Collagen Functionalized With Graphene Oxide Enhanced Biomimetic

Nov 26, 2018 - ACS Applied Materials & Interfaces. Balasubramanian, Annalakshmi, Chen, Sathesh, Peng, and Balamurugan. 2018 10 (50), pp 43543–43551...
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Applications of Polymer, Composite, and Coating Materials

Collagen Functionalized With Graphene Oxide Enhanced Biomimetic Mineralization and In Situ Bone Defect Repair Chuchao Zhou, Shaokai Liu, Jialun Li, Ke Guo, Quan Yuan, Aimei Zhong, Jie Yang, Jiecong Wang, Jiaming Sun, and Zhenxing Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b17636 • Publication Date (Web): 26 Nov 2018 Downloaded from http://pubs.acs.org on November 27, 2018

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Collagen Functionalized With Graphene Oxide Enhanced Biomimetic Mineralization and In Situ Bone Defect Repair

Chuchao Zhou#, †, Shaokai Liu#, †, Jialun Li†, Ke Guo†, Quan Yuan†, Aimei Zhong†, Jie Yang†, Jiecong Wang†, *, Jiaming Sun†, *, Zhenxing Wang†, *



Department of Plastic Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China

#

These authors contributed equally to this work.

*

Corresponding authors [email protected] (Jiecong Wang) [email protected] (Jiaming Sun) [email protected] (Zhenxing Wang)

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Abstract Biomimetic mineralization using simulated body fluid (SBF) can form a bone-like apatite (Ap) on the natural polymers and enhance osteoconductivity, biocompatibility and reduce immunological rejection. Nevertheless, the coating efficiency of bone-like apatite layer on natural polymers is still needed to be improved. Graphene oxide (GO) is rich in functional groups, such as carbonyls (-COOH) and hydroxyls (-OH), which can provide more active sites for biomimetic mineralization and improve the proliferation of the rat bone marrow stromal cells (r-BMSCs). In this study, we introduced 0% W/V, 0.05% W/V, 0.1% W/V and 0.2% W/V concentrations of GO into collagen (Col) scaffolds and immersed the fabricated scaffolds into SBF for 1, 7 and 14 days. In vitro environment scanning electron microscopy (ESEM), energy dispersive spectrometer (EDS), thermogravimetric analysis (TGA), micro CT, calcium quantitative analysis and cellular analysis were used to evaluate the formation of bone-like apatite on the scaffolds. In vivo implantation of the scaffolds into the rat cranial defect was used to analyse the bone regeneration ability. The resulting GO-Col-Ap scaffolds exhibited a porous and interconnected structure coated with a homogeneous distribution of bone-like apatite on their surfaces. Ca/P ratio of 0.1% GOCol-Ap group was equal to that of natural bone tissue based on EDS analysis. More apatites were observed in 0.1% GO-Col-Ap group through TGA analysis, micro CT evaluation, and calcium quantitative analysis. Furthermore, the 0.1% GO-Col-Ap group showed significantly higher r-BMSCs adhesion and proliferation in vitro and more than two-fold higher bone formation than Col-Ap group in vivo. Our study provides a new approach of introducing graphene oxide into bone tissue engineering scaffolds to enhance biomimetic mineralization.

Keywords : graphene oxide, collagen, bone-like apatite, biomimetic mineralization, simulated body fluid

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1. Introduction Large bone defects caused by traumatic injury, infection or tumour resection pose a huge clinical burden worldwide1. Current clinical treatments for large bone defects including autogenous grafts, allografts and xenografts are generally limited by their inherent disadvantages such as donor site morbidity, disease or virus transmission, immunogenicity and host-donor junction complications2. Therefore, the use of bone graft substitutes as scaffolds to regenerate the bone tissue has attracted the interests of researchers.

Type I collagen (Col) is one of the most prevalent collagen type found in the extracellular matrix (ECM), especially in tendon and bone tissues. It has been broadly used as a graft substitue due to its good biocompatibility and biodegradability3, 4. However, the mechanical strength and osteoconductivity of collagen scaffolds are still unsatisfactory5. The presence of functional groups in collagen makes it convenient to modify, crosslink or coat with other bioactive molecules to create collagen-based materials with tailored mechanical and biological properties6.

Graft substitutes coated with CaP have been widely used due to their outstanding intrinsic bioactivity and osteoconductivity. CaP coating was first introduced to treat the surface of titanium (Ti) metal implants using plasma sparying method in the early 1950s. Since then, various types of methods aimed to prepare bioactive CaP coatings have been developed, including thermal spraying7, sputter coating8, sol-gel deposition9, dip coating10, and hot

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isostatic pressing11. A common limitation of these methods is the high processing temperature which is unsuitable for polymers with low melting temperature, such as natural polymers and biological materials. Biomimetic mineralization using simulated body fluid (SBF) is a promising method that provides suitable conditions of temperature, ions concentration and pH which are similar to human blood plasma. These biomimetic conditions could promote deposition of bone-like apatite (Ap) covered on graft substitutes12. However, there are still some improvements required to develope an uniform and thick-enough coating on the natural polymers13.

Graphene oxide (GO), a single layer of sp2-bonded carbon atoms, has emerged as a promising substrate for constructing graft substitutes due to its superior mechanical and biological properties14. The presence of epoxides, carbonyls and hydroxyls on the surface of GO makes it easily dispersed in aqueous solutions and can provide anchor sites for binding with polymers or nanoparticles in a scaffold15. Recent studies have shown that GO can serve as an effective reinforcement filler by enhancing the network structure of the scaffolds16, 17 and as a biological activator in natural polymers such as collagen and chitosan through introducing a plethora of functional groups to more closely mimic the properties of native bone and coordinate ions to mineralize the scaffolds18.

Therefore, we hypothesized that GO nanosheets might significantly improve the biomineralization efficiency of collagen in SBF solutions. In this study, crosslinked and

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porous GO-Col scaffold was prepared by amidation and lyophilization of GO and Col. To improve the osteoconductivity of the scaffold, the bone-like apatite was linked to GO and Col on the surface of the scaffold through soaking in SBF for 7 days (Fig. 1). Then, environmental scanning electron microscopy (ESEM), energy dispersive spectrometer (EDS), fourier transform infrared spectrometer (FTIR), thermogravimetric analysis (TGA), micro CT, calcium quantitative analysis were used to evaluate the formation of bone-like apatite on the scaffolds. Systematic in vitro experiments determining the cellular response (cell viability and cell proliferation) to the scaffolds were carried out to evaluate their cytocompatibility. Moreover, in vivo experiments evaluating the ability of scaffolds to repair critical-size bone defects in rat skull were performed and further analyzed by CT scanning and histological examination.

2. Materials and Methods Graphene (500 meshes) was obtained from Acros Organic Company. Collagen, N-(3Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride crystalline (EDC) and Nhydroxysuccinimide (NHS) were purchased from Sigma-Aldrich (Shanghai, China).. Reagents NaNO3, H2SO4, HCl, CaCl2, Na2SO4, KMnO4, H2O2, HAc, C2H6O, NaCl, NaHCO3, KCl, K2HPO4•3H2O, MgCl2•6H2O, and CNH2(HOCH2)3 were purchased from Sinopharm Chemical Reagent Co., Ltd, China. SBF solution was prepared according to Kokubo’s method19

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Fetal bovine serum (FBS) was purchased from Hyclone. Phosphate buffered saline (PBS) and Trypsin-EDTA were purchased from Servicebio technology Co., Ltd, China. Lowglucose Dulbecco’s modified Eagle medium (L-DMEM) and 1% penicillin and streptomycin were purchased from Hyclone (Logan, UT, USA). Fluorescein diacetate (FDA), 3-[4,5-dimehyl-2-thiazolyl]-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (Shanghai, China).

2.1. Fabrication of the GO-Col-Ap scaffolds 2.1.1. Synthesis of GO-Col scaffolds The GO-Col scaffolds were synthesized by chemical crosslinking as illustrated in Fig. S1. Briefly, GO was prepared by the modified Hummer’s method20. 4% Col solution (4% W/V in 0.1M HAc) and GO solutions (0%, 0.1%, 0.2%, 0.4% W/V in 0.1M HAc) were mixed at 1:1 volume ratio (the final concentrations of GO were 0% W/V, 0.05% W/V, 0.1% W/V, and 0.2% W/V) under sonication for 30 min. Then, the obtained solutions were cast in molds (5 mm in diameter and 1 mm in height). The molds were frozen overnight at -20℃ and subsequently lyophilized for 24 h at -50℃ under vacuum. Thereafter, the freeze-dried plates were taken out of the molds and immersed in 95% ethanol solution, followed by addition of EDC (10 mg/mL) and NHS (4 mg/mL). The scaffolds were kept for 24 h at room temperature to allow the collagen to crosslink with GO and form a stable GO-Col scaffold. The collagen scaffolds were formed using the same procedures described above. To remove the by-products of the chemical reactions, the scaffolds were washed with

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ddH2O three times, followed by freezing overnight at -20℃ and lyophilizing for 24 h at 50℃ under vacuum.

2.1.2. Biomimetic apatite deposition The crosslinked scaffolds were immersed into CaCl2 and K2HPO4 solutions to accelerate biomimetic apatite deposition. Next, the scaffolds were immersed in 20 mL of 0.2M CaCl2 solution for 3 min and then soaked in 20 mL of ddH2O for 10s, followed by soaking in 20 mL of 0.2M K2HPO4 solution for 3 min and then soaked in 20 mL of ddH2O for another 10s. The entire experiment was repeated three times. These alternately soaked scaffolds were subsequently immersed in SBF for biomimetic apatite deposition (10 mL of SBF was poured into one well of 6-welled culture plate containing one alternately soaked scaffold). After a 5 min of vacuum treatment, the scaffolds were kept at 37℃ for 1 day, 7 days, 14 days, and the SBF was renewed every 24 h to maintain a consistent ionic strength throughout the experiment. The SBF solution was prepared by dissolving NaCl, NaHCO3, KCl, K2HPO4•3H2O, MgCl2•6H2O, CaCl2, Na2SO4 in ddH2O and buffering to PH 7.40 at 36.5℃ with Tris (hydroxymethyl) aminomethane and aqueous 1M HCl solution. The scaffolds were removed from the SBF, gently washed with ddH2O and subsequently lyophilized at -50℃ for 24 h under vacuum.

2.2. Characterization of the scaffolds 2.2.1. Mass increase of the scaffolds after biomimetic apatite deposition

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The mass increase (MI) of the scaffolds after biomimetic apatite deposition in SBF was measured using an electronic analytical balance (accurate to 10-5 g). The mass increment was expressed as the difference between the M1 (mass of the scaffolds before alternately soaking in CaCl2 and K2HPO4 solution) and M2 (mass of the scaffolds after biomimetic immersion in SBF), described by equation (1): (1)

2.2.2. ESEM and EDS evaluation The surfaces of the scaffolds were characterized before and after the biomimetic coating process by environmental scanning electron microscopy (ESEM; Quanta 200, FEI company, Holland) at 10 kV. The ratio of the calcium and phosphate of the biomimetic coating was performed by the energy dispersive spectrometer (EDS). Prior to ESEM observation and EDS analysis, the scaffolds were sputter-coated with gold for 1 min.

2.2.3. XRD、FTIR and TGA evaluation Crystalline phases were assayed using X-ray diffractometer (XRD; Empyrean, PANalytical B.V.) at a scanning rate of 0.013° s-1 in a 2θ range from 5° to 40° with CuKα radiation (k = 1.540598 nm). Functional groups were confirmed using FTIR spectroscopy (VERTEX 70; Bruker company, German) from 4000 cm-1 to 500 cm-1 at a resolution of 0.4 cm-1. The amount of apatite in the 3D scaffolds was determined using TGA (Diamond TG/DTA; PerkinElmer Instruments, Shanghai, China). The scaffolds were heated at a

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heating rate of 10℃/min from 25℃ to 800℃ in the N2 atmosphere.

2.2.4. Mechanical evaluation The elastic modulus was tested to evaluate the mechanical properties of the scaffold using All-Electric Dynamic Test Instrument (ElectroPuls E1000, INSTRON, British) at a loading rate of 1 mm/min until the 80% compression of the samples was achieved. The slope of the initial linear portion of the stress-strain curves were claculated to obtain the elastic modulus.

2.2.5. Micro CT evaluation To observe the biomimetic apatite deposition in the scaffolds, micro-CT scanner (SkyScan 1176; Broker) was used with the following scanning parameters: 58 kV, 385 mA, and 18 μm slice thickness. Corresponding 3D images were reconstructed using VG studio software (Volume Graphics GmbH, Heidelberg, Germany). Apatite volume (AV) and the percentage of apatite volume relative to total volume (AV/TV) were determined by the micro-CT assistant software (Scanco Medical, Zurich, Switzerland).

2.2.6. Calcium quantitative analysis The calcium content assay was performed by dissolving the mineralized scaffolds in 0.4 mL 0.5M acetic acid overnight and quantifying them with a calcium assay kit (BioAssay Systems, Hayward, CA, http://www.bioassaysys.com) according to the manufacturer’s instruction.

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2.3. In vitro cellular evaluation 2.3.1. Rat BMSCs isolation and in vitro culture Rat bone marrow stromal cells (r-BMSCs) were isolated and harvested as described previously21. In brief, newborn Sprague Dawley rats (3-5 days old) were euthanized by cervical dislocation, followed by soaking in 75% alcohol for 15 min. Femurs and tibias were separated from attached muscles and soft tissues. Then, cartilages at both end of the bones were cut off, followed by repeated washing of the cavities with the culture medium until the cavities appeared white. Finally, the fresh bone marrow tissues were seeded on 10 cm culture dishes with 7 mL of culture medium (L-DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin). The culture dishes were incubated (Thermo Scientific, Heracell 150i) at 5% CO2, 37℃ and the culture medium was changed every 3 days. When the attached r-BMSCs became confluent, the BMSCs were passaged.

2.3.2. Cell seeding on scaffolds and Cell adhesion capability To evaluate the cytocompatibility of the scaffolds, each scaffold (n=5) were seeded 2×105 r-BMSCs in 20 μL of culture medium. The plates containing scaffolds were cultured in an incubator at 5% CO2, 37℃ and the culture medium was changed every 3 days.

After 2h of initial attachment period, the r-BMSCs seeded on scaffolds were transferred to a new 6-well plate. For assessment of cell adhesion capability, the number of r-BMSCs

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attatched to the bottom of the plate was collected and counted (N). Cell adhesion capablity (CAC) was calculated using the following equation (2): (2) where 2×105 in Eq. (2) was the total amount of cells loaded.

2.3.3. Evaluation of cell viability and morphology The cell viability and morphology of r-BMSCs seeded on the scaffolds were assessed by FDA cell imaging Kit (Sigma-Aldrich, China) staining. In brief, the scaffolds were washed three times with phosphate buffered saline (PBS) and subsequently immersed in 2 μM fluorescein diacetate (FDA) for 30 min at 37℃. After 1, 7, and 14 days of incubation, scaffolds were examined by confocal fluorescence microscope.

To further evaluate the cell morphology, the cell-seeded scaffolds were washed thrice with PBS, fixed with 2.5% glutaraldehyde for 3 h and washed again with PBS. Then, the scaffolds were dehydrated with a graded series of ethanol solutions, lyophilized at -50℃ under vacuum, and sputter-coated with gold for ESEM analysis.

2.3.4. The proliferation of r-BMSCs on the scaffolds 2×105 r-BMSCs were seeded on each scaffold in 6-well plates. After 1, 7 and 14 days of incubation, the cell proliferation rate was evaluated using MTT assay based on the manufacturer’s instructions.

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2.4. In vivo cranial defect study 2.4.1. In vivo rat cranial defect orthotopic bone formation To explore the ability of the GO-Col-Ap implants to form new bone in vivo , 24 male adult SD rats (age:6 weeks, weight:180-220 g; supplied by the animal center of Tongji medical college, Huazhong University of science and technology) were randomly divided into four groups: (1) Col-Ap; (2) 0.05% GO-Col-Ap; (3) 0.1% GO-Col-Ap; (4) 0.2% GO-Col-Ap. (n=6 rats per group). The rats were anesthetized by inhaled isoflurane and subcutaneous buprenorphine injection, and a 2.0 cm sagittal incision was subsequently made on the middle of the scalp. Two parallel 5.0 mm critical cranial defects were drilled into each rat using a 5.0 mm diameter trephine (Nouvag AG, Goldach Switzerland). After the scaffolds were implanted into the defect areas, the incision was closed.

2.4.2. Micro CT evaluation Animals were sacrificed at 4 and 12 weeks postoperation and the skulls were collected for fixation with 10% formalin (n=3 rats per group). The bone tissue in the defect areas (diameter: 5mm; height: 2mm) was evaluated by Micro-CT scanning. 3D images (mean threshold value = 226) of the samples were reconstructed by VG studio software and the quantitative morphometric analyses were performed using micro-CT assistant software to determine the bone volume (BV), bone volume/tissue volume (BV/TV), and bone mineral density (BMD). The total soft tissue and bone tissue in the 5mm rat cranial bone defect

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area is defined as tissue volume (TV).

All surgical procedures in the experiments were approved by the Huazhong University of Science and Technology Animal Care and Use Committee.

2.4.3. Histological observation At the end of micro-CT evaluations, all samples were formalin-fixed for 7 days and subsequently decalcified in EDTA for 4 weeks. Thereafter, the samples were dehydrated through graded ethanol series and embedded in paraffin, followed by sectioning to 3 μm pieces and staining with hematoxylin and eosin (Sigma) and Masson’s Trichrome (Sigma) separately to examine the tissue morphology and new bone formation under the microscope (Eclipse Ni-E; Tokyo, Japan).

For immunohistochemical analyses, sections were blocked by diluted ghost serum antibody and then incubated with OCN primary antibody (1:100 dilution, Abcam). Next, the sections were incubated with HRP-labeled goat antibody/mouse secondary antibody (Abcam) and subsequently colored by DAB reagent (DAKO). The sections were then stained with hematoxylin. The immunohistochemical staining samples were observed under a microscope.

2.5. Statistical analysis

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All data presented in this study are expressed as the mean ± SD. The means of two groups were compared using Student’s t-test and the means of multiple-groups were compared using one-way analysis of variance (ANOVA) tests with post hoc contrasts by StudentNewman-Keuls test. P < 0.05 was considered to be a statistically significant difference.

3. Results 3.1. Macro and micro images of scaffolds before and after biomimetic apatite deposition. The colors of the collagen scaffold and three groups of GO-Col scaffolds were white and brown, respectively. The brown color of GO-Col scaffolds increased with the GO concentration from 0.05% W/V to 0.2% W/V (Fig. 2A). After 7 days of soaking in SBF, white crystals were deposited on the scaffolds and the color of GO-Col scaffolds changed to white (Fig. 2B). The SEM images revealed the surface morphologies of scaffolds and crystals at the micro scale level. Porous and interconnected structures were observed in the four groups of scaffolds. The surface of Col scaffold was smooth while folded and uneven surfaces were observed in three groups of GO-Col scaffolds (Fig. 2C). After biomimetic apatite deposition in SBF, homogeneous crystals were deposited on the surface of scaffolds (Fig. 2D), which led to a remarkable increase of mass at each time-point in the quantitative assay (Fig. 2E). Significant differences were observed (p < 0.001) at day 7 (Col-Ap: 174.2% ± 13.8%, 0.05% GO-Col-Ap: 170.8% ± 21.3%, 0.1% GO-Col-Ap: 220.0% ± 18.3%, 0.2% GO-Col-Ap: 225.8% ± 7.9%) relative to day 1 (Col-Ap: 82.9% ± 8.2%, 0.05% GO-Col-

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Figure 1. Schematic illustration of the fabrication of GO-Col-Ap scaffolds and their application in vivo. (1) The GO-Col scaffolds were fabricated by the crosslinking of collagen and graphene oxide. (2) Apatite crystals were deposited on the scaffolds after soaking in simulated body fluid (SBF) for 7 days. (3) The GO-Col-Ap scaffolds were implanted directly into rat cranial defects sites to evaluate orthotopic bone formation.

Ap: 79.3% ± 7.0%, 0.1% GO-Col-Ap: 95.3% ± 7.1%, 0.2% GO-Col-Ap: 101.8% ± 9.1%), but no significant differences were observed between day 7 and day 14. Given these findings, the 7 days period of SBF treatment was chosen for the following in vitro and in vivo experiments.

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Figure 2. Macro and micro images of the scaffolds before and after biomimetic apatite deposition. The color of collagen and graphene oxide-collagen scaffolds were white and brown, respectively (A). White crystals were deposited on the scaffolds after the biomimetic apatite deposition (B). As shown in the SEM images, the scaffolds surfaces before biomimetic apatite deposition were smooth (C), while crystals were observed on the scaffolds after biomimetic apatite deposition at 1, 7, 14 days (D). The mass increases of the four groups were remarkable between 1 day and 7 days of incubation in SBF, but no significant difference was observed between 7 days and 14 days of incubation in SBF (E). (Scale bars in C, D: 10 μm, ***P