The in-vitro cell viability, the protein adsorption assay and the antibacterial test results indicated that the nanocomposite possesses a good osseous cell adhesion, a suf ficient protein[r]
Trang 1Original Article
Novel poly (methyl methacrylate) grafted guar gum/mineral
substituted apatite nanocomposites for orthopedics applications:
In vitro physicochemical and biochemical studies
G Priyaa,b,*, N Vijayakumaria, R Sangeethaa,b
a Department of Chemistry, Govt Arts College for Women, Salem-8, India
b Department of Chemistry, Shri Sakthikailassh Women's College, Salem, India
a r t i c l e i n f o
Article history:
Received 8 April 2018
Received in revised form
12 June 2018
Accepted 20 June 2018
Available online 25 June 2018
Keywords:
Nanocomposite
Bacterial activity
Guar gum
Poly (methyl methacrylate)
Osteoblast cell adhesion
a b s t r a c t
A poly (methyl methacrylate) grafted guar gum/mineral substituted hydroxyapatite (PMMA-GG/M-HA) nanocomposite is reported to show enhanced physico-chemical and bio-chemical properties This nanocomposite offers a possible bone cell integration around it with augmenting fresh bone develop-ment, thus declining the risk of cartilages' structural collapse In this study, the as-fabricated nano-composite was characterized by using physicochemical strategies The cell-material boundary of the nanocomposite was observed in vitro with human osteoblast cells, and the cell replication was tested The nanocomposite promoted the bone cell adhesion and proliferation, improved the mechanical strength and repressed the growth of bacterial cells
© 2018 Publishing services by Elsevier B.V on behalf of Vietnam National University, Hanoi This is an
open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
1 Introduction
Osteoporosis is a chronic infection that steadily influences bone
after some time This age-linked ailment influences men and ladies
over the age of 50[1] Bone mass is step by step lessened,
aban-doning debilitated and weak bones The spine and hip, made for the
most part out of trabecular cartilage, encounter constant
compressive stacking and are liable to an expanded danger of
disappointment As osteoporotic crack rates increment with the
maturing populace, there will be a requirement for creative
or-thopedic devices and bone growth arrangements[2]
Commercially, bone reinforces are acrylic and non-static, being
mostly made out of PMMA The major drawback of the PMMA is
their comparative non-bioactivity, severe exothermic
polymeriza-tion reacpolymeriza-tion, monomers toxicity and frailty[3,4] To this end,
at-tempts have been carried out to reinforce the PMMA polymer graft
with other bio-polymers and nanoparticles Grafting with diverse
bio-polymers is a potential approach to vary PMMA and enhance its
utilization The grafting of PMMA with biodegradable polymeric substances, for example, chitosan, guar gum, psyllium, and carbo-hydrate have good degradability and excellent biological properties
[5e7] Among these natural polymers, guar gum (GG) is an inex-pensive, hydrophilic, and non-toxic amorphous biopolymer GG is isolated from the endospermic seed of the plant Cyamopsis tetragonolobus [6] Recently, many researchers have introduced biopolymer grafted PMMA composites, due to their bio-inert properties
Since there is not yet a single substance that fulfills all the essential obligations for clinical applications, nanocomposites have been developed Consequently, there is an increasing curiosity in nanocomposite fabrics Currently, much attention has been paid to ceramic/polymer nanocomposites Especially those containing bio-active ceramics such as hydroxyapatite (HA)[8] The substituted apatite nanoparticles with synthetic polymers have been exten-sively employed in various orthopedic surgeries [9e11] A large number of works have been published with respect to substituting
Ca with other di and trivalent cations In particular, the substitution
of Ce and Zn bioactive ions has stimulated a growing curiosity given their valuable effects on the cartilage development and avoidance
of bone suction[12]
In contrast with other mineral ions, cerium ions have been utilized as bactericidal agents in solution for a while, because of the
* Corresponding author Department of Chemistry, Govt Arts College for Women,
Salem-8, India.
E-mail address: priya88chemistry@gmail.com (G Priya).
Peer review under responsibility of Vietnam National University, Hanoi.
Contents lists available atScienceDirect Journal of Science: Advanced Materials and Devices
j o u r n a l h o m e p a g e : w w w e l s e v i e r c o m / l o c a t e / j s a m d
https://doi.org/10.1016/j.jsamd.2018.06.002
2468-2179/© 2018 Publishing services by Elsevier B.V on behalf of Vietnam National University, Hanoi This is an open access article under the CC BY license ( http://
Journal of Science: Advanced Materials and Devices 3 (2018) 317e322
Trang 2high security and expansive scope of antibacterial action
Cerium-based antibacterials have attracted much consideration, on
ac-count of the low cerium ions to human cells being an enduring
biocide with high wellbeing and high warm dependability[13] Ce
can act like Ca in living beings, so it gathers in bones in little sums
In this way, cerium containing mixes can empower metabolism in
organisms[14,15] Besides, being one of the vital minerals
associ-ated with the metabolism of hard tissues, ionic zinc (Zn) has
appeared to render proliferative properties of osteoblasts as well as
an inhibitory impact on the bone reclamation by osteoclasts[16]
Therefore, Ce and Zn as imperative mineral particles can play a key
part in upgrading the bioactivity and antibacterial activity of HA for
biomedical applications
The motivation of the present study is to fabricate a novel
nanocomposite by reinforcing M-HA with PMMA-GG The
struc-ture, phase, tensile strength, antimicrobial, protein adsorption
properties and cells propagation capability were investigated Our
study shows that the newly fabricated PMMA-GG/M-HA
nano-composite can be used as a promising substrate for cell affection
and movement in osseous tissue engineering
2 Experimetal
2.1 Raw materials
Chemicals used were PMMA, Ca(NO3)2$4H2O (99%),
Ce(N-O3)2$6H2O (99%), Zn(NO3)2$H2O (99%), (NH4)2HPO4 (98%), guar
gum (99%) and methyl methacrylate (99%) All the chemicals were
purchased from Sigma Aldrich India, analytical grade
2.2 Fabrication of M-HA and PMMA-GG
The fabrication of Ce and Zn substituted HA, solutions of calcium
(0.9 M), cerium (0.05 M), zinc (0.05 M) and 0.6 M phosphate
independently attuned at pH-10 via the inclusion of NH3solution
The phosphate solution was gradually included drop wise into the
mineral (Ca, Ce& Zn) solutions to fabricate a colloidal solution The
colloidal solution was set aside in a microwave synthesizer at
500 W for 20 min To acquire the precipitate, excess un-reactive
ions were detached by rinsing the precipitate constantly by DD
water,filtered as well as after that dehydrated at 150C for 12 h in a
burning air oven
In short, for the fabrication of the PMMA-GG grafted polymer,
the solution of GG (0.5 g in 20 ml water) was mixed with the PMMA
solution (2.5 g in 10 ml water) Then, the mixer solution was set
aside in a microwave synthesizer at 900 W for 90 min Finally, the
gel like precipitated (PMMA-GG) grafted polymer was gathered and
was desiccated in a blistering air oven[6]
2.3 Fabrication of PMMA-GG/M-HA
The M-HA nano-particles were suspended in a solvent by
stir-ring for 30 min Afterward, PMMA-GG was dissolved in the M-HA
dispersed solvent to make homogeneous solutions with a 1: 1
volume ratio Then, a 5 ml PMMA-GG/M-HA solution was poured
into a Teflon vessel, stirred continuously (30 min) and subsequently
shifted into a freezer at 5C to encourage a solideliquid phase
separation The specimens were freeze-dehydrated for 24 h to
remove solvent[17]
2.4 Characterization
2.4.1 Physico-chemical characterization
The physico-chemical properties of the as-fabricated
nano-composite were evaluated via FTIR spectrophotometer (Nicolet
380), XRD (Seifert, X-ray diffractometer Siemens D500 Spectrom-eter), FESEM-EDX (Curl Jdiss Supra 40-2007, Germany)
2.4.2 Biological characterization The as-fabricated samples having identical weights were used and put into a culture anxious disc with the MEM (supplemented with a 10% FBS) culture solution and hatched at 25C in 2e6 h The samples were then washed in PBS medium three times to eliminate the un-adsorbed proteins on the exterior The entire protein vol-ume was measured with a BCA analyzer[18]
The bacterial properties of the as-fabricated samples have been researched against both gram-negative microorganisms via the inhibition zone method[19] The inoculums of the two microorganisms were setup from the crisp overnight medium (Tripton soy medium with 0.6% yeast remove) that was brooded
at 37C The subsequent medium cultures were utilized for the dispersion tests The agar dissemination test was performed at Muller-Hinton agar and it was done by emptying agar into petri plates to shape 4 mm thick layers and including 2 ml thick inoculums of the tried bacteria's so as to get the semi-blended development The petri plates were left to desiccate at the air and from that point onward, the fabricated bio-composite tests
to be tried with various concentrations were impregnated into the well against the vaccinated bacteria's on the agar exterior and were hatched for one day at RT The concentrations of the utilized bio-composite were 25 um individually The distinctive concentrations of the fabricated material tests were taken from
a 1 wt.% centralization of the bio-composite which was set up
by dissolving 0.2 g of material tests in 2 ml of dimethyl sulphoxide (DMSO) At last, the hindrance zone was checked
by measuring the width of the zone of restraint (mm) around the well
The cytocompatibility of the fabricated samples was evaluated
by MG63 system purchased from National Center for Cell Science (NCCS), Pune, India The cells were developed in Dulbecco's Modified Eagle Medium (Hi Media Laboratories) supplemented with 10% fetal bovine serum, streptomycin (100 U mL1) and penicillin (100 U mL1) The medium was revived each day The cells were hatched in a humidified atmosphere with CO2at 37C The samples were sterilized in an autoclave at 80C for 120 min and then aliquot into 96-well cell growth plates The cytotoxicity of the as-fabricated bio-composite was evaluated in vitro by using MTT test[20] In short, the MTT store reagent in PBS was included in every well to attain a concluding volume of 0.5 mg mL1 After 4 h, the excess MMT was detached and the absorbance of all the wells was then deliberated at 570 nm by a microplate reader The
qual-ified cell feasibility was calculated using the subsequent equation:
% of cell feasibility
¼ absorbance of composite=absorbance of control 100
2.5 Statistical analysis All tests were completed in triplicate and the consequences were revealed as the mean± standard deviation and were exam-ined by utilizing one-way ANOVA
3 Results and discussion 3.1 FTIR analysis
Fig 1shows the symmetric stretching vibrations of the phos-phate group of apatite are viewed at 568, 723, 876 and 1090 cm1in the M-HA nanoparticle[21] From the FTIR spectrum of the
PMMA-GG composite, it is found that the bands at 3590, 2936, 1665, 1023,
Trang 3879 & 818, cm1 correspond to the OeH (stretching), CeH (stretching), CeO (stretching), OeH (in-plane bending), CeOeC (stretching), and C¼O (stretching) vibrations Therefore, the 1665 and 1023 bands in the PMMA-GG composite are well clarified via the existence of an embedded PMMA matrix and the substantiation
of the grafting [6] The FTIR spectrum of the PMMA-GG/M-HA nanocomposite scaffold was established to hold extra bands at
560, 719, 869 and 1082 cm1, which are assigned to the phosphate group stretching vibrations of M-HA The interaction between the M-HA and polymer can be explained as follows The intensity of the distinguishing phosphate group bands of M-HA was reported to reduce when included in a polymer composite[21] The phosphate band of M-HA at 1090 shifted to 1082 cm1in the case of PMMA-GG/M-HA In other words, the FTIR analysis confirmed the devel-opment of a nanocomposite among PMMA-GG and M-HA
3.2 Phase detection
Fig 2shows the XRD patterns of M-HA, GG, and PMMA-GG/M-HA nanocomposite The XRD pattern of the M-HA nano-particles by JCPDS (09-0432) card for apatite shows a distinctive crystalline character of the M-HA nano-particles[22] The
PMMA-GG does not demonstrate any crystalline nature in its place; a wide peak is viewed consequently validating the amorphous character of the as-fabricated polymer composite Moreover, the wide planes detected in the 2theta range 25e35for the PMMA-GG/M-HA nanocomposite suggest the existence of M-HA nano-particles, with the less crystalline character of the nanocomposite
[23]
3.3 Morphological analysis The morphology of the as-fabricated samples is revealed in
Fig 3 The SEM micrographs of the M-HA nano-particles signify that the beads are textured (Fig 3a and b) The addition of M-HA into the polymer composite (PMMA-GG/M-HA) resulted inflat and porosity with interconnectivity surfaces (Fig 3d and e) The as-fabricated nanocomposite shows pores at surface, which are generally beneficial for biomedical applications[24] Moreover, the mineral components are present in the M-HA and PMMA-GG/M-Fig 1 FTIR spectra of the fabricated samples.
Fig 2 XRD patterns of the fabricated samples.
Fig 3 SEM-EDX images of M-HA (aec) and PMMA-GG/M-HA (def).
G Priya et al / Journal of Science: Advanced Materials and Devices 3 (2018) 317e322 319
Trang 4HA nanocomposite phases, including Ca, P, O, C, Ce, and Zn, as
revealed with EDX for every sample (Fig 3c, f)
3.4 Mechanical and protein adsorption properties
The mechanical properties of the PMMA-GG/M-HA
nano-composite are the key features together with their medical function
and bone healing capacity[25] The mechanical properties of the
M-HA loaded PMMA-GG have been improved for tendon
rejuve-nation, analogous to the PMMA-GG polymer matrixes (Fig 4a) The
highest load of the PMMA-GG/M-HA nanocomposite was 165 N,
which was superior to that of the PMMA-GG polymer matrix 103 N
The modulus of the PMMA-GG/M-HA nanocomposite was 126 MPa,
which is comparable to that of an ordinary human tendon modulus
value [26] This incidence was similar to the mechanism of the
PMMA-GG polymer, which strengthened high tensile potency and
is the resolution of the interface contact, which mostly refers to the
chemical absorption of the polymer matrix to the M-HA
nano-particles exterior
The protein adsorption statistics of the as-fabricated samples
are shown inFig 4b The adsorbed protein volume was enlarged on
occasion from 1 to 6 h The enhancement in the protein adsorption
for the PMMA-GG/M-HA nanocomposite as compared to the
PMMA-GG (control) composite can be due to the circulation of
M-HA nano-particles on the nanocomposite facades, which augment
the binding situates on the fabric exterior for proteins[18]
3.5 Antibacterial properties
One system to advance a host tissue mix over microorganisms
biofilm arrangement on implantable biomaterials is the
consoli-dation of anti-infection parts[27] In this regard, cerium and zinc
metals, nanoparticles and edifices have been consolidated into a
scope of wound-care and implantable medical devices to misuse
the wide range of antibacterial properties[12] The consequences of the antibacterial hindrance zone tests utilizing S aureus and E coli are given inFig 4c Particular clear zones were noted around the PMMA-GG/M-HA and M-HA composite layers in contact with all bacteria
3.6 Biocompatible and cell adhesion assay Basically, the bioactive PMMA-GG/M-HA nanocomposite is cytocompatible with osseous tissues The cell feasibilities for cul-tures in contact with expanding amounts of PMMA-GG, M-HA, and PMMA-GG/M-HA nanocomposite are contrasted with those of the control (without the as-fabricated samples), as shown in
Fig 5(aee) This demonstrates that there is no critical loss of cell suitability for the cultures in contact with the as-fabricated speci-mens In this case, the type of Ce-Zn-HA (M-HA) in the PMMA-GG nanocomposite can exhibit the antimicrobial movement without the negotiational cytocompatibility regarding osseous cells Additionally,Fig 5(f,g) demonstrates the multi-layered polyg-onal structure and strong cellecell interactions, showing a large cell cytoskeleton texture on the PMMA-GG/M-HA nanocomposite for
24 h This result is consistent with those reported previously on osseous cells that possessed a great spreading and cell attachment onto the PMMA-GG/M-HA nanocomposite[28]
4 Conclusion
We have demonstrated the facile fabrication of the PMMA-GG/ M-HA nanocomposite The fabricated sample was characterized chemically (via FT-IR, XRD), morphologically (by FESEM) and bio-logically (antibacterial, protein adsorption & cell viability) The FESEM examination showed the existence of M-HA nano-particles
in the nanocomposite composition, and these particles also modi-fied the exterior morphology Interfaces appeared to occur at the
Trang 5interaction between the M-HA and PMMA-GG composite enhanced
the mechanical performance of the PMMA-GG/M-HA
nano-composite The in-vitro cell viability, the protein adsorption assay
and the antibacterial test results indicated that the nanocomposite
possesses a good osseous cell adhesion, a sufficient protein
adsorption, and repressed the growth of bacterial cells Therefore,
the PMMA-GG/M-HA nanocomposite is a potential candidate for
orthopedic applications
Acknowledgements
The authors thank The South Indian Textile Research
Associa-tion, Coimbatore for providing all the required facilities
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