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Graphene nanoparticles as osteoinductive and osteoconductive platform for stem cell and bone regeneration
Graphene nanoparticles as osteoinductive and osteoconductive platform for stem cell and bone regeneration Hoda Elkhenany, Shawn Bourdo, Silke Hecht, Robert Donnell, David Gerard, Ramadan Abdelwahed, Andersen Lafont, Karrer Alghazali, Fumiya Watanabe, Alexandru S. Biris, David Anderson, Madhu Dhar PII: DOI: Reference:
S1549-9634(17)30095-3 doi: 10.1016/j.nano.2017.05.009 NANO 1593
To appear in:
Nanomedicine: Nanotechnology, Biology, and Medicine
Received date: Revised date: Accepted date:
3 October 2016 27 April 2017 18 May 2017
Please cite this article as: Elkhenany Hoda, Bourdo Shawn, Hecht Silke, Donnell Robert, Gerard David, Abdelwahed Ramadan, Lafont Andersen, Alghazali Karrer, Watanabe Fumiya, Biris Alexandru S., Anderson David, Dhar Madhu, Graphene nanoparticles as osteoinductive and osteoconductive platform for stem cell and bone regeneration, Nanomedicine: Nanotechnology, Biology, and Medicine (2017), doi: 10.1016/j.nano.2017.05.009
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ACCEPTED MANUSCRIPT Graphene nanoparticles as osteoinductive and osteoconductive platform for
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stem cell and bone regeneration
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Hoda Elkhenanya,b, Shawn Bourdoc, Silke Hechtd, Robert Donnelld, David Gerarde, Ramadan Abdelwahedb, Andersen Lafontc, Karrer Alghazalic, Fumiya Watanabec, Alexandru S. Birisc,
Tissue Regeneration Laboratory, Department of Large Animal Sciences, College of Veterinary
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a
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David Andersona,d, *Madhu Dhara,d
Medicine, University of Tennessee, Knoxville, Tennessee 37996, USA.
Center for Integrative Nanotechnology Sciences, University of Arkansas at Little Rock, Little
Rock, Arkansas 72204, USA.
Small Animal Clinical Sciences and Biomedical and Diagnostic Services, College of Veterinary
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Department of Surgery, Faculty of Veterinary Medicine, Alexandria University, Egypt 22785.
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b
e
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Medicine, University of Tennessee, Knoxville, Tennessee 37996, USA. Department of Oral and Maxillofacial Surgery, University of Tennessee Graduate School of
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Medicine, Knoxville, Tennessee 37920, USA
*Corresponding author. Email: [email protected]; phone number: 865-974-5703 Word count for abstract: 147 Word count for manuscript: 4,347 Number of references: 45 Number of figures: 7
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ACCEPTED MANUSCRIPT Funding: This work was supported by the Egyptian Cultural and Educational Bureau (ECEB), the University of Tennessee, Center of Excellence in Livestock Diseases and Human Health, the
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Center for Advanced Surface Engineering, under the National Science Foundation Grant No. IIA1457888 and the Arkansas EPSCoR Program, ASSET III and the Food and Drug Administration
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though the memorandum of Understanding with the State of Arkansas.
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Conflicts of interest: The authors declare that there are no known conflicts of interest associated with this publication. The authors also confirm that the results presented in this manuscript are
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entirely original, and have not been either published or under submission/review process for
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another journal.
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ACCEPTED MANUSCRIPT Abstract The potential of graphene-based nanoparticles (GNPs) has recently gained significant attention
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in biomedicine, especially in tissue engineering. In this study, we investigated the osteoinductive and osteoconductive effects of low oxygen content graphene (LOG) nanoparticles on adult
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mesenchymal stem cells (MSCs) in vitro and in vivo. We showed that adult goat MSCs were viable in the presence of 0.1 mg/mL LOG and retained their stem cell properties. A 3D scaffold
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made from agarose was used to encapsulate MSCs and LOG nanoparticles. Scanning electron
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microscopy demonstrated the cell morphology and adherence of MSCs to LOG in the 3D form. The LOG and MSCs in the 3D scaffold were xenogenically implanted into a rat unicortical tibial
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bone defect. The combination of MSCs and LOG nanoparticles resulted in improved active bone formation and increased mineralization. These results strengthen the applicability of LOG
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nanoparticles as an adjunct treatment for bone tissue engineering.
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Keywords: graphene, nanoparticles, MSCs, osteogenesis, cytotoxicity
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ACCEPTED MANUSCRIPT Background Adult mesenchymal stem cells (MSCs) are plastic-adherent cells that express specific
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cluster-of-differentiation (CD) surface protein markers and have the potential to differentiate into various lineages, including, bone, cartilage, and fat1. Bone marrow and adipose tissue are the two
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most widely used and studied sources of MSCs2-11. Adipose tissue (fat) offers a promising, easy, and relatively noninvasive source of MSCs; in human medicine, it is considered the preferred
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source over bone marrow. The stem cell properties of adipose-derived MSCs (AdMSCs) have
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been described in horses, rodents, goats, sheep, dogs, and humans12-16. These MSCs can be readily isolated, characterized, and expanded finitely in vitro to generate enough numbers for
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implantation in vivo17, 18.
Graphene-based materials have been suggested as the next generation of biologically active
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nanomaterials, able to provide an environment for stem cells to adhere, proliferate, and
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differentiate towards osteogenic lineage19. However, graphene presents many challenges as a biomaterial due to having physicochemical properties that vary based on its source and
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processing techniques20. The bioactive potential, efficacy, and toxicity of graphene-based materials in biomedicine have been discussed in several reviews and individual in vitro studies2125
. Their in vivo potential in bone regeneration, however, remains unexplored. Numerous in vitro
studies—including one from our laboratory—have reported the osteogenic potential of mouse pre-osteoblasts and adult MSCs proliferating on 2D and 3D graphene-based nanomaterials, but few have tested these constructs in vivo26-28. In cell-based applications, commercially available graphene can be challenging because of its hydrophobicity. As a result, functionalized forms of graphene are used. Functionalization improves the solubility and biocompatibility and reduces the toxicity of graphene. Altering the 4
ACCEPTED MANUSCRIPT level of oxygen on the surface of graphene is one modification that can significantly affect the downstream applications. As described recently, our group is working with low oxygen
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functionalized graphene (LOG), which has an oxygen content higher than normal graphene
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(2.5%) but less than graphene oxide (31%). LOG is used with the goal of improving the physiochemical properties of graphene and, thus, making it more amenable for biomedical
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applications23. Though graphene and its derivatives are not novel and may be an efficient
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platform for stem cell and tissue engineering, only a single report evaluating the osteogenic potential of LOG nanoparticles on MSCs in vitro exists26. Furthermore, studies evaluating LOG
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nanoparticles’ potential in vivo in long skeletal bone defects have not been published. In our laboratory, we use goats as large animal preclinical models to assess the bone
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healing capacity of biomaterial implants and prostheses29, 30. We have reported that, in vitro over
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a 10-day period, bone marrow-derived goat MSCs (BMMSCs) adhered to, were viable, and proliferated on LOG nanoparticle films. We also observed that in the presence of graphene,
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BMMSCs underwent osteogenic differentiation without any osteogenic inducer, suggesting that
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LOG can serve as osteoinducers and do not require chemical induction to undergo osteogenesis26. In another study, we demonstrated that low passaged goat BMMSCs displayed superior osteogenic potential in vitro compared to AdMSCs17. Specifically, the BMMSCs and AdMSCs underwent osteogenesis via different signal transduction pathways, and we concluded that AdMSCs might require an osteoinducer to improve their osteogenic regenerative potential. Based on previously published literature and our earlier data, we hypothesize that LOG nanoparticles will be cytocompatible and support the osteogenic differentiation of AdMSCs on 2D graphene films in vitro and, subsequently, on 3D scaffolds in an in vivo rat tibial bone defect.
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ACCEPTED MANUSCRIPT To prove our hypothesis, we evaluated the cellular effects of LOG nanoparticles on the proliferation and osteogenic differentiation of goat AdMSCs in vitro and demonstrated that the
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nanoparticles were cytocompatible, as the MSCs maintained their proliferation and osteogenic
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differentiation. For in vivo testing, LOG nanoparticles were synthesized in the bulk form, mixed with MSCs, encapsulated in a 3D agarose scaffold, and implanted into a unicortical defect
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(simple weight bearing model)31 in rat tibia. The combination of nanoparticles and MSCs
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resulted in improved active bone formation (day 21) and significantly higher mineralization (day 45).
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Methods
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All chemicals, biochemicals, and disposables were purchased from Thermo Scientific unless reported otherwise.
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Synthesis and characterization of LOG nanoparticles
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Graphene was functionalized as described previously26. 150mL of concentrated sulfuric acid (95.0-98.0 w/w%) and 50mL of concentrated nitric acid (70%) were added to a mixture of
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100mg of graphene [1.0-1.2 nm thick (3-4 layer), ≤10µm in XY-dimension, Angstron Materials, N002-PDR] and 75mL of distilled water, and the mixture was stirred for 48 hours. The mixture was then diluted with water, and a vacuum filtration setup with a 0.2 GTTP Millipore membrane filter was used to isolate the solids. The filtration was complete after the pH of the filtrate was neutral. The sample was then removed and placed in an oven at 110°C overnight to dry. Subsequently, 20ml of 0.1mg/ml LOG nanoparticles were sprayed on tissue culture dishes to form a 2D film for in vitro studies. The 3D bulk form of LOG nanoparticles for the scaffold was prepared by adding 4mg of LOG powder to 4mL 1X phosphate buffered saline. The 1mg/mL dispersion was bath sonicated 6
ACCEPTED MANUSCRIPT for 2 hours, and the liquid was evaporated at 100°C. When the composite was completely dry, 100µL of 1X PBS was added to form a slurry.
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Elemental composition of the LOG nanoparticles was analyzed using x-ray photoelectron
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spectroscopy (XPS) (Thermo Scientific K-Alpha Instrument). The sample was affixed to a glass substrate, placed on the sample holder, and loaded into the instrument. The data were collected
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using a monochromatic Al Kα (hυ = 1436.6 eV) x-ray source. An x-ray beam of 36W, 400 µm in
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diameter was used. Survey scans (0–1350 eV) were taken at a pass energy (CAE) of 200 eV and 1 eV step size. The relative abundance of elements was determined using Avantage software by
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integrating and comparing the area under the respective photoemission peaks. Spectral comparisons from 3 different spots of the graphene sample processed with PBS as a matrix
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material were performed, and the spots were averaged to give the representative composition.
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Isolation, adhesion, and viability of goat AdMSCs on LOG nanoparticles
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Adipose tissue-derived goat MSCs described earlier were used in this study17. Adult MSCs of passages 2 to 3 were used in all the experiments described below.
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Cell adhesion and viability was assessed in vitro using the MTS assay (Promega) over a period of 10 days and was confirmed microscopically using calcein-AM (Invitrogen) and propidium iodide (PI) (Invitrogen) at day 7. Goat AdMSCs were seeded on LOG-coated plates at a density of 1.0×103 cells/well in a 24-well plate. Cells were stained according to the manufacturer's protocols and subsequently visualized using a Zeiss Axiovert 40C microscope (Carl Zeiss) equipped with a Nikon Digital Sight DS-Qi1Mc camera (Nikon). Equal numbers of AdMSCs were simultaneously seeded on polystyrene and graphene surfaces for comparison. Trilineage differentiation of goat AdMSCs on LOG nanoparticles
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ACCEPTED MANUSCRIPT Osteogenesis, chondrogenesis, and adipogenesis were performed on nanoparticle-coated plates using identical passages and media as described earlier
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. All assays were carried out
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simultaneously on the polystyrene and graphene surfaces for comparison.
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Preparation of the 3D scaffold
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A 3D construct for in vivo implantation was prepared by embedding cells and the nanoparticles in agarose. The agarose has been chosen for its biodegradable and biocompatible
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properties and commonly used in the biomedical research field32-34. To prepare the scaffold, 30
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µl of 2% aqueous solution of agarose was mixed with 0.4mg of LOG nanoparticles and 3x106 MSCs, and the complex was allowed to gel on ice. Scaffolds of nanoparticles alone, cells alone,
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and agarose alone were prepared using the same procedure and transported on ice packs to the animal facility for implantation. All the scaffolds were freshly prepared before the surgery.
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To confirm the viability and properties of cells in the presence of agarose, calcein-am/PI
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and CD44 (a mesenchymal stem cell marker) staining were carried out. The scaffolds were embedded in OCT and snap frozen in an isopentane-dry ice bath. 5 µm tissue sections were cut
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and mounted on super frost plus slides. Samples were blocked with 5% normal serum for 30 minutes at room temperature and incubated with 5µg of anti-CD44. CD44 was detected using Alexa Fluor 488 IgG. All samples were mounted with SlowFade Gold Antifade with DAPI (Life Technologies), and images were taken with a laser scanning spectral confocal microscope (Leica TCS SP2). Cell adhesion and morphology in the agarose scaffold was also assessed using SEM. Animals and surgery 8–10 week-old Sprague Dawley rats (Harlan Laboratories) weighing 20–24g were used35. All animal procedures were carried out according to approved protocols by the University of Tennessee, Institutional Animal Care and Use Committee. All rats were housed at a stable 8
ACCEPTED MANUSCRIPT temperature (22 ± 2 ºC) with a 12 h light/dark cycle. They had ad libitum access to drinking water and standard laboratory rat pellet diet.
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To create a unicortical tibial bone defect, an incision approximately 2 to 3 cm in length was
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made over the tibia, the periosteum covering the tibia was gently reflected, and 4 mm diameter defects were created in each tibia using a micro drill (Stoelting). The defects were washed with
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sterile saline before the implants were placed. All tissue layers were then apposed using suitable
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suture materials and pattern.
Rats were randomly divided into 3 treatment groups (n=6 per group): Group 1: LOG-
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nanoparticles alone; Group 2: AdMSCs alone; and Group 3: LOG-nanoparticles + AdMSCs. In all groups, the right limb was used as an untreated limb which received agarose only and the
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contralateral limb was used as the treated defect. All the rats were observed closely for any
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reaction, general or local at the site of surgery once per day for the first week following the operation and then twice per week.
At 45 days post implantation, the animals were sacrificed
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and tibial bones were harvested.
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Computed tomography
Computed tomography was carried out using a 40-slice helical CT scanner (Philips Brilliance-40). Images were captured directly after implantation (T0) and after sacrifice at 45 days (T45) to evaluate bone healing. Animals were positioned in sternal recumbency, and multidetector helical CT scans of the defects were acquired with 120 kV, 139 mA, 0.67 mm slice thickness, spacing between slices -0.33, 512 x 512 matrix, 500 mm scan field of view, and 64 mm display field of view. Images were reconstructed using either an edge-enhancing or standard algorithm and were displayed in a bone window with 600 HU center and 2300 HU width. Results were exported to a picture archiving and communication system (PACS) for evaluation.
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ACCEPTED MANUSCRIPT Multi-planar reconstruction (MPR) was performed to achieve tangential orientation of the reconstructed slice with the bone defect. PACS measuring tools were utilized to determine the
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area of the bone defect on the MPR images. Each measurement was made 3 times and averaged.
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Quantitatively, the percentage healing in the treated defect was determined relative to the
were blindly analyzed and illustrated graphically.
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Assessment of the new bone formation
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untreated defect. These data were compared and calculated in each animal in each group and
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All rats were injected subcutaneously with a dose of 25 mg/kg of a 20mg/mL solution of calcein in isotonic sodium bicarbonate solution (150 mEq/L) (Sigma-Aldrich) on day 21 post-
midpoint of the study.
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Histomorphometric analysis
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therapy. Calcein was used as the fluorescent label to assess dynamic bone formation at the
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After euthanasia on day 45, the tibias from both sides were dissected and processed for histological evaluation. Slides from all specimens were stained using Von Kossa and
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Haematoxylin & Eosin (H&E) stains. The area of the defect (ROI) was analyzed. The analyst was blinded to the samples. Calcein labeling was visualized under UV, and images were captured using NIS Elements 3.10 (Nikon). The ROIs from the same sections were visualized for Von Kossa and H&E staining under bright light. Image J 1.44P was used for all image analyses. Statistical analysis Data was analyzed using a mixed model design with the group as the whole plot factor and treatment as the split plot factor. Statistical significance was identified at p< 0.05. All analyses were conducted using the SAS system (SAS Institute Inc.). Ranked transformation was applied if 10
ACCEPTED MANUSCRIPT diagnostics analysis showed violation of ANOVA assumptions such as non-normality and unequal variance.
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Results Characterization of LOG nanoparticles
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The LOG nanoparticle films were generated and confirmed for uniformity in thickness and
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integrity as described earlier26. Like commercially available graphene, LOG retains much of its extensive π-network of carbon‒carbon bonds. By introducing the oxygen functional groups, we
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increased the dispersion of graphene in aqueous media, making it easy to handle. The chemical composition and surface features of nanoparticles in the bulk state, which was used for the 3D
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scaffold, were analyzed (Figure 1). The XPS analysis of the 3D scaffold prepared for this study showed that the nanoparticles were composed of approximately 88% carbon and 4.5% oxygen,
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confirming that the functionalized form of graphene is the LOG form. The presence of sodium
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(3.04%), chlorine (3.64%), and phosphorous (0.87%) could be attributed to the PBS, which was used to disperse the nanoparticles into a slurry (Figure 1a). The SEM showed that graphene
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flakes agglomerate into a high surface area material with a complex, convoluted nanostructure (Figure 1b). The addition of agarose smoothened the surface of the matrix considerably, but some underlying wavy features of graphene were still visible (Figure 1c). Cells were detected in SEM, confirming their adherence on the surface of the nanoparticle/agarose scaffold (Figure 1d). Biocompatibility of LOG nanoparticles Cell viability of the isolated, characterized, and cryopreserved goat AdMSCs17 was evaluated on the LOG films in vitro using the MTS proliferation assay. The AdMSCs retained their proliferation and maintained their viability on LOG films, similar to the polystyrene-coated surface (Figure 2a). Their viability was further confirmed by the live-dead fluorescent staining of 11
ACCEPTED MANUSCRIPT cells (Figure 2b). As judged by the green fluorescence (calcein-AM), cells adhered to the graphene films and retained their viability. Similarly, the green fluorescent AdMSCs in the 3D
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construct of MSCs, graphene, and agarose demonstrated their viability in the bulk form prior to
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implantation (Figure 2c). Lack of red fluorescent (propidium iodide) cells confirmed that both the nanoparticles and the agarose were not cytotoxic.
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Mesenchymal cell properties in presence of LOG nanoparticles
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In vitro tri-lineage differentiation of MSCs in the presence of LOG nanoparticles was
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carried out to determine their multipotency. As judged by the alizarin red, alcian blue, and oilred-o staining patterns, goat AdMSCs underwent osteogenesis, chondrogenesis, and
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adipogenesis, respectively, on the 2D LOG in the presence of lineage-specific media (Figure 3). Additionally, the expression of a previously tested and validated MSC protein marker,
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CD44, was evaluated when the 3D construct of AdMSCs and LOG was formed. Cells retained
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the expression of CD44 (Figure 4)1. Collectively, these data confirm that the stem cell properties of goat AdMSCs were maintained in the presence of the 2D and 3D forms of LOG nanoparticles.
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LOG nanoparticles promote bone healing in vivo Next, to test whether the MSCs and the graphene construct have in vivo osteogenic potential similar to that in vitro, a unicortical tibial bone defect model in rats was established. The nanoparticles and MSCs were encapsulated in inert agarose gel to form a scaffold that could be implanted into the defect relatively quickly and without technical difficulty. CT analyses of the defects were carried out by comparing the region-of-interest (ROI)–the defect site–at the time of surgery (T0) and at sacrifice (T45) in each rat (Figure 5). This strategy was used to control the individual variations, if any, in creating the defect at T 0. Subjective and quantitative analyses of the ROI at T45 showed a marked filling of the AdMSCs and the 12
ACCEPTED MANUSCRIPT AdMSCs+nanoparticle-treated tibias relative to the group implanted with nanoparticles alone (Figure 5A). Even though there was a visual filling of the defect in both groups, the percentage
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healing calculated from the CT data (Figure 5B) was not statistically different, suggesting that
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both the MSCs alone or in combination with nanoparticles had the potential to heal bone. To further evaluate bone healing and new bone formation, histomorphometric analyses at
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T45 were performed (Figure 6). Agarose was still visible at the defect site, particularly in the
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control limbs treated with agarose only, where no visible filling of the defect was observed. Comparatively, the defect site implanted with LOG nanoparticles alone showed black particles
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without any staining, helping us identify the ROI with confidence. Subjectively, the ROI and new mineralized bone formed in each section was identified using Von Kossa staining (Figure
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6a). The defects treated with AdMSCs and with AdMSCs+LOG nanoparticles showed marked
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healing and new mineralized bone formation. Interestingly, the defect filled with the AdMSCs+LOG nanoparticles showed relatively more vasculature and mineralized bone in the
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defect. Bone healing patterns were further supported by the active bone labeling at T 21 (Figure
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6b). The calcein green fluorescence marked the active bone formed at T21 (i.e., midway through the study period), strengthening the model and the strategy employed in this study. H&E staining did not show any multinucleated inflammatory or abnormal cells in the defects treated with agarose, AdMSCs, and the nanoparticles, whether alone or in combination, confirming lack of immunologic response to any of the substances in the rats (Figure 6c). Both the active bone at T21 and mineralized bone at T45 were further quantitated and statistically analyzed (Figure 7). As observed in Figure 7A, there was a trend towards an increase in active bone formed in the AdMSCs+nanoparticles group; data was not significantly different from the group treated with AdMSCs alone at T21. However, as shown in Figure 7B, the relative amount of mineralized bone
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ACCEPTED MANUSCRIPT formed at T45 was significantly higher in the rats treated with AdMSCs+nanoparticles compared to the control and the AdMSCs alone groups, suggesting potential osteoinductive and
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osteoconductive effects of graphene nanoparticles on AdMSCs.
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Discussion
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In this study, we present data to show the osteoinductive and osteoconductive properties of LOG-nanoparticles in an in vivo rat model of unicortical bone defect. This data strengthens the
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notion that the use of specific forms of graphene nanoparticles to improve or enhance the
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osteogenic potential of MSCs is a promising bone tissue regeneration therapy. Specifically, our data indicates that LOG nanoparticles have the potential for use in the healing of trauma-induced
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defects in long bones while supporting the biological activity of stem cells. The use of graphene nanoparticles as bioactive materials in vivo in bone tissue engineering
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can be challenging because of their complex physicochemical properties. Handling graphene
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itself can prove challenging, since it is very light with high surface area. Incorporating graphene into an aqueous environment is also difficult, as the material is inherently hydrophobic. For this
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reason, the graphene in this study underwent a light oxidative treatment, which introduced oxygen functional groups on the surface to allow for better dispersion and create a biological environment conducive to osteogenesis. In this study, in vitro data demonstrated that goat AdMSCs proliferate and remain viable in presence of 2D and 3D forms of LOG nanoparticles. A linear increase in cell viability as well as fluorescent imaging over a period of 10 days confirms that the LOG nanoparticles are not toxic to goat AdMSCs (Figure 2), which is in agreement with reports published by us and other investigators that graphene nanoparticles are not cytotoxic to MSCs26, 36, 37.
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ACCEPTED MANUSCRIPT While the nanoparticles used with goat AdMSCs were confirmed to be similar in their physical and chemical properties to those previously used with goat BMMSCs26, the osteogenic
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differentiation process was not totally identical. Unlike the goat BMMSCs, nanoparticles alone
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were not sufficient for the AdMSCs to undergo osteogenesis; the osteogenic inducers dexamethasone, beta-glycerophosphate, and ascorbic acid were required. There was no
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significant osteogenic differentiation in the fetal bovine serum-containing media without these
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factors, suggesting that the in vitro mechanism of osteogenesis of BMMSCs in the presence of LOG nanoparticles may be different from that of AdMSCs. This supports our recent observation
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that without any biomaterial, goat BMMSCs and AdMSCs undergo osteogenesis via two different MAPK proteins17.
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It has recently been shown that the gold nanoparticles and GNPs interact with the
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extracellular matrix, up-regulate β1-integrin in vitro, and, as a result, generate mechanical stress on the MSCs, leading to the activation of the p38 MAPK pathway, which in turn may induce
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spontaneous osteogenic differentiation36-38. Since goat AdMSCs undergo osteogenesis via the
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p44/42 MAPK and goat BMMSCs do so via p38 MAPK, it is possible that the BMMSCs and AdMSCs trigger different cues when they undergo osteogenesis on LOG, making it imperative to identify the signaling mechanisms involved in these processes. Hence, further studies to identify the molecular targets that are triggered when LOG nanoparticles interact with AdMSCs are crucial to understanding this process39. One of the challenges in using a combination of nanoparticles + MSCs in an animal model is the fabrication of the 3D form. In this study, we were able to implant a construct of goat AdMSCs and LOG into a rat unicortical tibial bone defect using agarose as an inert scaffold. As observed in the histomorphometric data (Figure 6), the use of agarose did not interfere with the
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ACCEPTED MANUSCRIPT bone healing potential of either the MSCs or the LOG. It is possible that agarose alone inhibited the migration of osteoblasts in healing the defect, whereas the presence of MSCs or
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MSCs+nanoparticles with agarose showed significant healing. The varying effects of agarose
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need to be determined. In future studies or clinical applications, 3D forms of LOG nanoparticles with an optimal scaffold should be considered.
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This report also represents an excellent initial step towards using characterized MSCs as
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universal donor cells. MSCs are known to exhibit low immunogenicity, including immunomodulatory protective and repair effects. Of all sources of MSCs, AdMSCs are
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abundant, readily available, ethically acceptable, easy to obtain, and most importantly, have low MHC-I expression and lack MHC-II molecules, suggesting that they are not likely to induce
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rejection40. Therefore, it was not surprising that local or systemic rejection reaction was not
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detected following implantation and during the course of the study. No animal displayed any signs of inflammation in the area of the surgical wound. These data are in agreement with similar
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published reports and strengthen the use of xenogenic MSCs in rodent models41, 42.
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Multiple studies, including data from our laboratory, have shown that various forms of graphene can promote the in vitro proliferation and osteogenic differentiation of MSCs. Osteogenic differentiation is enhanced spontaneously as well as in the presence of chemical inducers26, 38,43-45. Supported by results from our laboratory and published by others, we suggest that LOG nanoparticles can guide bone tissue engineering by creating a microenvironment in which MSCs adhere and undergo osteogenesis. Specifically, in this study, by introducing oxygen groups in pristine graphene, we created a biological environment for MSCs to stay viable and undergo osteogenesis in vitro and in vivo. These nanomaterial-derived signals remain unknown, so the exact effects of graphene on cells, tissues, or organs require further studies. More research
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ACCEPTED MANUSCRIPT is needed regarding the potential applicability of graphene and its derivatives as biocompatible, transferable, and implantable platforms for stem cell culture and delivery. Using the strategy
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described in this paper, experiments to address the above issues can now be initiated.
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(inset); scanning electron micrograph images of (b) graphene, (c) graphene + agarose, and (d) graphene + agarose + cells.
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Figure 2. Assessment of cell viability in presence of LOG nanoparticles by (a) MTS assay, the data is expressed as the mean ±SD based on at least triplicate observations and (b) live-dead
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fluorescent staining using calcein-AM and propidium iodide. (c) Cell viability was also assessed
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in the presence of the 3D scaffold using live-dead staining. AdMSCs were seeded on the 2D graphene surface over a period of 10 days and in the agarose scaffold in the presence of 3D
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graphene for a period of 20 days. Scale bar = 100µm.
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Figure 3. Tri-lineage differentiation of AdMSCs was evaluated in the absence (a, b & c) and presence of 2D LOG nanoparticles (d, e & f). Representative Images showing alizarin red (a&d),
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alcian blue (b&e), and oil-red-o (c&f) staining of osteocytes, chondrocytes, and adipocytes,
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detected at day 21 post-differentiation. Scale bar = 100µm. Figure 4. Representative images showing the expression of CD44 in the presence of 3D LOG nanoparticles. The panels on the left show the agarose scaffold alone (a), LOG in the scaffold (c), and the scaffold of agarose with AdMSCs + LOG (e). Corresponding images viewed through the fluorescent microscope are shown (b, d, f). Isotype control for CD44 was used as the control. The inset in f shows the presence of cells stained with DAPI. Scale bar = 100µm. Figure 5. Computed tomography. (A) CT images of the tibias depicting the ROI were taken at T0 and T45. Representative images showing LOG nanoparticles alone (a), AdMSCs alone (b), and AdMSCs + LOG (c) are shown. The right limbs (indicated by R) were used as control, and the 24
ACCEPTED MANUSCRIPT contralateral limb was the treated limb. The arrows indicate the ROI in the treated limbs. (B) Quantitative analysis of the CT images. The ROI and, thus, the % healing was quantitated as
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described, and the data is represented. Y-axis indicated the fold change in the defect area at T45,
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which was calculated relative to the defect area at T0. The data is expressed as the mean ±SD based on at least two observations from four independent images. Different letters denote a
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significant difference, p < 0.05.
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Figure 6. Representative images of histological sections from each group are shown. Von Kossa
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staining at 2x magnification at T45 (a), H&E staining at 2x magnification at T45 (b), and Calcein green fluorescent staining at 5x magnification on T21 (c) are depicted. Various treatment groups,
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control and the treated limbs, are labeled accordingly. Figure 7. Graphs representing (a) the percentage of the active bone labeled with calcein green in
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the treated limbs relative to the controls at T21 and (b) the percentage of new mineralized bone
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formed in the treated limbs relative to the controls at T45 are shown. The data is expressed as the mean ±SD based on at least duplicate observations from at least 4 independent specimens. The
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different letters denote a significant difference, p < 0.05.
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ACCEPTED MANUSCRIPT Graphical abstract A conclusive study on the cytotoxicity and bone regenerative capacity of low oxygen content
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graphene nanoparticles in vitro and in vivo in rat models. Here in we propose the LOG
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nanoparticles to potentiate and enhance the osteogenic differentiation capacity of the Adipose derived mesenchymal stem cells. Unicortical defect model in rats was used to study the regenerative effect in bone defect. The in vitro results were matching the in vivo results and
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supports the proposal of LOG nanoparticles potentiating effect.
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Graphical abstract
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S1549-9634(17)30095-3 doi: 10.1016/j.nano.2017.05.009 NANO 1593
To appear in:
Nanomedicine: Nanotechnology, Biology, and Medicine
Received date: Revised date: Accepted date:
3 October 2016 27 April 2017 18 May 2017
Please cite this article as: Elkhenany Hoda, Bourdo Shawn, Hecht Silke, Donnell Robert, Gerard David, Abdelwahed Ramadan, Lafont Andersen, Alghazali Karrer, Watanabe Fumiya, Biris Alexandru S., Anderson David, Dhar Madhu, Graphene nanoparticles as osteoinductive and osteoconductive platform for stem cell and bone regeneration, Nanomedicine: Nanotechnology, Biology, and Medicine (2017), doi: 10.1016/j.nano.2017.05.009
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Graphene nanoparticles as osteoinductive and osteoconductive platform for
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stem cell and bone regeneration
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Hoda Elkhenanya,b, Shawn Bourdoc, Silke Hechtd, Robert Donnelld, David Gerarde, Ramadan Abdelwahedb, Andersen Lafontc, Karrer Alghazalic, Fumiya Watanabec, Alexandru S. Birisc,
Tissue Regeneration Laboratory, Department of Large Animal Sciences, College of Veterinary
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a
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David Andersona,d, *Madhu Dhara,d
Medicine, University of Tennessee, Knoxville, Tennessee 37996, USA.
Center for Integrative Nanotechnology Sciences, University of Arkansas at Little Rock, Little
Rock, Arkansas 72204, USA.
Small Animal Clinical Sciences and Biomedical and Diagnostic Services, College of Veterinary
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Department of Surgery, Faculty of Veterinary Medicine, Alexandria University, Egypt 22785.
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e
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Medicine, University of Tennessee, Knoxville, Tennessee 37996, USA. Department of Oral and Maxillofacial Surgery, University of Tennessee Graduate School of
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Medicine, Knoxville, Tennessee 37920, USA
*Corresponding author. Email: [email protected]; phone number: 865-974-5703 Word count for abstract: 147 Word count for manuscript: 4,347 Number of references: 45 Number of figures: 7
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ACCEPTED MANUSCRIPT Funding: This work was supported by the Egyptian Cultural and Educational Bureau (ECEB), the University of Tennessee, Center of Excellence in Livestock Diseases and Human Health, the
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Center for Advanced Surface Engineering, under the National Science Foundation Grant No. IIA1457888 and the Arkansas EPSCoR Program, ASSET III and the Food and Drug Administration
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though the memorandum of Understanding with the State of Arkansas.
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Conflicts of interest: The authors declare that there are no known conflicts of interest associated with this publication. The authors also confirm that the results presented in this manuscript are
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entirely original, and have not been either published or under submission/review process for
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another journal.
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ACCEPTED MANUSCRIPT Abstract The potential of graphene-based nanoparticles (GNPs) has recently gained significant attention
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in biomedicine, especially in tissue engineering. In this study, we investigated the osteoinductive and osteoconductive effects of low oxygen content graphene (LOG) nanoparticles on adult
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mesenchymal stem cells (MSCs) in vitro and in vivo. We showed that adult goat MSCs were viable in the presence of 0.1 mg/mL LOG and retained their stem cell properties. A 3D scaffold
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made from agarose was used to encapsulate MSCs and LOG nanoparticles. Scanning electron
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microscopy demonstrated the cell morphology and adherence of MSCs to LOG in the 3D form. The LOG and MSCs in the 3D scaffold were xenogenically implanted into a rat unicortical tibial
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bone defect. The combination of MSCs and LOG nanoparticles resulted in improved active bone formation and increased mineralization. These results strengthen the applicability of LOG
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nanoparticles as an adjunct treatment for bone tissue engineering.
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Keywords: graphene, nanoparticles, MSCs, osteogenesis, cytotoxicity
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ACCEPTED MANUSCRIPT Background Adult mesenchymal stem cells (MSCs) are plastic-adherent cells that express specific
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cluster-of-differentiation (CD) surface protein markers and have the potential to differentiate into various lineages, including, bone, cartilage, and fat1. Bone marrow and adipose tissue are the two
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most widely used and studied sources of MSCs2-11. Adipose tissue (fat) offers a promising, easy, and relatively noninvasive source of MSCs; in human medicine, it is considered the preferred
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source over bone marrow. The stem cell properties of adipose-derived MSCs (AdMSCs) have
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been described in horses, rodents, goats, sheep, dogs, and humans12-16. These MSCs can be readily isolated, characterized, and expanded finitely in vitro to generate enough numbers for
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implantation in vivo17, 18.
Graphene-based materials have been suggested as the next generation of biologically active
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nanomaterials, able to provide an environment for stem cells to adhere, proliferate, and
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differentiate towards osteogenic lineage19. However, graphene presents many challenges as a biomaterial due to having physicochemical properties that vary based on its source and
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processing techniques20. The bioactive potential, efficacy, and toxicity of graphene-based materials in biomedicine have been discussed in several reviews and individual in vitro studies2125
. Their in vivo potential in bone regeneration, however, remains unexplored. Numerous in vitro
studies—including one from our laboratory—have reported the osteogenic potential of mouse pre-osteoblasts and adult MSCs proliferating on 2D and 3D graphene-based nanomaterials, but few have tested these constructs in vivo26-28. In cell-based applications, commercially available graphene can be challenging because of its hydrophobicity. As a result, functionalized forms of graphene are used. Functionalization improves the solubility and biocompatibility and reduces the toxicity of graphene. Altering the 4
ACCEPTED MANUSCRIPT level of oxygen on the surface of graphene is one modification that can significantly affect the downstream applications. As described recently, our group is working with low oxygen
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functionalized graphene (LOG), which has an oxygen content higher than normal graphene
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(2.5%) but less than graphene oxide (31%). LOG is used with the goal of improving the physiochemical properties of graphene and, thus, making it more amenable for biomedical
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applications23. Though graphene and its derivatives are not novel and may be an efficient
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platform for stem cell and tissue engineering, only a single report evaluating the osteogenic potential of LOG nanoparticles on MSCs in vitro exists26. Furthermore, studies evaluating LOG
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nanoparticles’ potential in vivo in long skeletal bone defects have not been published. In our laboratory, we use goats as large animal preclinical models to assess the bone
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healing capacity of biomaterial implants and prostheses29, 30. We have reported that, in vitro over
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a 10-day period, bone marrow-derived goat MSCs (BMMSCs) adhered to, were viable, and proliferated on LOG nanoparticle films. We also observed that in the presence of graphene,
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BMMSCs underwent osteogenic differentiation without any osteogenic inducer, suggesting that
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LOG can serve as osteoinducers and do not require chemical induction to undergo osteogenesis26. In another study, we demonstrated that low passaged goat BMMSCs displayed superior osteogenic potential in vitro compared to AdMSCs17. Specifically, the BMMSCs and AdMSCs underwent osteogenesis via different signal transduction pathways, and we concluded that AdMSCs might require an osteoinducer to improve their osteogenic regenerative potential. Based on previously published literature and our earlier data, we hypothesize that LOG nanoparticles will be cytocompatible and support the osteogenic differentiation of AdMSCs on 2D graphene films in vitro and, subsequently, on 3D scaffolds in an in vivo rat tibial bone defect.
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nanoparticles were cytocompatible, as the MSCs maintained their proliferation and osteogenic
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differentiation. For in vivo testing, LOG nanoparticles were synthesized in the bulk form, mixed with MSCs, encapsulated in a 3D agarose scaffold, and implanted into a unicortical defect
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(simple weight bearing model)31 in rat tibia. The combination of nanoparticles and MSCs
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resulted in improved active bone formation (day 21) and significantly higher mineralization (day 45).
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Methods
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All chemicals, biochemicals, and disposables were purchased from Thermo Scientific unless reported otherwise.
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Synthesis and characterization of LOG nanoparticles
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Graphene was functionalized as described previously26. 150mL of concentrated sulfuric acid (95.0-98.0 w/w%) and 50mL of concentrated nitric acid (70%) were added to a mixture of
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100mg of graphene [1.0-1.2 nm thick (3-4 layer), ≤10µm in XY-dimension, Angstron Materials, N002-PDR] and 75mL of distilled water, and the mixture was stirred for 48 hours. The mixture was then diluted with water, and a vacuum filtration setup with a 0.2 GTTP Millipore membrane filter was used to isolate the solids. The filtration was complete after the pH of the filtrate was neutral. The sample was then removed and placed in an oven at 110°C overnight to dry. Subsequently, 20ml of 0.1mg/ml LOG nanoparticles were sprayed on tissue culture dishes to form a 2D film for in vitro studies. The 3D bulk form of LOG nanoparticles for the scaffold was prepared by adding 4mg of LOG powder to 4mL 1X phosphate buffered saline. The 1mg/mL dispersion was bath sonicated 6
ACCEPTED MANUSCRIPT for 2 hours, and the liquid was evaporated at 100°C. When the composite was completely dry, 100µL of 1X PBS was added to form a slurry.
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Elemental composition of the LOG nanoparticles was analyzed using x-ray photoelectron
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spectroscopy (XPS) (Thermo Scientific K-Alpha Instrument). The sample was affixed to a glass substrate, placed on the sample holder, and loaded into the instrument. The data were collected
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using a monochromatic Al Kα (hυ = 1436.6 eV) x-ray source. An x-ray beam of 36W, 400 µm in
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diameter was used. Survey scans (0–1350 eV) were taken at a pass energy (CAE) of 200 eV and 1 eV step size. The relative abundance of elements was determined using Avantage software by
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integrating and comparing the area under the respective photoemission peaks. Spectral comparisons from 3 different spots of the graphene sample processed with PBS as a matrix
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material were performed, and the spots were averaged to give the representative composition.
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Isolation, adhesion, and viability of goat AdMSCs on LOG nanoparticles
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Adipose tissue-derived goat MSCs described earlier were used in this study17. Adult MSCs of passages 2 to 3 were used in all the experiments described below.
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Cell adhesion and viability was assessed in vitro using the MTS assay (Promega) over a period of 10 days and was confirmed microscopically using calcein-AM (Invitrogen) and propidium iodide (PI) (Invitrogen) at day 7. Goat AdMSCs were seeded on LOG-coated plates at a density of 1.0×103 cells/well in a 24-well plate. Cells were stained according to the manufacturer's protocols and subsequently visualized using a Zeiss Axiovert 40C microscope (Carl Zeiss) equipped with a Nikon Digital Sight DS-Qi1Mc camera (Nikon). Equal numbers of AdMSCs were simultaneously seeded on polystyrene and graphene surfaces for comparison. Trilineage differentiation of goat AdMSCs on LOG nanoparticles
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. All assays were carried out
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simultaneously on the polystyrene and graphene surfaces for comparison.
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Preparation of the 3D scaffold
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A 3D construct for in vivo implantation was prepared by embedding cells and the nanoparticles in agarose. The agarose has been chosen for its biodegradable and biocompatible
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properties and commonly used in the biomedical research field32-34. To prepare the scaffold, 30
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µl of 2% aqueous solution of agarose was mixed with 0.4mg of LOG nanoparticles and 3x106 MSCs, and the complex was allowed to gel on ice. Scaffolds of nanoparticles alone, cells alone,
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and agarose alone were prepared using the same procedure and transported on ice packs to the animal facility for implantation. All the scaffolds were freshly prepared before the surgery.
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To confirm the viability and properties of cells in the presence of agarose, calcein-am/PI
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and CD44 (a mesenchymal stem cell marker) staining were carried out. The scaffolds were embedded in OCT and snap frozen in an isopentane-dry ice bath. 5 µm tissue sections were cut
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and mounted on super frost plus slides. Samples were blocked with 5% normal serum for 30 minutes at room temperature and incubated with 5µg of anti-CD44. CD44 was detected using Alexa Fluor 488 IgG. All samples were mounted with SlowFade Gold Antifade with DAPI (Life Technologies), and images were taken with a laser scanning spectral confocal microscope (Leica TCS SP2). Cell adhesion and morphology in the agarose scaffold was also assessed using SEM. Animals and surgery 8–10 week-old Sprague Dawley rats (Harlan Laboratories) weighing 20–24g were used35. All animal procedures were carried out according to approved protocols by the University of Tennessee, Institutional Animal Care and Use Committee. All rats were housed at a stable 8
ACCEPTED MANUSCRIPT temperature (22 ± 2 ºC) with a 12 h light/dark cycle. They had ad libitum access to drinking water and standard laboratory rat pellet diet.
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To create a unicortical tibial bone defect, an incision approximately 2 to 3 cm in length was
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made over the tibia, the periosteum covering the tibia was gently reflected, and 4 mm diameter defects were created in each tibia using a micro drill (Stoelting). The defects were washed with
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sterile saline before the implants were placed. All tissue layers were then apposed using suitable
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suture materials and pattern.
Rats were randomly divided into 3 treatment groups (n=6 per group): Group 1: LOG-
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nanoparticles alone; Group 2: AdMSCs alone; and Group 3: LOG-nanoparticles + AdMSCs. In all groups, the right limb was used as an untreated limb which received agarose only and the
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contralateral limb was used as the treated defect. All the rats were observed closely for any
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reaction, general or local at the site of surgery once per day for the first week following the operation and then twice per week.
At 45 days post implantation, the animals were sacrificed
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and tibial bones were harvested.
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Computed tomography
Computed tomography was carried out using a 40-slice helical CT scanner (Philips Brilliance-40). Images were captured directly after implantation (T0) and after sacrifice at 45 days (T45) to evaluate bone healing. Animals were positioned in sternal recumbency, and multidetector helical CT scans of the defects were acquired with 120 kV, 139 mA, 0.67 mm slice thickness, spacing between slices -0.33, 512 x 512 matrix, 500 mm scan field of view, and 64 mm display field of view. Images were reconstructed using either an edge-enhancing or standard algorithm and were displayed in a bone window with 600 HU center and 2300 HU width. Results were exported to a picture archiving and communication system (PACS) for evaluation.
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area of the bone defect on the MPR images. Each measurement was made 3 times and averaged.
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Quantitatively, the percentage healing in the treated defect was determined relative to the
were blindly analyzed and illustrated graphically.
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Assessment of the new bone formation
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untreated defect. These data were compared and calculated in each animal in each group and
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All rats were injected subcutaneously with a dose of 25 mg/kg of a 20mg/mL solution of calcein in isotonic sodium bicarbonate solution (150 mEq/L) (Sigma-Aldrich) on day 21 post-
midpoint of the study.
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Histomorphometric analysis
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therapy. Calcein was used as the fluorescent label to assess dynamic bone formation at the
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After euthanasia on day 45, the tibias from both sides were dissected and processed for histological evaluation. Slides from all specimens were stained using Von Kossa and
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Haematoxylin & Eosin (H&E) stains. The area of the defect (ROI) was analyzed. The analyst was blinded to the samples. Calcein labeling was visualized under UV, and images were captured using NIS Elements 3.10 (Nikon). The ROIs from the same sections were visualized for Von Kossa and H&E staining under bright light. Image J 1.44P was used for all image analyses. Statistical analysis Data was analyzed using a mixed model design with the group as the whole plot factor and treatment as the split plot factor. Statistical significance was identified at p< 0.05. All analyses were conducted using the SAS system (SAS Institute Inc.). Ranked transformation was applied if 10
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Results Characterization of LOG nanoparticles
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The LOG nanoparticle films were generated and confirmed for uniformity in thickness and
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integrity as described earlier26. Like commercially available graphene, LOG retains much of its extensive π-network of carbon‒carbon bonds. By introducing the oxygen functional groups, we
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increased the dispersion of graphene in aqueous media, making it easy to handle. The chemical composition and surface features of nanoparticles in the bulk state, which was used for the 3D
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scaffold, were analyzed (Figure 1). The XPS analysis of the 3D scaffold prepared for this study showed that the nanoparticles were composed of approximately 88% carbon and 4.5% oxygen,
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confirming that the functionalized form of graphene is the LOG form. The presence of sodium
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(3.04%), chlorine (3.64%), and phosphorous (0.87%) could be attributed to the PBS, which was used to disperse the nanoparticles into a slurry (Figure 1a). The SEM showed that graphene
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flakes agglomerate into a high surface area material with a complex, convoluted nanostructure (Figure 1b). The addition of agarose smoothened the surface of the matrix considerably, but some underlying wavy features of graphene were still visible (Figure 1c). Cells were detected in SEM, confirming their adherence on the surface of the nanoparticle/agarose scaffold (Figure 1d). Biocompatibility of LOG nanoparticles Cell viability of the isolated, characterized, and cryopreserved goat AdMSCs17 was evaluated on the LOG films in vitro using the MTS proliferation assay. The AdMSCs retained their proliferation and maintained their viability on LOG films, similar to the polystyrene-coated surface (Figure 2a). Their viability was further confirmed by the live-dead fluorescent staining of 11
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construct of MSCs, graphene, and agarose demonstrated their viability in the bulk form prior to
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implantation (Figure 2c). Lack of red fluorescent (propidium iodide) cells confirmed that both the nanoparticles and the agarose were not cytotoxic.
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Mesenchymal cell properties in presence of LOG nanoparticles
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In vitro tri-lineage differentiation of MSCs in the presence of LOG nanoparticles was
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carried out to determine their multipotency. As judged by the alizarin red, alcian blue, and oilred-o staining patterns, goat AdMSCs underwent osteogenesis, chondrogenesis, and
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adipogenesis, respectively, on the 2D LOG in the presence of lineage-specific media (Figure 3). Additionally, the expression of a previously tested and validated MSC protein marker,
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CD44, was evaluated when the 3D construct of AdMSCs and LOG was formed. Cells retained
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the expression of CD44 (Figure 4)1. Collectively, these data confirm that the stem cell properties of goat AdMSCs were maintained in the presence of the 2D and 3D forms of LOG nanoparticles.
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LOG nanoparticles promote bone healing in vivo Next, to test whether the MSCs and the graphene construct have in vivo osteogenic potential similar to that in vitro, a unicortical tibial bone defect model in rats was established. The nanoparticles and MSCs were encapsulated in inert agarose gel to form a scaffold that could be implanted into the defect relatively quickly and without technical difficulty. CT analyses of the defects were carried out by comparing the region-of-interest (ROI)–the defect site–at the time of surgery (T0) and at sacrifice (T45) in each rat (Figure 5). This strategy was used to control the individual variations, if any, in creating the defect at T 0. Subjective and quantitative analyses of the ROI at T45 showed a marked filling of the AdMSCs and the 12
ACCEPTED MANUSCRIPT AdMSCs+nanoparticle-treated tibias relative to the group implanted with nanoparticles alone (Figure 5A). Even though there was a visual filling of the defect in both groups, the percentage
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healing calculated from the CT data (Figure 5B) was not statistically different, suggesting that
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both the MSCs alone or in combination with nanoparticles had the potential to heal bone. To further evaluate bone healing and new bone formation, histomorphometric analyses at
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T45 were performed (Figure 6). Agarose was still visible at the defect site, particularly in the
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control limbs treated with agarose only, where no visible filling of the defect was observed. Comparatively, the defect site implanted with LOG nanoparticles alone showed black particles
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without any staining, helping us identify the ROI with confidence. Subjectively, the ROI and new mineralized bone formed in each section was identified using Von Kossa staining (Figure
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6a). The defects treated with AdMSCs and with AdMSCs+LOG nanoparticles showed marked
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healing and new mineralized bone formation. Interestingly, the defect filled with the AdMSCs+LOG nanoparticles showed relatively more vasculature and mineralized bone in the
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defect. Bone healing patterns were further supported by the active bone labeling at T 21 (Figure
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6b). The calcein green fluorescence marked the active bone formed at T21 (i.e., midway through the study period), strengthening the model and the strategy employed in this study. H&E staining did not show any multinucleated inflammatory or abnormal cells in the defects treated with agarose, AdMSCs, and the nanoparticles, whether alone or in combination, confirming lack of immunologic response to any of the substances in the rats (Figure 6c). Both the active bone at T21 and mineralized bone at T45 were further quantitated and statistically analyzed (Figure 7). As observed in Figure 7A, there was a trend towards an increase in active bone formed in the AdMSCs+nanoparticles group; data was not significantly different from the group treated with AdMSCs alone at T21. However, as shown in Figure 7B, the relative amount of mineralized bone
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Discussion
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In this study, we present data to show the osteoinductive and osteoconductive properties of LOG-nanoparticles in an in vivo rat model of unicortical bone defect. This data strengthens the
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notion that the use of specific forms of graphene nanoparticles to improve or enhance the
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osteogenic potential of MSCs is a promising bone tissue regeneration therapy. Specifically, our data indicates that LOG nanoparticles have the potential for use in the healing of trauma-induced
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defects in long bones while supporting the biological activity of stem cells. The use of graphene nanoparticles as bioactive materials in vivo in bone tissue engineering
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can be challenging because of their complex physicochemical properties. Handling graphene
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itself can prove challenging, since it is very light with high surface area. Incorporating graphene into an aqueous environment is also difficult, as the material is inherently hydrophobic. For this
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reason, the graphene in this study underwent a light oxidative treatment, which introduced oxygen functional groups on the surface to allow for better dispersion and create a biological environment conducive to osteogenesis. In this study, in vitro data demonstrated that goat AdMSCs proliferate and remain viable in presence of 2D and 3D forms of LOG nanoparticles. A linear increase in cell viability as well as fluorescent imaging over a period of 10 days confirms that the LOG nanoparticles are not toxic to goat AdMSCs (Figure 2), which is in agreement with reports published by us and other investigators that graphene nanoparticles are not cytotoxic to MSCs26, 36, 37.
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differentiation process was not totally identical. Unlike the goat BMMSCs, nanoparticles alone
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were not sufficient for the AdMSCs to undergo osteogenesis; the osteogenic inducers dexamethasone, beta-glycerophosphate, and ascorbic acid were required. There was no
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significant osteogenic differentiation in the fetal bovine serum-containing media without these
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factors, suggesting that the in vitro mechanism of osteogenesis of BMMSCs in the presence of LOG nanoparticles may be different from that of AdMSCs. This supports our recent observation
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that without any biomaterial, goat BMMSCs and AdMSCs undergo osteogenesis via two different MAPK proteins17.
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It has recently been shown that the gold nanoparticles and GNPs interact with the
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extracellular matrix, up-regulate β1-integrin in vitro, and, as a result, generate mechanical stress on the MSCs, leading to the activation of the p38 MAPK pathway, which in turn may induce
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spontaneous osteogenic differentiation36-38. Since goat AdMSCs undergo osteogenesis via the
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p44/42 MAPK and goat BMMSCs do so via p38 MAPK, it is possible that the BMMSCs and AdMSCs trigger different cues when they undergo osteogenesis on LOG, making it imperative to identify the signaling mechanisms involved in these processes. Hence, further studies to identify the molecular targets that are triggered when LOG nanoparticles interact with AdMSCs are crucial to understanding this process39. One of the challenges in using a combination of nanoparticles + MSCs in an animal model is the fabrication of the 3D form. In this study, we were able to implant a construct of goat AdMSCs and LOG into a rat unicortical tibial bone defect using agarose as an inert scaffold. As observed in the histomorphometric data (Figure 6), the use of agarose did not interfere with the
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ACCEPTED MANUSCRIPT bone healing potential of either the MSCs or the LOG. It is possible that agarose alone inhibited the migration of osteoblasts in healing the defect, whereas the presence of MSCs or
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MSCs+nanoparticles with agarose showed significant healing. The varying effects of agarose
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need to be determined. In future studies or clinical applications, 3D forms of LOG nanoparticles with an optimal scaffold should be considered.
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This report also represents an excellent initial step towards using characterized MSCs as
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universal donor cells. MSCs are known to exhibit low immunogenicity, including immunomodulatory protective and repair effects. Of all sources of MSCs, AdMSCs are
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abundant, readily available, ethically acceptable, easy to obtain, and most importantly, have low MHC-I expression and lack MHC-II molecules, suggesting that they are not likely to induce
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rejection40. Therefore, it was not surprising that local or systemic rejection reaction was not
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detected following implantation and during the course of the study. No animal displayed any signs of inflammation in the area of the surgical wound. These data are in agreement with similar
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published reports and strengthen the use of xenogenic MSCs in rodent models41, 42.
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Multiple studies, including data from our laboratory, have shown that various forms of graphene can promote the in vitro proliferation and osteogenic differentiation of MSCs. Osteogenic differentiation is enhanced spontaneously as well as in the presence of chemical inducers26, 38,43-45. Supported by results from our laboratory and published by others, we suggest that LOG nanoparticles can guide bone tissue engineering by creating a microenvironment in which MSCs adhere and undergo osteogenesis. Specifically, in this study, by introducing oxygen groups in pristine graphene, we created a biological environment for MSCs to stay viable and undergo osteogenesis in vitro and in vivo. These nanomaterial-derived signals remain unknown, so the exact effects of graphene on cells, tissues, or organs require further studies. More research
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ACCEPTED MANUSCRIPT Figure legends Figure 1. XPS and SEM analyses: (a) x-ray photoelectron spectrum and elemental composition
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Figure 2. Assessment of cell viability in presence of LOG nanoparticles by (a) MTS assay, the data is expressed as the mean ±SD based on at least triplicate observations and (b) live-dead
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fluorescent staining using calcein-AM and propidium iodide. (c) Cell viability was also assessed
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in the presence of the 3D scaffold using live-dead staining. AdMSCs were seeded on the 2D graphene surface over a period of 10 days and in the agarose scaffold in the presence of 3D
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graphene for a period of 20 days. Scale bar = 100µm.
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Figure 3. Tri-lineage differentiation of AdMSCs was evaluated in the absence (a, b & c) and presence of 2D LOG nanoparticles (d, e & f). Representative Images showing alizarin red (a&d),
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alcian blue (b&e), and oil-red-o (c&f) staining of osteocytes, chondrocytes, and adipocytes,
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detected at day 21 post-differentiation. Scale bar = 100µm. Figure 4. Representative images showing the expression of CD44 in the presence of 3D LOG nanoparticles. The panels on the left show the agarose scaffold alone (a), LOG in the scaffold (c), and the scaffold of agarose with AdMSCs + LOG (e). Corresponding images viewed through the fluorescent microscope are shown (b, d, f). Isotype control for CD44 was used as the control. The inset in f shows the presence of cells stained with DAPI. Scale bar = 100µm. Figure 5. Computed tomography. (A) CT images of the tibias depicting the ROI were taken at T0 and T45. Representative images showing LOG nanoparticles alone (a), AdMSCs alone (b), and AdMSCs + LOG (c) are shown. The right limbs (indicated by R) were used as control, and the 24
ACCEPTED MANUSCRIPT contralateral limb was the treated limb. The arrows indicate the ROI in the treated limbs. (B) Quantitative analysis of the CT images. The ROI and, thus, the % healing was quantitated as
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which was calculated relative to the defect area at T0. The data is expressed as the mean ±SD based on at least two observations from four independent images. Different letters denote a
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significant difference, p < 0.05.
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Figure 6. Representative images of histological sections from each group are shown. Von Kossa
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staining at 2x magnification at T45 (a), H&E staining at 2x magnification at T45 (b), and Calcein green fluorescent staining at 5x magnification on T21 (c) are depicted. Various treatment groups,
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control and the treated limbs, are labeled accordingly. Figure 7. Graphs representing (a) the percentage of the active bone labeled with calcein green in
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the treated limbs relative to the controls at T21 and (b) the percentage of new mineralized bone
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formed in the treated limbs relative to the controls at T45 are shown. The data is expressed as the mean ±SD based on at least duplicate observations from at least 4 independent specimens. The
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different letters denote a significant difference, p < 0.05.
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ACCEPTED MANUSCRIPT Graphical abstract A conclusive study on the cytotoxicity and bone regenerative capacity of low oxygen content
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graphene nanoparticles in vitro and in vivo in rat models. Here in we propose the LOG
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nanoparticles to potentiate and enhance the osteogenic differentiation capacity of the Adipose derived mesenchymal stem cells. Unicortical defect model in rats was used to study the regenerative effect in bone defect. The in vitro results were matching the in vivo results and
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supports the proposal of LOG nanoparticles potentiating effect.
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