Silk Fibroin

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Bioengineered Osteoinductive Broussonetia Kazinoki/ Silk Fibroin Composite Scaffolds for Bone Tissue Regeneration Do Kyung Kim, Jeong In Kim, Tae In Hwang, Bo Ra Sim, and Gilson Khang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b14351 • Publication Date (Web): 21 Dec 2016 Downloaded from http://pubs.acs.org on December 22, 2016

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ACS Applied Materials & Interfaces

Bioengineered osteoinductive Broussonetia kazinoki/ Silk fibroin composite scaffolds for bone tissue regeneration

†

†

2

Do Kyung Kim,1, Jeong In Kim,2, , Tae In Hwang, Bo Ra Sim,1 Gilson Khang1, *

1

Department of BIN Convergence Technology, Department of Polymer Nano Science &

Technology and Polymer BIN Research Center, Chonbuk National University, Deokjin-gu, Jeonju 561-756, Republic of Korea 2

Department of Bionanosystem Engineering, Graduate School, Chonbuk National University, Jeonju 561-756, Republic of Korea

† Equally-Contributed Authors

Corresponding author. Phone: 82-63-270-2848; E-mail: [email protected] Keywords: plant extract; broussonetia kazinoki; silk fibroin; bone regeneration; scaffold

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Abstract In this article, Broussonetia kazinoki (BK) powdery extract is utilized to modify silk fibroin (SF) scaffold and applied to bone defect area. The BK/SF scaffold is an efficient cell-carrier which promotes cell proliferation and osteogenic differentiation of rBMSCs. We confirmed biocompatibility and osteogenic differentiation capacity of BK/SF scaffolds compared to pristine SF scaffold in both in vitro and in vivo evaluation. Gene expression related to osteogenic differentiation and bone regeneration significantly up-regulated in BK/SF scaffold group. The implanted scaffolds were attached well to the surface of bone defect region and integrated with surrounding tissues without significant inflammatory reaction. Furthermore, almost 45 % of bone volume has been recovered at 8 weeks post-surgery while the SF and control group showed 20 % recovery. These results suggest BK powdery extract incorporated with SF scaffold might be a suitable substitute for alternative bone graft for bone regeneration. 1. Introduction Engineering of regenerative new bone tissue to repair bone fracture caused by tumor resection, skeletal diseases, osteitis and traumatic injury is one of the most challenge field.1 Bone defects occurring in elderly patients lead to morbidity and disability with overall health problems and decrease quality of life.2 In the United States, 6.3 million bone fractures occur and 2 million patients suffer from osteoporosis associate with bone fracture which costs 20 billion dollars per year approximately. Autografts and allografts were suggested for bone reconstruction and demonstrated successfully. However, autografting requires patients to meet standards which accompanied by risks and limited availability. On the other hand, allografting can transmit disease and occur inflammation responses by nonunion with host tissues.3 This led to the significance of efficient alternative bone graft made with high quality


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biocompatible materials as well as proper physical and mechanical properties. Bone marrow derived mesenchymal stem cells (BMSCs) are multipotential stem cells which can differentiate into osteoblasts, myoblasts, chondrocytes, hepatocytes, astrocytes, endothelial cells, fibroblasts, cardiomyocytes, and neurons.4–6 BMSCs have the capacity of culture expansion, low tumorigenic hazards, minimal immunogenic properties and ease genetic manipulation.7,8 In this regard, BMSCs thought to be an available cell source for bone regeneration such as dental, craniofacial reconstruction and orthopedic repairs.9,10 The BMSC-based bioengineered scaffolds for bone regeneration has been studied from many research groups.8 Selection of biomaterial for tissue engineered scaffold is an important factor to support and guide cells into functional tissue through enhancing cell adhesion, migration, proliferation, and differentiation.11–13 We chose SF isolated from silk cocoons from Bombyx mori as a base material for the osteoinductive scaffold. SF offers high biocompatibility, controllable biodegradability, impressive mechanical superiority and ease of handling.11,14 SF is nature derived protein consists of two main proteins: sericin and fibroin. Unlike other natural proteins, the inner part of SF called fibroin does not occur immune rejection.15 SF has been used as a base biomaterial for bone regeneration scaffold previously and many papers have been published on the study of SF scaffold incorporated with different osteoinductive sources such as bone growth factors and cytokines. However, the studies of its direct use of plant extract for complete bone regeneration are sparse. Plant extracts have been used in various diseases with its herbal remedies. Approximately, 25 % of available drugs which are allowed to prescript are derived from plants, trees and herbs.16 In this study, we used BK which quickly spread out in the forest by its great adaptability and growth.17 BK has been used in China for a long time as a traditional herbal medicine to treat inflammatory disorder patients such as septic inflammation and chronic bronchitis. Flavonoids, broussochalcone and papyriflavonol-A 3 ACS Paragon Plus Environment


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found in BK demonstrate anti-inflammatory, anti-fungal, antibacterial and anti-oxidant effects.18–20 Moreover, high calcium hydroxide content in BK powdery extract induces proliferation,

migration,

osteogenic

differentiation

of

stem

cells

and

promotes

osteoconductivity.21 The use of BK extract as a biomaterial for bone regeneration instead of using growth factors and cytokines reduces the cost and the risks. The whole BK powdery extract was a main target for promoting bone growth in the bone defect region by enables its use in the clinical field. The ultimate goal of this study is to design an efficient alternative bone graft which promotes the proliferation and osteogenic differentiation of BMSCs. We evaluated whether the BK/SF scaffolds could promote osteogenic differentiation of rBMSCs in in vitro environment and form new bones in rat critical size calvaria defects and under subcutaneous region of athymic nude mice in vivo. Fabricated SF and BK/SF scaffolds were examined for chemical, thermal and mechanical properties analysis such as compressive strength, water uptake, ICP-MS, XRD, SEM, DSC TGA and FTIR. Fabricated scaffolds were evaluated its biocompatibility using MTT assay, Live and Dead assay, total DNA content assay, ALP assay and Real-time PCR. Furthermore, bone regeneration capacity of BK/SF scaffold was assessed after implantation in rat calvaria defects for 8 weeks in vivo. 2. Materials and Methods 2.1. Preparation of silk fibroin solution Silkworm cocoons were cut into pieces and boiled in 0.02 M Na2CO3 (Showa Chemical, Japan) with distilled water for 30 min to remove sericin. Boiled silkworm cocoons were washed with distilled water for 3 times and fully dried under the fume hood. After drying, silkworm cocoons were dissolved in the oven with 9.3 M LiBr (Kanto chemical, Japan) at 60 ℃ for 4 h. To remove LiBr, dissolved solution was dialyzed using dialysis tube (Snake


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Skin Dialysis Tubing 3,500 MWCO (molecular weight cut-off), Thermo SCIENCE, USA) for 72 h in distilled water. SF solution was stored at 4 ℃ until use. The final concentration of SF solution was 8 wt/vol.%, determined by gravimetric analysis. 2.2. BK powdery extraction The bough of fully grown BK was collected from the garden. The fresh bough of BK was boiled in distilled water for 5 h. After boiling, barks and black skins around the BK were removed. To remove the non-fibrous materials, 10 g of BK was boiled in 0.1 M NaOH dissolved in distilled water for 4 h. Boiled BK was washed 5 times with distilled water to remove NaOH. The BK was lyophilized and prepared as powdery extract using freezemilling machine (SPEX SamplePrep, USA). The powder was stored in deep freezer at -70 ℃ until use. 2.3. Fabrication of scaffold SF and BK/SF scaffolds were fabricated by freeze-drying method. 1 wt.% of BK powdery extracts was blended with SF aqueous solution and poured into 96-well plate. Later, solution was kept in deep freezer overnight and lyophilized. Lyophilized scaffolds were cross linked with Methanol for 1 h and washed with distilled water. Scaffolds were cut with a thickness of 3 mm and a diameter of 6 mm. The average pore size of the scaffolds was under 150 ㎛, measured by Image J program. (n=5) Mechanical properties 2.4. FTIR spectroscopy Infrared spectra of SF and BK/SF scaffolds were measured using FTIR (Perkin Elmer, USA) in spectra range of 4000 to 400 cm-1 wave numbers. For FTIR measurement, samples were examined in the solid state. 5 ACS Paragon Plus Environment


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2.5. Thermal characterization The thermaldynamic property of the scaffolds were analyzed by using Differential Scanning Calorimeter (DSC, Q20, TA Instruments, South Korea) and Thermo Gravimetric Analyzer (Q600, TA Instruments, South Korea) under N2 atmosphere at heating rate of 10 ℃/min. 2.6. Compressive strength Compressive strength of bare and cell-cultured scaffolds was measured using Texture Analyzer (FTC, Sterting, Virginia, USA), loaded with a crosshead speed of 10mm/min and a force of 1 N. rBMSCs were cultured for 28 days on SF and BK/SF scaffolds and compressive strength was determined. 2.7. Swelling degree Swelling degree of SF and BK/SF scaffolds were evaluated by weighing method. To measure the dry weight (Wd) of the scaffolds, scaffolds were kept in the oven at 60℃ for 4 h and weighed. Dried scaffolds were immersed in phosphate buffer saline (PBS) and wet weigh was taken (WW) at each time point. Swelling ratio was calculated using formula A. A. Swelling ratio (%) = (WW - Wd)/ Wd x 100 2.8. The characterization of BK extract The elements in the BK were analyzed using Inductively Coupled Plasma Mass Spectrometry (ICP-MS, 7500a, Agilent, USA) and X-ray Diffraction (XRD, MAX-2500, Rigaku). For ICPMS analysis, powdery extract of BK (0.0192g) was prepared in liquid state dissolved in 0.6 ml of 70 % nitric acid solution. Before measurement, extract solution was diluted in 10 ml distilled water. For XRD analysis, BK powdery extract was prepared in solid state, without further preparation. 2.9. Isolation of rBMSCs 6 ACS Paragon Plus Environment

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New Zealand white rabbit was sacrificed to isolate the rBMSCs. Rabbit femur was collected and straightly moved to PBS. Rabbit femur was washed 3 times with PBS. The surrounding tissues around the femur were removed and washed with 70 % alcohol under the clean bench. Bone marrow in the rabbit femur was collected using 20 G needle. Bone marrow with Alpha MEM medium (Lonza, USA) was centrifuged at 1200 rpm for 3 min. Pellets were washed with PBS and re-suspended using 70 ㎛ cell strainer with medium containing 20 % fetal bovine serum (FBS) and 1 % penicillin/streptomycin. rBMSCs were cultured into dishes (Corning, USA) and medium was added. Medium was changed every 3 days. Primary passage 2 of rBMSCs were used for this study. 2.10. Cytotoxicity and cell proliferation on scaffolds The cytotoxicity of BK/SF scaffolds was analyzed after 4 h of cell seeding (5 x104 rBMSCs /scaffold) using Live/Dead Cell Imaging kit (Invitrogen, USA). Cells were stained with calcein AM and ethidum homodimer for 15 min. Live and dead cell images were taken under confocal laser scanning microscope (LSM 510 META, Carl Zeiss, Germany). The viability of cultured rBMSC was evaluated after 3, 5, 7, 14 and 28 days of culture using MTT (3-[4,dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide;thiazolyl blue) assay. The rBMSCs (5 x104) were seeded on each scaffold for cell viability evaluation. (n=3) Cultured media was removed and replaced with 1 ml fresh media, and then 100 µL MTT solution (5 mg/ml in PBS) was added to each well. Scaffolds were stored at 37 ℃ in a humidified 5 % CO2 incubator for 4 h to allow formazan crystal formation. After incubation, supernatant was removed and 1 ml of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. Dissolved solution was placed in 96-well plate and read at absorbance of 570 nm. 2.11. ALP activity of rBMSCs Alkaline phosphatase (ALP) activity of rBMSCs was measured using ALP assay kit (Takara 7 ACS Paragon Plus Environment


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Bio, Japan). The ALP activity of cultured rBMSCs was evaluated after 3, 5, 7, 14 and 28 days of culture. The rBMSCs (5 x104) were seeded on each BK/SF scaffold for ALP assay. (n=3) Cultured media was removed and washed 1 time with PBS. Cultured rBMSCs were extracted using 1 ml of extraction solution and 50 µL of p-nitrophenylphosphate (pNPP) was added into 24-well plate. Well plate was incubated at 37 ℃ in a humidified 5 % CO2 incubator for 1 h. After incubation, reaction was stopped by 50 µL 0.9 N NaOH stop solution and read at absorbance of 405 nm. Total DNA content was determined Quant-iT™ HighSensitivity dsDNA Assay Kit (Thermo scientific, USA), read at 480 nm and calculated according to ds DNA HS standards. ALP levels were normalized to total DNA content at the end of the experiment. 2.12. Quantitative real-time polymerase chain reaction Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted after rBMSC cultured on scaffolds for 28 days. The rBMSCs (5 x104 cells/scaffold) were seeded on the scaffolds for qRT-PCR analysis. (2The mRNA was extracted from cell cultured scaffolds using Trizol (Invitrogen, USA) and Chloroform (Sigma-Aldrich, USA) and centrifuged at 12000 rpm for 15 min at 4 ℃. After centrifugation, supernatant was collected carefully and Iso-propanol (Sigma-Aldrich, USA) was added. Samples were kept at 4 ℃ overnight and mRNA from rBMSC was dissolved in RNase-DNase free water (Gibco, USA). The concentration of mRNA in each sample was determined using BioSpectrophotometer (Eppendorf, USA). To prepare cDNA samples, reverse transcription reaction was conducted using cDNA Synthesis kit (BioRad, USA). qRT-PCR was performed by StepOnePlus RealTime PCR system (Applied Biosystems, USA) using SYBR Green Master Mix (Applied Biosystems, USA). The expression of osteogenic and anti-inflammatory markers such as Collagen type 1 (COL1), Osteocalcin (OCN), Vascular endothelial growth factor (VEGF), Cyclooxygenase 2 (COX-2), Tumor necrosis factor-alpha (TNF- α) and Runt-related 8 ACS Paragon Plus Environment

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transcription factor 2 (RUNX-2). The expression level was determined by pristine SF scaffold group, normalizing expression of an endogenous reference transcript β-actin. 2.13. Scanning electron microscopy (SEM) The surface of the scaffolds and morphology of rBMSCs cultured on SF and BK/SF scaffolds were observed by SEM (JEOL JSM 5900LV, Oxford EDS). Suspension of rBMSCs (2 x104 cells/scaffold) was seeded on SF and BK/SF scaffolds and cultured for 28 days. Cultured scaffolds were washed with PBS and fixed with 2.5 % glutaraldehyde (Sigma-Aldrich, USA) for 24 h at 4 ℃. After fixation, scaffolds were dehydrated by changing the graded series of ethanol (50, 60, 70, 80, 90, 100 %) for 30 min each. Before the SEM examination, all samples were dried under the fume hood at room temperature. 2.14. Biocompatibility and osteoconductibility of the scaffolds in rat calvaria defect All experiment procedures were performed with the approval of Chonbuk National University Animal Care Committee, Jeonju, South Korea. The scaffolds for in vivo test were cut into a diameter of 4 mm and a thickness of 1 mm for implantation in rat calvaria defect area using 4 mm diameter biopsy punch (Kai medical, Japan). To examine the bone regeneration ability of SF and BK/SF scaffold, eighteen 6 week old female SD rat weighing 150 g - 250 g were used in the experiment. Rats were anesthetized intramuscularly by injection of 3 cc for each rat with a mixture of Zoletil 50 (30 mg/kg, Virbac, France) and Domitor (1 mg/kg, Orion, Finland). The 10 mm incision near calvaria bone was penetrated with slit knife and circular shape was made on each side of cranial bone using 4 mm biopsy punch (Kai industry, Japan). Two circular defects were made on the SD rat cranium using micro drill (Saeshin precision co., South Korea). On the right side, scaffold with cultured rBMSCs (2 x104 cells/scaffold) was implanted. On the left side, defect area was remained without treatment as a control group. The SD rats were divided into two groups. First group



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had implantation of pristine SF scaffold with rBMSC cultured for 3 days. Second group was implanted BK/SF scaffold with rBMSC cultured on. After surgery, one dose of Oxytetracycline dihydrate (2 mg/kg, Eagle Vet, South Korea) was given to each animal intramuscularly. All animals were sacrificed at 8 weeks post-surgery and whole cranium was harvested. Harvested cranium was fixed with 10 % formalin solution (Sigma-Aldrich, USA) to prepare histological and immunohistological analysis. 2.15. Micro-Computered Tomography analysis Bone regeneration in the bone defected region was visualized by Micro-Computered Tomography (Micro-CT) systems (Skyscan, Kontich, Belgium) at 2, 4 and 8 weeks postsurgery. The bone defect was quantified by using CTAn software (Bruker microCT, USA) and 3D images were obtained using CTvox software (Bruker microCT, USA). 2.16. Histological analysis of BK/SF scaffolds implanted in rat calvaria defect area For evaluation of bone regeneration in the rat cranium defect model, eighteen SD rats were sacrificed at 8 weeks post-surgery. Sacrificed rat cranium was harvested and fixed with 10 % formalin solution. Samples were decalcified using decalcifying solution (Sigma-Aldrich, USA) and embedded in paraffin. The paraffin blocks were sectioned into 7㎛ and stained with hematoxylin and eosin (H&E, Sigma-Aldrich, USA) and masson’s trichrome (MTS, Merck KGaA, Germany).

For immunohistochemistry analysis, paraffin sections were

deparaffinized and stained with collagen type I (COL 1) and osteopontin (OPN). For nonspecific blocking, protein blocking solution (DAKO) was added to each sample for 12 min under dark room at room temperature. After washing, samples were incubated with anticollagen type I (1:150, Santa Crux Biotechnology, USA) and anti-osteopontin (1:100, Santa Crux Biotechnology, USA) as primary antibodies for 90 min at room temperature. As secondary antibody, Alexa Fluor 594-conjugated AffiniPure Donkey Anti-Rabbit IgG (1:300, 10 ACS Paragon Plus Environment

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Jackson Immuno Research Laboratories, Inc., USA) was used for OPN and COL 1 detection. At last, all samples for immunohistochemistry were mounted with mounting medium with DAPI (Santa Cruz Biotechnology, USA) and immunofluorescence images were taken using confocal laser scanning microscope (LSM 510 META, Carl Zeiss, Germany). 2.17 Immunohistochemistry analysis of BK/SF scaffolds implanted under subcutaneous area of athymic nude mice To evaluate the potential of BK/SF scaffolds for osteogenic differentiation ability and biocompatibility. rBMSCs (5 x104 cells/scaffold) loaded BK/SF scaffolds were implanted under subcutaneous area of 6 weeks old nude athymic mice. The limited number of eight nude mice were used for in vivo experiment. Nude mice were anesthetized intramuscularly 0.06 cc for each nude mouse with a mixture of Zoletil 50 (30 mg/kg, Virbac, France) and Domitor (1 mg/kg, Orion, Finland). Nude mice were sacrificed for further examination at 3 weeks post-surgery. The implanted scaffolds were collected and fixed in 4 % formalin solution (Sigma Aldrich, USA) for immunohistochemistry analysis. The scaffolds were embedded in paraffin and sectioned into 7 ㎛. Sectioned scaffold samples were deparaffinized using Xylen and stained with Hematoxylin and Eosin (H&E, Sigma-Aldrich, USA) and von Kossa (Millipore, USA). For immunohistochemistry analysis, deparaffinized samples were washed with PBS and treated with 0.3 % hydrogen peroxide (Showa, Japan) in distilled water for 10 min at room temperature. After washing, samples were blocked with blocking solution (Ultra Tek HRP, ScyTek, USA) for 10 min. Scaffold sections were treated with appropriate concentration of primary antibodies: Collagen type I (COL1,1:100, Santa cruz, USA) and Anti-CD68 (ED1, 1:100 Abcam, USA) for 1 h at room temperature. For secondary antibodies, Goat Anti-mouse IgG-HRP (1:200, AB frontier, South Korea) and Rabbit Anti-Goat IgG-HRP (1:200, Santa cruz, USA) were used for ED-1 and COL1



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detection. After secondary antibody detection, streptoavidine-perioxidase was added to each sample for 30 min under dark room. Perioxidase activity was detected using AEC Substrate Chromogen (Dako, USA) and washed with PBS. Sections were mounted and examined under microscopy (Nikon, Japan). 2.17. Statistical analysis All results are presented as mean±standard deviation. Statistical analysis was carried out based on one-way analysis of variance (one-way ANOVA test) and the differences were considered significant at P

Silk Fibroin

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