Saxena Et Al. 2019

Materials Research Express
ACCEPTED MANUSCRIPT
Optimization of the bio-mechanical properties of Ti-8Si-2Mn alloy by

1393B3 bioactive glass reinforcement
To cite this article before publication: Vipul Saxena et al 2019 Mater. Res. Express in press https://doi.org/10.1088/2053-1591/ab1280
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Page 1 of 28 AUTHOR SUBMITTED MANUSCRIPT - MRX-112842.R1
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3 Optimization of the bio-mechanical properties of Ti-8Si-2Mn alloy by 1393B3
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6 bioactive glass reinforcement
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8 Vipul Saxenaa, Vijay Kumara, Amrendra Raia, Rishikesh Yadava, Uttam Guptab,
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10 Vinay Kumar Singha, Partha Pratim Mannab
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Affiliations: a Department of Ceramic Engineering, IIT (BHU), Varanasi 221005, India
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b Immunobiology Laboratory, Department of Zoology, Institute of Science,
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17 Banaras Hindu University, Varanasi- 221005, India.
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Abstract
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22 The low cost Ti-8Si-2Mn alloy matrix has been synthesized by powder metallurgical route.
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24 The alloy is reinforced with 1393 B3 bioactive glass to optimize the mechanical and
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biological properties. The reinforced weight percentage of glass is 0% (S1), 5% (S2), 10%
(S3) and 20% (S4). The physical, mechanical, biological and corrosion properties of these
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31 composites were investigated. The cell culture suggests that composite S1 and S3 support
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33 the growth of the tumor cells when used at lower concentration. The hardness of samples
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decreases with the increasing percentage of reinforced glass; however, the respective
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38 hardness is still superior to the cortical bone. The composite S3 shows better compressive
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40 strength (339 MPa), low Young's modulus (76 GPa), optimum density (2.71 gm/cm3) and
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hardness (381 Hv) as compared to pre-existing Ti implants. So it can be proposed as a new
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45 novel load bearing bio implant which will potentially reduce the stress shielding effect,
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47 toxicity and will improve the Osseo conductivity.
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AUTHOR SUBMITTED MANUSCRIPT - MRX-112842.R1 Page 2 of 28
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3 Keywords: Alloys; Bioactive glass; Composites; Corrosion/corrosion resistance; Young's
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6 modulus; Cell culture.
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1. Introduction
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15 The rise in population and the use of technology in daily life have raised demand for a
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17 better aspect of life which puts pressure on humans to do daily routine activities in a rapid
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manner. This leads to degradation of physio-mechanical properties of the bones. So the
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22 need of novel implant biomaterials for soft and hard tissues is continuously increasing.
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24 Within the above domain, the metallic materials, e.g., Co-Cr alloys, 316 stainless steel,
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titanium (Ti) alloys are widely used as load-bearing implants (hard tissues). [1] Among the
above metallic materials, commercial pure titanium (C.P. Ti) and its alloy have attracted
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31 great attention in the medical field due to their biological compatibility and good corrosion
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33 resistance. These characteristics are because of the natural formation of a protective and
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stable oxide layer on the surface of the implant. [2,3].
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38 Currently Ti-6Al-4V alloy is used in the implant because of its enhanced
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40 mechanical properties, e.g., tensile strength, hardness, yield strength, and high resistance to
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corrosion in biological environments. [4] Despite these advantages, there are some pre-
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45 existing problems in Ti-6Al-4V alloy like high Young’s modulus as compared to cortical
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47 bones and the expensive cost of the implant [5]. The mismatch between the elastic
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49 modulus of the cortical bone and implant causes stress shielding, which results in
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52 weakening of bone. Further, literature suggests that vanadium (V) and aluminum (Al)
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54 released in the Ti-6Al-4V alloy may cause allergic reaction and neurological disorders like
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56 Alzheimer’s disease. [6, 7, 8].
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3 To overcome these drawbacks, one of the ways is to replace the toxic elements in
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6 titanium alloy with non-toxic elements. Earlier works suggest that titanium may be alloyed
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8 with elements like Cr, Mo, Zr, Mn, which can lead to the solid solution hardening, as well
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10 as lowering the melting temperature. [9, 10]. By decreasing the melting temperature of
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titanium, it is possible to reduce its reactivity with oxygen. In response to these facts,
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15 titanium alloys free from Al and V such as Ti-Mo [11, 12], Ti-Cr [13, 14], Ti-Sn [15, 16],
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17 Ti-Zr [17, 18] have been developed recently for medical implants.
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Ti–Si alloy is a good choice for the implant materials in tissue and damaged organs
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22 because of its good biological compatibility. The phase diagram of Ti-Si alloy reveals that
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24 the melting temperature of titanium (16700 C) decreases with an increasing percentage of
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26 Si and reaches approximately 13300C at the eutectic composition (8.3 wt % of Si) [19, 20].
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Manganese (Mn) is a low-cost material which reduces α to β transformation temperature of
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31 titanium and is considered as a β stabilizer element. It also positively affects the
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33 remodeling of the bone, osteogenesis and other functional mechanisms in the body. It has
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been found in the literature that Mn concentration less than 8 wt % in titanium shows
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38 negligible effects on the cell proliferation of osteoblasts cells and on the metabolic activity.
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40 So it could be used as an alloying element in a smaller amount for biomedical applications.
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[21, 22].
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45 The production of Ti alloys is difficult because of its high reactivity at elevated
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47 temperature as well as the possibility of contamination. Ti alloys production via powder
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49 metallurgy (PM) route is preferred because of the ability to produce net-shape components.
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52 In powder metallurgy route, the mechanical alloying is achieved by planetary ball mill
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54 where high-energy collision occurs among the balls to induce diffusion at the atomic level.
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56 This increases the limit of solid solubility in the alloy.
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3 The efforts are being made to improve the osseointegration and host response by altering
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6 the surface of the titanium[24], In spite of all the above facts, it is still difficult to achieve a
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8 chemical bonding with the bone tissue and form a new bone on its surface. To overcome
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10 this issue, the implant surface had been coated with bioactive materials like
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hydroxyapatite, bioglass, etc. These materials have an excellent capacity to bond with the
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15 living tissues. [5, 23]. But the problems associated with the coated alloys are poor
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17 adhesiveness, non-uniform coating and the difference in the thermal coefficient of the
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metal substrate and coating material, [25]
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22 Bio-active glasses are the richest class of biomaterials which are commonly based
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24 on borate, silicate, and amorphous phosphate structures. [26] Hench has invented the first
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bioactive silicate glass known as 45S5 Bioglass which has good bonding characteristics
with the bone and also encourages the growth of bone. [27-28]. The silicate glasses have
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31 the slow rate of degradation due to SiO2 rich layer formed within the body, whereas the
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33 borate base glass like 1393 B3 has the high rate of degradation because of high leachability
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[26, 29]. Along with the fast degradation rate, it shows good bioactivity within the cells.
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38 However, the low level of strength and brittle nature of glasses puts the limit on its
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40 application in soft tissues. [30, 31].
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Current work comprises the synthesis of newly developed low cost Ti-8Si-2Mn
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45 alloy matrix by powder metallurgical route which is reinforced with 1393 B3 bioactive
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47 glass in the percentage range of 5 - 20%. Borate bioactive glass has been chosen because
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49 of the quick degradation and its conversion into hydroxyapatite. The main aim of current
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52 work is to optimize the mechanical and biological properties of 1393 B3 bioactive glass to
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54 match the resultant biomechanical properties with the cortical bone and to remove the
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56 drawback of coated alloys by incorporating the glass into the matrix.
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2. Experimental
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3 2.1 Raw Material Used
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6 Commercially pure titanium (98%, Loba Chemie) Silicon (99%, Loba Chemie) and
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Manganese (98%, Loba Chemie) metal powders were used in this study for the preparation
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11 of alloy powder. AR grade Quartz(99% SiO2 ), Magnesium Oxide (99%MgO) Calcium
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13 Carbonate (99%CaCO3), Sodium Carbonate (99%Na2CO3) , Potassium carbonate
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15 (98%K2CO3) and Boric acid (99%B2O3) raw chemicals were used to synthesize the
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18 bioactive glass. Reagent-grade chemicals of NaCl, NaHCO3, KCl, K2HPO4 3H2O, MgCl2
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20 6H2O, CaCl2, and Na2SO4 tris ((CH2OH) 3CNH2) and 1 M hydrochloric acid are used for
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22 the preparation of simulated body fluid for the testing of samples. Deionized water was
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used for the preparation of simulated body fluid and in washing the precipitates.
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2.2 Alloy Preparation
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30 Titanium (Ti) metal powder is mechanical alloyed (MA) with eight weight percent of
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Silicon (Si) and two weight percent of Manganese (Mn) metal powders in a high energy
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35 ball mill containing stainless steel vial along with tungsten balls for the synthesis of
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37 titanium alloy powder at room temperature. The powder-to-ball weight ratio was
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39 maintained at 10:1. The vial of the mill was loaded with powder in an argon-filled glove-
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42 box to prevent oxidation.
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The mill was run up to 15 h for powder alloy preparation. Because of the ductility of metal
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47 powders; the milling is carefully controlled by adding a small amount of process control
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49 agent, (PCA), i.e., two weight % ethanol to the milling process was added. The PCA
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enhance milling efficiency by refreshing metal surface contacts. It also reduces the welding
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54 of the powder particles to each other and prevents the oxidation and contamination of
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56 material. [32].
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59 2.3 Glass Preparation
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3 The glass batch containing following composition 56.6 % B2O3,18.5 % CaO, 5.5%Na2O,
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6 11.1% K2O,4.6 %MgO,3.7% P2O5, as weight percentage were prepared taking the starting
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8 material as Quartz(99% SiO2 ), Magnesium Oxide (99%MgO) Calcium Carbonate
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10 (99%CaCO3), Sodium Carbonate (99%Na2CO3), Potassium carbonate (98%K2CO3) and
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Boric acid (99%B2O3) . The materials were mixed for 30 minutes in a mortar pestle. These
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15 reagents were then melted in a platinum crucible for 4.5 h in an electric furnace at 13000C.
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17 Further, the glass melt was water quenched. Resultant glass was crushed and ball milled in
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the pot mill to bring in the powder form.
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22 2.4 Composite preparation
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24 The mechanically alloyed metal powder matrix was reinforced with 5, 10 and 20
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percentage of melt-quenched 1393 B3 bioactive glass (Table 1) and ball milled for 20
minutes for homogeneous mixing, and 0.3 % of carboxyl methyl cellulose (CMC) is added
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31 as an organic binder. A uniaxial cold press machine is used to consolidate the composites
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33 under the pressure of 450 MPa in the form of bars and pallets. The green compacts were
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pre-sintered first at 7000C for 1 h for binder removal and then sintered in a high vacuum
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38 tube furnace with the holding time of 4 h at 1250 °C with the heating rate of 10 °C/min.
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40 The cooling is achieved first at 10 °C/min till 900°C and then allowed to cool at a faster
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rate of 40°C/min.
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48 Sample S1 S2 S3 S4
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50 Alloy (Ti-8Si-2Mn) 100 95 90 80
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52 BAG (1393 B3 ) 00 05 10 20
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55 Table 1 Alloys –Bioactive glass Composite composition
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57 3. Characterization
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1
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3 Major phases in the composites were identified by compact powder XRD machine
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6 (Rigaku, Japan) using copper Kα radiation. Phase identification was completed by
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8 comparing the respective powder XRD patterns with the standard JCPDS database (PDF-2
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10 Database 2003).
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For microstructure determination, all samples were polished by emery papers of grades
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15 1/0, 2/0, and 3/0 and then polish with the diamond paste of grade 1/4-OS-475. Samples
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17 were chemically etched with Kroll solution: 3 ml HF, 6 ml HNO3, 100 ml H2O and
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Scanning Electron Micrograph (SEM) were carried out using INSPECT 50 FEI instrument.
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22 Compressive strength measurements were done according to ASTM C78M using a
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24 universal testing machine at a strain rate of 2 mm/min at room temperature. Elastic moduli
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were evaluated by ultrasonic measurement gauge (45MG, Olympus, USA), by measuring
density, velocities of the longitudinal and transversal wave are measured by ultrasonic
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31 pulse-echo technique. These measurements were taken with the help of the piezoelectric
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33 transducer (10 MHz), which makes contact with the sample via coupling gel. Vickers’s
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hardness of the samples was evaluated by the indentation method with the help of
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38 Vickers’s hardness tester (RVM 50) under the load of 1 kgf, and indentation is seen under
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40 an optical microscope. For all mechanical testing, four samples for each group (S1, S2, S3,
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and S4) are tested, and the mean and standard deviation were calculated. The results were
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45 shown as a mean value ± SD with the corresponding error bars.
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49 Archimedes method is used for the density measurement as it is more reliable (ASTM
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52 C373-88) and simple. The samples were immersed in water with the help of steel wire.
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54 Following mathematical expressions were used for the calculation of Bulk density and
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56 apparent porosity [33].
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59 W−D
60 Apparent porosity (A.P %) = X 100, Where W=suspended weight in gm
W−S
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3 D
4 Bulk Density (B.D gm/cm3) = W−S S= Soaked weight in gm
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6 D=Dry weight in gm
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9 All the samples were immersed in SBF for bioactive analysis. The SBF was prepared
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12 according to Kokubo et al. [34] method which contains the same inorganic ion
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14 concentrations as presents in human body fluid. The preparation of SBF was carried out at
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16 37°C by using all analytical grade reagent NaCl, KCl, NaHCO3, MgCl2·6H2O, CaCl2 and
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19 KH2PO4 in distilled water. TRIS (tris-hydroxy methyl amino methane) and 1N HCl are
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21 used as a buffering agent to kept pH value at 7.2. In the sterilized container, the composites
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23 were immersed in SBF and incubated for seven days at 37 °C. The pH of SBF was
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measured regularly for seven days using a universal Bio-microprocessor pH meter. The
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samples were further dried at 100 °C for 1 h and were subject to SEM (Inspect S50, FEI)
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33 The corrosion study has also been done with the help of weight loss method, where the
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composites were dipped into SBF solution at 370C for 42 days. Initially, the weight (dry
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38 weight) of all the samples was taken, and after immersion, the samples were taken out of
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40 SBF one by one at a time after 24, 48, 72, 96, 120, 144, 192, 216 and 240 hours. They were
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washed with the tap water to remove any corrosion products, and the weight (wet weight)
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45 of the samples were measured at a defined interval. The corrosion rate is calculated with
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47 the help of following mathematical expression [35].
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50 𝑊
51 Corrosion Rate in mili-meter per year =87.6
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𝐷𝐴𝑇
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54 Where A= Area of samples in cm2
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56 D= Density of the sample in g/cm3.
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The w=weight loss in mg
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3 T=Time of immersion in hours
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6 3.1 Cytotoxicity assay
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9 The lytic activity of the free compound S1, S2, S3, and S4 against the U2-OS cells was
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11 measured by cytotoxicity assay (CytoTox 96 cytotoxicity assay kit, Promega, USA) [36].
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13 Tumor target cells (5×103) were co-cultured in the presence of increasing concentrations of
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16 the indicated formulations in a 96 well plate. The cells were incubated for 18 hours in a
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18 CO2 incubator (370C, 5% CO2). Percentage of cytotoxicity was ascertained from the
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20 undermentioned formula:
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23 (Experimental - Effector Spontaneous - Target Spontaneous)
24 % Cytotoxicity = ×100
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3.2 Cell growth inhibition assay
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31 MTT assay was performed to analyze growth inhibitory potential for the samples S1, S2,
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34 S3 or S4 against the U2-OS. The treatment of tumor target cells (5×103 cells /well) has
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36 been carried out with serial concentrations of the compounds in a 96 well culture plate and
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38 incubated for 48 hours at 370C, 5% CO2. The proliferation of the tumor cells was also
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41 observed by MTT assay using Cell Titer 96 kit (Promega, USA). The absorbance (OD
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43 values) is measured at 570 nm in a micro plate reader (BioTek, USA) [37]. The
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45 undermentioned formula has been used for the calculation of percent inhibition of the
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tumor cells:
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51 Experimental OD570
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% Growth Inhibition =[1- ]×100

52 Target OD570
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55 The Experimental OD indicate the values of the tumor cells in the presence of the
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compounds S1-S4 whereas the Target OD represents the corresponding values of the tumor
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60 cells, grown alone in culture media.
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3 4. Result and Discussions
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6 4.1 Phase Analysis
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8 Fig.1 shows the X-Ray diffraction pattern of the samples after sintering. The X-ray pattern
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10 reveals that metal alloy without bioactive glass (S1) has hexagonal α Ti (PCPDF No
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892762) as a significant phase with some sort of BCC β Ti (PCPDF No-894913), Si
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15 (PCPDF No 800018) and α Mn (PCPDF No 892412). In addition to alpha and beta
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17 titanium, the brittle intermetallic compound Ti5Si3 (PCPDF No 893721) also formed. With
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the reinforcement of bioactive 1393 B3 glass in Ti alloy matrix (S2, S3, and S4), some
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22 additional peaks of Ti5P3 (PCPDF No.658413), Ti (Bo3) (PCPDF No.850168) are also
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24 observed in the pattern as a reaction between metal and glass constituents. XRD pattern of
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29 of reinforced BAG.
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composite suggests that the intensity of these peaks increases with the weight percentage
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49 Fig.1 XRD spectra of composites containing 1393 B3 bioactive glass
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4.2 Scanning Electron Microscopy (SEM)

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54 The SEM micrographs of composites are shown in Fig 2. Fig (2a) and (2b) shows the
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56 micrograph of pure alloy phase, where, the structure is plate-like with some sort of brittle
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58 intermetallic compound between them. The plate-like structure is due to the fact that
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3 during ball mill, material suffers random collision with steel balls and become flattered
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6 (plate-like) due to the ductility of metals. The lower amount of glassy phase which
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8 encapsulates the alloy (S2) is also visible in microstructures 2c and 2d where the
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10 reinforcement of BAG is small (5%). Figure 2 (e, f, g, h) reveal that the alloy particles are
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embedded more in the glassy phase as the percentage of bioactive glass changes from 10%
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15 (S3) to 20 % (S4). The more embedded metal particles in glassy phase signify the
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17 homogeneous distribution of melted glass on the surface of the composite. The enhanced
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non-homogenous pores having approx. 80-110 µm sizes with the reinforced percentage of
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22 bioactive glass are also clearly visible in micrographs.
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42 Fig.2 SEM micrographs of (a,b) pure alloy S1 (c,d)Alloy + 5%BAG S2 (e,f) Alloy
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44 +10%BAG S3 (g,h)Alloy + 20%BAG S4

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46 4.3 Physio-mechanical Properties
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Fig 3 (a) and 3 (b) shows apparent porosity and bulk density of the samples
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51 respectively. With the increasing amount of bioactive glass from 5% to 20%, the porosity
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53 of the synthesized porous samples increases while bulk density decreases. The maximum
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55 porosity i.e. 22 % is obtained from the reinforcement of sample containing 20 % of
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58 bioactive glass. This can be attributed to the increase in weight percentage of bioactive
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60 glass that replaces the Ti alloy matrix. A conceivable reason for this behaviour is that when
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3 bioactive glass replaces the original alloy matrix in composite, an interplay between
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6 particles of glass and alloy at interface increases. A little proportion of the reinforcing
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8 phase may occupy the internal voids of alloy matrix easily, so the densification affects by
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10 smaller amounts but as the percentage of reinforcement increases, the internal friction and
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resulting bridging effect during single-action pressing effectively suppresses the
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15 densification [38, 39]. This leads to lower density of the composites. The SEM
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17 micrographs (Fig 2) also confirm the discussion given above.
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38 Fig.3 (a) Apparent porosity of all the samples (S1, S2, S3, S4)
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59 Fig.3 (b) Bulk Density of all the samples (S1, S2, S3, S4).
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21 Fig.4.Vickers’s Hardness of all the samples (S1, S2, S3, and S4)
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24 The results of Vickers's hardness measurement of the reinforced alloy are shown in
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26 figure 4. The hardness of pure alloy matrix (S1) increases (422 Hv) as compared to
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commercial pure Ti and Ti6Al4V alloy because of the formation of intermetallic
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compounds in the composite. Further, the graph also reveals that hardness decreases with
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33 the reinforced percentage of bioactive glass, reaching to the value of 358 Hv at 20% of
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35 reinforcement but it is still superior to the hardness of the cortical bone [40]. Although the
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37 amount of intermetallic increases in composites S2, S3, and S4 which would have raised
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40 the hardness of the composites but due to increased porosity, the hardness decreases.
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3 Fig.5 Elastic Modulus of all the samples (S1, S2, S3, and S4)
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6 The measured elastic modulus of prepared composites is represented in fig.5. It
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8 shows that the pure alloy matrix (S1) possesses the elastic modulus of 97 GPa which is
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10 lower than the commercially available Ti-6Al-4V alloy (110 GPa) [41]. Fig. 5 also shows
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the modulus value decreases on increasing the reinforcement of 1393 B3 bioactive glass.
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15 The value reaches up to 68 GPa at 20 % reinforcement which is an essential requirement of
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17 the implant to minimize the stress shielding effect [42].
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34 Fig. 6 (a) Compressive Stress-Strain relationship for all the samples.
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37 Fig 6(a) shows the stress-strain relationship for the different composites for the
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39 compressive strength measurement. The corresponding compressive strengths of different
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41 samples in relation to the reinforcement percentage are depicted in fig 6(b). Compression
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43
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44 tests were performed up to the strain value of about 30%. Fig 6(b) shows that the pure
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46 alloy matrix (S1) has the maximum compressive strength of 418 Mpa which decreases up
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48 to 318 Mpa (S4) with the increased porosity, even though, it is superior to the human
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cortical bones.[43]
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22 Fig. 6 (b) Compressive Strength of all the samples (S1, S2, S3, and S4)
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4.4 In vitro Bioactivity
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To analyze the formation of hydroxyl carbonated apatite (HCA) layer on immersing the
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31 samples in SBF solution, the pH behavior of SBF solution has been noted down during
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34 different immersion time. Figure 7 shows the change of pH in different immersion time up
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36 to 5 days. It is clear from the graph that the pH value of all the composite samples (S2, S3,
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38 S4) except for S1, increases to 7.82, 8.30, 8.49 respectively within five days as compared
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41 to the initial pH (7.4) of SBF solution at 370C under physiologic conditions.
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59 Fig. 7 Variation of pH for all samples with respect to the immersing time
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3 The increase in pH in composites S2, S3 and S4 illustrate the dissolution of cations
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6 i.e boron ions and the network modifiers (such as Na+ and K+) from the surface of the
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8 glass.[44] The different value of pH after 5 day for the S1, S2 and S3 composites are due to
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10 the difference in weight percentage of bioactive glass in the composition. These cations
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from the glass along with the phosphate ions released from the SBF solution give rise to
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15 the formation of the hydroxyl appetite layer [45].
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42 Fig.8 SEM of (a) sample S1 (b) sample S2 (c) sample S3 (d) sample S4 (e) EDS spectra
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of S1 (f) EDS spectra of S3 after immersion in SBF for 5 days
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47 Elements Ca Na P Cl O Si Ti Mg Al Fe Mn
48 (Weight %)
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50 S1 2.09 13.33 1.1 12.46 31.41 1.59 17.28 1.32 1.56 7.66 10.20
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S3 29.00 24.60 19.00 11.00 08.40 04.20 03.80 -- -- -- --
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55 Table 2. EDS elemental analysis of sample S1 and S3 after immersion in SBF
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1
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3 The graph also depicts that the rate of change of pH also increases with the reinforcing
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6 percentage of 1393 B3 bioactive glass. The growth of hydroxyl carbonated apatite layer
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8 formed on the surface of samples can also be seen in SEM micrographs which are taken
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10 after five days of immersion (fig 8). The change of morphologies is seen in the
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micrographs when compared with the initial surface of samples (fig 2). The EDX spectra
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15 of S1 and S3 samples are shown in Fig 8 (e) and 8 (f ) respectively, and the corresponding
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17 elemental analysis was given in Table 2, which indicates the presence of Ca, P, Na, Cl, Si,
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and Ti element on the surface of the samples. The Ca to P ratio in the layer formed on the
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22 surface of the S3 sample calculated from table 2 is 1.52 which further confirms the
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24 formation of HCA layer on the surface. Fig 8 also shows that the irregular shape of
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carbonated-hydroxyl apatite on the surface of the samples and as the percentage of
bioactive glass increases in samples from 5 % to 20%, crystallization of HCA particles are
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31 grown better in the form of agglomerates.
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4.5 Cell culture
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38 Human osteosarcoma cells U2-OS were procured from American Type Culture Collection
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40 (ATCC), Manassas, USA and was maintained in RPMI 1640 (Invitrogen, Carlsbad, CA),
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supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 100 U/ml penicillin and
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45 100 μg/ml streptomycin (Invitrogen, Carlsbad, CA), henceforth considered as complete
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47 medium.
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49 Treatment of U2-OS cells with the increasing concentration of the compound S1, S2, S3,
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52 and S4 shows a dose-dependent augmentation of the cytotoxicity in the tumor cells (Fig.9
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54 A-D). Among the products, S1 and S3 are more tolerant compared to the other two
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56 formulations. Comparative analysis of the cytotoxicity at concentrations of 25 and 100 µg,
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1
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3 suggests that the compounds at a higher level are severely toxic to the cells and only
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6 relatively safe at the lower concentration tested.
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9 3.3 Statistical analysis
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12 For the comparison between the groups, unpaired student's t-test or one way ANOVA
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14 followed by Tukey's post hoc test have been used. The data represented as mean ± SD
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(standard deviation) and the test was conducted three times for each sample. The
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19 significant differences were considered for ‘p’ value < 0.05. p< 0.001 (****).
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47 Fig.9. The concentration-dependent killing of the U2-OS cells in the presence of S1,
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50 S2, S3, and S4.
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53 We tested the proliferation of the cells in the presence of varying concentration of the
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55 above compounds. Our data suggested that compound S1 and S3 supports the growth of
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the tumor cells at a concentration of 25 µg (Figure 10). However, both S2 and S4 are not
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60 tolerant to the tumor cells and hinder the proliferation of the cells. Increasing the
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3 concentration of the compound largely detrimental for the tumor cells growth for all the
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6 compounds tested (Figure 10).
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36 Fig.10. The proliferation of the U2-OS cells in the presence of varying concentration
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38 of S1, S2, S3, and S4.
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41 From the above results, it is clear that compounds are relatively safe at low concentration;
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44 however, at higher concentration the formulations may be detrimental for the cells.
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47 Besides the concentration-dependent kinetics, we have also performed time-dependent
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49 effect of a fixed concentration of the alloy/BAG complex against the U2-OS cells. At a
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concentration of 100 µg, the effect of the alloy/BAG complex was studied up to 72 hours.
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54 Our data suggested that as time progresses, the proliferation of the cells in the presence of
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56 the compound was affected.. The effect of the compounds on the dividing cells like U2-OS
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58 indicates that the alloy/BAG complexes are relatively tolerant to the cells and the
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1
2
3 compounds are cytocompatible (Fig 11). This also suggests that the compound may be
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6 used for the implant since osteocytes like U2-OS are present in association with bone
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8 tissues where the possible implantation could be made. This indicates that the compound
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10 may be biologically compatible for real-life scenario.
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34 Fig 11. Time dependent proliferation of the U2-OS cells in the presence of varying
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37 concentrations of S1, S2, S3, and S4.
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40 4.6 Corrosion Study
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43 The corrosion study has been carried out from weight loss method to study the effect of
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45 SBF on the prepared samples. Fig 12 depicts the relation between the corrosion rate of
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tested samples and immersion time. This graph shows that the composites S2, S3, and S4
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50 comprise 1393 B3 BAG initially have a high corrosion rate as compared to pure alloy S1.
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52 This is probably because of the fast degradation rate of borate bioactive glass which leads
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54 to the weight loss of the samples [46]. Due to this fact, initially the corrosion rate becomes
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high, but as the time passes, the rate of weight loss decreases and hence the corrosion rate
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59 decreases and approaches nearly to .0015 millimetre per year (mmpy).
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21 Fig.12 corrosion rate of pure alloy (S1) and all composites containing BAG (S2, S3,
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24 S4)
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5. Conclusions
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30 The effect of reinforcement of bioactive glass on the physical, mechanical, biological and
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32 corrosion properties of the biomedical composite was investigated and led to the following
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findings.
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38 1. With the increased percentage of 1393 B3 bioactive glass, the porosity of composites
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40 increases and attain the value of 11% (S2), 15%(S3) and 22% (S4) due to the friction
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42 between glass and alloy particles at the interface which will be beneficial in the biological
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fixation and Osseointegration.
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47 2. The Hardness of pure alloy S1 (422 Hv) increases as compared to Ti-6Al-4V alloy
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49 because of inclusion of intermetallic and decreases with an increasing percentage of
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reinforcement due to increasing porosity. However, the respective harnesses are still
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54 comparable to cortical bone and Ti-6Al-4V reported in the literature.
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56 3. Among the studied samples, S3 and S4 exhibit the significant reduction in Young's
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58 modulus (76 and 68 Gpa) as compared to the reported modulus (110 Gpa) of Ti-6Al-4V
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1
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3 alloy. This lower value of Young's modulus comes much closer to that of cortical bones
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6 which will be beneficial to reduce the stress shielding effect between implant and bone.
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8 4. The compressive strength of pure alloy S1 is 418 Mpa. The addition of bioactive glass
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10 05% BAG (S2), 10%BAG (S3) and 20% BAG (S4) give the compressive strength of 323,
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339 and 318 Mpa respectively which are superior to cortical bone.
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15 5. HCA layer formation in SBF is more prominent in S3 and S4 composite where the
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17 reinforcement percentage reaches 10 % and 20 % respectively.
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6. The cell culture including cytotoxicity and proliferation essay suggest that composite S1
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22 and S3 are biocompatible and support the growth of the tumor cells at a lower
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24 concentration.
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7. Composites having bioactive glasses initially have a high rate of corrosion in SBF due to
the degradation of borate glass, and as degradation slows down, the corrosion rate reaches
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31 to the .0015 mmpy making the composite suitable for permanent implant.
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The above results infers that Composite S3 having 10 % reinforcement of 1393 B3
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38 bioactive glass in Ti-8Si-2Mn alloy matrix comes out to be optimized composition because
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40 of greater mechanical properties i.e. superior compressive strength (339 MPa), low
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Young's modulus (76 GPa), optimum density (2.71 gm/cm3) and hardness (381 Hv) as
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45 compared to pre-existing Ti implants and more closely matched mechanical and biological
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47 properties with that of cortical bone. So it can be proposed as a new novel load bearing bio
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49 implant which will potentially reduce the stress shielding effect, toxicity and will improve
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52 the Osseo conductivity.

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Tables and figures caption:
5
6 List of Table
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8 Table 1. Alloys –Bioactive glass Composite composition.
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11 Table 2. Elemental analysis by EDS after SBF immersion
12
13 List of Figures
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15 Fig.1 XRD spectra of composites containing 1393 B3 bioactive glass
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Fig.2 SEM micrographs of (a,b) pure alloy S1 (c,d)Alloy + 5%BAG S2 (e,f) Alloy
19
20 +10%BAG S3 (g,h)Alloy + 20%BAG S4
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22 Fig.3 (a) apparent porosity of all the samples (S1, S2, S3, S4)
23
24
Fig.3 (b) Bulk Density of all the samples (S1, S2, S3, S4).
25
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Fig.4.Vickers’s Hardness of the all the samples (S1, S2, S3&S4)
Fig.5 Elastic Modulus of the all the samples (S1, S2, S3&S4)
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31 Fig. 6 (a) Compressive Stress-Strain relationship for all the samples.
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34 Fig. 6 (b) Compressive Strength of all the samples (S1, S2, S3 & S4)
35
36 Fig. 7 Variation of pH for all samples with respect to the immersing time
37
38 Fig.8 SEM of samples (a) sample S1 (b) sample S2 (c) sample S3 (d) sample S4 after
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40
41
immersion in SBF for 7 days
42
43 Fig.9. Concentration dependent killing of the U2-OS cells in the presence of S1, S2, S3
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45 and S4.
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47
48 Fig.10. Proliferation of the U2-OS cells in the presence of varying concentration of S1,
49
50
51 S2, S3 and S4.
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54 Fig.11 Time dependent proliferation of the U2-OS cells in the presence of varying
55
56 concentrations of S1, S2, S3, and S4.
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3 Fig.12 Corrosion rate of pure alloy (S1) & all composites containing BAG (S2, S3,
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6 and S4)
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Materials Research Express

Optimization of the bio-mechanical properties of Ti-8Si-2Mn alloy by

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30 for surface morphology.

% Growth Inhibition =[1- ]×100

4.2 Scanning Electron Microscopy (SEM)

44 +10%BAG S3 (g,h)Alloy + 20%BAG S4

52 the Osseo conductivity.

44 [27] Tripathi H, Rath C, et al. Structural, physico-mechanical and in-vitro bioactivity

52 material. Ceramics International 2015 41: 11756–11769.

57 phosphate glasses. J of Non-Crystalline Solids 2010; 356: 1432–1438.

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