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Carbon Nanomaterials for Treating

Osteoporotic Vertebral Fractures

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Citation de Carvalho, Jancineide Oliveira et al. "Carbon Nanomaterials for Treating Osteoporotic Vertebral Fractures." Current Osteoporosis Reports 16, 5 (September 2018): 626–634. © 2018 Springer Science Business Media

As Published https://doi.org/10.1007/s11914-018-0476-2

Publisher Springer Science and Business Media LLC

Version Author's final manuscript

Citable link https://hdl.handle.net/1721.1/128503

Terms of Use Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use.

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SpringerLink Header: Regenerative Biology and Medicine in Osteoporosis (T. Webster, Section Editor)

To appear in Volume 16, Issue 5, October 2018

Carbon nanomaterials for treating osteoporotic vertebral fractures

Jancineide Oliveira de Carvalhoa,b, Francilio de Carvalho Oliveiraa,b, Sérgio Antonio Pereira Freitasb, Liana Martha Soaresc, Rita de Cássia Barros Limab, Licia de Sousa Gonçalvesb, Thomas Jay Websterd, Fernanda Roberta Marcianoa,d and Anderson Oliveira Loboa,e,f*

a

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

b

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

c

Hospital Universitário de Teresina, Campus Universitário Ministro Petrônio Portela, SG 07, s/n - Ininga, Teresina - PI, 64049-550, Brazil

d

Nanomedicine Laboratory, Department of Chemical Engineering, Northeastern University, Boston, Massachusetts, 02115, United States

e

Programa de Pós-Graduação em Ciência e Engenharia dos Materiais, Universidade Federal do Piauí, Campus Universitário Ministro Petrônio Portella, Bairro Ininga, Teresina, Piauí, CEP: 64049-550, Brazil

f

Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Ave, 18-393, Cambridge, Massachusetts, 02139, United States

*Corresponding Author: Prof. Dr. Anderson de Oliveira Lobo. E-mail: aolobo@pq.cnpq.br and lobo.aol@gmail.com

Keywords: Graphene nanoribbons; biomineralization; osteoporosis; oophorectomy; and histological analysis

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Abstract:

Purpose of review: To identify the use of carbon nanomaterials in bone regeneration and present new data on the regenerative capacity of bone tissue in osteopenic rats treated with graphene nanoribbons (GNR).

Recent findings: The results show that the physical and chemical properties of the

nanomaterials are suitable for the fabrication of scaffolds intended for bone regeneration. The

in vitro tests suggested a non-toxicity of the GNR as well as improved biocompatibility and

bone mineralization activity.

Summary: Here, for the first time, we evaluated the potential of GNR in remodeling and repairing bone defects in osteoporotic animal models in vivo. Interestingly, bone

mineralization and the initiation of the remodeling cycle by osteoclasts/osteoblasts was observed after the implantation of GNR. Thus, implying healthy bone remodeling when using GNR. This study, therefore, has opened our perspectives and certainly calls for more

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Introduction: A perspective of carbon nanomaterials as a filler to induced bone growth during osteoporosis

Osteoporosis is a disease characterized by a reduction in bone mass and deterioration of bone tissue microarchitecture, which increases bone fragility and subsequent susceptibility to fractures. It is one of the most prevalent health problems of modern society, affecting people’s quality of life, especially that of the elderly and is associated with decreased life expectancy. It is a systemic disease that occurs more frequently in the female population [1]. Osteoporosis is classified as primary or secondary. Primary osteoporosis may be type I or type II; type I is known as postmenopausal osteoporosis and is associated with the loss of ovarian function, with an increase in bone resorption mainly during the first few years after menopause. Primary type II osteoporosis is associated with the aging process and is

characterized by a deficiency in bone formation. Secondary osteoporosis is caused by an inflammatory process such as rheumatoid arthritis; endocrine changes such as

hyperthyroidism and adrenal disorders; the use of drugs such as heparin, alcohol, vitamin A, and corticosteroids; multiple myeloma; and bone disuse [2.3].

Several biomaterials, incorporating sensors and a capability of promoting bone regeneration without negative impacts on health (for example, carbon nanomaterials) have been developed for the purpose of monitoring the bone loss process [4,5]. However, it is important that their physical, mechanical, physicochemical, and biological properties are fully developed to optimize performance and allow the generation of innovative compositions for better bone regeneration.

Along these lines, the characterization and functionalization of carbon-based

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nanocomposites [6]. Recently, a number of interesting reviews have focused on the use of carbon materials for tissue engineering applications, showing much promise [7–9].

Carbon nanotubes (CNT) are among the carbon materials that can be used to prepare nanocomposites for bone regeneration. When used in regenerative medicine, these materials allow for the preservation of biological-inspired properties, and adhesion of cells, in addition to displaying excellent cytocompatibility, supporting the growth of osteoblasts, and

stimulating the production of new bone matrices [10]. However, several ways of improving the biocompatibility of CNT have been demonstrated, including constructing them in a vertically aligned manner and/or functionalizing them with hydrophilic groups; that is, forming nanotubes, referred to as superhydrophilic CNT and graphene oxide, with surfaces structurally organized with groups related to graphene oxide (GO) [11]. Certainly, the functionalization of GO with nano-hydroxyapatite (nHAp) increases their applicability in bone regeneration applications and the crystallinity of the deposited nHAp particles improves biocompatibility compared with both a pure HAp coating and a pure underlying Ti substrate [12]. Moreover, vertically aligned CNT mimic the vertically aligned natural hydroxyapatite in long bones of the body for anisotropic mechanical, piezoelectric, and electrical properties.

Of course, numerous studies have aimed to create biomaterials that are capable of promoting the regeneration of the bone matrix. The continuous search for scaffolds that can restore the biological and physical function of individuals has resulted in the emergence of several biomaterials; one of them is nano-hydroxyapatite (nHAp) as proposed by Zhao et al. (2017) [13] in aged mice models with induced osteoporosis. However, shortfalls have been observed, including the mechanical properties of nHAp. Thus, the incorporation of new matrices, such as multiwalled CNTs and GO in combination with nHAp, has been

investigated to improve the mechanical and other properties of nHAp [14,15]. For example, Ricci. et al. investigated the in vitro bioactivity, cell viability, osteogenic differentiation, and

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matrix mineralization activities of five genes directly associated with bone repair and bactericidal effects of high concentrations of CNT and graphene oxide (O-GNR). No

cytotoxic effects were detected for up to 100 μg mL-1, and no detrimental effects to the bone repair process were reported [16]. Therefore, O-GNRs appear to be promising nanomaterials for biomedical applications, especially in the treatment of osteoporosis.

Significant advances in nanotechnology have been made, especially in the

manufacturing of devices, which have facilitated the use of a wide-range of nanomaterials in various applications. However, osseointegration of adequate bone substitutes has been difficult to achieve in osteoporotic patients, even though GNR possesses suitable properties for the osseointegration process because of its ability to preserve biological properties, in addition to promoting cell growth, distribution, adhesion, and differentiation [17,18].

In vitro studies have demonstrated an improvement in the osseointegration process,

either with the functionalization with other organic matrices or with nanotubes, when there was an improvement in the chemical, physical, and toxicological properties of the

biomaterials [19,20] or incorporation of metals such as zinc and silver [21]. In studies involving the incorporation of silver into carbon nanotubes, the carbon nanotubes showed antimicrobial activity against some pathogenic bacteria such as gram-positive Staphylococcus

aureus and gram-negative rod-shaped Escherichia coli and Proteus mirabilis [22]. Similar

results were obtained by Song et al. (2018) and Farid et al. (2018) when they used only graphene oxide with antimicrobial agents, emphasizing that carbon-based biomaterials can be practice effective alternatives to conventional antibacterial agents [18,23].

Nonetheless, even though carbon-based biomaterials are being widely used and numerous studies have been conducted with them, their toxicity in humans is still is an issue of concern. In vivo studies with rats disclosed that doses between 25 µg/animal and 400

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to inflammatory processes, with an increase in the production of reactive oxygen species (ROS) [24]. In vitro studies have reported CNT toxicity levels higher than expected in humans; however, because there is still controversy on the minimum and maximum dosages that can be tolerated by humans without affecting health. Then, a strategy for toxicity

reduction in humans has been developed involving the functionalization of the surface of the carbon-based nanotube with oxygen groups that can maximize biocompatibility and

dissolution properties [23].

In particular, our group has expertise in the synthesis and application of carbon nanomaterials for biological applications. In this short review, for the first time, we assessed the effectiveness of a GNR powder as a scaffold for bone tissue regeneration in the tibias of osteopenic rats, based on its ability to accelerate mineralization of the bone matrix. The produced GNR was obtained through chemical and plasma treatment of vertically aligned CNT and, as will be described, showed much promise for bone regeneration.

Vertically aligned carbon nanotubes were synthesized on the walls of quartz tubes using a chemical vapor deposition (CVD) method at an atmospheric pressure at 1123,15 K. Camphor (C10H16O, 84% of the total mass) and an Fe catalyst (ferrocene, Fe (C5H5)2, 16% of

the total mass) were evaporated at 473,15 K and transferred into a quartz tube under a N2

flow rate of 1.5 L/min. After 5 min, the flow of vapor was stopped and the oven was cooled to room temperature under N2. Thus, nanomaterials were produced by pyrolysis of the

camphor/ferrocene mixture and purified by high-temperature annealing in an oxygen-free atmosphere (N2). For further purification, the Fe in the nanomaterials was removed with an

acid bath containing a solution of H2SO4:HNO3 (3:1). The vertically aligned CNT were

mixed with this solution and sonicated for 5 h using an Elmasonic S10H ultrasonic cleaner. Next, the solution was filtered using a Millipore membrane (pore size, 0.45 μm) to obtain purified vertically aligned CNT, which were subsequently washed with deionized water, until

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a neutral pH was attained. Finally, the powder form of the vertically aligned CNT was dried in an oven at a temperature of 100 °C for 12 h.

To couple GNR to the tips of the vertically aligned CNT, CNT functionalization was accomplished to possess oxygen-containing groups using a pulsed-direct-current plasma reactor (oxygen flow rate, 1 sccm; pressure, 85 mTorr; target voltage, 700 V; pulse frequency, 20 kHz; pulse duty cycle, 50%; time, 40 min). This resulted in the production of GNR (Fig. 1). Firstly, the vertically aligned CNT were grown using a CVD technique. Next, the vertically aligned CNT were unzipped and their tips were oxidized, both GOs were exposed, and carboxylic groups were formed to produce the GNR, a hybrid carbon nanomaterial.

[Figure 1]

GNR in vivo analysis

Twenty Wistar albino rats weighing 0.19-0.27 kg were obtained from the Animal Facility of the University Center for Health, Human Sciences and Technology of Piauí (UNINOVAFAPI). The animals were maintained in air-conditioned rooms at an ambient temperature of 25 ± 2 °C and a 12-h light:12-h dark photoperiod. The rats were maintained in collective cages (four rats/cage) and were fed with standard rat chow (Labina) and water at

ad libitum. Next, they were subjected to oophorectomy in the Laboratory of Experimental

Surgery in UNINOVAFAPI. During the oophorectomy, the animals were maintained under ketamine and xylazine anesthetics, administered intraperitoneally. A subcutaneous midline incision (1.5 cm in length) was made on the back of the animals, below the last rib, to remove the ovaries. The animals were allowed to rest for a period of 45 days (the time required for the onset of osteopenia). Next, the rats were divided into four groups (Table 1) and evaluated on days 21 and 45.

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For the GNR implant group, the animals were subjected to dissociative anesthesia via intramuscular administration of ketamine (40 mg/kg body weight) and xylazine (5 mg/kg body weight). In all the rats, the surgery was performed in the right tibia; the surgery was initiated by trichotomy of the region. The surgical procedures were the same for all animals and involved the implantation of the GNR (3.0 µg/animal) powder in the area corresponding to the bone defect (Fig. 2). An elliptical bone defect extending up to the spinal canal was created. This procedure was performed in the proximal third of the tibias using a carbide drill with a diameter of 1.5 mm, a surgical micromotor (Aseptico AEU-707A; Woodinville, WA, USA), and saline solution to irrigate the wounds created during the surgical procedure. The surgical incision was sutured using silk threads, thereby allowing the periosteum to lie on top of the implants.

[Figure 2]

After implantation, the right tibia of each rat in the control and implant groups were removed and fixed in formaldehyde for 24 h. Soft tissue was removed by dissection and bone segments were subjected to radiographic examination. To obtain the radiographic images, the GE 100 X-ray machine (General Electric Company, Milwaukee, USA) was used, under operating conditions of 50 kVp, 10 mA, and 12 impulses. The film-focus distance was set at 30 cm, perpendicular to the film-object plane. Direct digital imaging was performed using the DIGORA digital imaging plate system (Soredex Orion Corp., Helsinki, Finland). During the radiographic examination, a nine-step aluminum step wedge/penetrometer (6063 alloy, ABNT) was used as the densitometry reference. A bone segment was placed on each optical plate. The sensitized optical plates were scanned using a laser scanner and the images were manipulated using Digora software for Windows 2.51. Standardized images for selected bone defect areas were acquired. The area of the bone defect was limited to a square of 23 × 23

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pixels. The mean bone density was analyzed using the software. Gray-tone values of the radiographs were calculated and converted to millimeters.

The tibial sections were dissected, fixed in formaldehyde solution (10%), and

decalcified in an ethylenediamine tetra-acetic acid solution (EDTA, 10% solution, pH 7) for 30 days. Subsequently, the sections were dehydrated using a sequential alcohol dilution (70 to 100%), diaphanized in xylol, and embedded in paraffin blocks. Approximately 5-μm-thick sections were cut using a microtome and stained with H&E. The stained sections were analyzed using a Nikon E200 light microscope (magnification, 100×). The NIKON COOLPIX S8100 camera (resolution, 12 megapixels) was used to perform

photomicrography. The sections were quantitatively analyzed by measuring the cortical photometry of the injured bones with a millimeter rule. The results from osteoblast counting were statistically analyzed with the Kolmogorov–Smirnov test, as shown in Fig. 4d. Next, the biochemical analysis was performed. For this, the concentration of serum alkaline

phosphatase was determined. The blood samples were collected by vena cava venipuncture and transferred into glass tubes (without anticoagulant). For serum separation, the samples were centrifuged at 3500 rpm for 15 min. Dose measurements were performed using a fixed-time kinetic method and an end-point assessment was performed using an Alkaline

Phosphatase kit (Reference 40; Labtest Diagnostica SA). Absorbance was measured at 590 nm. The results were presented as normality test graphs.

The data were statistically analyzed using the Minitab 16 software. The data were subjected to one way (unstacked) ANOVA, which was used for comparison between the groups, followed by a post-hoc Tukey’s test (level of significance at a 95% confidence interval, p < 0.05). After confirming that the data followed a normal distribution pattern, the Kolmogorov–Smirnov test was performed to analyze statistical significance.

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Fig. 3 shows the detailed structure of the synthesized vertically aligned CNT, before and after the exfoliation process that was carried out to expose the GO sheets at the ends of the nanotubes to obtain the GNR. In general, the CNT had diameters between 50 nm and 70 nm, whereas the GO sheets had ~20 nm diameters. Details of the Fe nanoparticles are shown in Fig. 3A. As described previously, when produced with the thermal CVD process [25], the vertically aligned CNT had a high catalytic nanoparticle content in the nanotubes (arrows, Fig. 3A). Using EDS analysis, we chemically characterized the Fe and the contaminants obtained from the CVD process. After the purification and functionalization steps (using the oxygen plasma treatment), we observed that the graphene oxide sheets were exposed (circle, Fig. 3C) at the ends of the nanomaterials (but they did not show any contamination after the treatment). Additionally, the GNRs also displayed their characteristic features possessing multiple walls (arrows, Fig. 3C). Thus, we assigned the nomenclature GNR, since it represented a hybrid carbon-based nanomaterial.

Fig. 3. Micrographs and chemical analysis of the MWCNTs and MWCNT-GOs. (A) The detailed structure of the as-grown MWCNTs showing the excess Fe nanoparticles enclosed by its walls. (B) EDS analysis showing the Fe nanoparticles enclosed inside the as-grown MWCNTs (arrows). (C) Details of the GO sheets (circle), walls, and bamboo-like structures (arrows) present in the GNR.

Fig. 4. presents the radiographic images of the oophorectomized tibias, implanted with the GNR post-operatively on days 21 and 45, obtained using the Digora imaging system. No deformation in the bone structures was observed for any of the treatment groups. An increase in the bone density of the tibias of the osteopenic rats was observed. This was evident by the filling of the bone defect area in these animals (circle, Fig. 4A–C). Fig. 4D shows the

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radiographic density analysis (using the Digora software for Windows 1.5) of the rat tibias belonging to all treatment groups.

Fig. 4 Radiographic images of the oophorectomized control rats (A) and rats implanted with the GNR, on postoperative days 21 (B) and 45 (C), obtained with the Digora imaging system. The circles indicate the location of the bone defect. Statistical analysis of the bone

regeneration data (D); GI, GIII, control groups and GII, GIV, experimental groups 21 and 45 days respectively. Values are recorded as mean ± SD (n = 5). Different letters showing significant differences for p <0.05

Fig. 5 shows the photomicrographs of the sections of surgical defects of the tibias in the control rats (Fig. 5A and Fig. 5B) and the rats with implants, post-operatively on days 21 and 45 (Fig. 5C and Fig. 5D). All control and experimental groups presented vascularized regions throughout the bone defects (dark square) [20,21]. The presence of GNRs, in the form of clusters, were evident in the experimental groups (dark circle). Compared with the control groups (white triangle), regions with a thicker cortical and more compact bone structure were also observed in the experimental groups.

[Figure 5]

Quantitative analysis of bone healing was performed by measuring the cortical thickness of each bone. Significant differences in the bone densities of different treatment groups were observed (Fig. 6A). An increase in cortical thickness was observed in group GII compared with group GI. On postoperative day 45, the opposite trend was observed in group GIV (the group with implants) compared with the control group GIII. Upon analysis of the control group, the level of the alkaline phosphatase (Fig. 6B) in GIII was higher than the level in GI. For the experimental groups, the level of alkaline phosphate in GII was higher than the level in GIV.

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Quantitative analysis of the mean bone density in the bone defect area was performed (Fig. 4D). The results showed an increase in the mean bone density for group GIV compared with the other groups. This observation may be due to the prolonged exposure of the GIV group to the GNR, which resulted in better bone remodeling mediated by osteoclasts via organized induction of bone precipitates in the lesions [5,25,26].

Fig. 4A and 4B indicate spontaneous bone mineralization in the control groups that was evident by the formation of a compacted and a medullary bone structure over the entire surface of the sample. A reminiscence of the highly vascularized connective tissue was also observed (Fig. 5C) which indicated the presence of differentiating cells [23]. Subsequently, these cells formed differentiated osteoblasts in the implanted groups on postoperative day 21, compared with the GI control group (Fig. 4A). On postoperative day 45, the GNR implanted group displayed no differences in the cortical thickness compared with the other groups, thereby supporting the remodeling action of osteoclasts (Figs. 5C and 5D).

The data related to the cortical thickness (Fig. 6) initially indicated the induction of osteoporosis in the control groups (GI and GIII), which was promoted by oophorectomy [27]. This observation was supported by the low levels of alkaline phosphatase in these groups (Fig. 5B) and showed increased spontaneous bone tissue formation (Fig. 5A and 5B). A decrease in cortical thickness was observed in groups GI and GIII (Fig. 6A), indicating the initial phase of mineralization. This observation corroborated the presence of low levels of alkaline phosphatase in these groups (Fig. 6B). However, an increased level of alkaline phosphatase was observed in group GIII. Thus, we hypothesized that mineralization with calcium from the surrounding fluids occurred mainly in group GIII, suggesting a spontaneous regeneration of calcified tissue [28].

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The maximum cortical thickness was observed in group GII compared with the other groups (Fig. 6A). This suggested the continuation of endochondral ossification, implying that the healing process was incomplete. This corroborated the high level of alkaline phosphatase observed in this group (Fig. 6B). In group GIV, a reduction in the cortical thickness, along with a decrease in the level of alkaline phosphatase was observed. This indicated a reduction in the healing process and an increase in bone tissue remodeling, suggesting the effectiveness of GNR implantation after 21 days.

Summary and future directions

Currently, new alternative bone fillers to induce bone formation in osteoporotic bones are needed. Carbon nanomaterials have gained recognition as alternatives because of their chemical and physical properties, as already discussed. However, the importance of new carbon nanomaterials is evident because of new strategies and biological properties. For this purpose, GNR has been introduced in this review as an interesting alternative because of its combined attractive properties derived from carbon nanotubes and graphene. To date, there are many controversial reports regarding the effects of carbon nanomaterials on the body and cells. For orthopedic applications, it is especially evident that the carboxylic groups can stimulate in vitro and in vivo biomineralization and formation of calcium carbonate. However, the greatest problem preventing the approval of carbon nanomaterial for bone tissue engineering purposes by NIH stems from their biodegradability, agglomeration, and bioavailability of their transition metals. Here, for the first time, we evaluated the potential of GNR (a hydrophilic and a metal-free material) in remodeling and repairing bone defects in osteoporotic animal models in vivo. Interestingly, bone mineralization and the initiation of the remodeling cycle by osteoclasts/osteoblasts was observed after the implantation of GNR. Thus, implying healthy bone remodeling when using GNR. This study, therefore, has opened

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our perspectives and certainly calls for more attention to the use of carbon nanomaterials for a wide range of osteoporosis applications.

Acknowledgments

AOL and FRM would like to thank to National Council for Scientific and Technological Development (CNPq grants numbers AOL#303752/2017-3 and

FRM#304133/2017-5), Coordination for the Improvement of Higher Education Personnel (CAPES, grant numbers AOL#88881.120138/2016-01 and FRM#88881.120221/2016-01) and Universidade Brasil for the scholarships. JCO and FCO would like to thank

UNINOVAFAPI for the scholarship. The authors are also grateful to Tayná Cabral for the SEM images.

Compliance with Ethical Guidelines

Conflict of Interest

Jancineide Oliveira de Carvalhoa, Francilio de Carvalho Oliveira, Sérgio Antonio Pereira Freitas, Liana Martha Soares, Rita de Cássia Barros Lima, Licia de Sousa Gonçalves, Thomas Jay Webster, Fernanda Roberta Marciano and Anderson Oliveira Lobo declare no conflict of interest.

Human and Animal Rights and Informed Consent

This article does not contain any studies with human or animal subjects performed by any of the authors.

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Fig. 2. An illustration of the surgical procedure. (A) Preparation of the bone defect in the right tibia of the rats and (B) The white circle indicates the site created to insert the GNR materials.

Fig. 3. Micrographs and chemical analysis of the MWCNTs and MWCNT-GOs. (A) The detailed structure of the as-grown MWCNTs showing the excess Fe nanoparticles enclosed by its walls. (B) EDS analysis showing the Fe nanoparticles enclosed inside the as-grown MWCNTs (arrows). (C) Details of the GO sheets (circle), walls, and bamboo-like structures (arrows) present in the GNR.

Fig. 4. presents the radiographic images of the oophorectomized tibias, implanted with the GNR post-operatively on days 21 and 45, obtained using the Digora imaging system. No deformation in the bone structures was observed for any of the treatment groups. An increase in the bone density of the tibias of the osteopenic rats was observed. This was evident by the filling of the bone defect area in these animals (circle, Fig. 4A–C). Fig. 4D shows the radiographic density analysis (using the Digora software for Windows 1.5) of the rat tibias belonging to all treatment groups.

Fig. 4 Radiographic images of the oophorectomized control rats (A) and rats implanted with the GNR, on postoperative days 21 (B) and 45 (C), obtained with the Digora imaging system. The circles indicate the location of the bone defect. Statistical analysis of the bone

regeneration data (D); GI, GIII, control groups and GII, GIV, experimental groups 21 and 45 days respectively. Values are recorded as mean ± SD (n = 5). Different letters showing significant differences for p <0.05

Fig. 5. Photomicrographs showing sections of the surgical tibia defects in oophorectomized control rats (A and B), and rats with GNR implants, post-operatively on days 21 and 45 (C and D). A magnification of 10 was used. The circles indicate the GNR deposits, and the

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square arrows indicate vascularization, white triangle - compacted and trabecular bone mineralization

Fig. 6A. Quantitative analysis of the cortical thickness of the tibias of the oophorectomized control rats and rats with GNR implants on postoperative days 21 and 45. Fig. 6B:

Quantitative analysis of alkaline phosphatase in oophorectomized control and MWCNT-GO-implanted rats on postoperative days 21 and 45. Values are recorded as mean ± SD (n = 5). Different letters showed significant differences for p <0.05

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Table 1. Distribution of rats in different experimental groups. GI and GIII (control groups) represent oophorectomized rats (OVX) without implants at postoperative days 21 and 45, respectively; GII and GIV (experimental groups) represent oophorectomized rats implanted with GNR at postoperative days 21 and 45, respectively.

Experimental group Number of animals Post-operative day Treatment GI 05 21 OVX GII 05 21 OVX + GNR21d GIII 05 45 OVX GIV 05 45 OVX + GNR45d Total 20

Figure

Table 1. Distribution of rats in different experimental groups. GI and GIII (control groups)  represent oophorectomized rats (OVX) without implants at postoperative days 21 and 45,  respectively; GII and GIV (experimental groups) represent oophorectomized

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