Acta Polytechnica CTU Proceedings doi:10.14311/APP.2017.7.0029 Acta Polytechnica CTU Proceedings 7:29–32, 2017 © Czech Technical University in Prague, 2017 available online at http://ojs.cvut.cz/ojs/index.php/app DEFORMATION BEHAVIOUR OF GELLAN GUM BASED ARTIFICIAL BONE STRUCTURES UNDER SIMULATED PHYSIOLOGICAL CONDITIONS Nela Krčmářováa, ∗, Jan Šleichrta, Tomáš Fílab, Petr Koudelkab, Daniel Kytýřb a Czech Technical University in Prague, Faculty of Transportation Sciences, Department of Mechanics and Materials, Konviktská 20, 120 00 Prague 1, Czech Republic b Institute of Theoretical and Applied Mechanics AS CR, v.v.i., Prosecká 76, 190 00 Prague 9, Czech Republic ∗ corresponding author: krcmarova@fd.cvut.cz Abstract. The paper deals with investigation of deformation behaviour of gellan gum (GG) based structures prepared for regenerative medicine purposes. Investigated material was synthesized as porous spongy-like scaffold reinforced by bioactive glass (BAG) nano-particles in different concentrations. Deformation behavior was obtained employing custom designed experimental setup. This device equipped with bioreactor chamber allows to test the delivered samples under simulated physiological conditions with controlled flow and temperature. Cylindrical samples were subjected to uniaxial quasi-static loading in tension and compression. Material properties of plain GG scaffold and reinforced scaffold buffered by 50 wt% and 70 wt% BAG were derived from a set of tensile and compression tests. The results are represented in form of stress-strain curves calculated from the acquired force and displacement data. Keywords: gellan gum scaffold, reinforcement, uni-axial loading, simulated physiological conditions. 1. Introduction The worldwide incidence of bone disorders and con- ditions have trended steeply upward. Especially high income regions are expected twofold increase between 2010 and 2020 [1]. This is the tribute for populations aging coupled with improper nutrient consumption and poor physical activity. Globally more than 40 % of women and 30 % of men are at increased risk of emergence of bone disorders [2]. Annually in the USA only, more than half a million bone defects are re- ported. Worldwide the treatment cost reaches more than $2.5 billion. The bone disorders treatment using engineered bone tissue has been viewed promising and yet not fully exploited potential alternative to conventional use of autografts and allografts. Artificial tissue is overcom- ing problems with donor site morbidity, loss of bone inductive factors and/or resorption during healing [3]. In general, several essential demands are placed on artificial structure: i) chemical biocompatibility with- out toxic effect ii) reduction of the stress shielding effect iii) successful diffusion of nutrients and oxygen iv) controlled degradation and resorption [4]. Presented paper deals with uni-axial quasi-static testing of artificial spongy-like structure [5] proposed for bone tissue engineering purposes as a bone scaffold. The investigated gellan gum - bioactive glass (GG- BAG) material combines organic (polysaccharitic) component with inorganic (Silicon-Calcium based) nanoparticles. This approach effectively enables for adaptation of physical and mechanical properties of the synthesized material according to the desired ap- plication [6]. The studied material was subjected to quasi-static loading in both tension and compres- sion to evaluate its expected deformation response in interaction with human body. Therefore the experi- ment was carried out under simulated physiological conditions using bioreactor with circulating synthetic plasma. 2. Material Bioactive-glass-reinforced gellan-gum is a promis- ing material for wide use in bone tissue engineer- ing [7]. Originally the Gellan-gum (microbial ex- tracted polysaccharide) was used in food and phar- maceutical industry [8]. GG is composed of repeating units consisting of two D-glucose and one of each L- rhamnose and D-glucuronic acid [9]. Its main advan- tage is in ability to form highly porous 3D structures when properly cross-linked and fabricated [10]. Material investigated in this study was synthesized at Jozef Stefan Institute (Slovenia) as porous spongy- like structure buffered by bioactive glass (BAG) nano- particles [11]. During the production process gellan gum was dissolved in ultra-pure water by heating the solution for 30 minutes at 90 ◦C. To the hot GG solu- tion a dispersion of BAG was admixed and 0.18 wt% CaCl2 was added. Kept at high temperatures this mixture was then poured into required mould and let there to spontaneously jellify. Finally the samples were frozen at −80 ◦C and freeze-dried. 29 http://dx.doi.org/10.14311/APP.2017.7.0029 http://ojs.cvut.cz/ojs/index.php/app N. Krčmářová, J. Šleichrt, T. Fíla et al. Acta Polytechnica CTU Proceedings 3. Methods For initial awareness about deformation characteristics of synthesized material set of quasi-static experiments was performed. The first goal was to demonstrate possibilities of in house developed experimental infras- tructure for this purpose. Expected collapse forces was in range of single newtons and precise loading plate positioning was required as well. To obtain more relevant results some modifications of the experimen- tal devices in detail presented in 3.2 was carried out. Using this adapted devices basic material properties and stress–strain response were obtained. 3.1. Experimental procedure Cylindrical samples with height h = 8.6 ± 0.4 mm, diameter d = 5.0 ± 0.1 mm and weight m ≈ 11 mg, ≈ 16 mg and ≈ 24 mg for plain GG scaffold, GG- BAG reinforced scaffold with 50 wt% and 70 wt% BAG respectively were subjected to tensile and compres- sive loading under wet condition. For wet condition simulating physiological environment of human body infusion solution Plasmalyte (Bartex, Czech Repub- lic) was used. Loading plate displacement was set typically for 1000 µm corresponding to deformation approx. 11 − 12 % sufficient for significant sample damage. Loading rate was set to 2 µms−1. Force and position was read-out with sampling frequency 50 sps. 3.2. Instrumentation In house developed indentation device for low-force indentation was adapted for tensile and compression test. Originally the device was designed using mod- ular aluminum profile (30 × 30 mm) frame bearing i) X and Y motorized axis KK40 (HIWIN, Japan) for sample positioning with repeatability 10 µm ii) inden- tation axis based on linear stage MGW12 (HIWIN, Japan) and linear actuator 43 series (Haydon Kerk, USA) with position accuracy 3 µm and mounting for U9B/C series (HBM, Germany) load-cell. This axis was upgraded using the linear actuator with position accuracy 1.5 µm, encoder with resolution 0.5 µm and load cell with nominal force 50 N (the most precise force transducer in U9B/C series). For testing un- der wet condition testing device was equipped with bioreactor with controlled flow and temperature of circulating fluid. From the heated reservoir is the fluid pumped to the basin surrounding the samples and loading plates. 3.3. Strain calculation The investigated material exhibits very low stiffness, which, coupled with high porosity and suboptimal geometry of the samples, induces high potential for significant boundary effects. Unfortunately full-field optical strain measurement of the wet samples placed in fluid basin was not possible using the available setup. Therefore comparative measurements for contact and contactless strain evaluation methods was performed in previous studies [12, 13]. However production pro- cess of the GG-BAG samples does not allow to reliably produce cylindrical samples, the diameter of specimens varied in average ±100 µm, and the loaded faces were rough and not plan-parallel strain-stress curve derived directly from force transducer and encoder indicate relevant results. 3.4. Stress calculation The stress σ in all experimental analysis was consid- ered as engineering stress obtained using σ = F Ac (1) where Ac is cross-sectional area of the specimen calcu- lated from minimal sample diameter measured before deformation. Force F was acquired by the load-cell. For the purpose of stress calculations, samples were considered ideally cylindrical, neglecting all geometri- cal irregularities. 4. Results Material properties and deformation behaviour of plain GG scaffold and GG-BAG reinforced scaffold with 50 wt% and 70 wt% BAG content were studied in tensile and compression tests under dry and wet conditions. Five experiments for each type of material and loading mode were performed. Young’s modu- lus was calculated using linear regression applied on the elastic part of stress–strain diagrams. The calcu- lated elastic properties and yield stresses are listed in Tabs. 2–6. sample E [kPa] GG00 1 88.588 ± 0.147 GG00 2 119.507 ± 0.156 GG00 3 126.159 ± 0.142 GG00 4 93.571 ± 0.144 GG00 5 141.089 ± 0.146 GG00 mean 113.783 ± 22.219 Table 1. Elastic properties of plain GG samples for compression test sample E [kPa] GG00 1 56.343 ± 0.843 GG00 2 33.403 ± 0.209 GG00 3 36.383 ± 0.412 GG00 4 69.077 ± 0.945 GG00 5 70.436 ± 0.951 GG00 mean 53.129 ± 17.562 Table 2. Elastic properties of plain GG samples for tensile test 30 vol. 7/2017 DEFORMATION BEHAVIOUR OF WET GELLAN GUM SCAFFOLD sample E [kPa] GG50 1 81.4518 ± 0.145 GG50 2 131.882 ± 0.137 GG50 3 70.8462 ± 0.141 GG50 4 113.892 ± 0.281 GG50 5 140.221 ± 0.201 GG50 mean 107.659 ± 30.528 Table 3. Elastic properties of GG samples with 50 wt% BAG for compression test sample E [kPa] GG50 1 24.406 ± 0.090 GG50 2 27.156 ± 0.085 GG50 3 24.287 ± 0.080 GG50 4 23.719 ± 0.081 GG50 5 21.376 ± 0.089 GG50 mean 24.18 ± 2.061 Table 4. Elastic properties of GG samples with 50 wt% BAG for tensile test sample E [kPa] GG70 1 134.424 ± 0.120 GG70 2 63.566 ± 0.077 GG70 3 79.592 ± 0.078 GG70 4 67.376 ± 0.083 GG70 5 65.131 ± 0.077 GG70 mean 82.018 ± 29.967 Table 5. Elastic properties of GG samples with 70 wt% BAG for compression test sample E [kPa] GG70 1 22.7629 ± 0.080 GG70 2 23.4423 ± 0.082 GG70 3 20.9057 ± 0.081 GG70 4 21.1495 ± 0.101 GG70 5 22.7019 ± 0.079 GG70 mean 22.192 ± 1.105 Table 6. Elastic properties of GG samples with 70 wt% BAG for tensile test All obtained results in form of enveloped stress– strain curves are plotted in Figs. 1, 2. The stress– strain area for each type of the scaffold represents minimum and maximum stress for each strain value. 5. Conclusion GG-BAG samples with 0, 50 and 70 wt% fraction of reinforcing BAG particles were subjected to tensile and compressive loading test to evaluate deformation response in simulated physiological condition. It was found out, that the ambient environment has signifi- cant influence on mechanical response of the material as the measured properties. The scaffolds wetted by 0 0.002 0.004 0.006 0.008 0.01 0.012 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 C o m p re s s iv e s tr e s s [ M P a ] Compressive strain [−] EGG00 = 0.113±0.022 MPa EGG70 = 0.082±0.029 MPa EGG50 = 0.107±0.030 MPa Figure 1. Stress–strain curves envelope of scaffolds under compressive loading 0 0.005 0.01 0.015 0.02 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 T e n s il e s tr e s s [ M P a ] Tensile strain [−] EGG00 = 0.053±0.017 MPa EGG50 = 0.024±0.002 MPa EGG70 = 0.022±0.001 MPa Figure 2. Stress–strain curves envelope of scaffolds under tensile loading the synthetic plasma solution exhibited radical loss of stiffness, where the elastic modulae decreased more than ten times [12]. No significant reinforcement effect of BAG particles was observed during compression test. In case of tensile loading BAG buffered samples unexpectedly exhibited lower elastic modulae and ultimate stresses compared to the plain GG samples. That could be given by scaffold cell-wall heterogenity and disintegrity caused by rigid nature of glass particles. It can be concluded that presented analysis proved use of the considered experimental methods together with available experimental infrastructure for the test- ing of GG based scaffolds. The most limiting part of the experimental setup is load-cell signal-to-noise ratio at desired loading level and generally suboptimal geometrical characteristics of the samples inducing shear stresses during loading. Acknowledgements The research was supported by Grant Agency of the Czech Technical University in Prague (grant no. SGS15/225/OHK2/3T/16), by InterReg project Com3d- XCT (ATCZ38) and by institutional support RVO: 31 N. Krčmářová, J. Šleichrt, T. Fíla et al. Acta Polytechnica CTU Proceedings 68378297. We would like to express our special thanks to Ana Grantar for sample synthesization. References [1] W. H. Organization. World health statistics. WHO Press, 2015. [2] W. H. Organization. Global Recommendations on Physical Activity for Health. WHO Press, Switzerland, 2010. [3] A. R. Vaccaro, K. Chiba, J. G. Heller, et al. Bone grafting alternatives in spinal surgery. The Spine Journal 2(3):206 – 215, 2002. doi:10.1016/S1529-9430(02)00180-8. [4] A. R. Amini, C. T. Laurencin, S. P. Nukavarapu. Bone tissue engineering: Recent advances and challenges. Critical Reviews in Biomedical Engineering 40(5):363– 408, 2012. doi:10.1615/CritRevBiomedEng.v40.i5.10. [5] L. Polo-Corrales, M. Latorre-Esteves, J. E. Ramirez- Vick. Scaffold design for bone regeneration. Journal of nanoscience and nanotechnology 14(1):15–56, 2014. [6] E. R. Morris, K. Nishinari, M. Rinaudo. Gelation of gellan – a review. Food Hydrocolloids 28(2):373 – 411, 2012. doi:10.1016/j.foodhyd.2012.01.004. [7] M. Bououdina. Emerging Research on Bioinspired Materials Engineering. IGI Global, 2016. doi:10.4018/978-1-4666-9811-6. [8] D. Hoikhman, Y. Sela. Gellan gum based oral controlled release dosage forms- a novel platform technology for gastric retention, 2005. WO Patent App. PCT/IL2004/000,654. [9] J. T. Oliveira, L. Martins, R. Picciochi, et al. Gellan gum: A new biomaterial for cartilage tissue engineering applications. Journal of Biomedical Materials Research Part A 93A(3):852–863, 2010. doi:10.1002/jbm.a.32574. [10] N. Drnovšek, S. Novak, U. Dragin, et al. Bioactive glass enhances bone ingrowth into the porous titanium coating on orthopaedic implants. International Orthopaedics 36(8):1739–1745, 2012. doi:10.1007/s00264-012-1520-y. [11] A. Gantar, L. Da Silva, J. Oliveira, et al. Nanoparticulate bioactive-glass-reinforced gellan-gum hydrogels for bone-tissue engineering. Materials Science and Engineering C 43:27–36, 2014. Cited By 13, doi:10.1016/j.msec.2014.06.045. [12] D. Kytýř, T. Doktor, O. Jiroušek, et al. Deformation behaviour of a natural-shaped bone scaffold. Materiali in Tehnologije 50(3):301–305, 2016. Cited By 0, doi:10.17222/mit.2014.190. [13] J. Šleichrt, M. Adorna, M. Neuhäuserová, et al. Deformation characteristics of chopped fibre composites subjected to quasi–static tensile loading. Acta Polytechnica CTU Proceedings 3:71–74, 2016. doi:10.14311/APP.2016.3.0071. 32 http://dx.doi.org/10.1016/S1529-9430(02)00180-8 http://dx.doi.org/10.1615/CritRevBiomedEng.v40.i5.10 http://dx.doi.org/10.1016/j.foodhyd.2012.01.004 http://dx.doi.org/10.4018/978-1-4666-9811-6 http://dx.doi.org/10.1002/jbm.a.32574 http://dx.doi.org/10.1007/s00264-012-1520-y http://dx.doi.org/10.1016/j.msec.2014.06.045 http://dx.doi.org/10.17222/mit.2014.190 http://dx.doi.org/10.14311/APP.2016.3.0071 Acta Polytechnica CTU Proceedings 7:29–32, 2017 1 Introduction 2 Material 3 Methods 3.1 Experimental procedure 3.2 Instrumentation 3.3 Strain calculation 3.4 Stress calculation 4 Results 5 Conclusion Acknowledgements References