Accepted Manuscript Title: SYNTHESIS AND CHARACTERIZATION OF NEW COMPOSITE MATERIALS BASED ON POLY(METHACRYLIC ACID) AND HYDROXYAPATITE WITH APPLICATIONS IN DENTISTRY Author: Andrei Tiberiu Cucuruz Ecaterina Andronescu Anton Ficai Andreia Ilie Florin Iordache PII: DOI: Reference:
S0378-5173(16)30061-8 http://dx.doi.org/doi:10.1016/j.ijpharm.2016.01.061 IJP 15527
To appear in:
International Journal of Pharmaceutics
Received date: Revised date: Accepted date:
21-10-2015 19-1-2016 23-1-2016
Please cite this article as: Cucuruz, Andrei Tiberiu, Andronescu, Ecaterina, Ficai, Anton, Ilie, Andreia, Iordache, Florin, SYNTHESIS AND CHARACTERIZATION OF NEW COMPOSITE MATERIALS BASED ON POLY(METHACRYLIC ACID) AND HYDROXYAPATITE WITH APPLICATIONS IN DENTISTRY.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2016.01.061 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.
SYNTHESIS AND CHARACTERIZATION OF NEW COMPOSITE MATERIALS BASED ON POLY(METHACRYLIC ACID) AND HYDROXYAPATITE WITH APPLICATIONS IN DENTISTRY ANDREI – TIBERIU CUCURUZ1*, ECATERINA ANDRONESCU1, ANTON FICAI1, ANDREIA ILIE1, FLORIN IORDACHE2 1
2
POLITEHNICA University of Bucharest, Faculty of Applied Chemistry and Material Science; 1-7 Polizu Str., 011061 Bucharest, Romania
Institute of Cellular Biology and Pathology “Nicolae Simionescu”of Romanian Academy, Department of Fetal and Adult Stem Cell Therapy, 050568, Bucharest, Romania *
Corresponding author: Andrei CUCURUZ, POLITEHNICA University of Bucharest, 1-7 Gh. Polizu str., 011061 Bucharest, Romania, e-mail: [email protected] Graphical abstract
1
Abstract The use of methacrylic acid (MAA) in medicine was poorly investigated in the past but can be of great importance because the incorporation of hydroxyapatite (HA) can lead to new composite materials with good properties due to the strong electrostatic interactions between carboxylate groups of polymer and Ca2+ ions from HA. The scope of this study was to determine the potential of using composite materials based on poly(methacrylic acid) (PMAA) and hydroxyapatite in dentistry. Two routes of synthesis were taken into account: i) HA was synthesised in situ and ii) commercial HA was used. Fourier transform infrared spectroscopy and X-ray diffraction were used for compositional assessments. Scanning electron microscopy was performed to determine the morphology and differential thermal analysis (DTA) coupled with thermogravimetric analysis (TG) was used to study the thermal behaviour and to observe quantitative changes. In-vitro tests were also performed in order to evaluate the biocompatibility of both PMAA/HA composites by monitoring the development potential of human endothelial cells using MTT assay and fluorescent microscopy. Keywords: poly(methacrylic acid), hydroxyapatite, biocompatibility, dental material .
1. Introduction Nanocomposites are a promising new class of advanced materials that are made of two or more components in which at least one of them has one or more dimensions (width, length, thickness) in nanoscale. Polymethacrilic acid based nanocomposites exhibit enhanced optical thermal, mechanical and electrochemical properties compared to the conventional composites [1]. The synthesis of high-performance dental composites made from an organic matrix and inorganic filler/reinforcing agent has attracted interest both for research as well as for the applicative purposes. This can be explained by combining the advantageous properties of inorganic nanoparticles with those of organic polymers. Nevertheless, producing polymer nanocomposites with optimal properties is a challenge for the researchers due to surface energy differences between nanoparticles and polymers. Thus, nanoparticles surface modification is many times required especially for biological properties to overcome the incompatibility issue and to improve interactions between inorganic nanoparticles and polymer which leads to a good nanoparticle dispersion as well as better properties for polymer nanocomposites. Traces of unreacted monomers lead to side effects and poor properties of the final products which are not acceptable in the medical field [2]. Nowadays, dental composites have been accepted as the restorative material of choice; as compared to dental amalgams, the composites have better aesthetics, less safety concerns and have demonstrated good clinical results. Dental composites consist of resin matrices and different inorganic fillers [3-10]. Calcium phosphates are mainly used as bone substitutes in biomedical applications due to their biocompatibility, low density, chemical stability and their compositional resemblance to the mineral phase of bone [11-18]. Hydroxyapatite (HA) is the prime component of bone cements because it can be characterized as osteoconductive, biocompatible, noninflammatory, nontoxic, nonimmunogenic agent and bioactive, hence the
2
ability to form chemical bonds with living tissues. However, the poor strength of HA restricts its clinical applications under loadbearing conditions [19]. Methacrylic acid is a colourless, viscous organic compound with the chemical formula C4H6O2 whose use in dentistry was poorly investigated so far. In order to use poly(methacrylic acid) (PMAA) in dental applications, is mandatory to reinforce the polymer with ceramic particles [20]. Also, the incorporation of hydroxyapatite to PMAA can confer bioactivity to the composite [21]. For these reasons, polymer matrices reinforced with bioactive phases such as HA, combine the typical bioactive behavior of bioceramics with enhanced mechanical properties and make the composites comparable with human hard tissues [22]. Resin composites have a large area of applications in dentistry such as restorative materials, crowns, cements for tooth prostheses, cavity liners, pit and fissure sealants, cores, inlays, provisional restorations, endodontic sealers, and root canal posts. Because of their versatility, the use of this type of materials will continue to grow in frequency and application. The fast moving process of these materials suggests constant changes in this field [23]. In this study, polymethacrylic acid/HA composite materials were prepared via in situ precipitation/radical polymerization method.
2. Experimental 2.1. Materials Methacrylic acid (MAA) purrum monomer ≥98% and sodium hydroxide pellets ACS reagent ≥ 97% were supplied by Sigma-Aldrich. Hydroxyapatite (HA) puriss was supplied by Riedel-de Haën. Benzoyl peroxide (initiator) purrum ≥ 97% was supplied by Fluka. The precursors for hydroxyapatite: calcium hydroxide 98 % extrapure was supplied by Acros Organics and monosodium phosphate was supplied by Chimopar. The human endothelial cells line (EAhy923, ATCC, Virginia, USA) was used to evaluate the biocompatibility of PMAA/HA composites. The cells were cultured in DMEM medium (Sigma-Aldrich, Missouri, USA) supplemented with 10% fetal bovine serum, 1% penicillin and 1% streptomycin antibiotics (Sigma-Aldrich, Missouri, USA). To maintain optimal culture conditions, medium was changed twice a week. 2.2. Methods The composite materials based on PMAA and HA were synthesised starting from methacrylic acid and i) in situ obtained HA and ii) commercial available hydroxyapatite. The synthesis procedure for both types of composites is described below in Figure 5. The composites made with in situ synthesized hydroxyapatite were obtained from calcium hydroxide (Ca(OH)2) suspension and monosodium phosphate (NaH2PO4.2H2O) solution. First, the monomer was introduced, then the precursors and finally, the initiator – benzoyl peroxide, in order to begin the radical polymerization reaction. The polymerization process lasted for 3 hours at 90°C. The pH of the entire volume was fixed to 7.5 with NaOH solution 0.1 N, and then the sample was centrifuged, dried and grinded. At the end, the samples were subjected to the freeze-drying process of lyophilization, a conditioning technique that does not cause denaturation of the materials. A molar ratio of 1.67 between Ca/P was used for the precipitation of hydroxyapatite. For the composites made with commercial hydroxyapatite, the synthesis procedure is the same as for the synthesized hydroxyapatite mentioning that first was introduced a
3
minimum volume of distilled water and then commercial hydroxyapatite, monomer and the catalyst. The mass ratio between the polymer matrix and the inorganic compound was 1:1. Also, it was used a content of 1% of initiator reported to the monomer. The microstructures, composition and thermal behaviour of the synthesized composite materials were determined by FT-IR, XRD, SEM and DTA-TG. Also, pH and conductivity time evolution in distilled water and simulated body fluid were determined and biocompatibility was evaluated by monitoring the potential for cellular growth. IR spectroscopic measurements were performed using a Shimadzu 8400 FT-IR spectrometer. The spectra were recorded on a wavenumber range of 400–4000cm-1 with a resolution of 2cm-1. X-ray diffraction analysis was done using a Shimadzu XRD 6000 diffractometer at room temperature. It was used the Cu Kα radiation from a Cu X-ray tube. The samples were scanned in the Bragg angle, 2θ range of 10 – 87o at a scan rate of 2o.min−1. All samples were ground to fine powders before analysis. XRD analysis is useful to identify crystalline phases and to determine their concentrations. Scanning Electron Microscopy (SEM) was used to determine the morphology and was also useful to show the compatibility between phases. SEM images were taken with a HITACHI S2600N with an EDAX probe. All samples were covered with a silver layer prior to imaging. Differential thermal analysis (DTA) coupled with thermogravimetric analysis (TGA) was performed in air atmosphere with a Shimadzu DTG-TA-50H equipment at a heating rate of 10oC. Thermal analysis was used to determine the behaviour of the composites and to observe quantitative changes by increasing temperature. Conductivity and pH measurements were performed with an Eutech Instruments Cyberscan PCD 6500 pH/conductivity meter. The cytotoxicity and biocompatibility of human endothelial cells on PMAA/HA was assessed using MTT assay (CellTiter 96® Non-Radioactive Cell Proliferation Assay, Promega, Wisconsin, USA). Briefly, the human endothelial cells were grown in 96-well plates, with a seeding density of 3000 cells/well in the presence of PMAA/HA for 72 hours. Then, 15 ml Solution I was added and incubated at 37°C for 4 hours. After that, Solution II was added and pipetted vigorously to solubilize formazan crystals. After 1 hour, the absorbance was read using a spectrophotometer at 570 nm (TECAN, Männedorf, Switzerland). Furthermore we use a second method to evaluate the biocompatibility of these composite materials based on fluorescent microscopy using a RED CMTPX fluorophore (Life Technologies, Invitrogen, USA), a cell tracker for long-term tracing of living cells. The CMTPX was added in the cell culture and after 3 days, the viability and morphology of the endothelial cells was observed. The CMTPX fluorophore was added in the culture medium at a final concentration of 5 μM, incubated for 30 min in order to allow the dye penetration into the cells. Then, the endothelial cells were washed with PBS and visualized by fluorescent microscopy. The photomicrographs were taken with a digital camera driven by Axio-Vision 4.6 software (Carl Zeiss, Germany).
3. Results and discussions 3.1. Fourier transform infrared spectroscopy – FTIR
4
FTIR is an essential tool for characterizing materials being applicable for both crystalline and amorphous materials. Both composite materials (Figure 6) exhibit the main absorption bands of phosphate groups [24]. The shift of some vibrations of phosphate groups can be attributed to different interactions between organic and inorganic components, the stronger shift corresponding to stronger interactions. Based on this fact, it can be concluded that in situ formation of HA followed by polymerisation leads to a slightly better interaction between the two phases, the shift of the ν3 (PO43-) band being ~20cm-1 for the composite material synthesized in situ compared with the composite material obtained with commercial HA (the shift is only ~19cm-1). The spectrum of the PMMA/HA sample obtained by in situ precipitation of HA exhibits the vibration of the carbonate group, this result being also visible for other HA based materials [25]. Based on the absence of the characteristic peak of C=C double bond from around 1633cm-1 (vinyl group, – CH=CH2) as well as the deformation bands from ~900 and 1000cm-1 (vinyl group, =C-H) assigned to the monomer, it can be assumed that the polymerization has occurred with a high conversion degree. The two composite samples exhibit different FTIR spectra because of the distinct composition induced by synthesis route. The in situ precipitation of HA lead to a high degree of ionized carboxyl groups (COO-). The ionization of the carboxyl groups, the absorption band from ~1700cm-1 disappears and two new bands appear at 1544 and 1417cm-1 which are characteristic to asymmetric and symmetric vibrations of the R-COO- group. For the composite based on commercial HA, this ionization is not at all important because HA has a low solubility Ks and consequently the neutralization of COOH does not occur. Table presents the main characteristic bands of PMAA/HA composites.
3.2. X-Ray diffraction The crystalline phases were analysed by XRD, the results being complementary to the information obtained by FTIR (Figure 7). The diffractograms were useful in order to identify hydroxyapatite phases through the presence of diffraction peaks. Thereby, in situ formation of synthesized hydroxyapatite is confirmed according to ASTM [74-0566] and the presence of commercial hydroxyapatite complies with ASTM [72-1243]. Also, the sharp peaks of synthesized hydroxyapatite indicate a larger size of crystallites, compared to the commercial hydroxyapatite and, more important, the crystallinity of PMAA/HAs is lower than that of PMAA/HAc.
Scanning electron microscopy Based on SEM images (Figure 8), some differences can be noticed. First of all, the microstructure of the two composite materials is quite different. The in situ formation of HA leads to a homogenous, fibrillar-like composite while, the mixing of monomer with commercial HA followed by polymerization leads to agglomerates and heterogeneities. The agglomerates are not uniform, have different spherical or polyhedral shapes and can reach up to 20µm, in length/diameter. Also a different behaviour in the electron beam can be observed, the sample obtained by in situ precipitation of HA being more stable.
Differential Thermal Analysis – DTA-TG
5
DTA-TG determinations were performed for both PMAA/HA composites in order to observe the thermal stability of the samples (Figure 1). TG analysis indicated no major differences between the two composites in what it concerns the thermal behaviour with a minor distinction regarding mass loss; 54.35% in case of PMAA/HAc and 40.94 % for PMAA/HAs. In the range 96-388°C, DTA curve for PMAA/HAc indicates two endothermic effects (at 173°C and 353°C) accompanied by mass loss that can be attributed to the decomposition of the organic component. PMAA/HAs composite shows an exothermic effect at 84°C and two endothermic peaks at 223 and 310°C. Also, in the range 550-750°C, PMAA/HAs exhibits an exothermic effect due to carbonate group, its presence being useful from the biocompatibility point of view. Figure 1.
3.3. Conductivity /pH measurements Conductivity and pH measurements were performed over 2 days both in distilled water and simulated body fluid (SBF). As can be seen from Figure 2, pH values have a gradual decrease in distilled water and tend to stabilize after 4 hours in case of PMAA/HAs composites and after approx. 6 hours for the PMAA/HAc composites. On the other hand, both composites tend to reach constant values of pH much later in SBF, after approx. 43 hours. PMAA/HAs induce the slightest change in pH which leads to lower stress on the tissue in which the implantation will occur, so lower inflammatory reactions. Figure 2.
As can be observed from Figure 11, conductivity of the composites immersed in distilled water has a gradual increase until it reaches the threshold after approx. 43 hours. The same can be noticed for immersion in SBF mentioning a more pronounced conductivity increase especially in the case of PMAA/HAs composites.
3.4. In-vitro cell development The viability of PMAA/HA composites was performed by MTT assay, based on the biochemical reactions that measure the metabolic activity of living cells. The MTT assay demonstrated that the human endothelial cells present a normal metabolism and growth in the presence of PMAA/HA. The measured values of absorbance at 570 nm showed that endothelial cells grow better on both tested composite materials. After 24h of incubation, the proliferation of endothelial cells was 2 times faster on modified surfaces comparing with the control. The same effect it was also observed after 72h of incubation (Figure 8). These results demonstrate the biocompatibility potential of both composites and confirm their applications as new surfaces for in vitro culture tissues development. The in situ synthesis of the PMAA/HA composite material is beneficial on long term because the cell proliferation is improved even compared with the PMAA/HAc, this result being a consequence of the in vitro behaviour of these composite materials. The fluorescent microscopy images confirm the viability biochemical test showing that the endothelial cell viability is maintained after 3 days in the presence of PMAA/HA. The endothelial cells retain the normal morphology, were adherent and have a relatively uniform distribution on all investigated surfaces (Figure 9). These results are in consent with other researchers that showed PMAA/HA composites
6
present good biocompatibility, osteoinductive properties and improve the surface compatibility between HA and cellular matrix [26]. Domingo et al., 2001 analyzed the behaviour in water as well as the mechanical and surface properties of composite materials containing HA designed for dental restoration. They demonstrated that the materials containing bisphenol-alpha-glycidyl methacrylate:triethyleneglycol dimethacrylate and micrometric-HA coated with citrate, acrylate, or methacrylate displayed the most favourable results [27]. Figure 3. Figure 4.
4. Conclusions In this work, two PMAA/HA composite materials with different microstructures were obtained; the microstructural differences being induced only by the synthesis route. It is expected that such differences can strongly modify the mechanical properties of the materials as well as their interaction with the body fluids. FT-IR analysis indicated that are no bands assigned to the monomer, which proves that polymerisation occured with a high conversion degree. SEM analysis revealed that in situ formation of HA leads to a more homogenous composite with a good distribution of HA particles in the polymer matrix while the commercial form of HA causes the appearance of agglomerates. In-vitro testing of PMAA/HA composites showed a great biocompatibility that could be quantified by the development of eukaryotic cells after 24, 48 and 72 hours. These results bring new insights in the research of biocompatible surfaces engineering and may lead to the development of new surfaces used for facilitating in vitro culture tissues development. Also, the analyzed composite materials indicated a good potential for application in dentistry as cements or resins. Further works will be dedicated to appropriate, comparative testing of these materials, focusing on their mechanical properties.
Acknowledgements This work has been funded by the Sectorial Operational Programme Human Resources Development 2007-2013 of the Romanian Ministry of Labour, Family and Social Protection through the Financial Agreement POSDRU/107/1.5/S/76903.
7
References: 1.
Yan Bao, Jian-zhong Ma. Polymethacrylic acid/Na-montmorillonite/SiO2 nanoparticle composites structures and thermal properties. Polym. Bull. (2011) 66:541–549. SpringerVerlag 2010. 2. Wantinee Viratyaporn, Richard L. Lehman. Spectroscopic analysis of poly(methyl methacrylate)/alumina polymer nanocomposites prepared by in situ (bulk) polymerization. J Mater Sci (2010) 45:5967–5972. Springer Science+Business Media, LLC 2010. 3. M. Kostic, N. Krunic, L. Nikolic, V. Nikolic, S. Najman, I. Kostic, et al.; Influence of Residual Monomer Reduction on Acrylic Denture Base Resins Quality; Hemijska Industrija 2011, 65(2), 171. 4. L.F. Zhang, Y. Gao, Q. Chen, M. Tian and H. Fong; Bis-GMA/TEGDMA dental composites reinforced with nano-scaled single crystals of fibrillar silicate; Journal of Materials Science 2010, 45(9), 2521. 5. J. Xu, Y. Li, T. Yu and L. Cong; Reinforcement of denture base resin with short vegetable fiber; Dental Materials 2013, 29(12), 1273. 6. W.C. Kuo, Y.S. Lin, C.L. Lin and J.C. Wang; Biomechanical Investigation of Long-span Glass-fiber-reinforced Acrylic Resin Provisional Fixed Partial Denture: a Finite Element Analysis; Journal of Medical and Biological Engineering 2012, 32(5), 357. 7. L.S. Acosta-Torres, I. Mendieta, R.E. Nunez-Anita, M. Cajero-Juarez and V.M. Castano; Cytocompatible antifungal acrylic resin containing silver nanoparticles for dentures; International Journal of Nanomedicine 2012, 7, 4777. 8. W.L. Tham, W.S. Chow and Z.A.M. Ishak; Effects of titanate coupling agent on the mechanical, thermal, and morphological properties of poly(methyl methacrylate)/hydroxyapatite denture base composites; Journal of Composite Materials 2011, 45(22), 2335. 9. J.P. Zheng, Q. Su, C. Wang, G. Cheng, R. Zhu, J. Shi, et al.; Synthesis and biological evaluation of PMMA/MMT nanocomposite as denture base material; Journal of Materials Science-Materials in Medicine 2011, 22(4), 1063. 10. W. Viratyaporn and R.L. Lehman; Spectroscopic analysis of poly(methyl methacrylate)/alumina polymer nanocomposites prepared by in situ (bulk) polymerization; Journal of Materials Science 2010, 45(21), 5967. 11. M. Hautamaki, V.V. Meretoja, R.H. Mattila, A.J. Aho and P.K. Vallittu; Osteoblast response to polymethyl methacrylate bioactive glass composite; Journal of Materials Science-Materials in Medicine 2010, 21(5), 1685. 12. M.H. Fathi, A. Hanifi, V. Mortazavi. Preparation and bioactivity evaluation of bone-like hydroxyapatite nanopowder. Journal of materials processing technology 202 (2008) 536– 542. Elsevier. 13. A. Ilie, E. Andronescu, D. Ficai, G. Voicu, M. Ficai, M. Maganu, et al.; New approaches in layer by layer synthesis of collagen/hydroxyapatite composite materials; Central European Journal of Chemistry 2011, 9(2), 283. 14. A. Ficai, E. Andronescu, G. Voicu and D. Ficai. Advances in collagen/hydroxyapatite composite materials. In: Attaf B, editor. Advances in Composite Materials for Medicine and Nanotechnology: INTECH, 2011. 15. M. Ficai, E. Andronescu, D. Ficai, G. Voicu and A. Ficai; Synthesis and characterization of COLL-PVA/HA hybrid materials with stratified morphology; Colloids and Surfaces B: Biointerfaces 2010, 81(2), 614. 16. A. Ficai, E. Andronescu, G. Voicu, C. Ghitulica, B.S. Vasile, D. Ficai, et al.; Self assembled collagen/ hydroxyapatite composite materials; Chemical Engineering Journal 2010, 160(2), 794.
8
17. A. Ficai, E. Andronescu, G. Voicu, C. Ghitulica and D. Ficai; The influence of collagen support and ionic species on the morphology of collagen/hydroxyapatite composite materials; Materials Characterization 2010, 61(4), 402. 18. A. Ficai, E. Andronescu, G. Voicu, M.G. Albu and A. Ilie; Biomimetically synthesis of collagen/hydroxyapatite composite materials; Materiale Plastice 2010, 47(2), 205. 19. E. Dinu, M. Bîrsan, C. Ghiţulică, G. Voicu, E. Andronescu, Synthesis and characterization of hydroxyapatite obtained by sol-gel method, Romanian Journal of Materials, 2013, 43, 1, 55-60. 20. M. Ficai, E. Andronescu, A. Ficai, G. Voicu, B.S. Vasile, Poly bis-GMA/HA based hybrid composite materials, UPB Scientific Bulletin, Series B, 2011, 73, 75-84. 21. T.G. Tihan, M.D.Ionita, R.G. Popescu, D. Iordachescu; Effect of hydrophilic– hydrophobic balance on biocompatibility of poly(methyl methacrylate) (PMMA)– hydroxyapatite (HA) composites; Materials Chemistry and Physics 2009, 118, 265-269. 22. C.H. Navarro, K.J. Moreno, A. Chávez-Valdez, F. Louvier-Hernández, J.S. GarciaMiranda, R. Lesso, A. Arizmendi-Morquecho; Friction and wear properties of poly(methyl methacrylate)–hydroxyapatite hybrid coating on UHMWPE substrates; Wear 2012, 282-283, 76-80. 23. A. Ficai, E. Andronescu, C. Ghitulica, G. Voicu, V. Trandafir, D. Manzu, et al.; Colagen/Hydroxyapatite Interactions in Composite Biomaterials; Materiale Plastice 2009, 46(1), 11. 24. Jack L. Ferracane. Resin composite - State of the art (Review) - Dental materials 27 (2011) 29–38. Elsevier. 25. A. Ficai, M.G. Albu, M. Birsan, M. Sonmez, D. Ficai, V. Trandafir, et al.; Collagen hydrolysate based collagen/hydroxyapatite composite materials; Journal of Molecular Structure 2013, 1037, 154. 26. J. Gao, B. Huang, J. Lei, Z. Zheng. Photografting of methacrylic acid onto hydroxyapatite particles surfaces; Journal of Applied Polymer Science; 2010 (115) 2156– 2161. 27. C. Domingo, R.W. Arcís, A. López-Macipe, R. Osorio, R. Rodríguez-Clemente, J. Murtra, M.A. Fanovich, M. Toledano, Dental composites reinforced with hydroxyapatite: mechanical behavior and absorption/elution characteristics, J Biomed Mater Res, 2001, 56(2), 297-305.
9
FIGURES
Figure 5. Schematic representation for the synthesis routes of PMAA/HA composite materials
10
0.55 PMAA/HAc PMAA/HAs 0.50 0.45 0.40
Absorbance
0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 -0.05 4000
3500
3000
2500 2000 Wavenumbers (cm-1)
1500
1000
Figure 6. FT-IR spectra of PMAA/HA composite materials
800
PMAA/HAs PMAA/HAc
700
Intensity, a.u.
600 500 400 300 200 100 10
20
30
40
50
2 Theta Figure 7. XRD spectra of PMAA/HA composite materials
11
60
12
Figure 8. SEM images of the PMAA/HA composite materials at different magnifications: a. x100; b. x500; c. x1000; d. x5000.
Figure 9. DTA, TGA and DTG curves for: a) PMAA/HAc and b)PMAA/HAs
Figure 10. pH evolution of PMAA/HA composites in distilled water and simulated body fluid
Figure 11. Conductivity evolution of PMAA/HA composites in distilled water and simulated body fluid
13
Figure 12. Endothelial cells proliferation profiles after growing on control (green) and composite materials for up to 72 h
Figure 13. Fluorescence microscopic images of the cell monolayer treated for 72 h composite materials (x100): a) PMAA/HAs, b)PMAA/HAc and c) Ctrl
14
Table
Table 1. Main vibrations of PMAA/HA samples HA com.
PMAA/HAc
PMAA/HAs
Assignment
Band position, cm-1
568
560
556
ν4 (PO43-)
604
598
599
ν4 (PO43-)
1043
1024
1023
ν3 (PO43-)
15
S0378-5173(16)30061-8 http://dx.doi.org/doi:10.1016/j.ijpharm.2016.01.061 IJP 15527
To appear in:
International Journal of Pharmaceutics
Received date: Revised date: Accepted date:
21-10-2015 19-1-2016 23-1-2016
Please cite this article as: Cucuruz, Andrei Tiberiu, Andronescu, Ecaterina, Ficai, Anton, Ilie, Andreia, Iordache, Florin, SYNTHESIS AND CHARACTERIZATION OF NEW COMPOSITE MATERIALS BASED ON POLY(METHACRYLIC ACID) AND HYDROXYAPATITE WITH APPLICATIONS IN DENTISTRY.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2016.01.061 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.
SYNTHESIS AND CHARACTERIZATION OF NEW COMPOSITE MATERIALS BASED ON POLY(METHACRYLIC ACID) AND HYDROXYAPATITE WITH APPLICATIONS IN DENTISTRY ANDREI – TIBERIU CUCURUZ1*, ECATERINA ANDRONESCU1, ANTON FICAI1, ANDREIA ILIE1, FLORIN IORDACHE2 1
2
POLITEHNICA University of Bucharest, Faculty of Applied Chemistry and Material Science; 1-7 Polizu Str., 011061 Bucharest, Romania
Institute of Cellular Biology and Pathology “Nicolae Simionescu”of Romanian Academy, Department of Fetal and Adult Stem Cell Therapy, 050568, Bucharest, Romania *
Corresponding author: Andrei CUCURUZ, POLITEHNICA University of Bucharest, 1-7 Gh. Polizu str., 011061 Bucharest, Romania, e-mail: [email protected] Graphical abstract
1
Abstract The use of methacrylic acid (MAA) in medicine was poorly investigated in the past but can be of great importance because the incorporation of hydroxyapatite (HA) can lead to new composite materials with good properties due to the strong electrostatic interactions between carboxylate groups of polymer and Ca2+ ions from HA. The scope of this study was to determine the potential of using composite materials based on poly(methacrylic acid) (PMAA) and hydroxyapatite in dentistry. Two routes of synthesis were taken into account: i) HA was synthesised in situ and ii) commercial HA was used. Fourier transform infrared spectroscopy and X-ray diffraction were used for compositional assessments. Scanning electron microscopy was performed to determine the morphology and differential thermal analysis (DTA) coupled with thermogravimetric analysis (TG) was used to study the thermal behaviour and to observe quantitative changes. In-vitro tests were also performed in order to evaluate the biocompatibility of both PMAA/HA composites by monitoring the development potential of human endothelial cells using MTT assay and fluorescent microscopy. Keywords: poly(methacrylic acid), hydroxyapatite, biocompatibility, dental material .
1. Introduction Nanocomposites are a promising new class of advanced materials that are made of two or more components in which at least one of them has one or more dimensions (width, length, thickness) in nanoscale. Polymethacrilic acid based nanocomposites exhibit enhanced optical thermal, mechanical and electrochemical properties compared to the conventional composites [1]. The synthesis of high-performance dental composites made from an organic matrix and inorganic filler/reinforcing agent has attracted interest both for research as well as for the applicative purposes. This can be explained by combining the advantageous properties of inorganic nanoparticles with those of organic polymers. Nevertheless, producing polymer nanocomposites with optimal properties is a challenge for the researchers due to surface energy differences between nanoparticles and polymers. Thus, nanoparticles surface modification is many times required especially for biological properties to overcome the incompatibility issue and to improve interactions between inorganic nanoparticles and polymer which leads to a good nanoparticle dispersion as well as better properties for polymer nanocomposites. Traces of unreacted monomers lead to side effects and poor properties of the final products which are not acceptable in the medical field [2]. Nowadays, dental composites have been accepted as the restorative material of choice; as compared to dental amalgams, the composites have better aesthetics, less safety concerns and have demonstrated good clinical results. Dental composites consist of resin matrices and different inorganic fillers [3-10]. Calcium phosphates are mainly used as bone substitutes in biomedical applications due to their biocompatibility, low density, chemical stability and their compositional resemblance to the mineral phase of bone [11-18]. Hydroxyapatite (HA) is the prime component of bone cements because it can be characterized as osteoconductive, biocompatible, noninflammatory, nontoxic, nonimmunogenic agent and bioactive, hence the
2
ability to form chemical bonds with living tissues. However, the poor strength of HA restricts its clinical applications under loadbearing conditions [19]. Methacrylic acid is a colourless, viscous organic compound with the chemical formula C4H6O2 whose use in dentistry was poorly investigated so far. In order to use poly(methacrylic acid) (PMAA) in dental applications, is mandatory to reinforce the polymer with ceramic particles [20]. Also, the incorporation of hydroxyapatite to PMAA can confer bioactivity to the composite [21]. For these reasons, polymer matrices reinforced with bioactive phases such as HA, combine the typical bioactive behavior of bioceramics with enhanced mechanical properties and make the composites comparable with human hard tissues [22]. Resin composites have a large area of applications in dentistry such as restorative materials, crowns, cements for tooth prostheses, cavity liners, pit and fissure sealants, cores, inlays, provisional restorations, endodontic sealers, and root canal posts. Because of their versatility, the use of this type of materials will continue to grow in frequency and application. The fast moving process of these materials suggests constant changes in this field [23]. In this study, polymethacrylic acid/HA composite materials were prepared via in situ precipitation/radical polymerization method.
2. Experimental 2.1. Materials Methacrylic acid (MAA) purrum monomer ≥98% and sodium hydroxide pellets ACS reagent ≥ 97% were supplied by Sigma-Aldrich. Hydroxyapatite (HA) puriss was supplied by Riedel-de Haën. Benzoyl peroxide (initiator) purrum ≥ 97% was supplied by Fluka. The precursors for hydroxyapatite: calcium hydroxide 98 % extrapure was supplied by Acros Organics and monosodium phosphate was supplied by Chimopar. The human endothelial cells line (EAhy923, ATCC, Virginia, USA) was used to evaluate the biocompatibility of PMAA/HA composites. The cells were cultured in DMEM medium (Sigma-Aldrich, Missouri, USA) supplemented with 10% fetal bovine serum, 1% penicillin and 1% streptomycin antibiotics (Sigma-Aldrich, Missouri, USA). To maintain optimal culture conditions, medium was changed twice a week. 2.2. Methods The composite materials based on PMAA and HA were synthesised starting from methacrylic acid and i) in situ obtained HA and ii) commercial available hydroxyapatite. The synthesis procedure for both types of composites is described below in Figure 5. The composites made with in situ synthesized hydroxyapatite were obtained from calcium hydroxide (Ca(OH)2) suspension and monosodium phosphate (NaH2PO4.2H2O) solution. First, the monomer was introduced, then the precursors and finally, the initiator – benzoyl peroxide, in order to begin the radical polymerization reaction. The polymerization process lasted for 3 hours at 90°C. The pH of the entire volume was fixed to 7.5 with NaOH solution 0.1 N, and then the sample was centrifuged, dried and grinded. At the end, the samples were subjected to the freeze-drying process of lyophilization, a conditioning technique that does not cause denaturation of the materials. A molar ratio of 1.67 between Ca/P was used for the precipitation of hydroxyapatite. For the composites made with commercial hydroxyapatite, the synthesis procedure is the same as for the synthesized hydroxyapatite mentioning that first was introduced a
3
minimum volume of distilled water and then commercial hydroxyapatite, monomer and the catalyst. The mass ratio between the polymer matrix and the inorganic compound was 1:1. Also, it was used a content of 1% of initiator reported to the monomer. The microstructures, composition and thermal behaviour of the synthesized composite materials were determined by FT-IR, XRD, SEM and DTA-TG. Also, pH and conductivity time evolution in distilled water and simulated body fluid were determined and biocompatibility was evaluated by monitoring the potential for cellular growth. IR spectroscopic measurements were performed using a Shimadzu 8400 FT-IR spectrometer. The spectra were recorded on a wavenumber range of 400–4000cm-1 with a resolution of 2cm-1. X-ray diffraction analysis was done using a Shimadzu XRD 6000 diffractometer at room temperature. It was used the Cu Kα radiation from a Cu X-ray tube. The samples were scanned in the Bragg angle, 2θ range of 10 – 87o at a scan rate of 2o.min−1. All samples were ground to fine powders before analysis. XRD analysis is useful to identify crystalline phases and to determine their concentrations. Scanning Electron Microscopy (SEM) was used to determine the morphology and was also useful to show the compatibility between phases. SEM images were taken with a HITACHI S2600N with an EDAX probe. All samples were covered with a silver layer prior to imaging. Differential thermal analysis (DTA) coupled with thermogravimetric analysis (TGA) was performed in air atmosphere with a Shimadzu DTG-TA-50H equipment at a heating rate of 10oC. Thermal analysis was used to determine the behaviour of the composites and to observe quantitative changes by increasing temperature. Conductivity and pH measurements were performed with an Eutech Instruments Cyberscan PCD 6500 pH/conductivity meter. The cytotoxicity and biocompatibility of human endothelial cells on PMAA/HA was assessed using MTT assay (CellTiter 96® Non-Radioactive Cell Proliferation Assay, Promega, Wisconsin, USA). Briefly, the human endothelial cells were grown in 96-well plates, with a seeding density of 3000 cells/well in the presence of PMAA/HA for 72 hours. Then, 15 ml Solution I was added and incubated at 37°C for 4 hours. After that, Solution II was added and pipetted vigorously to solubilize formazan crystals. After 1 hour, the absorbance was read using a spectrophotometer at 570 nm (TECAN, Männedorf, Switzerland). Furthermore we use a second method to evaluate the biocompatibility of these composite materials based on fluorescent microscopy using a RED CMTPX fluorophore (Life Technologies, Invitrogen, USA), a cell tracker for long-term tracing of living cells. The CMTPX was added in the cell culture and after 3 days, the viability and morphology of the endothelial cells was observed. The CMTPX fluorophore was added in the culture medium at a final concentration of 5 μM, incubated for 30 min in order to allow the dye penetration into the cells. Then, the endothelial cells were washed with PBS and visualized by fluorescent microscopy. The photomicrographs were taken with a digital camera driven by Axio-Vision 4.6 software (Carl Zeiss, Germany).
3. Results and discussions 3.1. Fourier transform infrared spectroscopy – FTIR
4
FTIR is an essential tool for characterizing materials being applicable for both crystalline and amorphous materials. Both composite materials (Figure 6) exhibit the main absorption bands of phosphate groups [24]. The shift of some vibrations of phosphate groups can be attributed to different interactions between organic and inorganic components, the stronger shift corresponding to stronger interactions. Based on this fact, it can be concluded that in situ formation of HA followed by polymerisation leads to a slightly better interaction between the two phases, the shift of the ν3 (PO43-) band being ~20cm-1 for the composite material synthesized in situ compared with the composite material obtained with commercial HA (the shift is only ~19cm-1). The spectrum of the PMMA/HA sample obtained by in situ precipitation of HA exhibits the vibration of the carbonate group, this result being also visible for other HA based materials [25]. Based on the absence of the characteristic peak of C=C double bond from around 1633cm-1 (vinyl group, – CH=CH2) as well as the deformation bands from ~900 and 1000cm-1 (vinyl group, =C-H) assigned to the monomer, it can be assumed that the polymerization has occurred with a high conversion degree. The two composite samples exhibit different FTIR spectra because of the distinct composition induced by synthesis route. The in situ precipitation of HA lead to a high degree of ionized carboxyl groups (COO-). The ionization of the carboxyl groups, the absorption band from ~1700cm-1 disappears and two new bands appear at 1544 and 1417cm-1 which are characteristic to asymmetric and symmetric vibrations of the R-COO- group. For the composite based on commercial HA, this ionization is not at all important because HA has a low solubility Ks and consequently the neutralization of COOH does not occur. Table presents the main characteristic bands of PMAA/HA composites.
3.2. X-Ray diffraction The crystalline phases were analysed by XRD, the results being complementary to the information obtained by FTIR (Figure 7). The diffractograms were useful in order to identify hydroxyapatite phases through the presence of diffraction peaks. Thereby, in situ formation of synthesized hydroxyapatite is confirmed according to ASTM [74-0566] and the presence of commercial hydroxyapatite complies with ASTM [72-1243]. Also, the sharp peaks of synthesized hydroxyapatite indicate a larger size of crystallites, compared to the commercial hydroxyapatite and, more important, the crystallinity of PMAA/HAs is lower than that of PMAA/HAc.
Scanning electron microscopy Based on SEM images (Figure 8), some differences can be noticed. First of all, the microstructure of the two composite materials is quite different. The in situ formation of HA leads to a homogenous, fibrillar-like composite while, the mixing of monomer with commercial HA followed by polymerization leads to agglomerates and heterogeneities. The agglomerates are not uniform, have different spherical or polyhedral shapes and can reach up to 20µm, in length/diameter. Also a different behaviour in the electron beam can be observed, the sample obtained by in situ precipitation of HA being more stable.
Differential Thermal Analysis – DTA-TG
5
DTA-TG determinations were performed for both PMAA/HA composites in order to observe the thermal stability of the samples (Figure 1). TG analysis indicated no major differences between the two composites in what it concerns the thermal behaviour with a minor distinction regarding mass loss; 54.35% in case of PMAA/HAc and 40.94 % for PMAA/HAs. In the range 96-388°C, DTA curve for PMAA/HAc indicates two endothermic effects (at 173°C and 353°C) accompanied by mass loss that can be attributed to the decomposition of the organic component. PMAA/HAs composite shows an exothermic effect at 84°C and two endothermic peaks at 223 and 310°C. Also, in the range 550-750°C, PMAA/HAs exhibits an exothermic effect due to carbonate group, its presence being useful from the biocompatibility point of view. Figure 1.
3.3. Conductivity /pH measurements Conductivity and pH measurements were performed over 2 days both in distilled water and simulated body fluid (SBF). As can be seen from Figure 2, pH values have a gradual decrease in distilled water and tend to stabilize after 4 hours in case of PMAA/HAs composites and after approx. 6 hours for the PMAA/HAc composites. On the other hand, both composites tend to reach constant values of pH much later in SBF, after approx. 43 hours. PMAA/HAs induce the slightest change in pH which leads to lower stress on the tissue in which the implantation will occur, so lower inflammatory reactions. Figure 2.
As can be observed from Figure 11, conductivity of the composites immersed in distilled water has a gradual increase until it reaches the threshold after approx. 43 hours. The same can be noticed for immersion in SBF mentioning a more pronounced conductivity increase especially in the case of PMAA/HAs composites.
3.4. In-vitro cell development The viability of PMAA/HA composites was performed by MTT assay, based on the biochemical reactions that measure the metabolic activity of living cells. The MTT assay demonstrated that the human endothelial cells present a normal metabolism and growth in the presence of PMAA/HA. The measured values of absorbance at 570 nm showed that endothelial cells grow better on both tested composite materials. After 24h of incubation, the proliferation of endothelial cells was 2 times faster on modified surfaces comparing with the control. The same effect it was also observed after 72h of incubation (Figure 8). These results demonstrate the biocompatibility potential of both composites and confirm their applications as new surfaces for in vitro culture tissues development. The in situ synthesis of the PMAA/HA composite material is beneficial on long term because the cell proliferation is improved even compared with the PMAA/HAc, this result being a consequence of the in vitro behaviour of these composite materials. The fluorescent microscopy images confirm the viability biochemical test showing that the endothelial cell viability is maintained after 3 days in the presence of PMAA/HA. The endothelial cells retain the normal morphology, were adherent and have a relatively uniform distribution on all investigated surfaces (Figure 9). These results are in consent with other researchers that showed PMAA/HA composites
6
present good biocompatibility, osteoinductive properties and improve the surface compatibility between HA and cellular matrix [26]. Domingo et al., 2001 analyzed the behaviour in water as well as the mechanical and surface properties of composite materials containing HA designed for dental restoration. They demonstrated that the materials containing bisphenol-alpha-glycidyl methacrylate:triethyleneglycol dimethacrylate and micrometric-HA coated with citrate, acrylate, or methacrylate displayed the most favourable results [27]. Figure 3. Figure 4.
4. Conclusions In this work, two PMAA/HA composite materials with different microstructures were obtained; the microstructural differences being induced only by the synthesis route. It is expected that such differences can strongly modify the mechanical properties of the materials as well as their interaction with the body fluids. FT-IR analysis indicated that are no bands assigned to the monomer, which proves that polymerisation occured with a high conversion degree. SEM analysis revealed that in situ formation of HA leads to a more homogenous composite with a good distribution of HA particles in the polymer matrix while the commercial form of HA causes the appearance of agglomerates. In-vitro testing of PMAA/HA composites showed a great biocompatibility that could be quantified by the development of eukaryotic cells after 24, 48 and 72 hours. These results bring new insights in the research of biocompatible surfaces engineering and may lead to the development of new surfaces used for facilitating in vitro culture tissues development. Also, the analyzed composite materials indicated a good potential for application in dentistry as cements or resins. Further works will be dedicated to appropriate, comparative testing of these materials, focusing on their mechanical properties.
Acknowledgements This work has been funded by the Sectorial Operational Programme Human Resources Development 2007-2013 of the Romanian Ministry of Labour, Family and Social Protection through the Financial Agreement POSDRU/107/1.5/S/76903.
7
References: 1.
Yan Bao, Jian-zhong Ma. Polymethacrylic acid/Na-montmorillonite/SiO2 nanoparticle composites structures and thermal properties. Polym. Bull. (2011) 66:541–549. SpringerVerlag 2010. 2. Wantinee Viratyaporn, Richard L. Lehman. Spectroscopic analysis of poly(methyl methacrylate)/alumina polymer nanocomposites prepared by in situ (bulk) polymerization. J Mater Sci (2010) 45:5967–5972. Springer Science+Business Media, LLC 2010. 3. M. Kostic, N. Krunic, L. Nikolic, V. Nikolic, S. Najman, I. Kostic, et al.; Influence of Residual Monomer Reduction on Acrylic Denture Base Resins Quality; Hemijska Industrija 2011, 65(2), 171. 4. L.F. Zhang, Y. Gao, Q. Chen, M. Tian and H. Fong; Bis-GMA/TEGDMA dental composites reinforced with nano-scaled single crystals of fibrillar silicate; Journal of Materials Science 2010, 45(9), 2521. 5. J. Xu, Y. Li, T. Yu and L. Cong; Reinforcement of denture base resin with short vegetable fiber; Dental Materials 2013, 29(12), 1273. 6. W.C. Kuo, Y.S. Lin, C.L. Lin and J.C. Wang; Biomechanical Investigation of Long-span Glass-fiber-reinforced Acrylic Resin Provisional Fixed Partial Denture: a Finite Element Analysis; Journal of Medical and Biological Engineering 2012, 32(5), 357. 7. L.S. Acosta-Torres, I. Mendieta, R.E. Nunez-Anita, M. Cajero-Juarez and V.M. Castano; Cytocompatible antifungal acrylic resin containing silver nanoparticles for dentures; International Journal of Nanomedicine 2012, 7, 4777. 8. W.L. Tham, W.S. Chow and Z.A.M. Ishak; Effects of titanate coupling agent on the mechanical, thermal, and morphological properties of poly(methyl methacrylate)/hydroxyapatite denture base composites; Journal of Composite Materials 2011, 45(22), 2335. 9. J.P. Zheng, Q. Su, C. Wang, G. Cheng, R. Zhu, J. Shi, et al.; Synthesis and biological evaluation of PMMA/MMT nanocomposite as denture base material; Journal of Materials Science-Materials in Medicine 2011, 22(4), 1063. 10. W. Viratyaporn and R.L. Lehman; Spectroscopic analysis of poly(methyl methacrylate)/alumina polymer nanocomposites prepared by in situ (bulk) polymerization; Journal of Materials Science 2010, 45(21), 5967. 11. M. Hautamaki, V.V. Meretoja, R.H. Mattila, A.J. Aho and P.K. Vallittu; Osteoblast response to polymethyl methacrylate bioactive glass composite; Journal of Materials Science-Materials in Medicine 2010, 21(5), 1685. 12. M.H. Fathi, A. Hanifi, V. Mortazavi. Preparation and bioactivity evaluation of bone-like hydroxyapatite nanopowder. Journal of materials processing technology 202 (2008) 536– 542. Elsevier. 13. A. Ilie, E. Andronescu, D. Ficai, G. Voicu, M. Ficai, M. Maganu, et al.; New approaches in layer by layer synthesis of collagen/hydroxyapatite composite materials; Central European Journal of Chemistry 2011, 9(2), 283. 14. A. Ficai, E. Andronescu, G. Voicu and D. Ficai. Advances in collagen/hydroxyapatite composite materials. In: Attaf B, editor. Advances in Composite Materials for Medicine and Nanotechnology: INTECH, 2011. 15. M. Ficai, E. Andronescu, D. Ficai, G. Voicu and A. Ficai; Synthesis and characterization of COLL-PVA/HA hybrid materials with stratified morphology; Colloids and Surfaces B: Biointerfaces 2010, 81(2), 614. 16. A. Ficai, E. Andronescu, G. Voicu, C. Ghitulica, B.S. Vasile, D. Ficai, et al.; Self assembled collagen/ hydroxyapatite composite materials; Chemical Engineering Journal 2010, 160(2), 794.
8
17. A. Ficai, E. Andronescu, G. Voicu, C. Ghitulica and D. Ficai; The influence of collagen support and ionic species on the morphology of collagen/hydroxyapatite composite materials; Materials Characterization 2010, 61(4), 402. 18. A. Ficai, E. Andronescu, G. Voicu, M.G. Albu and A. Ilie; Biomimetically synthesis of collagen/hydroxyapatite composite materials; Materiale Plastice 2010, 47(2), 205. 19. E. Dinu, M. Bîrsan, C. Ghiţulică, G. Voicu, E. Andronescu, Synthesis and characterization of hydroxyapatite obtained by sol-gel method, Romanian Journal of Materials, 2013, 43, 1, 55-60. 20. M. Ficai, E. Andronescu, A. Ficai, G. Voicu, B.S. Vasile, Poly bis-GMA/HA based hybrid composite materials, UPB Scientific Bulletin, Series B, 2011, 73, 75-84. 21. T.G. Tihan, M.D.Ionita, R.G. Popescu, D. Iordachescu; Effect of hydrophilic– hydrophobic balance on biocompatibility of poly(methyl methacrylate) (PMMA)– hydroxyapatite (HA) composites; Materials Chemistry and Physics 2009, 118, 265-269. 22. C.H. Navarro, K.J. Moreno, A. Chávez-Valdez, F. Louvier-Hernández, J.S. GarciaMiranda, R. Lesso, A. Arizmendi-Morquecho; Friction and wear properties of poly(methyl methacrylate)–hydroxyapatite hybrid coating on UHMWPE substrates; Wear 2012, 282-283, 76-80. 23. A. Ficai, E. Andronescu, C. Ghitulica, G. Voicu, V. Trandafir, D. Manzu, et al.; Colagen/Hydroxyapatite Interactions in Composite Biomaterials; Materiale Plastice 2009, 46(1), 11. 24. Jack L. Ferracane. Resin composite - State of the art (Review) - Dental materials 27 (2011) 29–38. Elsevier. 25. A. Ficai, M.G. Albu, M. Birsan, M. Sonmez, D. Ficai, V. Trandafir, et al.; Collagen hydrolysate based collagen/hydroxyapatite composite materials; Journal of Molecular Structure 2013, 1037, 154. 26. J. Gao, B. Huang, J. Lei, Z. Zheng. Photografting of methacrylic acid onto hydroxyapatite particles surfaces; Journal of Applied Polymer Science; 2010 (115) 2156– 2161. 27. C. Domingo, R.W. Arcís, A. López-Macipe, R. Osorio, R. Rodríguez-Clemente, J. Murtra, M.A. Fanovich, M. Toledano, Dental composites reinforced with hydroxyapatite: mechanical behavior and absorption/elution characteristics, J Biomed Mater Res, 2001, 56(2), 297-305.
9
FIGURES
Figure 5. Schematic representation for the synthesis routes of PMAA/HA composite materials
10
0.55 PMAA/HAc PMAA/HAs 0.50 0.45 0.40
Absorbance
0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 -0.05 4000
3500
3000
2500 2000 Wavenumbers (cm-1)
1500
1000
Figure 6. FT-IR spectra of PMAA/HA composite materials
800
PMAA/HAs PMAA/HAc
700
Intensity, a.u.
600 500 400 300 200 100 10
20
30
40
50
2 Theta Figure 7. XRD spectra of PMAA/HA composite materials
11
60
12
Figure 8. SEM images of the PMAA/HA composite materials at different magnifications: a. x100; b. x500; c. x1000; d. x5000.
Figure 9. DTA, TGA and DTG curves for: a) PMAA/HAc and b)PMAA/HAs
Figure 10. pH evolution of PMAA/HA composites in distilled water and simulated body fluid
Figure 11. Conductivity evolution of PMAA/HA composites in distilled water and simulated body fluid
13
Figure 12. Endothelial cells proliferation profiles after growing on control (green) and composite materials for up to 72 h
Figure 13. Fluorescence microscopic images of the cell monolayer treated for 72 h composite materials (x100): a) PMAA/HAs, b)PMAA/HAc and c) Ctrl
14
Table
Table 1. Main vibrations of PMAA/HA samples HA com.
PMAA/HAc
PMAA/HAs
Assignment
Band position, cm-1
568
560
556
ν4 (PO43-)
604
598
599
ν4 (PO43-)
1043
1024
1023
ν3 (PO43-)
15
