mineral substituted apatite nanocomposites for orthopedics applications: In vitro physicochemical and biochemical studies

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]

Original 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

aDepartment of Chemistry, Govt Arts College for Women, Salem-8, India bDepartment of Chemistry, Shri Sakthikailassh Women's College, Salem, India

a r t i c l e i n f o Article history:

Received 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

high 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 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 150
C 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 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 Afterward, PMMA-GG was dissolved in the M-HA dispersed solvent to make homogeneous solutions with a 1: volume ratio Then, a ml PMMA-GG/M-HA solution was poured into a Teflon vessel, stirred continuously (30 min) and subsequently shifted into a freezer at 5
C 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 25
C 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 37
C 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 mm thick layers and including 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 wt.% centralization of the bio-composite which was set up by dissolving 0.2 g of material tests in 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 37
C The samples were sterilized in an autoclave at 80
C for 120 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 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

879 & 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 25
e35
for 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 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 FTIR spectra of the fabricated samples

Fig XRD patterns of the fabricated samples

HA 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 to 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

interaction 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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http://creativecommons.org/licenses/by/4.0/ ScienceDirect 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 T.N Shamsi, R Parveen, S Fatima, Characterization, biomedical and agricul-tural applications of protease inhibitors: a review, Int J Biol Macromol 91 J Sarin, L Bjorkvik, M Hiltunen, L Hupa, J Pulkkinen, P.K Vallittu, The effectof S.B Aziz, T.J Woo, M.F.Z Kadir, H.M Ahmed, A conceptual review on polymerelectrolytes and ion transport models, J Sci Adv Mater Dev (2018) 117 M.P Bostrom, J.M Lane, Future directions Augmentation of osteoporoticvertebral bodies, Spine 22 (1997) 38S42S C Radhakumary, P.D Nair, S Mathew, C.P Reghunadhan Nair, Biopolymercomposite of chitosan and methyl methacrylate for medical applications, M Mahinroosta, Z Jomeh Farsangi, A Allahverdi, Z Shakoori, Hydrogels asintelligent materials: a brief review of synthesis, properties and applications, S Mishra, S Sinha, K.P Dey, G Sen, Synthesis, characterization and applica-tions of polymethylmethacrylate grafted psyllium as R Kumar, S.K Ha, K Verma, S.K Tiwari, Recent progress in some selected bio-nanomaterials and their engineering applications: an overview, J Sci Adv. L.A Adams, E.R Essien, A.T Adesalu, M.L Julius, Bioactive glass 45S5 fromdiatom biosilica, J Sci Adv Mater Dev (2017) 476e482 A Munaz, R.K Vadivelu, J St John, M Barton, H Kamble, N.-T Nguyen, Three-dimensional printing of biological matters, J Sci Adv Mater Dev (2016) J Sonamuthu, S Samayanan, A.R Jeyaraman, B Murugesan, B Krishnan,S Mahalingam, Influences of ionic liquid and temperature on the tailorable 12851299. 78e82. O Yousheng, X Yushan, T Shaozao, S Qingshan, Structure, antibacterial ac-tivity of Ce A.M Deliormanl, Electrospun cerium and gallium-containing silicate based13-93 bioactive glass S Sathiskumar, S Vanaraj, D Sabarinathan, K Preethi, Evaluation of anti-bacterial and antibiofilm activity of synthesized zinc-hydroxyapatite R Zhang, P.X Ma, Poly(alpha-hydroxyl acids)/hydroxyapatite porous com-posites for bone-tissue engineering I Preparation and morphology, J Biomed. H.N Wang, Y.B Li, Y Zuo, J.H Li, S.S Ma, L Cheng, Biomaterials 28 (2007)33384334 S Karayil, S.D Deshpande, G.V Koppikar, Effect of honey on multidrugresistant organisms and its synergistic action with three common antibiotics, D Govindaraj, M Rajan, M.A Munusamy, A A.Alarfaj, A Higuchi, S SureshKumar, Carbon nanotubes/pectin/minerals substituted apatite nanocomposite R Govindan, G Suresh Kumar, E.K Girija, Polymer coated phosphate glass/hydroxyapatite composite scaffolds for bone tissue engineering applications, S Yang, H He, L Wang, X Jia, H Feng, Oriented crystallization of hydroxy-apatite by the biomimetic amelogenin nanospheres from self-assemblies of Y Zhou, H Yao, J Wang, D Wang, Q Liu, Z Li, Greener synthesis of elec-trospun L.C Gerhardt, A.R Boccaccini, Bioactive glass and glass-ceramic scaffolds forbone tissue engineering, Materials (2010) 3867e3910 H Peng, N Poovaiah, M Forrester, E Cochran, Q Wang, Ex vivo culture ofprimary intestinal stem cells in collagen gels and foams, ACS Biomater Sci. G Wu, X Deng, J Song, F Chen, Enhanced biological properties of biomimeticapatite fabricated polycaprolactone/chitosan nanofibrous bio-composite for A.D Pye, D.E.A Lockhart, M.P Dawson, C.A Murray, A.J Smith, A review ofdental implants and infection, J Hosp Infect 72 (2009) 104e110 S.M Baek, M.H Shin, J Moon, H.S Jung, S.A Lee, W.B Hwang, J.T Yeom,S.K Hahn, H.S Kim, Superior pre-osteoblast cell response of etched ultra