In vitro and in vivo biocompatibility of orthopedic bone plate nano-coated with vancomycin loaded niosomes

Accepted Manuscript In vitro and in vivo biocompatibility of orthopedic bone plate nano-coated with vancomycin loaded niosomes Anupma Dwivedi, Anisha Mazumder, Norased Nasongkla PII:

S1773-2247(19)30366-1

DOI:

https://doi.org/10.1016/j.jddst.2019.04.018

Reference:

JDDST 1024

To appear in:

Journal of Drug Delivery Science and Technology

Received Date: 12 March 2019 Revised Date:

10 April 2019

Accepted Date: 14 April 2019

Please cite this article as: A. Dwivedi, A. Mazumder, N. Nasongkla, In vitro and in vivo biocompatibility of orthopedic bone plate nano-coated with vancomycin loaded niosomes, Journal of Drug Delivery Science and Technology (2019), doi: https://doi.org/10.1016/j.jddst.2019.04.018. 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.

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ACCEPTED MANUSCRIPT

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In vitro and in vivo biocompatibility of orthopedic bone plate nano-coated with vancomycin

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loaded niosomes

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Anupma Dwivedi, Anisha Mazumder, Norased Nasongkla*

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Department of Biomedical Engineering, Faculty of Engineering, Mahidol University, Nakorn Pathom, 73170

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Thailand.

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*

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Abstract

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The focus of current study was to evaluate the in vitro and in vivo biocompatibility of nano-coated

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orthopedic bone plate. In our previous work this nano-coating was designed, characterized and its

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antibacterial properties and cell toxicity were assessed. This new strategy of nano-coating designed with

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antibacterial niosomes, were coated on orthopedic bone plate via layer-by-layer deposition. Therefore in

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the present study it was of paramount importance to measure its biosafety before it can be further

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evaluated for clinical studies. Fibroblast morphology, proliferation and cell adhesion properties were

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examined. The results demonstrated that coated bone plate exhibited no adverse effects and enhanced cell

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adhesion and proliferation than the uncoated bone plate. The extracts from the coated bone plate were

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tested against human blood cells and demonstrated excellent hemocompatibility. In vivo skin toxicities

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such as skin sensitization and irritation were investigated on guinea pigs and rabbits, which revealed no

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toxicity or any signs of inflammation and suggested its superior biocompatibility. The data gathered from

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these studies are promising and proposed that these antibacterial nano-coatings are non-toxic and has

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potential to utilize in various implant coating applications.

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Corresponding author, e-mail: [email protected]

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Keywords: niosome, biocompatibility, cell adhesion, skin irritation, skin sensitization, nano-coating.

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Declarations of interest: none

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1. Introduction

Biocompatibility is an impediment in joint replacements, stabilizing bone fracture and defect fixation due

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to the toxicity of components from the metal implant and failure or loosening of the implanted devices

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over period of time. Both prevention of infection and integration of the implant with the host-tissue are

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challenging during the design of coating strategy on implant. Biocompatibility is the pivotal characteristic

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that an orthopedic implant should possess. Ideally, it must not inimically affect the local and systemic

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host milieu of interaction and also it is achieved when utility of the implant is accomplished without

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eliciting a foreign body reaction within the tissues1. Metal implants represent an important class for their

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excellent mechanical and biomedical properties. Stainless steel (316 L) has reported to be an ideal

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material for metal implant and has been used for trauma surgery2. These implants are featured by

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improved mechanical properties like; ductility, elasticity and stiffness, they can be easily produced and

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are cost effective3.

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Osseointegration portrays the direct anchorage and incorporation of an implant within the bone4,5. In order

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to acquire an optimal osseointegration, the material properties of metallic implants are of eminence,

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wherein; both biocompatibility and mechanical tolerance are very significant. There is a phenomenon

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known as “the race for the surface” suggests that, there is a competition between bacterial colonization

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and tissue integration following implantation of an orthopedic device to inhabit the surface of the implant6

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and therefore biocompatibility testing is of equal precedence for implant integration into the tissues. An

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orthopedic implant with surface treatment or anti-infective coating provides an ideal substrate for bone

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aposition which facilitates improved implant fixation and prolonged life span7.Polymers have been

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exploited by the layer-by-layer (LbL) assembly procedure to generate controlled release systems with

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biomedical devices8. However in a fairly new approach, nanoparticles have also been utilized for coating

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technology. Previously, our research group has endeavored coating of nanoparticles on medical

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devices9,10,11,1213. Giglio et al designed coating system utilizing antibiotic loaded chitosan nanoparticles

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for titanium implants for antibacterial and biocompatibility studies14.

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ACCEPTED MANUSCRIPT 3 To eliminate all potential hazards to the patients, it is indispensable that biocompatibility must be

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conducted for medical devices that have tissue contact and also evaluate safety assessment of a device or

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an implant and is driven and steered by ISO 10993 standards15,16. The cutaneous reaction of biomaterials

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can be categorized into; primary irritation (irritation dermatitis) and sensitization of the skin (allergic

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dermatitis)17. Guinea pig maximization method was introduced by Magnusson and Kligman that exploits

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Freund’s Complete Adjuvant18 and this method determines delayed contact hypersensitivity. This assay is

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known to be the most sensitive procedure for detecting the ability of a substance to create contact

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hypersensitivity in guinea pigs19,20. Modified draize rabbit skin irritation test in 1980’s and 1990’s was

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incorporated in the Tripartite Agreement test matrix and ISO 10993 standards for anticipating the skin

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irritation ability of medical devices21,22. Skin irritancy testing along with cytotoxicity and sensitization

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now recommended for all medical devices in biological risk assessments23. Animal irritation testing is

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deployed to investigate whether a patient-contacting medical device could trigger an irritation response as

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spotted by erythema and edema. This is achieved in conformity with the ISO 10993-10 and ISO 10993-12

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standards24. Rabbit intracutaneous irritation test is the most renowned skin irritation test for medical

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devices or implants25.Some of the biocompatibility studies have been performed in the past, e.g.,

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vancomycin-poly(D,L)-lactic acid (PDLLA) loaded titanium plates for medical implants were

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investigated by Tang et al26and concluded with no toxicity and exerting good biocompatibility. Surgical

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sutures biocompatibility was examined to evaluate local irritant effects and was observed to be non-

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irritant to the skin of rabbits27.

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Niosomes are nonionic surfactant vesicles that can either be unilamellar, bilamellar or multilamellar that is

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formed from the self-assembly of amphiphiles28,29. Niosomes have been proved to enhance the drug efficacy

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and are non toxic to the cell30,31. Niosomes biocompatibility was investigated by Kumbhar et al and it did

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not exhibit any irritation when applied to the skin and there were no signs of erythema and edema32.

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Creatinine based niosomes loaded with clarithromycin were designed and their safety assessments were

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conduced and found to be non toxic and biocompatible33.

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ACCEPTED MANUSCRIPT 4 Formerly, we have fabricated antibacterial nano-coating deploying layer by layer coating technique with

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alternative layers of antibiotic (vancomycin), polymer (PLA) and antibiotic-loaded niosomes on

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orthopedic bone plate, which has been evidently proved to be superior to the uncoated bone plate. This

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designed antibacterial nano-coating has prolonged the vancomycin release and has significantly

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suppressed the growth of S.aureus13.This kind of nano-coating was not explored in the past and has

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potential in combating implant associated infection. Hence, these finding lead us to further scrutinize the

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biosafety and risk assessment of these coated bone plate before it can be translated into clinical studies.

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Thus, in the current study, in vitro and in vivo biocompatibility and adverse toxic effects of nano-coated

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bone plate have been explored.

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2. Materials and method

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2.1 Materials

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Span 60 and cholesterol were purchased from Tokyo Chemical Industry Co., Ltd, (Tokyo, Japan).

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Vancomycin was procured from Gold Biotechnology (St. Louis, USA) and Poly(lactic acid)(PLA) was

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procured from NatureWorks LLC (Minnetonka, USA). Hoescht 33342 was purchased from Thermo

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scientific (Massachusetts, USA). Solvents were purchased from RCI Labscan Ltd (Thailand). Bone plate

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(LC-LCP 4.5/5.0, narrow 6 holes) was procured from The Bangkok Unitrade Co. Ltd. Bangkok,

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Thailand. Cell culture supplies were purchased from Merck (Germany).

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2.1.1 Animals

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Guinea pigs and Rabbits were purchased from National Laboratory Animal Center, Thailand. The animals

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were kept under standard conditions 12:12 (light: dark cycles) at 22 ± 3 0C and 30-70% relative humidity.

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The animals were housed individually in stainless steel cages. All animals were allowed to acclimatize for

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at least 5 days prior to the study. Guidelines of “Guide for the care and use of laboratory animals”

ACCEPTED MANUSCRIPT 5 (Institute of laboratory animal resources, National academic press 2011; NIH publication number #85-23,

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revised 2011) were strictly followed throughout the study. The study was approved by National

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Laboratory Animal Center Animal Care and Use Committee (NLAC-ACUC), Mahidol University,

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Thailand, and approval number for sensitization study is GLP2018-23 and for irritation study is

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GLP2018-26.

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2.2 Methods

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2.2.1 Niosome formation and coating onto the orthopedic implant

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Niosomes were formulated by thin film hydration method as described previosly31. Briefly, span 60 and

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cholesterol in the ratio 3:1 were mixed in chloroform after which chloroform was allowed to evaporate.

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The obtained thin film was hydrated with DI water in which vancomycin was dissolved and probe

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sonicated to obtain niosomes loaded vancomycin. The entrapment efficiency was calculated to be 50.47±

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3.66%. These niosomes were then coated onto the original orthopedic implant made of stainless steel. The

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implant used in this study was bone plate with dimension 11.5 x 1.5 x 0.5 cm. The bone plates were

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dipped in three different solutions; vancomycin, PLA and vancomycin encapsulated in niosomes. Layer-

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by-layer nano-coating was deposited by automatic dipping machine with 40 cycles of continuous dipping.

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Detailed method of this nano-coating has been described previously12.

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2.2.2Cell adhesion and morphology

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Fibroblast (L929) cells were purchased from JCRB Cell bank, Osaka, Japan and cultured in Minimum

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Essential Media (MEM) medium supplemented with 10% FBS and 1% penicillin-streptomycin. These

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cells were seeded into 6 well plates with 5 x 104 cells per well and incubated with the coated and uncoated

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bone plates (BP). After 24 hr cells were observed and implants were removed, washed with PBS and then

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stained with hoechst (12 µg/ml) in the dark for 30 min. Following staining, the implants were rinsed with

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PBS and allowed to dry in the dark for visualization under fluorescent microscope (Eclipse Ti-S Nikon,

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ACCEPTED MANUSCRIPT 6 Japan). The L929 cells in the wells were also stained with crystal violet in order to assess the cell

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morphology and viability34. To accomplish this, the cells were washed with PBS and stained with crystal

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violet (0.5 %) for 20 min at room temperature after which the cells were again washed with PBS three

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times to remove unbound stain and were visualized under inverted light microscope. After visual analysis,

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methanol was added to the well plate and incubated for 20 min subsequently optical density was

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measured at 570 nm with plate reader (Lab Systems Multiskan RC, USA).

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For SEM analysis, the coated bone plates were cultured with L929 cells at the concentration of 30,000

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cell/cm2 and after 48 h, the implants were washed with PBS. Cells on the bone plates were fixed with

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2.5% glutaraldehyde (in phosphate buffer) for 2 h and then these colonized implants were dehydrated

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with increasing concentrations of ethanol (up to 100%) thereafter, theses bone plates were coated with

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gold for observation under SEM (JEOL, JSM-IT-500HR).

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2.2.3 Hemocompatibility

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To evaluate hemocompatibility, hemolytic test was conducted according to ISO 10993-4. Briefly, blood

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was collected from three healthy human subjects. The blood was then diluted to a concentration of 10

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mg/ml with PBS. The samples, i.e., coated and uncoated bone plates, were extracted by soaking the bone

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plates in PBS for 24 h at 37 °C shaker. The blood (1 ml) was then incubated with extracted sample (7 ml)

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at 37 °C for 3 h while gently mixing the tubes after every 20 min. After incubation the tubes were

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centrifuged at 2700 rpm for 15 min and the color of supernatant and blood cell pellet was recorded. The

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supernatant of the samples were added with Drabkin’s reagent and after 10-15 min incubation at room

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temperature optical density was measured at 540 nm using spectrophotometer. The positive control used

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was water and negative control was PBS in this study.

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2.2.4

Skin sensitization

ACCEPTED MANUSCRIPT 7 This study was conducted according to the International Organization for Standardization 10993–

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1035.Thirty Dunkin Hartley albino male guinea pigs of body weight ranging from 300-500 g were selected

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for sensitization studies. The coated bone plates were extracted in polar (0.9% sodium chloride) and non-

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polar (propylene glycol) solvent. The extracted samples were used to perform the test procedure. Guinea

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pigs were randomly selected into 4 groups, two test groups, i.e., animals treated with samples extracted in

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polar and non-polar solvent and their respective controls. Guinea pigs maximization test consist majorly

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of three phases, i.e., intradermal induction phase; the animals in test group and control group were

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injected with 0.1 ml of polar and non polar extract of the sample in the clipped area for 7 days. Topical

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induction phase; after 7th day all test and control animals were patched with test item by topical

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application. Challenge phase; all test and control animals were topically exposed with the same

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concentration to sites that were not treated during the induction phase. By the end of challenge phase, skin

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was observed after 24 h and 48 h for any skin reactions. The scores were recorded in accordance with the

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magnusson and kligman grading scale. All animals (test groups and control groups) were observed daily

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for general health and their body weights were recorded weekly. The diagram below depicts the

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intradermal injection test site in guinea pigs.

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Figure 1. Location of intradermal injection sites

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1. Cranial end

A. Freunds complete adjuvant (FCA) + NaCl(0.9%) / propylene glycol

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0.1 ml intradermal injections

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3. Clipped intrascapular region

B. test extract + NaCl (0.9%) / propylene glycol C. FCA + extracts of NaCl (0.9%) / propylene glycol

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4. Caudal end

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2.2.5

Skin irritation

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This study was conducted according to the International Organization for Standardization 10993-

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1035.Three healthy young male New Zealand White rabbits of body weight in range 2,030-2,242g were

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obtained for skin irritation study. The test item (coated bone plate) was extracted in physiological saline

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(0.9% sodium chloride) and propylene glycol in a water bath at 37ºC for 72 hours. On the day of

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exposure, the clipped area was divided into 4 parts (two test parts and two control parts) on each animal.

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The test extracts and control samples were intracutaneously injected to the test sites and control sites,

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respectively. Approximately, 0.2 ml of each test extracts, i.e., physiological saline and propylene glycol

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extracts, were injected aseptically into five sites of two test parts. Similarly, both the control samples were

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also injected into five sites of two different parts of the same rabbit. Skin reaction (erythema and edema),

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at the site of injection was assessed and scored at 24, 48 and 72 hour after intracutaneous injection of the

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extracted samples. The diagram below depicts the location of sample application on rabbit’s skin site.

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Figure 2. Location of skin application site 1. Cranial end

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2. Test site (coated bone plate extract with 0.9% sodium chloride)

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3. Test site (coated bone plate extract with propylene glycol)

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4. Control site (0.9% sodium chloride)

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5. Control site (propylene glycol)

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2.2.6

Histology

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Guinea pigs and rabbit’s skin tissues were surgically excised after terminating the animals. These skin

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tissues were placed in 10% neutral buffered formalin. After which the skin tissues were washed several

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times with ethanol and xylene until the tissues were dehydrated and observed to be clear. Then, these skin

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tissues were embedded in paraffin wax using standard histological techniques. Tissue embedded wax

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sections (4–5 µm) were sliced by microtome and spread on glass slides. These slides were marked and

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stained using a standard haematoxylin–eosin (H&E) staining protocol. The images were captured under

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bright-field inverted microscope (Eclipse Ti-S Nikon, Japan).

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2.2.7

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Experiments were performed in triplicate and data was analyzed by GraphPad Prism™ software (version

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5.01, USA). Statistical significance was determined with one-way ANOVA. Data is presented as mean ±

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standard deviation (SD), and significance was set at P ≤ 0.05.

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Results and discussion

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Statistical analysis

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3.1 Cell proliferation and morphology with the coated bone plate

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The designed nano-coating was characterized before and the total drug loading (vancomycin) was

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calculated to be 6.93 mg. The coating was homogenous as analyzed with contact angle analysis and the

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thickness was 0.45µm on an average12. For improved orthopedic implant integration, cellular attachment

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is the first step in the process of cell surface interaction. Before examining cell adhesion, it was

ACCEPTED MANUSCRIPT 10 prerequisite to study the cell morphology and proliferation with the coated and uncoated bone plate and to

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assess the milieu surrounding the bone plate. Cell viability and changes in cell morphology were analyzed

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via phase contrast and electron microscope. In fig.3 A and B, L929 cells were cultured for 24 h and 48 h

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with the coated and uncoated bone plate. After 24 h, the cells cultured with coated and uncoated bone

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plate demonstrated normal and healthy morphology whereas after 48 h, cells burgeoned and exhibited

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high confluency similar to the untreated control group. It was noteworthy that after 48 h of culturing the

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cells with the coated bone plate spread rapidly and confluency of the cell population was increased than

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the uncoated bone plate population. In fig. 3 Ab and Bb (coated and uncoated) the dark area represents

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bone plate and it is evident that cells around the coated and uncoated bone plates were healthy which also

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suggests that the milieu around the bone plate was not toxic to the cells growing in that region and hence

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the morphology of the cells in the petri plate was normal and healthy.

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After 48 h, coated and uncoated bone plates were removed and cells were washed with PBS thereafter

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they were stained with crystal violet to assess the number of viable cells. From fig.4A, it has been noticed

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that the cell spreading and proliferation was higher with the coated bone plate than the uncoated one,

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suggesting that the coating is highly biocompatible and suitable for cell growth. Similarly, fig. 4B

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substantiates the data obtained in fig. 3A, B and fig. 4A. After qualitative analysis by visualizing the

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morphology of stained cells, these cells were quantitatively analyzed by determining its cell viability (fig.

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4B) which revealed that the nano-coated bone plate did not exert any toxic effect and supports cell

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growth, vital for implant integration.

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3.2 Cell adhesion and morphology on the coated and uncoated bone plate

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It is essential to understand and study the cell surface attachment to the coated bone plate in order to

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assess the biocompatibility or biosafety and may also assist in implant integration. Cell adhesion occurs

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rapidly and involves physiochemical linkages between the cells and the substrate (bone plate). Cellular

ACCEPTED MANUSCRIPT 11 attachment to the substrates at the contact sites are basically of three types, i.e., focal contacts, close

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contacts and extracellular contacts which generally depends on the distance between the cell and the

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substrate and participation of certain proteins36. Primarily the attachment of cells to the surface of a

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biomaterial is a fundamental requirement for implant integration and for further biological processes, such

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as cell survival, attachment, proliferation, differentiation, and migration37. In fig. 5A, coated and uncoated

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bone plate was allowed to culture with L929 cells and after 24 h these bone plates were washed few times

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and stained with Hoechst thereafter visualized under inverted fluorescent microscope. The image depicts

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that coated bone plate have higher cell adhesion than the uncoated bone plate. In fig. 5B and C, coated

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and uncoated bone plates were cultured for 48 h with L929 cells and visualized under SEM. In fig. 5B, it

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has been noted that cells attachment and proliferation was enhanced with coated bone plate than the

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uncoated one. Figure 5 C is the magnified image of fig. 5 B which demonstrates cells spreading with

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smoother cell surfaces. Long filopodia were noticed through which neighboring cells maintain physical

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contact with each other in image C. Cell adhesion is one of the parameters of biocompatibility where

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different surfaces were essentially tested for cell adhesion and usually if the cell attachment is minimized

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it could be considered as measure of toxicity or non-biocompatible surface. In this study, the coated bone

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plate was colonized with greater cell density and cells appeared to be healthy and proliferative than the

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uncoated bone plate. Hence, it was concluded that the nano-coating has better performance than the blank

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surface of stainless steel bone plate.

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3.3 Hemocompatibility

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In vitro hemolysis test was conducted in order to examine the toxicity of nano-coated bone plate towards

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blood cells. Since the orthopedic implant remains in direct contact with the blood therefore it was

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necessary to test the human blood compatibility. Blood was collected from healthy human subjects and

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hemolysis test was conducted according to the ISO10993-4 protocol38. From fig. 6A, data demonstrated

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that coated BP (coated bone plate) and control BP (uncoated bone plate) were non-hemolytic to human

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blood cells. Both test groups, i.e., coated and uncoated extract of bone plate demonstrated similar

ACCEPTED MANUSCRIPT 12 percentage of hemolysis as the negative control (all showed less than 4% hemolysis).The data from fig.

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6A corroborated fig. 6B where the positive control showed the lysis of red blood cells whereas both test

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groups and negative control displayed clear solution and the intact red blood cells was noticed at the

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bottom of the tubes. Figure 6 C reveals the hemolytic index of coated and uncoated bone plate, i.e., 0.59

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and 1.81, respectively and according to ASTM F756 standard grading scale these values are considered

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to be non-hemolytic grade. The results suggested that the coated bone plate was not hemolytic to the

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human blood further confirming the non toxic nature of the coating. These results serve as a preliminary

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indication of the biocompatibility of the coated bone plate.

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3.4 Skin sensitization

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Delayed hypersensitization study is usually conducted to evaluate the medical devices that may cause

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adverse local or systemic effect after repeated or prolonged exposure of the test item or implants. Guinea

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pigs have been the preferred animal for investigating sensitization test since past many years and their

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skin are known to be as responsive to dermal sensitizers as humans. In this prolonged hypersensitivity test

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for the period of 21 days, there were no harmful effect noted to the test group compared to the control

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group. There were 10 guinea pigs in treatment groups and 5 guinea pigs in control groups and all were

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noticed to be healthy and there was no sign of skin sensitization during the entire study period. All guinea

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pigs demonstrated normal behavior and their treated skin showed no sign of erythema or edema. They

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were healthy and their body weight (fig. 7) was normal during the study. From fig. 7A-B, the body weight

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of guinea pigs increased during the different phases of study and remained under healthy weight range.

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For sensitization score, guinea pigs were treated with coated bone plates extract in 0.9% NaCl and in

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propylene glycol, demonstrated no visible changes in the skin following treatment for 24 h and 48 h of

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both induction phase and challenge phase. It was noticed that after 24 h and 48 h of intracutaneous

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induction, topical induction and challenge phase the sensitization score remained zero in all treatment and

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control groups. These test samples did not elicit any signs of delayed hypersensitization (redness and

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ACCEPTED MANUSCRIPT 13 swelling) in guinea pigs compared to the control group. Hence, suggesting the biosafety and

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biocompatibility of the coated bone plate. In a previous study, vancomycin-poly (D,L-lactic acid)

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(PDLLA) coated titanium plates were evaluated for hemolysis and skin sensitization on guinea pigs and

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data revealed that coated plate was non-hemolytic and did not elicited any skin reaction. The data in this

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study is in accordance with our study26.

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3.5 Skin irritation

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In order to inspect the biocompatibility of this anti-infective nano-coated bone plate, it was necessary to

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test this coating for skin irritation before any human exposure. Skin irritation assessment is required for

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any comprehensive biosafety and biocompatibility test. For this study, New Zealand albino rabbits were

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employed for skin irritation evaluation. The most preferable animal model is albino rabbits due to its

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highly sensitive, light skin which makes it easier to identify slight skin irritation caused by exposure of

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any material39. Body weight of all animals were recorded in every phase of the study and presented in fig.

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8. It has been noticed that the weight of animals were almost consistent throughout the study and the

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rabbits were healthy and their behavior was normal. For skin irritation score, after the intracutaneous

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injection of coated bone plates extracted in 0.9% NaCl and propylene glycol, rabbit’s skin did not show

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any cutaneous reaction after 24, 48 and 72 h of administration, which was similar to the control group

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(negative control). Each rabbit had five test sites and five control sites which revealed that the Primary

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Irritation Index (PII) of zero (0.0) for all rabbits. Hence, it can be concluded that there were no signs of

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erythema and edema at any point of the study. Kojic et.al studied the irritant properties of calcium

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phosphate ceramic, α-tricalcium phosphate (α-TCP) which has been used in orthopedic, reconstructive

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and maxillofacial surgery. The data revealed that there was negligible irritability39. In another study

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biodegradable poly (D,L-lactic acid) (PDLLA) film was constructed and its skin irritation potential was

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tested on rabbits skin. Their data suggested that there was no skin reaction elicited by these films40.The

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skin irritation data from our study was found to be congruent with the above studies. Thus, the nano-

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coated bone plate has higher degree of biocompatibility hence, the data indicates about the potential of the

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coated bone plate. Therefore, the coated bone plate is safe to use in humans.

318 3.6. Histological analysis

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Histological analysis was essential to microscopically observe the skin tissues for any severe signs of

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inflammation, damage skin architecture or necrosis. The histological examination of the hematoxylin and

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eosin (H&E) sections of skin revealed normal architecture of skin cells. These skin sections were excised

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from the control and treatment groups of animals in sensitization and irritation studies. Results showed

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that cells were normal and healthy and there were no signs of allergic reaction compared to the control

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group (negative control). In fig. 9A, B, C and D is the guinea pig skin section of sensitization study

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whereas E, F, G and H is the rabbit skin section of irritation study. Data demonstrated no sign of severe

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inflammation as no infiltration of granulocytes and lymphocytes or necrosis was noticed in the dermal

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stroma of treatment group in comparison to their control group. Hence, the findings from histological

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analysis indicated no severe dermal toxicity and suggested the biosafety of these coated bone plate.

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4. Conclusion

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The overall data suggested that the nano-coated bone plate exhibited no toxicity to human blood cells and

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higher cell adhesion was noticed with the coated bone plate than the uncoated one. This confirms its non-

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toxic and osteointegrative properties. The cell morphology was normal and healthy and no signs of

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toxicity were observed. Also, there were no indications of dermal toxicity and hence confirming its

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biosafety. From the in vitro and in vivo biocompatibility results, the designed nano-coated bone plate is

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superior to the uncoated bone plate and extremely biocompatible. Thus, our findings support the

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nanocoating strategy which can be tested further for implantation and clinical studies. This antibacterial

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nano-coating technique could be applied to various implants that are prone to infection.

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Acknowledgment

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This research was conducted under the postdoctoral research sponsorship of Mahidol University,

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Thailand.

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Conflict of interest

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The authors have no conflict of interest to declare.

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Figure Captions Figure 1. Location of intradermal injection sites

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Figure 2. Location of skin application site

Figure 3. L929 cell morphology and proliferation cultured with the coated and uncoated bone plate and visualized under inverted light microscope. A after 24 h of culture and B after 48 h of culture.The dark

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area in Ab and Bb represents coated and uncoated bone plates and the images Aa and Ba are the areas

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away from coated and uncoated bone plate. The scale bar is 50 µm.

Figure 4. L929 morphology and cell viability, cultured for 48 h. A display the images obtained after stained with crystal violet and B is the percentage cell viability calculated after culturing with coated and

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uncoated bone plate. The scale bar is 50 µm.

Figure 5. Cell adhesion (L929) micrographs A after 24 h coated and uncoated bone plate with cells, stained with Hoechst and images were captured under fluorescent microscope. Scale bar is 25 µm. B and C are SEM micrographs after 48 h of cell attachment where C (x1000, scale bar 10 µm) is the magnified

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image of the B (x150, scale bar 100 µm).

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Bone Plate Figure 6. Percent hemolysis of A) the coated and uncoated bone plate, B) the hemolysis of different samples and C) the hemolysis index.

Figure 7. Body weight of guinea pigs treated with A) 0.9% NaCl extract and B) propylene glycol extract during different phases of sensitization studies (wt. is weight, ind. is induction and gr. is group).

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Figure 8. Body weight of rabbits treated with 0.9% NaCl and propylene glycol extract of coated bone plate during different phases in skin irritation study.

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Figure 9. Skin section of sensitization study stained with H&E, A and C is the guinea pig skin of control group treated with only 0.9% NaCl and propylene glycol, respectively. B and D is the guinea pig skin of test group treated with 0.9% NaCl extract and propylene glycol extract of coated bone plate. E and G is

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the rabbit skin for irritation test of control group treated with 0.9% NaCl and propylene glycol, respectively whereas F and H is the test group treated with 0.9% NaCl and propylene glycol extract of

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coated bone plate. Scale bar is 20 µm.

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