Graphene Nanoribbons Nanocomposites Induce

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Nano-hydroxyapatite/graphene nanoribbons nanocomposites induce in vitro osteogenesis and promote in vivo bone neoformation Joelson Silva Medeiros, Aureliano Oliveira, Jancineide Carvalho, Ritchelli Ricci, Maria do Carmo Martins, Bruno Vinicius Manzolli Rodrigues, Thomas Jay Webster, Bartolomeu Cruz Viana, Luana Vasconcellos, Renata Canevari, Fernanda Marciano, and Anderson Oliveira Lobo ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b01032 • Publication Date (Web): 05 Apr 2018 Downloaded from http://pubs.acs.org on April 6, 2018

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ACS Biomaterials Science & Engineering

Nano-hydroxyapatite/graphene nanoribbons nanocomposites induce in vitro osteogenesis and promote in vivo bone neoformation Joelson S. Medeiros1#, Aureliano M. Oliveira1#, Jancineide O. de Carvalho1,2, Ritchelli Ricci3, Maria do C. C. Martins4, Bruno V. M. Rodrigues1, Thomas J. Webster5 Bartolomeu C. Viana6,7, Luana M. R. Vasconcellos8, Renata A. Canevari3, Fernanda R. Marciano1,5 and Anderson O. Lobo1,6,9* 1

Instituto de Ciência e Tecnologia, Universidade Brasil, Rua Carolina da Fonseca, 584, Bairro Itaquera, São Paulo, CEP: 08230-030, Brazil. 2

Centro universitário Uninovafapi, Rua Vitorino Orthiges Fernandes, nº 6123, Bairro Uruguai, Teresina, Piauí, CEP: 64073-505, Brazil.

3

Laboratório de Biologia Molecular do Câncer, Universidade do Vale do Paraíba, Av.

Shishima Hifumi, nº 2911, Bairro Urbanova, São José dos Campos, São Paulo, CEP: 12244-000, Brazil. 4

Departamento de Biofísica e Fisiologia/CCS, Universidade Federal do Piauí, Campus

Universitário Ministro Petrônio Portella, Bairro Ininga, Teresina, Piauí, CEP: 64049550, Brazil. 5

Department of Chemical Engineering, Northeastern University, 360 Huntington Ave, Boston, Massachusetts, ZIP: 02115, United States of America.

6

Laboratório Interdisciplinar de Materiais Avançados, Programa de Pós-Graduação em Ciência e Engenharia dos Materiais, Universidade Federal do Piauí, Campus

Universitário Ministro Petrônio Portella, Bairro Ininga, Teresina, Piauí, CEP: 64049550, Brazil. 7

Departamento de Física, Centro de Ciências Naturais, Universidade Federal do Piauí,

Campus Universitário Ministro Petrônio Portella, Bairro Ininga, Teresina, Piauí, CEP: 64049-550, Brazil. 8

Departamento de Biociências e Diagnóstico Oral, Instituto de Ciência e Tecnologia, Universidade Estadual de São Paulo, Avenida Eng. Francisco José Longo, nº 777. Jardim São Dimas São José dos Campos, São Paulo, Brazil.

9

Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Ave, 18-393, Cambridge, Massachusetts, ZIP: 02139, United States of America. # *

These authors contributed equally.

Corresponding author: Professor Anderson de Oliveira Lobo. Email: [email protected] and [email protected]

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Abstract: Nanomaterials based on graphene oxide nanoribbons (GNR) and nanohydroxyapatite (nHAp) serve as attractive materials for bone tissue engineering. Herein, we evaluated the potential of nHAp/GNR towards in vitro analysis of specific genes related to osteogenesis and in vivo bone regeneration using animal model. Three different concentrations of nHAp/GNR composites were analyzed in vitro using a cytotoxicity assay, and osteogenic potential was determined by ALP, OPN, OCN, COL1 and RUNX2 genes and alkaline phosphatase assays. In vivo bone neoformation using a well-established in vivo rat tibia defect model was used to confirm the efficiency of the optimized composite. The scaffolds were non-toxic, and the osteogenesis process was dose-dependent (at 200 µg mL-1 of nHAp/GNR) compared to controls. The in vivo results showed higher bone neoformation after 15 days of nHAp/GNR implantation compared to all groups. After 21 days, both nHAp/GNR composites showed better lamellar bone formation compared to control. We attributed this enhanced bone neoformation to the high bioactivity and surface area presented by nHAp/GNR composites, which was systematically evaluated in previous studies. These new in vivo results suggest that nHAp/GNR composites can be exploited for a range of strategies for the improved development of novel dental and orthopedic bone grafts to accelerate bone regeneration.

Keywords Graphene oxide nanoribbons; nanohydroxyapatite; in vivo; bone neoformation; gene expression


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INTRODUCTION Around the world, 1.3 million deaths and many more disabilities arise annually due to road accidents. Bone injuries are one of the main results of these accidents, and they pose a major problem in public health. Specifically, open fractures pose particular challenges due to difficulties in stabilizing the fracture, covering them with new bone and the re-alignment of bone non-unions. Approximately one in ten road injuries involves a femoral shaft fracture that is most effectively treated with surgery. In Central America and Europe, a total of at least $52 million is spent due to bone injuries (commonly to forearm, femur and lower leg). In Brazil, the main factor leading to bone fractures result from road accidents, with data from the DATASUS database showing more than 356,000 surgical procedures due to bone fractures performed in 2015 alone. In this context, there has been a notable public health investment around the world seeking for the development of new and alternative synthetic biomaterials to be used as improved fillers for healing bone defects 1-3. Calcium phosphates, such as hydroxyapatite (HAp) and β-tricalcium phosphate (β-TCP), have been widely used as clinically available bone substitutes due to their excellent biocompatibility and osteoconductivity properties

4-6

. To date, several

nanobiomaterials based on carbon nanostructures and nanohydroxyapatite (nHAp) have been investigated as bone substitutes for enhancing in vivo lamellar formation 7. Among the ceramic materials usually used for biomedical applications, nHAp has been widely investigated as an emergent bioceramic due to its biomimetic properties including chemical, structural, and crystallographic similarity to the mineral component of natural bones. Nevertheless, the low tensile strength and fracture toughness of nHAp limit its application in large bone defects since nHAp is naturally incorporated in the body as a



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collagen-based (or carbon-based) matrix. In this context, the literature has introduced many different approaches to combine nHAp and carbon nanomaterials in order to promote the bioactivity for orthopedic applications 8. Superhydrophilic multiwalled carbon nanotubes (s-MWCNT), graphene oxide (GO) and, more recently, graphene nanoribbons (GNR), have emerged as excellent 2D and 3D layers and/or nanofillers

9-11

. However, to date, few papers have described the

application of GNR for tissue engineering. GNR are a novel nanomaterial and can be obtained from MWCNT exfoliation using chemical and/or acid treatment. In comparison to MWCNTs, GNR have higher surface area and functional oxygen groups12. Chowdhury et al. was the first to analyze the biological impacts of GNR using different cell lines

13

. Recently, Foreman et al oxidized GNR (O-GNR) for gene

delivery of double-stranded DNA into mammalian cells, showing an efficiency in loading DNA fragments

14

. Contributing to this, our group demonstrated that GNR

enhanced the bioactivity, cell viability, osteogenic differentiation, matrix mineralization and upregulated mRNA levels of the five genes related direct to bone repair and had a bactericidal effect at high concentrations of GNR (100 µg mL− 1)

15

. However, it is

evident that the combination of GNR with nHAp could be interesting for bone tissue engineering applications. In this context, nHAp and graphene derivatives, including GO and reduced GO (rGO), have been combined and examined extensively in the context of in vitro osteogenesis

8, 16

. Lee et al showed that different nano-carbons combined with HAp

stimulated the late osteogenic differentiation marker without any osteogenic agents.17 A recent study showed that the differentiation potential of human-adipose-derived MSCs to osteoblasts did not change significantly when treated with 2D GO nanostructures, including graphene nano-onions, GNR and GO nanoplatelets at a low (10 µg mL−1) and


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high (50 µg mL−1) concentrations for 24 h 18. Raucci et al investigated the application of biomineralized GO using two different approaches as nanofiller to associate with nHAp for bone tissue engineering applications

19

. Interestingly, the authors found that for

nHAp/GO nanocomposites containing different amounts of GO prepared by the sol–gel approach, the expression induced early osteogenic differentiation and ALP compared to the biomimetic method. Then, the authors opened possibilities to analyze different amounts of GNR to control or the non-bioactivity-promoting bone regeneration. Recently, we showed that nHAp/GNR composites were bioactive and suitable for biomedical applications, demonstrating biocompatibility and bactericidal properties against S. aureus and E. coli 20. However, to date, no reports have focused on in vitro osteogenesis evaluation and in vivo osteogenic potential using nHAp/GNR nanocomposites. Thus, we analyzed and correlated in vitro and in vivo studies using different concentrations of nHAp/GNR composites. We analyzed the potential of nHAp/GNR composites to upregulate some genes related to osteogeneses process and their in vivo bone formation (using a well-established rat tibia defect model). To the best of our knowledge, it is the first report that correlates the in vitro osteogenic process and in vivo bone regeneration using HAp/GNR as nanofiller for bone tissue regeneration, special to orthopedic applications.



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MATERIALS AND METHODS

Materials All chemicals used in this work were analytical grade and purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).

GNR production and further ultrasound-assisted preparation of nHAp/GNR nanocomposites The synthesis process of nHAp/GNR is illustrated in Fig. 1.

Briefly, we

prepared MWCNT using a mixture of camphor (85% wt) and ferrocene in a thermal chemical vapor deposition (CVD) furnace. The mixture was mixed (100 RPM) and vaporized at 220 °C in an antechamber, and then, the vapor was carried by argon gas at atmospheric pressure to the chamber of a CVD furnace set at 850 °C. Fe catalytic particles were removed using acid treatments as briefly described: MWCNT were subjected to ultrasound irradiation (UI, Vibracell Sonics, 500 W) for 5 h in a H2SO4:HNO3 (3:1) solution and then filtered using a Millipore membrane (0.45 µm), washed extensively with water and finally dried (100 ºC, 12 h). To obtain GNR, we performed an oxygen plasma treatment (plasma enhanced CVD reactor, 1 sccm, pressure of 85 mTorr, − 700 V, pulse frequency of 20 kHz and duration of 1 hour) to incorporate oxygen-containing groups and exfoliated MWCNT to open the tip to expose graphene sheets. The nHAp/GNR nanocomposites were prepared as described in previous work 20

. Briefly, calcium nitrate tetrahydrate [Ca(NO3)2·4H2O] [Ca(NO3)⋅4H2O] and

diammonium hydrogen phosphate [(NH4)2HPO4] were dissolved in 50 mL of deionized ACS Paragon Plus Environment

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water, and two different GNR amounts (40 mg and 80 mg) were added and sonicated (Vibracell Sonics, 500 W) for 30 minutes (Ca/P =1.67), separately. The pH was kept at ~10 by the dropwise addition of a NH4OH solution (25%). The remaining solid was allowed to settle for 120 h until maturation and then was filtered, water-washed, and dried at 60°C (48 h). For further analysis, nHAp/GNR nanocomposites were macerated in an analytical mill (IKA, model A11), as well as pristine nHAp, which was used as a control sample. For the characterization and biological in vitro studies, only the nHAp/GNR group produced using 40 mg of GNR was used. Meanwhile, both groups (containing 20 and 40 mg of GNR) were used for in vivo evaluation. For in vivo evaluation, the groups were named nHAp/GNR 1% (group prepared using 20 mg of GNR) and nHAp/GNR 2 % (group prepared using 40 mg of GNR).



Figure 1. Scheme illustrating the whole process to produce nHAp/GNR nanocomposites Characterization

High-Resolution Transmission Electron Microscopy (HR-TEM, FEI-Tecnai G2 F20 microscope) was used to characterize the GNR, nHAp and nHAp/GNR. The pore size distribution and specific surface area of the GNR, nHAp and nHAp/GNR were analysed using N2 absorption-desorption (BET and BJS) at 77K. Quantachrome Pore Master (version 7.01), Quantachrome NovaWin 2, and Quantachrome Instruments (version 2.2) software were used for data analysis. To collect isotherm curves, the samples were outgassed. The data were expressed using isotherm graphs (Fig. S1, page


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S1) and porous analysis (Fig. S2, page S2). Table S1-S3 (Table S1-S3, pages S3 – S5) show all extracted data from analysis.

In vitro tests Cell culture and groups A human osteoblast cell line MG-63 (ATCC® CRL-1427™, 2×105 cells mL-1) was cultivated in 24-well plates using Dulbecco’s Modified Eagle Medium (DMEM, with 10% fetal bovine serum, 100 IU mL-1 of penicillin and 100 µg mL-1 of streptomycin) and incubated for 24 hours (37ºC in a 5%CO2 humidified incubator). Then, two different groups (nHAp and nHAp/GNR) and three different concentrations of nHAp/GNR (100 µg mL-1, 200 µg mL-1 and 300 µg mL-1) were dispersed in DMEM (stock solution) and then inserted in individual wells. nHAp was used as control.

Total RNA isolation and synthesis of cDNA First, the total RNA from cells was extracted from each group and wells using a Trizol® Reagent (Life Technologies, Rockville, MD, USA). Next, the concentrations and integrity of the RNA samples were assessed using the ND-1000 spectrophotometer (NanoDrop Inc.,v.3.0.7, Labtrade) and 1.5% agarose gel electrophoresis, respectively. For cDNA synthesis, 2 µg of total RNA from each sample was used per the manufacturer's protocol ImProm-IITM Reverse Transcription System (Promega, São Paulo, Brazil) and reactions were carried out in a thermal cycler (Biocycler, MJ96G, USA).

Real time-qPCR



ALP (alkaline phosphatase), OPN (osteopontin), OCN (osteocalcin), COL I (Collagen type I) and RUNX2 (runt-related transcription factor 2) expression genes were evaluated in triplicate using GoTaq® qPCR Master Mix (Promega, São Paulo, Brazil) and analyzed by the ABI Prism 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) after 15 days of incubation with all analysed groups. IDT software (Integrated DNA Technologies/ available on: http://www.idtdna.com) and Primer-Blast software (available on: http://www.ncbi.nlm.nih.gov/tools/primer-blast) were used to determine the primers needed to amplify the five target and reference genes. In order to avoid amplification of contaminated genomic DNA, the primers were placed at the junction between the two exons or in a different exon. Table 1 shows the sequences of the primers and the main functions of the analyzed genes. GAPDH (glyceraldehyde 3-phosphate dehydrogenase), 18SrRNA (18S ribosomal RNA) and βactin (Actin smooth muscle-beta) housekeeping genes were tested. β-actin was selected as reference gene, used to normalize gene expression and to validate the accuracy of real time-qPCR. The following parameters were used: 5 min at 95 °C, 40 cycles of 15 seconds at 95°C and 1 min at 60 °C and finally 5 min at 72 °C. The dissociation curve was included in all experiments. Standard curves of the targets and reference genes showed similar amplification efficiencies (>90%) Results analysis The Delta-Delta Ct (∆∆Ct) method was used to calculate gene expression. For this, the average values of the cycle threshold (Cts) were obtained for the target genes and compared to the Cts reference gene 21. These values were normalized and expressed as the values of relative fold-change (Relative Quantification, RQ). Two-way ANOVA and Tukey's multiple comparison tests were used to analyze statistical difference of the expression levels between the groups analyzed (GraphPad Prism InStat software,


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version 6.1 San Diego, CA, USA). Values of P < 0.05 were considered significant. P < 0.01 and P < 0.001 were considered highly significant.

Table 1. Details of five targets and one reference gene used in the RT- qPCR assay Gene symbol/ (access number)

Gene name

5’-ACCAACTGGGACGACATGGAGAAA3’

β-actin / ACTB (NM_001101)

Primer sequences

Actin Beta 5’-TAGCACAGCCTGGATAGCAACGTA3

(reference


Function

Cell motility, structure, and integrity


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gene)

ALP (NM_000478.4)

Alkaline phosphatase

5’-CCGTGGCAACTCTATCTTTGG-3 Bone mineralization

5’-GCCATACAGGATGGCAGTGA-3

OPN /

Secreted phosphoprotei 5’-AGACACATATGATGGCCGAG-3 SPP1 n 1/ 5’-GGCCTTGTATGCACCATTCAA-3 Osteopontin (NM_1251830) OC /

Osteocalcin/ 5’-AAGAGACCCAGGCGCTACCT-3

BGLAP (NM_199173)

Bone gammacarboxygluta 5’-AACTCGTCACAGTCCCGGATTG-3 mate protein/

COL I / COL1A1

5’-CCCTGGAAAGAATGGAGATGAT-3

Collagen type 5’-ACTGAAACCTCTGTGTCCCTTCA-3 (NM_000088.3) I alpha 1

RUNX2

Runt related transcription factor 2

5’-AGCAAGGTTCAACGATCTGAGAT-3 5’-TTTGTGAAGACGGTTATGGTCAA-3

Mineralized bone matrix,

Highly abundant bone Regulates bone remodeling

Abundant in bone and tendons.

Osteoblastic differentiation and skeletal morphogenesis

(NM_004348)

Alkaline Phosphatase assay (ALP) The ALP assay was used to confirm the efficiency of the osteogenesis process. For this, MG-63 cells were cultured with the samples in a 24-well plate for 14 days. The wells were washed three times with PBS at 37 °C and incubated with 2 mL of 0.1% sodium lauryl sulfate (SLS) for 30 min. The SLS/cell solution was mixed with the Lowry solution (Sigma) and incubated for 20 min at room temperature. Folin-Ciocalteu phenol reagent (Sigma) was added for 30 min at room temperature to allow for color


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development. Absorbance was measured at 680 nm. The total protein content was calculated based on an albumin standard curve and expressed as µg mL-1. To determine ALP activity through the releasing of thymolphthalein monophosphate, we used an Alkaline Phosphatase Kit (Labtest Diagnostica, Belo Horizonte, BR) in accordance with the manufacturer's recommendations. First, 50 µL of thymolphthalein monophosphate were mixed with 0.5 mL of 0.3 M diethanolamine buffer for 2 min at 37 °C. The solution was then added to 50 µL of the lysates obtained from each well. After 10 min at 37°C, 2 mL of 0.09 M Na2CO3 and 0.25 M NaOH were added for color development for 30 min. The samples were centrifuged at 1500 rpm for 3 min, and the supernatant was added to a 96-well plate. Absorbance was measured at 590 nm using a UV 1203 spectrophotometer. ALP activity was correlated with total protein content and expressed as ALP µmol thymolphthalein/min/mL.

In vivo – experimental model Thirty 50-day old male rats (Rattus norvegicus) weighing between 400 to 450 g were used in this study. All rats were kept in cages with three animals each, and received food and water ad libitum, in a temperature-controlled environment (24-26 °C). This study was approved by the Ethics in Research Committee of the Graduate School of Dentistry of the Sao Jose dos Campos, UNESP (10/2015-CUA/ICT-CJSCUNESP).



The best material, determined from the in vitro tests, was used for in vivo analysis and compared to nHAp (control). Before surgery, nHAp, nHAp/GNR 1% and nHAp/GNR 2% were sterilized in absolute ethanol under an ultraviolet lamp for 3 h. These groups were then divided into two distinct times: 15 and 21 days. A control group using only gelatin was also evaluated. A critical defect was done (analyzed by Digora Imaging system, Fig. S3, page S6) using a 3 mm drill coupled at a motor (1,600 rpm) irrigated with 0.9% of saline solution. Then, a 100 µg sample of each group was implanted as cylindric scaffolds (homogenized in 10% gelatin in water, 2 mm of diameter and 2 mm of dept) using a curette, which completely filled the bone defect. After each test time period, the rats were anesthetized. Anesthesia was performed with an intramuscular injection of xylazine chloride 2% (0.1 mL 100 g-1 body weight). We used an antibiotic prophylactic treatment with procaine Benzylpenicillin (20,000,000 UI) associated with Dihidroestreptomycinesulphate at 8.0 g and 0.60 g of piroxicam at 0.1 mL 100 g-1. After anesthesia, a hind leg dissection was performed to provide access to the tibia. The fragments from the area of surgical defect and the surrounding tissues were removed in blocks, which were fixed in 10% neutral formalin, rinsed with water, and then processed by well-established histological techniques for wear. After, each fragment was divided longitudinally into two sections from the original surgical defect and submitted for histological evaluation. The histological analysis was performed by an examiner who was blinded with respect to the scaffold placed into the defect. The images of the histological sections were captured by a digital camera coupled to a light microscope at an original magnification of ×5. Micrographs were also collected using scanning electron microscope (FEI, Inspect s50). The image analyses were done using Image J® software. The amount of newly formed bone was quantified and expressed as


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a percentage of the total area of the surgical defect. The total area of the original surgical defect was marked by the margins of the surgical defect, and the newly formed bone area was delineated within the confines of the total area. Computer software GraphPadPrism (version 6) was used to analyze the data, using a one-way ANOVA parametric test, with p values set at < 0.05. A Tukey’s test was performed to compare several groups of the same treatment length, while the comparison of groups for different treatment lengths was completed by a two-way ANOVA.

RESULTS AND DISCUSSION

Fig. 2a shows bamboo-like structures of as-grown MWCNT. The MWCNT have between 50-70 walls and diameter between 50-80 nm. The oxygen plasma was sufficient to exfoliate and oxidize the MWCNT to produce GNR. Details of GNR exposed from MWCNT after oxygen plasma treatment are shown in Fig. 2b. The details of needle-like nHAp produced by the sonochemistry method are shown in Fig. 2c. The nHAp crystals presented diameters between 15-20 nm and lengths between 40-80 nm. Finally, the details of the nHAp/GNR nanocomposites are shown in Fig. 2c. The hydrophilicity (already reported previously by our group22) obtained after oxygen treatment was essential to incorporate functional groups presented at GNR and sufficient to incorporate nHAp onto them (Fig. 2d). Then, we could verify the predominance of needle-like apatites covering the GNR. Herein, we confirmed that the GNRs can be successfully used as a nanofeatured scaffold for nHAp nucleation (Fig. 2c and 2d).



BET and BJS analysis identified that all analyzed groups are considered mesoporous in accordance with IUPAC23 (lower than 50 nm) (Fig. S1 and Fig. S2, pages S1-S2; Table S1-S3, pages S3-S5).

nHAp/GNR also presented a typical

hysteresis loop in adsorption-absorption, indicating the presence of plate-like particles (typically observed by nHAp produced by sonochemistry method) and that the material is mesoporous (Fig. S1c, page S1)

24

. A discreet difference in hysteresis loop was

observed by GNR (Fig. S1a) due to either lower pore surface area (27.89 m2 g-1, Fig. S2d, page S2) or to their confined pores formed in aggregated structures, typically an attribute of MWCNTs25. nHAp/GNR has high porous surface area (Fig. S6d, 45.658 m2 g-1), volume (0.094 cm3 g-1, Fig. S2d, Page S2) and size (64.8 nm, Fig. S2d, page S2). Similar values were also observed for nHAp (Fig. S2d, page S2). These data are also consistent with TEM analysis (Fig. 2c).


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Figure 2. (a) TEM showing MWCNT structures. (b) TEM identifying details of GNR after oxygen plasma treatment. TEM illustrating the needle-like structures of nHAp obtained by the sonochemical method. (d) TEM showing details of the nHAp formed onto GNR. Expression of genes that are directly related to osteogenic pathway was also evaluated, and an in vivo model to analyze bone formation was used. Gene expression of key genes involved in the osteogenesis process is shown in Fig. 3. The values corresponding to nHAp/GNR were non-statistically comparable to control and composites groups (not included in graphs). Clearly, we detected that gene expression can be altered when MG-63 cells were incubated in different concentrations of



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nHAp/GNR composites. We analyzed non-specific and specific genes related to the osteogenesis process, collectively evaluated to highlight the application of nHAp/GNR as nanofillers for bone regeneration. ALP (Fig. 3a) is a marker for osteogenic activity, especially related to hard tissue formation, and is thought to be the first functional gene expressed in the process of calcification (especially regarding bone regeneration). All concentrations of nHAp/GNR analyzed expressed more ALP when compared to control. However, low concentration of nHAp/GNR (100 µg mL-1) was able to upregulate more than other two analyzed concentrations (p

Graphene Nanoribbons Nanocomposites Induce

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