as-synthesized Qtn (low-content)/SF/HAp scaffolds can be envisioned to provide a biomimetic bone-like microenvironment promoting rBMSCs differentiation into osteoblast, thus suggesting a potential alternative graft for high-performance regeneration of bone tissues. Keywords: Scaffolds, Osteogenic differentiation, Quercetin, Silk fibroin/hydroxyapatite, , Bone marrow-derived mesenchymal stem cells 1 ACS Paragon Plus Environment

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1. INTRODUCTION Bone tissue engineering, a promising approach provides suitable technology for designing bone substitutes/regeneration of new bone tissue for bone fracture repair occurring due to vehicular accidents, traumatic injury, skeletal diseases, tumor resection, bone tissue necrosis, or rheumatic disease. Bone substitutes/grafts (allogenous/autogenous) have limitations owing to lack of ideal bone substitute, osteogenic potential of grafts, immune rejection and secondary trauma1-2. Addressing such issues, several reports have highlighted the importance tissue engineered grafts for various tissue regeneration, which includes scaffolds/grafts/films (3D polymeric network), desired cells and signaling cues or growth inducer/promoters. Although all the elements are critical for an effective fabrication of grafts, however the scaffolds and its biomaterials is an important factor since it interact with cells directly and guides the cells into functional tissue. An ideal scaffold presents a favorable microenvironment and cues for accelerating cell growth, proliferation and differentiation via mimicking the extracellular matrix of the target tissues. Thus, scaffolds designed for bone tissue engineering must approximate the structure and composition of natural bone with high mechanical features, biocompatibility, and biodegradability3-6. In this regard, various studies were reported on fabrication of silk fibroin/hydroxyapatite composite/scaffolds via different approaches for bone tissue engineering7-8. Silk fibroin, a natural protein owing to its biocompatibility, tunable biodegradability, and easy fabrication protocols has considered as potential biomaterials for tissue regeneration, especially for bone tissue engineering9-11. On the other hand, hydroxyapatite (HA) as a vital mineral phase of bone has excellent osteoconductivity and therefore has been coupled with silk fibroin to obtain porous structured scaffold with high mechanical strength and enhanced osteoinduction12-13. Furthermore, BMSCs (multi-potential bone-marrow derived mesenchymal stem cells) have been studied in various studies to exhibit the culture expansion ability, negligible/low immunogenic and tumorigenic features, ease of genetic manipulation, and can potentially differentiate into hepatocytes, osteoblasts, astrocytes, cardiomyocytes, chondrocytes, endothelial cells, fibroblasts, etc14-15. Thus, BMSCs are one of the promising available cell sources for regeneration of bone studies including orthopedic repairs, craniofacial reconstruction and dental structures. Recently, the additive usages of bioactive phytochemicals have been reported for enhancing bone formation and inhibition of bone resorption through their action on cell signalling pathways, influencing osteoblast and osteoclast differentiation16. Flavonoids 2 ACS Paragon Plus Environment

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consist of various biological/pharmacological characteristics such as antitumor, antioxidant, anti-angiogenic, anti-inflammatory and antiviral properties, etc. Mostly, they were found to encourage bone formation via promoting osteoblastogenesis or, inhibit osteo-clastogenesis, ultimately preventing bone resorption and bone loss17-22. Specifically, Quercetin (Qtn), a plant flavonoid, has been emphasized for enhanced osteoblast via positive interaction with the bone cells and further efficiently slowing-down/inhibiting osteoclast activity (important for resorption of bone)22-24. Inspired by the properties of flavonoids compounds, in this study, we evaluated the effects of a bioactive phytochemical (quercetin) on the BMSCs osteogenic differentiation at different concentration inlaid in silk fibroin/hydroxyapatite scaffold (Qtn/SF/HAp scaffolds) as a proof of concept. Our results demonstrated that quercetin, incorporated in the engineered bone scaffold at appropriate concentration resulted in an increased osteogenic differentiation of BMSCs and bone tissue regeneration in in vivo and thus suggest the Qtn/SF/HAp scaffolds as an efficient osteoinductive and osteoconductive platforms.

2. EXPERIMENTAL DETAILS 2.1. Materials Silkworm cocoons (Bombyx mori) were purchased from Kyebong Farm (Cheongyang, Korea), poly(lactic-co-glycolic acid) (PLGA) (90,000 g/mole average molecular weight, mole ratio of lactide:glycolide = 75:25, Resomer® RG756) was received from Boehringer Ingelheim Chem. Co. Ltd (Germany). Hydroxyapatite (HAp) was obtained by CGBio and Quercetin (Qtn) was purchased from Sigma Aldrich (St Louise, MO, USA). All reagents utilized were of high-performance liquid chromatography (HPLC) grade.

2.2. Synthesis of quercetin/SF/HAp scaffolds The quercetin in laid SF/HAp scaffolds (Qtn/SF/HAp scaffolds) were fabricated using freeze drying method. First, the silk fibroin (SF) solution was prepared using our previously reported protocol25-26. In brief, silkworm cocoons were cut into 1 cm ⨯ 1 cm, added to 0.02 M Na2CO3 and boiled for 1 h followed by washing with distilled water to remove sericin and air dried at room temperature. Next, SF was dissolved in LiBr of 9.3 M at 60 °C for 4 h to form an aqueous silk, dialyzed with a cellulose dialysis membrane (3500 Mw, Thermo, USA) to obtain a pure aqueous silk solution, and finally stored at 4 °C. To synthesize quercetin/SF/HAp scaffold, quercetin (in DMSO) at different wt% (0.03, 3 ACS Paragon Plus Environment


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0.05, and 0.1) was blended with 3 wt % SF aqueous solution, poured in the mold (48 well plates) and lyophilized. Then methanol was added for 90 min to perform physical crosslinking. The as-prepared scaffold was further coated with HAp by stirring in simulated body fluid (SBF) at pH=7.4 for 24 h. The control group i.e. SF/HAp scaffold was synthesized by following the above procedure without the addition of quercetin. The size of scaffold was cut to a 7mm diameter and 3 mm height.

2.3. Characterizations of Qtn/SF/HAp scaffolds The structural morphology of the as-synthesized scaffolds was studied by scanning electron microscope (SEM; S-2250N, Hitachi, Tokyo, Japan). The samples purity and surface charge were analyzed using Fourier transform infrared spectroscopy (FTIR, Spectrum GX, Perkin Elmer, USA) in the range from 4000 cm-1 to 400 cm-1. Porosity of all as-fabricated scaffolds was evaluated using the following equation,porosity % =

⨯ 100, where, V1

is the initial volume of distilled water, V2 is the volume of DW immersed scaffold, V3 is the residual DW volume after removal of DW immersed scaffold. Furthermore, the mechanical properties of as-synthesized scaffolds were studied by evaluating the samples comprehensive strength using Material testing machine (TMS-pro, Food Technology Corporation, Sterling, Virginia, USA) where, the measurement distance value of the device was 1 mm, the measuring speed was 20 mm/min, and the measuring force was 0.5 N.

2.4. Osteogenesis measurement of BMSCs on Qtn/SF/HAp scaffolds 2.4.1. Isolation and culture of BMSCs BMSCs were obtained from 3 week old female New Zealand White rabbits (Hanil laboratory animal center, Wanju, Korea). Rabbit femurs were removed and subsequently bone marrow was extracted using syringe (needles gauge 18G). The isolated BMSCs were suspended and cultured using alpha-minimum essential medium (α-MEM) (Lonza, Walkersville, MD, USA) supplemented with 20% fetal bovine serum and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) at 37 °C and 5 % CO2. 2.4.2. BMSCs morphology and initial attachment study The morphology and initial attachment of BMSCs on the as-fabricated scaffolds were examined by SEM (SEM; S-2250N, Hitachi, Tokyo, Japan). Prior to the cell seeding, 4 ACS Paragon Plus Environment

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sterilization of the scaffolds were performed using 70% ethanol followed by washing in PBS (phosphate buffer saline) and hydrated in α-MEM in the incubator. BMSCs were seeded on a Qtn/SF/HAp scaffolds at a density 1×105/scaffold followed by 7 and 28 days of cell culturing. Then, the culture medium was removed and washed with PBS followed by sample fixation with 2.5% glutaraldehyde (Sigma-Aldrich, Saint Louis, USA) at room temperature for 24 h, dehydrated with ethanol solutions (50, 60, 70, 80, 90 and 100%) for 20 min each and then dried. The specimens were cut and fixed on a sample holder for observations.

2.4.4. Cellular metabolic activity The

BMSCs

metabolic

activity

on

the

scaffolds

was

evaluated

by

3-2,5-

diphenyltetrazoliumbromide (MTT, Sigma-Aldrich, USA) assay after 1, 7 ,14, 21, and 28 days of cell culturing. At each experiment time point, the samples were washed with 1X PBS and 1 mL

cell medium with MTT solution (5 mg/mL stock in PBS, Sigma-Aldrich, USA) was added and incubated at 37 oC and 5% CO2 for 4 h. Then, dimethyl sulfoxide (DMSO, SigmaAldrich, USA) was used to replace the medium and to solubilize the formazan crystals, kept for 30 min at 4 oC. The samples absorbance was monitored at 570 nm using SynergyTM Mx monochromator-based multi-mode microplate reader (Biotek instuments, inc., USA).

2.4.5. BMSCs osteogenic differentiation To confirm the osteogenic differentiation of BMSCs on Qtn/SF/HAp scaffolds, alkaline phosphatase (ALP) activity was measured using ALP assay kit (Takara Bio Inc., Tokyo, Japan). BMSCs (1×105/scaffold) were seeded and cultured on the scaffolds for 1, 7, 14, 21 and 28 days’ time points. Then at each experimental time point, the samples were rinsed with PBS (1X) and 5% extraction solution was added to extract BMSCs from scaffolds and paranitrophenyl phosphate (pNPP) solution. The extracted BMSCs were incubated at 37 °C and 5 % CO2 for 1 h and added 0.9 N NaOH stop solution. The absorbance was monitored at 405 nm with a microplate reader.

2.4.6. Real time-PCR analysis Osteogenic gene expression analyses were analyzed using RT-PCR (reverse transcription polymerase chain reaction), where the mRNA was extracted and analyzed from the BMSCs (1×105 cells/scaffolds) seeded scaffolds for 28 days. Scaffolds were added to 1 mL of trizol (RNAiso Plus, Takara, Japan) and 0.2 mL of chloroform and centrifuged at 12,000 rpm at 4 5 ACS Paragon Plus Environment


o

C for 15 min. The supernatant was precipitated with isopropanol (0.5 mL) and Polyacryl

CarrierTM (5 µL, Molecules Research Center, Inc., Cincinnati, USA). Complementary DNA (cDNA) templates from each sample were made from separated RNA with Oligo (dT) primer (Invitrogen, USA), 5× first strand buffer (Invitrogen, USA), dNTP (dGTP, dATP, dTTP, dCTP, Gibco, USA), RNase inhibitor (Invitrogen, USA), Super Script II RT (Invitrogen, USA), and RNase H reverse transcriptase (Invitrogen, USA) and DNas/RNase free water (Invitrogen, USA) by authorized thermal cycler (TP 600, Takara Bio Inc, Japan) reversed cDNA amplification of the specific gene of DNA. RT-PCR was performed to evaluate the expression of Osteocalcin (OCN), Collagen type I (Col I), Runt-related transcription factor 2 (Runx2), and housekeeping gene, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The obtained PCR products were analyzed by electrophoresis on 1% (v/v) agarose gel containing Ethidium Bromide and expression of band was observed by Flourchem™ HD2 (Alpha Innotech, San Leandero, USA). The normalization of mRNA expressions of Col I, OCN and Runx2 were performed according to housekeeping gene, GAPDH using ImageJ.

2. 5. Rat calvarial defect model and micro-computerized tomography The bone mineralization in in vivo was evaluated in female Sprague-Dawley rats (SD rat, Hanil laboratory animal center, Wanju, Korea 6 weeks, female) and anesthetized with intramuscular injection with 60 µL of Zoletil and Domitor (2:1 ratio). The surgical site was shaved using an electric shaver and washed with povidone-iodone. Midline coronal surgical incision was done and dissected to expose the calvarias. Bone defects of two 4 mm diameter sized circular full-thickness were created using a surgical drill. Then, Qtn/SF/HAp scaffolds with BMSCs at a density of 1 x 105 cells / scaffold, cultured for 3 days, were implanted at the defect site. After the implantation, the subcutaneous tissue was closed and the skin incisions were sutured. The implanted scaffolds in the defect sites were examined for bone formation for 6 weeks. At 6 weeks post-surgery, micro computerized tomography (µCT) analysis was performed using µCT system (Skyscan 1076, Optoscan, Belgium). Prior to photographing, experimental animals were anesthetized, placed on stand, and then taken under external surveillance. The scanner was equipped with the following settings: voltage 101 kV, current 98 µA, filtration 0.5 mm aluminum. The obtained data is evaluated using the Skyscan program (DataViewer, CTAn, CTvol), numerical analysis of bone mineral density (BMD), bone volume (BV), percent bone volume (BV/TV), bone surface (BS), trabecular number (Tb) 6 ACS Paragon Plus Environment

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and trabecular thickness (Tb.Th). All the animal experiments were performed in accordance with the guidelines and approval of Chonbuk National University Animal Care Committee, Jeonju, Republic of Korea (CBNU 2016-50).

2. 6. Histological analysis Histological analysis of the calvarial samples was carried out after 6 weeks post-surgery. Rats were sacrificed and the extracted bones coupled with the scaffolds were washed in PBS, and fixed in 10% formaldehyde. The constructs were then treated with decalcifying solution, dehydration was done using a series of graded ethanol and embedded in paraffin. 7 µm thickness of paraffin sections were cut using microtome (Thermo Scientific, USA) and fixed on poly-L-lysine (PLL) coating slide. After post de-paraffin process of the sections, the hematoxylin and eosin (H&E) and Masson’s trichrome (MTS) staining was performed for histological examination and analyzed under an optical microscope (Nikon TE-2000, Japan).

2. 7. Statistical analysis The data are calculated as mean ± standard deviation (SD) and analyzed with one-way ANOVA using GraphPad Prisme 5.0 (San Diego, CA, USA), respectively. A Probability (p) value of

Hydroxyapatite Scaffold Promotes

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