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Accepted Manuscript
Fabrication of poly (methyl methacrylate)/Ce/Cu substituted
apatite/Egg white (Ovalbumin) biocomposite owning adjustable
properties: Towards bone tissue rejuvenation
R. Sangeetha, D. Madheswari, G. Priya
PII:
DOI:
Reference:
S1011-1344(18)30631-6
doi:10.1016/j.jphotobiol.2018.08.015
JPB 11333
To appear in:
Journal of Photochemistry & Photobiology, B: Biology
Received date:
Revised date:
Accepted date:
12 June 2018
10 August 2018
13 August 2018
Please cite this article as: R. Sangeetha, D. Madheswari, G. Priya , Fabrication of poly
(methyl methacrylate)/Ce/Cu substituted apatite/Egg white (Ovalbumin) biocomposite
owning adjustable properties: Towards bone tissue rejuvenation. Jpb (2018), doi:10.1016/
j.jphotobiol.2018.08.015
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ACCEPTED MANUSCRIPT
Fabrication of poly (methyl methacrylate)/ Ce/Cu substituted apatite/Egg white
(Ovalbumin) biocomposite owning adjustable properties: towards bone tissue
rejuvenation†
a, b
,
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R. Sangeetha a, b, D. Madheswari ‫٭‬a , G. Priya
Department of Chemistry, Shri Sakthikailassh Women’s College, Salem, India
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b
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‫٭‬a Department of Chemistry, Govt. Arts College for Women, Salem, India
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ED
M
AN
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Email address: sangeetchem85@gmail.com
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*Author to correspondences
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Dr. D. Madheswari - Tel: +91 9600322055; Email: sangeetchem85@gmail.com
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Abstract
A new biocomposite including Poly (methyl methacrylate)/Ce-Cu substituted apatite/Egg
white (Ovalbumin) (PMMA/MHAP/EW) has been effectively manufactured via lyophilization
technique. The reason to plan such biocomposite is to investigate for a perfect simple which may
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bio-imitate the constitution of natural bone for hard tissue designing regarding appropriate
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biodegradability, biocompatibility, and bactericidal properties. The FTIR and XRD spectra of
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prepared biocomposite showed the considerable intermolecular interface between the different
constituents of the biocomposites. The examination of SEM pictures of the biocomposites
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demonstrated that the addition of EW affected the exterior morphology while a superior in-vitro
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biocompatibility has been seen in PMMA/MHAP/EW than in PMMA/MHAP in view of ALP,
hemocompatibility and mineralized extracellular matrix studies, referring a more superior
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probability for assembly straight attach to living hard tissues if embedded. Additionally,
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cytocompatibility assay exposed the superior non-lethal character of prepared biocomposites to
MG-63 cells when contrasted with PMMA/MHAP. The relative biodegradation studies of
biocomposites
exposed
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prepared
a
superior
degradation
rate
for
Comp-3.
Besides,
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PMMA/MHAP/EW showed enhanced antibacterial property against both E. Coli as well as S.
aureus microbes with respect to PMMA/MHAP. These findings have set PMMA/MHAP/EW
designing.
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biocomposites on the platform to be utilized as a prospective option biomaterial for hard tissue
Keywords: Biocomposite; Biodegradation; Bactericidal; Hydroxyapatite; PMMA; Egg white
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1. Introduction
Vast bone deformities induced by injury, innate abnormality, or tumor resection have for
quite some time been an exceptionally difficult medical issue [1]. They are normally described
by short recovery potential and subsequently will require more particular surgical administration.
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Bone destruction, as well as blood deficiency, is two major negative aspects of the cartilage
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reconstruction development [2, 3]. Bone destruction can be handled by embedding bone
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implants, comprising extensive segmental synthetic cartilage implants, allografts, and autografts.
However, the obstacle in the donor section, additional operation, infection spread and
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consumption create a demand to strengthen substitutes for synthetic cartilage implants, allografts
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and autografts [4-6].
Among the numerous engineered bone grafts fabrics, PMMA and its derivatives have
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been utilized effectively in orthopedic and orthodontic surgical treatment [7]. PMMA bone grafts
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fit in with the state of its environment, permits even dissemination of embed loads, and structures
a solid mechanical connection with inserts [8, 9]. Nevertheless, its extensive utilize is restricted
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by numerous difficulties. For instance, bioinert, weaker than spine, arms and legs bones, severe
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exothermic polymerization reaction, monomers toxicity and frailty [10, 11]. Even though bone
joint restorations have been around 90% effective in the course of the most recent 10 years,
[12].
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disappointments have happened, frequently because of aseptic slackening also cellular death
In such a manner, the inclusion of additive substance biomaterials into the PMMA matrix
has been perceived as a successful strategy to beat these inconveniences [13]. Among different
sorts of additives substances, apatite has been utilized as a part of various orthopedics and
orthodontic applications including bone concrete for craniofacial imperfection repair and
coatings for femoral segments of hip substitutions because of its solid mechanical properties and
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osteoinduction [14]. The biochemical characteristics of fabricated apatite can be enhanced by
substituting different cations and anions components into apatite crystal phase [15]. Among these
minerals, Cu is an inexpensive and plentiful resource on earth and is outstanding for its solid
expansive range antimicrobial activity. Cu is additionally a vital trace component in the
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mammalians body and improves osseous cells development [16]. Moreover, it was discovered
could
enhance/fortify
metabolism [17].
Conversely,
Ce particles likewise have
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apatite
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that Ce can act comparatively to Ca in the human bones and teeth. As per these outcomes, Ce-
antibacterial movement and exceptional osseous tissue regenerative properties [18]. Besides, late
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investigations demonstrated that the utilization of Ce in orthodontics may help anticipate
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cavities. These biochemical natures make Ce reasonable for applications in the clinical field.
How to exert the biodegradability and bioactivity of the parallel platform comprised of non-
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degradable polymer and bioceramic still remains a test. Though, binary constituent composites
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can't help and bioimitate every one of the properties of bone and consequently creating
multicomponent composites as an option for bone repair becomes mandatory.
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Lately, some multi-component composites have also been produced with enhanced
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bioactivity and osseous cell propagation on the composite as contrasted to binary composite [1921]. As a piece of our interest to improve the clinical characteristics of PMMA/HA composites,
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it was thought focusing to fabricate PMMA/MHA/EW biocomposites at three different
concentration of EW (Ovalbumin) in PMMA/MHA biocomposite to developed multi-component
composites through lyophilization approach. To the best of our knowledge, no scientific
examination about the independent utilization of EW (Ovalbumin) based multi-component
biocomposite with PMMA and MHA for orthopedics rejuvenation applications have been
investigated to date.
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2. Experimental Section
2.1. Materials
The chemicals used were Ca(NO 3 )2.4H2 O (99%), Ce(NO 3 )3 ·6H2 O (99%), Cu(NO 3 )2 ·H2 O
(99%) and (NH4 )2 HPO 4 (98%). All chemical were purchased from Sigma Aldrich India,
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analytical grade.
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2.2. Isolation of EW Ovalbumin
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Duck eggs were obtained from the nearby market (Salem, Tamil Nadu, India) and
vigilantly wrecked to isolated the yolk from EW medium. The fraction of EW poured into 250
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mL beaker and 50 mL DD water poured into the EW solution and sonicated for 1 hrs at a
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frequency of 30-40 KHz. White residual composed of globulin proteins are separated via
centrifugation as well as the supernatant solution of ovalbumin is allowed to lyophilization for 12
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hrs.
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2.3. Fabrication of the MHAP
MHAP nanoparticles were fabricated via sonochemical technique. Minerals precursors
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solution, as well as the phosphate precursors solution, were measured at the molar ratio of M(Ca,
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Ce, Cu )/P=1.67. About 20 mL phosphate precursor solution was added to the 20 mL of minerals
precursor solution slowly while it was being sonicated 2 hrs (at 30-40 KHz) to get a
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homogeneous reaction. The resulting solution was centrifuged at 2000 rpm for 30 min.
Subsequent to centrifugation; the precipitate was acquired and sintered for 1 hrs in the
temperature at 800°C.
2.4. Fabrication of the Scaffolds
Biocomposites were fabricated via blending of PMMA (Sigma Aldrich, India), MHAP
and EW in ethanol. The PMMA/MHAP/EW biocomposite (Comp-1) provided as a model, and
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its process was expressed as follows: 2 g of prepared MHAP nanoparticles were added into 20
mL of ethanol (Sigma Aldrich, India). The suspension was sonicated for 30 min. Afterward, 7 g
of PMMA particles and 1 g of EW (ovalbumin) were suspended suitably in 75 mL of ethanol
through sonication.
The prepared MHAP solution, as well as the PMMA/EW solution, were
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then mixed and sonicated for 2 hrs. Subsequently, the biocomposite solutions were placed in a
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freezer at −60 ◦ C. Frozen mixtures were lyophilized at −40 ◦ C and 5 Pa for 2 days. The
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biocomposite with diverse PMMA/MHA/EW weight fractions were fabricated as expressed
above. The PMMA/ MHAP/EW biocomposites were labeled as Comp-O, Comp-1, Comp-2 and
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Comp-3 biocomposites for the 8:2, 7:2:1, 5:2:3, and 3:2:5 ratio of
2.5. Characterization
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2.5.1. Physicochemical characterization
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correspondingly.
PMMA: MHAP: EW,
biocomposites were investigated
via FT-IR
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Functional groups of the prepared
spectroscopy (PerkinElmer RX1). Crystalline Phase of the prepared biocomposites was verified
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via XRD (Rigaku MiniFlex II powder). The morphologies and elemental compositions of
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prepared biocomposites were characterized using a scanning electron microscope with energy
dispersive X-ray spectroscopy (SEM-EDX, JSMa JEOL-6390). Mechanical characteristics of
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prepared biocomposites were assessed by a universal testing machine (UTM).
The bio-decomposable performances of the prepared biocomposites were observed in
phosphate-buffered saline (Sigma Aldrich, India) medium at RT. Biocomposites for each group
were measured as X0 , followed by submerged in phosphate-buffered saline at RT for 24, 72, 168
and 336 hours. Subsequent to the conclusion of each hatching time, the biocomposites were
vigilantly removed from the solution, washed with DD water for an elimination of ions sorption
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on biocomposites exterior, dehydrated at RT for one day to steady mass, and weighed as X1 . The
Mass loss (%) was verified via the formula:
% =(X0 -X1 )/X0 ×100%, where X0 and Xt are the dehydrated weight of the biocomposites
before as well as after submerged, correspondingly.
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2.5.2. Biological characterization
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To verify the protein sorption performance, the prepared biocomposites were pre-rinsed
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with phosphate-buffered saline, dried as well as hatched with 500 μL of fetal bovine serum at RT
for 120 min. The biocomposite was washed softly with phosphate-buffered saline to eliminate
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free proteins following incubation subsequent the process accounted earlier [22].
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In- vitro hemolysis examination was achieved as a beginning toxicity investigation
evaluated via calculated the hemoglobin discharged as a consequence of membrane effusion. The
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membrane interruption is caused by experience with a little dosage of the biocomposites as
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accounted in the article [23].
Minimum inhibitory concentration (MIC) is the most reduced
grouping of an
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antimicrobial aggravate that will hinder the noticeable development of microorganisms after a
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determined day and period. Minimum inhibitory concentrations are imperative in order to study
the obstruction of microorganisms against antimicrobial specialists and furthermore in the
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screening of action of new antimicrobial operators. For minimum inhibitory concentration
estimation, micro-dilution technique was done on two basic pathogenic strains to be specific E.
coli and S. aureus. The qualities were resolved on 96-well small scale weakening plates as per
the conventions grew already [23, 24].
The MG-63 cell suspension with a volume of 4 × 104 was included into a 24-well plate.
The samples were cultured in α-MEM medium contain prepared biocomposites. Estimation of
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the ALP action was led by the technique depicted by the ALP recognition pack.
Biomineralization of the MG-63 cell was encouraged in a mineralization medium with prepared
biocomposites for 24, 72, 168 and 336 hours. Calcium metabolisms were distinguished by
alizarin strategy when dark murky zones showed up on the cells slides.
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The biocompatibility of as-fabricated was evaluated by culturing human osteoblast HOS
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MG63 cells obtained from National Centre for Cell Science (NCCS), Pune, India. The
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cytotoxicity of the as-fabricated bio-composite was evaluated in vitro using MTT test [25]. In
short, MTT store reagent in PBS was included to every well to attain a concluding volume of 0.5
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mg mL-1 . Further 4 h, excess MMT was detached and the absorbance of all well was then
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deliberate at 570 nm by a microplate reader. The qualified cell feasibility was calculated using
the subsequent equation:
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% of cell feasibility = absorbance of composite/absorbance of control X100
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2.5.3. Statistical Analysis
All experiments were completed in triplicate and the consequences were revealed as the
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mean ± standard deviation and were examined by utilizing one-way ANOVA.
3. Results and discussion
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3.1. Functional groups and phase analysis
In Fig. 1A, isolated-EW exhibited main bands at 3311 cm-1 , 2947 cm–1 , 1666 cm–1 , 1389
cm–1
and 1555 cm–1 referred to stretching vibrations of –OH, -CH, -C=O, -C–O- and -N–H,
correspondingly [26]. As-received PMMA exhibited the major bands at 2,995 cm−1 , 1,443 cm−1 ,
and 1,245 cm−1 individually due to the stretching vibration of–CH3 –, C–O–C [27]. Prepared
MHAP revealed a band at 3455 cm -1 , 480 cm-1 and 1101 cm-1 that is referred to bending mode
of -OH and PO 4 3-, correspondingly [28, 29]. Additionally, the FTIR spectra of prepared
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PMMA/MHAP/EW biocomposite (Comp-3) displayed the fundamental bands of EW, PMMA
showing that the MHAP were very much installed in the macromolecular lattice.
In order to significantly verify the existence of PMMA, EW, MHAP nanoparticles,
PMMA/MHAP/EW biocomposite (Comp-3) were identified via XRD (Fig. 1B). The diffraction
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range of as-isolated EW, as well as as-received PMMA, indicated wide peaks revealed to the
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amorphous character of macromolecules [30, 31]. Furthermore, the diffraction range of as-
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fabricated MHA nanoparticles indicated that 29°, 31°, 34 and 39° phase of apatite. In addition,
the diffraction of PMMA/MHAP/EW biocomposite indicated 18°, 20°, 22° and 28° were
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compared to PMMA [32], as well as MHAP (29°, 31°, 34° and 39°) [27, 33]. The outcomes
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demonstrate that PMMA/MHAP/EW biocomposite was effectively fabricated via intermolecular
hydrogen response.
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3.2. Morphological analysis
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As appeared in Fig. 2, the achieved prepared biocomposites (Comp-O to Comp-3)
showed excellent three-dimensional construction as well as uniform surfaces, suggesting that
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MHAP were completely enclosed in the PMMA as well as EW macromolecular network in view
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of their little sizes with great dispersion character (Fig. 2). Furthermore, their fine characterized
pores with the diameter varying from 50 to 250 μm could be seen through SEM pictures (Fig. 2
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a-d). Interestingly, the lyophilizer procedure made pores more regular because of the
arrangement of biocomposite [34], as well as the joining of EW into PMMA/MHA network
brought about the incensement of the pore volume which may attribute to the fortifying impact
of EW through the permeation arrangement held by hydrogen bonds. Furthermore, the porosity
of Comp-3 was 79.1% which was higher than Comp-2 (72.9%) and Comp-1 (67.3%), suggesting
that the great definite exterior field, mechanical reinforcement as well as barrier properties of
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EW could dramatically increase the porous network of the biocomposites. Moreover, the
composition of minerals verified via EDX spectrum (Fig. 2 e) validates the existence of Ce, Cu,
Mg, Ca, P and C in Comp-3.
3.3. Mechanical properties
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Mechanical experiments were attended to examine the compressive strength and
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elongations at different as-fabricated bio-composites (Fig. 3A). It could be observed that the
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Comp-O had the great capacity to prevent compressive damage with the greatest strength and
elongations. On the other hand, the Comp-3 had the most minimal strength as well as
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elongations. Furthermore, the strength, as well as elongations of Comp-1 and Comp-2, was
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intermediate between those of the Comp-O as well as Comp-3, which displayed a pattern of
decay with the expansion in EW substance. Additionally, it was fascinating to watch that the
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strength, as well as elongations of the biocomposites, varied moderately steady as EW substance
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expanded, whereas diminished drastically as EW substance additionally expanded. The strength,
as well as elongations of the Comp-2 biocomposites, was significantly superior to that of the
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Comp-3 biocomposite. Actually, PMMA had superb strength as well as elongations which were
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considerably superior to that of EW [35]. Subsequently, it was sensible to make a finale that
consolidation of EW into PMMA diminished the mechanical natures of biocomposites, and the
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mechanical character was predominantly overwhelmed by the consistent stage PMMA on
account of EW substance under 3 Wt% (Comp-2).
3.4. Bio-decomposable study
Perfectly, biocomposites should be bio-decomposable whereas fresh cartilage tissue is
developing, as well as have excellent bioactivity to stimulate the cartilage-connecting capacity
with osteoblast cell compatibility for cartilage reconstruction. It is notable that PMMA is a bio-
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static substance as well as consequently has restricted capacity to tie with characteristic cartilage
tissue [7]. Additions of biomaterials, (for example, bioglass and bioceramic, etcetera) are a
powerful technique for enhancing the biocompatibility and bioactivity [36]. Actually, PMMA
goes about as a layer in which biomaterials is totally enclosed subsequent to the
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liquefying/hardening method, in addition to PMMA nearly doesn't decomposable, thus the
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biocomposite can't show the bio-decomposable as well as bioactivity. Hence, in this current
MHAP (Fig. 3B).
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3.5. Protein adsorption and hemocompatibility assay
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examination, bio-decomposable EW was acquainted with applying the bio-compatibility of
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It is basic to assess the protein sorption evaluation on prepared biocomposites as the
tissues, as well as biomaterial association, starts with protein sorption [37]. As appeared in Fig.
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4A, in contrast with Comp-O to Comp-3 biocomposites, demonstrated altogether enhanced
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protein sorption. This might be comprehended by the prudence of improved porosity having
superior specificity for protein sorption with the expansion of EW in PMMA/MHA network
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supporting protein sorption. The Comp-3 biocomposite indicated relatively better protein
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sorption as compared to Comp-O, Comp-1, and Comp-2 in perspective of showing superior
exterior to volume proportion thus providing extra protein attaching sites reliable with its
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greatest porosity. Furthermore, the existence of EW expanded the protein sorption through
improved non-covalent interfaces among the three parts with the -NH2 functionality of the
proteins [38].
The
Hemolytic
capability of the prepared
biocomposites has additionally been
contemplated as appeared in Fig. 4B. The amount of hemolysis for all the biocomposites under
scrutiny were observed to be underneath the universal admissible level, demonstrating that these
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biocomposites don't generate the lethality against heme cells, along these lines justifying their
blood compatibility. The Comp-1 to Comp-3 biocomposites demonstrated lower hemolysis as far
as their great official as well as integrity nature between the MHAP and EW/PMMA
macromolecules
which
enhances
the
hemocompatibility
supporting
their
biomedical
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acknowledgment to be utilized as bio-implant material [39].
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3.6. Antimicrobial activity
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The prepared biocomposites were examined for its bactericidal action against gram
positive as well as negative microorganisms’ via the consommé dilution technique furthermore
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assessed on both microorganisms through agar diffusion process (Fig. 5). All the prepared
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biocomposites showed bactericidal action, however, the minimum inhibitory concentrations
significance for Comp-1 to Comp-3 on the above-mentioned microorganisms was greater than
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that of Comp-O. The bactericidal action of Comp-1 to Comp-3 was because of the attribute
dispersion
technique
with
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antimicrobial character of EW [40]. The consequences were additionally supported via agar
an
enlarged
area
of inhibition
detected
in
the
case
of
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PMMA/MHAP/EW biocomposite as revealed in Fig. 5B. These additional recommend the
tissue engineering.
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bactericidal character of the prepared biocomposites guaranteeing its functions in orthopedic
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3. 7. ALP activity and calcium deposition assay
Alkaline phosphate activity of MG-63 cells co-colonization with prepared biocomposites
was investigated at 24, 72 and 168 hours (Fig. 6A). Alkaline phosphate activities of prepared
biocomposites got enlarged with hours. Conversely, no huge distinction in alkaline phosphate
action has been detected for prepared biocomposites for 24 and 72 hours. Whereas by and large,
a higher alkaline phosphate action was detected for prepared biocomposites following co-
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colonization
for
alkaline phosphate
168 hours contrasted with Comp-O biocomposite where a most elevated
movement was seen for Comp-3 might be because of moderately more
permeable surface setting off an up directive of alkaline phosphate, associated with the principal
registration for osseous-genesis integrations. Consequently, it can be presumed that the support
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of bioactive prepared biocomposites gave a powerful substrate to cell integrations potentially
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owing to its MHAP mineralization character that enhanced the nucleation of apatite, bringing
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about the improvement of osseous-genesis [41].
Osseous development happens in the porous of cartilages tissue during calcification [42].
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In past investigation, it has been demonstrated that the porous in developing cartilages pulls in
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Ca2+ which carries on as nucleating specialists for the arrangement of apatite [43]. We
subsequently investigated the capability of the prepared biocomposites for development of Ca 2+
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coated as well as osteogenesis utilizing ARS test (Fig.6 B). The outcomes demonstrate that cells
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hatched with the prepared biocomposites framed more Ca 2+ coated than pre-ossification only at
24, 72 and 168 hours. In general, marginally higher Ca 2+ coated was seen within the sight of
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Comp-3 of biocomposites. As appeared in Fig. 6 C, the red recoloring of the biocomposites
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showing the arrangement of Ca2+ stores by the pre-ossification at 24, 72 and 168 hours.
3.8. Cell viability and Live/ dead cells assay
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The viability of the prepared biocomposites is indispensable for their use in the
orthopaedic as bone implants. Thus, the biocompatibility study of the prepared biocomposites
was performed with MG- 63 cells based on the MTT assay [44]. The assessment of
biocompatibility of the biocomposites was performed by incubating them for 24 h with varying
concentrations. As shown in Fig. 6C, that biocomposites exhibited considerably higher
cytocompatibility for the MG-63 cells even at larger concentrations as compared to Comp-O
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biocomposite. Hence, prepared biocomposites can be successfully incorporated as a bone
scaffold owing to its higher cytocompatibility which makes it a better host for the bone cells.
The biocompatibility of osteoblast cells on the prepared biocomposites was assessed with
a fluorescence microscope subsequent to being dyeing through AO/EB (Figure 7A). There were
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no huge departed cells on all prepared biocomposites subsequent to they were incubation for 24,
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72 and 168 hours. Therefore, this outcome demonstrates that all prepared biocomposites show
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agreeable biocompatibility. It could be viewed that cells developed remarkably successfully, as
well as a great percentage of alive cells attached to the biocomposites exterior. The number of
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alive cells expanded considerably with rising in EW substance in the biocomposites (Fig. 7B).
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The number of cells on biocomposites surface developed with incubation while continuing. The
% of multiple cells on the Comp-3 was considerably greater than that on the composite with
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Comp-1 and Comp-O, suggesting that the Comp-3 were higher profitable for the conversion of
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preosseous to bone tissues. The outcomes were predictable with that of the cell attachment and
multiplication examines.
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4. Conclusion
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A new conceivably bioactive PMMA/MHAP/EW biocomposites have been effectively
prepared via lyophilization method for hard tissue applications is the primary case including EW
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(Ovalbumin). The FTIR spectra of PMMA/MHAP/EW biocomposites exposed a major
intermolecular interface between diverse constituents whereas the outcomes of the XRD
information signified a diminish in size of PMMA/MHAP with the inclusion of EW. The
morphological
outcomes
exposed
permeable
and
interconnected
in
PMMA/MHAP/EW
biocomposite contrasted with the PMMA/MHAP composites. This was additionally upheld by
the biomineralization examine which showed a high calcification capability of the calcium
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particle. The prepared biocomposite showed a discriminated cell development and differentiation
alongside predominant non-harmful character validated from the aftereffects of ALP and
viability tests. The predominant antibacterial activities of PMMA/MHAP/EW biocomposites
were ascribed to its EW and Cu and Ce in MHAP. In this way, the conclusion drawn from the
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above-mentioned examinations interprets the fruitful intercession of EW (Ovalbumin) the bio-
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mimic composites, yielding capable develop superior to PMMA/MHAP biocomposites for hard
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tissue designing.
F. Cialdai, I. Landini, S. Capaccioli, S. Nobili, E. Mini, M. Lulli, M. Monici, In vitro
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Graphical abstract
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A novel fabrication of poly (methyl methacrylate)/ Ce/Cu substituted apatite/Egg white
(Ovalbumin) biocomposite
Development of morphology, mechanical and biological properties for the favor of bone tissue
rejuvenation
Excellent in vitro osteoblast proliferation of MG-63 cell on the biocomposite scaffolds
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CE
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M
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Very suitable biocomposite scaffolds material for bone tissue rejuvenation applications
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