Construction novel hydroxyapatite-nitinol nanocomposite for hard tissue applications

Construction novel hydroxyapatite-nitinol nanocomposite for hard tissue applications

Accepted Manuscript Construction novel hydroxyapatite-nitinol nanocomposite for hard tissue applications Samaneh Kamali, Sepideh Shemshad, Alireza Kha...

4MB Sizes 0 Downloads 92 Views

Accepted Manuscript Construction novel hydroxyapatite-nitinol nanocomposite for hard tissue applications Samaneh Kamali, Sepideh Shemshad, Alireza Khavandi, Shahram Azari PII:

S0254-0584(18)30742-9

DOI:

10.1016/j.matchemphys.2018.08.077

Reference:

MAC 20920

To appear in:

Materials Chemistry and Physics

Received Date: 3 April 2018 Revised Date:

22 July 2018

Accepted Date: 24 August 2018

Please cite this article as: S. Kamali, S. Shemshad, A. Khavandi, S. Azari, Construction novel hydroxyapatite-nitinol nanocomposite for hard tissue applications, Materials Chemistry and Physics (2018), doi: 10.1016/j.matchemphys.2018.08.077. 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.

ACCEPTED MANUSCRIPT

Construction Novel Hydroxyapatite-Nitinol nanocomposite for hard tissue applications Samaneh Kamali*a, Sepideh Shemshad b,Alireza Khavandi c, Shahram Azarid Masters student, department of Material Engineering, Iran University of Science and Technology, Tehran, Iran, Email: [email protected]. Phone numbers: 989136666335 b

RI PT

a

Masters student, department of Material Engineering, Iran University of Science and Technology, Tehran, Iran c Professor,

department of Material Engineering, IRAN University of science and Technology, Tehran, Iran. d

National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran.

SC

Abstract

Keywords

EP

TE D

M AN U

Natural Hydroxyapatite (HA)- Nitinol (NiTi) nanocomposites with different percentage of NiTi were fabricated by powder metallurgy. The structural stability of HA phase in HA- NiTi samples and mechanical strength of nanocomposites were studied by FTIR, XRD and compression test. In addition, biological behavior of the composites were investigated by in vitro studies. The existence of NiTi metal phase can promote dehydration and decomposition of HA ceramic phase into more stable calcium phosphate phases at high temperatures. According to results that obtained, the nanocomposite sample with 10wt.% NiTi has the maximum compressive strength (67.67 MPa) compare to pure HA ceramic (46 MPa) that manufactured under the same condition. Crack deflection is the chief strength mechanism in the nanocomposites. in vitro studies showed that MG-67 osteoblast cells attached and spread on the surface of the sample. The results revealed that nanocomposite with 10wt.% NiTi has a good mechanical strength and suitable biological behavior that can be used in medical applications.

AC C

Hydroxyapatite, Nitinol, ceramic, nanocomposite

1. Introduction

Hydroxyapatite (HA, Ca10(PO4)6(OH)2) is a unique ceramic material with chemical composition and crystallographic structure similar to the mineral phase of the bone[1]–[3]. It has excellent properties such as bioactivity, biocompatibility and the ability to form a strength bone- bond with other tissues through an osteoconductive mechanism[4]–[6]. Therefore, in terms of biocompatibility, hydroxyapatite seems an appropriate ceramic for hard tissue replacement[7]. But, its brittleness and low mechanical strength in tension, prevent its clinical use as

ACCEPTED MANUSCRIPT

RI PT

long term load- bearing applications[1], [2], [8]. There are several methods to improving the mechanical properties of HA. Such as, fabricate HA ceramic composites or decreasing the grain size (in the range of nano)[7], [9], [10]. Thus, making HA nanocomposite can achieve the new materials with Proper mechanical properties For bone replacement applications[11].

SC

In recent years, many reinforcements, including 316L stainless steel[5], Zirconia[12], bioactive glass[13], alumina[14] and titanium[7] have been used in HA ceramic to improve its mechanical reliability. Among them, metal reinforcements with an energy-absorbing mechanism by the plastic deformation at the tips of cracks, it seems more appropriate[7], [15].

M AN U

Nitinol is a nickel-titanium alloy with near equiatomic composition[16]. It is well known for its unique physical and mechanical properties, such as unique shape memory effect[17], as well as superelastic properties, low elastic modulus, high strength, corrosion resistance in seawater and excellent biocompatibility[17]– [21]. Due to these properties and similarity superelastic biomechanical properties with some human hard tissues such as bones and tendons, its promising for orthopedic implant applications and hard-tissue replacements[19], [22], [23].

EP

TE D

The present study aimed to improve the mechanical properties of natural HA by addition of NiTi with different weight percentage of NiTi( 5,10 and 15). samples fabricated by cold press and sintering method. The structural stability of HA phase in HA-NiTi composite by means of FTIR and XRD considered. Finally, the mechanical properties and biological behaviors composite were investigated by mechanical and invitro studies. 2. Materials and methods

AC C

2.1. Material preparation

In this study, HA was produced from bovine femoral bones. Head parts of the bones were cut and shafts of the bones were boiled in water to remove bone marrow. The remaining soft tissues on the surface of bones have been scraped off by using a knife. bones were cut into several pieces in order to obtain larger surface area that will be advantageous in subsequent calcination process. The cleaned bone pieces were calcined at 850 °C for 2 hours to obtain purely inorganic phase (mainly HA) by removing organic phase of the bone. An agate mortar and pestle have been used for HA powder production from calcinated bone parts. Then, the produced HA powders were ball milled in zirconia bowl and yttria-stabilized

ACCEPTED MANUSCRIPT

zirconia (YSZ) balls at 300 rpm running speed for 6 hours by using a laboratory planetary ball mill to obtain Nanoparticles HA powder(ethanol (C2H6O) was utilized as Process-Control Agent (PCA), the ball-to-powder weight ratio (BPR) was 10:1).

2.2. Production composite

M AN U

SC

RI PT

Nitinol powder used in this study was produced by mechanical alloying. For this purpose, The powders of Ti (99.5%) and Ni (99.5 %) were mixed with molar ratio of 1:1. Ni powder with a mean particle size of about 10 and Ti powder with a mean particle size of about 10 . The elemental powders were mechanically alloyed in a planetary ball mill under an argon atmosphere. The argon used was high purity grade to prevent TiO2 formation during MA (mechanically alloyed). The experiments were carried out in a hardened steel container with steel balls at room temperature. The ball-to-powder weight ratio and the rotational speed were 10:1 and 200 rpm, respectively. The maximum milling time was 40 h and, to avoid temperature increase during MA, 30 min periods of milling were alternated with 10 min periods of rest(ethanol (C2H6O) was utilized as Process-Control Agent (PCA)).

AC C

EP

TE D

The mixed powder of HA and NiTi with different percentages of NiTi( 5, 10 and 15 wt.%) were first blended by ball milling and then compacted at 300 MPa to produced green samples with a ratio of diameter to height 1 to 2. Finally, produced samples sintered via two step sintering process as shown in Fig.1 ( heating and cooling rate 10 ᵒC/min). The samples produced nomination H(pure HA), HN5(HA5%NiTi), HN10(HA-10%NiTi) and HN15(HA-15%NiTi).

ACCEPTED MANUSCRIPT

1400

t1= 30 min, T1= 1150 ᵒC

800

t2= 6 hr, T2= 950 ᵒC

600 400 200 0 0

100

200

300

400

500

600

700

M AN U

Time (min)

RI PT

1000

SC

Temperature (ᵒC)

1200

Figure. 1. Two step sintering process to compact the nanocomposite samples. 2.3. XRD analysis

2.4. FTIR analysis

TE D

To investigate the phase composition of extracted HA powder and HA-NiTi composite, X ray diffraction (XRD: PW3830) analysis was carried out. This system works with CuK2 incident radiation (1.5405 Aᵒ). The XRD peaks are recorded in 2 theta range of 0-90ᵒ).

EP

To identify the existence of organic species and also the degree of probable dehydroxylation of HA, Fourier transform infrared (FTIR: S 8400) analysis was performed. The measurements were carried out with wave numbers from 400-4000 cm-1 with a resolution of 4 cm-1.

AC C

2.5. SEM and EDS analysis

To study the surface morphology and microstructure of the sample, and cell adhesiveness Scanning Electron Microscopy (TESCANVEGA//XXMU ) was used. All the samples are coated with thin film of gold (Au) to reduce charging of the sample. Finally, a scanning electron microscope equipped with energy dispersive spectroscopy (EDS). 2.6. Mechanical testing Mechanical characteristics of the samples was evaluated with compressive test according to ASTM C 1424-04. The compressive tests were performed on

ACCEPTED MANUSCRIPT

cylindrical samples (5 mm in diameter and 10mmin length) with using a universal testing machine (Zwick/Roell Z100). The crosshead speed was 0.5 mm/min. 2.7. MTT assay

2.8. Alkaline phosphatase activity

M AN U

SC

RI PT

MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) colorimetric assay was used to investigate cell viability 7 and 14 days after seeding. For this purpose, the first 1×104 cells in 96 wells cell culture plate was poured. Then for 24 hours were at incubator 37ᵒC to the cells stick to the bottom plate. Extract taken from each sample was added to the wells and the cells cultured for 24 hours in the presence of the extract. Then medium was removed from the samples and 100 microliters of MTT at a concentration of 0.5 mg/ml was spilled to each well. After 4 hours, solution on the cells was removed and isopropanol solution was added to the purple crystals created to be solved. Then concentration material dissolved in isopropyl alcohol of in wavelength 545 nm was calculated. Well, have more cells, optical density (OD) higher than in wells with lower cell shows.

EP

2.9. Cell adhesion

TE D

Osteogenic differentiation on various samples was characterized by the activity of alkaline phosphatase (ALP) after 7 and 14 days. For this purpose, cells were seeded on the samples and control disc in 6 well plates at a density of ~50×103/well. Than samples were placed in an incubator and at certain times some medium was added to each well. Finally, after 7 and 14 days from culture, the sample medium was removed and evaluated to determining the ALP.

AC C

In order to investigate the cell adhesion on the HA-NiTi composites, MG63 cell lines were cultured on the samples containing culture medium supplemented with 10% fetal bovine serum (FBS). The fabricated specimens were placed into a 24-well plate, which was followed by plating 3*104 cells/ cm2 on to those. The cells were cultured for 4-5 h in a humidified incubator under an atmosphere of 5% CO2 at 37 C. the culture medium was removed and the samples for 30 seconds with PBS (phosphate buffered saline) was washed. For the SEM observations, the cultured cells were fixed with 3.5% glutaraldehyde and then dehydrated with graded ethanol (50%, 60%, 70%, 80% and 96%). 3. Results and discussion

ACCEPTED MANUSCRIPT

RI PT

Fig .2 shows XRD patterns for NiTi powder after 40h mechanical alloying. The presence of the intermetallic phases NiTi is confirmed by comparing the 2 position peaks with the standard JCPDS 0351281(Martensitic Nitinol) and 0650917( Nitinol austenitic). The XRD spectrum of NiTi demonstrated peaks at 34.88° and 43.91° that are important for NiTi.

500

NiTi (B2) NiTi (B19')

450

SC

350 300 250 200 150 100 50 0 10

20

30

M AN U

intensity(a. u.)

400

40

50

60

70

80

TE D

2 Theta (degree)

Figure. 2. X-ray diffraction patterns of Ni50Ti50 powder at 40h mechanical alloying.

AC C

EP

Fig. 3 shows XRD spectra of raw HA powder extracted from bovine cortical bone. By comparing the pattern with the standard JCPDS 090432[4], The peaks of the synthesized nHA particles showed peaks at 26.172°, 28.25°, 29.1°, 31.9°, 32.35°, 33°, 34.2°, 39.96°, 46.85°, 48.25°, 50.65°, 51.4°, 52.35°, 63.1° and 53.38° which are specific to HA. this indicated that the powder produced is hydroxyapatite.

ACCEPTED MANUSCRIPT

1400

1000 800 600 400 200 0 10

15

20

25

30

35

40

45

50

55

60

2 Theta (degree)

RI PT

intensity(a. u.)

1200

65

70

75

80

85

90

Figure. 3. XRD pattern of Bone ash powder.

630.68

981.70-

EP

60

3421.48 3382.91

3569.99

70

AC C 50

603.6 570.8

40 30

1089.71

20 10 0

3900

3400

2900

2400

1900

1400

1045.

Transimittance(arbitrary units)

80

1988.4

90

2360.71 2337.56

100

2921.9 2856.38

TE D

M AN U

SC

Accomplished FTIR analysis to confirm the results obtained from the XRD analysis of hydroxyapatite powder. The chemical groups in the FTIR spectrum of synthesized HA are PO43-,OH-, as well as CO32- that are characteristic of HA. The FTIR spectrum of raw HA powders shown in fig. 4 exhibit most bonds that have been reported by Gheisari[24] and Heidari[25]. The vibration peak of OH- group is relatively wide, from 2800 to 3600 cm-1, with a sharp peak at 3569 cm-1; also a weaker peak of OH- is formed at 630 cm-1. A small quantity of CO32- groups at 1400 cm-1 could be found in raw natural HA powders. It is reported that the carbonate ions increases the sinterability of hydroxyapatite if they replace phosphate groups in HA lattice[7]. The bands located at 570, 603, 1045 and 1089 cm-1 originate by PO43- Ions, which represent natural HA (NHA).

900

Wavenumber(cm^-1)

Figure. 4. FTIR spectrum of produced hydroxyapatite powder.

400

ACCEPTED MANUSCRIPT

The SEM images of HA powder are shown in Fig. 5(a). In order to check the grain size with more precision, MIP software was used. According to the results; the majority of grains length and width are below 370 nm and HA exhibits spherical

RI PT

morphology. The Nitinol grain size and surface morphology of the particles after mechanical alloying are shown in Fig. 5(b) As can be seen, the particle size is below 500nm. Due to the difficulty for the nanoparticles to be dispersed homogeneously due to their extremely high surface areas. There might be errors in

M AN U

SC

the calculation because of a high tendency of nano particles to agglomerate.

EP

AC C

b

TE D

a

Figure. 5. SEM images of (a) pure nHA and (b) pure NiTi

ACCEPTED MANUSCRIPT

Fig. 6 shows SEM image and EDS analysis of HA-NiTi composite. According to the analysis EDS (figure 6(a, b)) Nitinol phase in the composite is well observed

RI PT

(The presence of a peaks gold is related to gold coat).

SC

a

M AN U

b

AC C

b

EP

TE D

a

Figure. 6. SEM image of HA-NiTi composite, and EDS analysis of (a) and (b) points. 3.1. Structural stability of HA phase in HA-NiTi composite

ACCEPTED MANUSCRIPT

RI PT

Fig. 7 shows XRD spectra of HA and HA-NiTi composites sintered at 1150ᵒC. In all samples, main peaks of hydroxyapatite (26.172°, 32.35°, 33, 34.2°, 46.85°) and slightly phase decomposition to Ca4O (PO4) 2, (28, 47) was observed in XRD. Furthermore, the peaks at 38.23ᵒ and 60.45ᵒ in the samples is due to Nitinol. As can be seen, with the increase in the amount of Nitinol, the intensity of the peaks NiTi and Ca4O (PO4) 2 is also increased. in addition, any new compound was observed in XRD that indicates a lack of chemical reaction between hydroxyapatite and Nitinol. 4500

SC

H

4000

3000

2500

2000

HN15 NiTi HA Ca4O(PO4)2

TE D

intensity(a.u.)

HN10

M AN U

3500

HN5

1500

EP

1000

500

AC C

0 10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

2 Theta (degree)

Figure. 7. XRD spectra of HA and HA- NiTi composites.

The presence of OH- ions in the hydroxyapatite structure enables it to heat temperatures above 1400 °C. But, because of a lower partial pressure of water at high temperature, dehydration reactions or phase decomposition occurs[7]. Further, amplifier components also can reduce the structural stability of HA and promote dehydration reactions and decomposition of hydroxyapatite phase. This can reduce the ability to densification HA composites[7][26].

ACCEPTED MANUSCRIPT

H HN5 HN10 HN15

AC C

EP

TE D

Transimittance(arbitrary units)

M AN U

SC

RI PT

Fig. 8 shows FTIR spectra of pure HA and HA-NiTi composites after sintering at 1150 ᵒC. In the HA sample, it can be seen the intensity of OH- peak has decreased slightly (compared with raw HA). The IR spectra of HA-NiTi composites Showed that by increasing the amount of Nitinol, the intensity of OHGroup reduced. So that the composite HA-15NiTi, It is observed that the intensity of vibration peaks for OH- group is very low and a weak shoulder-shape peak presents at 630 cm-1. These indicate that OH- ions in HA phase have escaped clearly. According to what explained before, the existence of NiTi metal phase promotes the dehydration and decomposition reaction of HA phase in HA-NiTi composites.

3900

3400

2900

2400 1900 Wavenumber(cm^-1)

1400

900

400

Figure. 8. FTIR spectra of HA-NiTi composites. 3.2. Mechanical properties Table. 1. shows the characteristics of pure HA ceramic and HA-NiTi composites. The presence of pores in the final product depends on the initial

ACCEPTED MANUSCRIPT

porosity of the green compacts, and process characteristics. The total porosity (PT) was calculated according to the theoretical and apparent densities as follows (Eq. (1)): ) × 100

(1)

RI PT

(%) = (1 −

The density of compacts ( ) was calculated from the volume (V) and mass (m) measurement according to the well-known formula (Eq. (2)): =

(2)

M AN U

SC

and Theoretical densities (ρT) of all the samples were calculated by using the rule of mixture. According to the data reported in the table. 1, can be seen that by increasing the amount of Nitinol to 10 wt% the density has been increased. Since the pores provide a mechanical interlock leading to a firm fixation of the material; by decreasing density, probably improve mechanical bonding. But because porosity decreases mechanical properties, an implant is suitable that along with porosity, it exhibits good mechanical properties. For this reason, samples with 10% Nitinol, shows better properties among other samples.

AC C

EP

TE D

According to this topic that, the existence of reinforcements can decrease the stability of HA and promote the dehydration and decomposition of HA phase, which can affect the compressibility behavior of HA composites[7]. can be said that adding of Nitinol, effects on the compressibility behavior of composites. According to results of XRD and FTIR analysis, it is observed that by adding Nitinol stability of HA phase is reduced. As it was observed in the FTIR analysis, With the addition of Nitinol intensity of the absorption bands of OH group dropped. so that the shoulder peak at 15% Nitinol in 630 cm-1 completely removed. Also in XRD spectra of HA-15NiTi maximum decomposition phase was observed. Due to the fact that the products of decomposition reaction prevent the sintering ability of HA phase, while the product of dehydration has a better sintering ability than HA phase[7]. Can be said up to 10% Nitinol that more reaction was for dehydration, sintering ability increased. This is owing to the increased strength and density with increased Nitinol. But in 15% Nitinol because of removal, a large percentage of OH- ions more phase decomposition happened (compared to other samples). Products produced by the decomposition reactions prevents the appropriate sintering so density was reduced[7].

ACCEPTED MANUSCRIPT

RI PT

decomposition of HA to some calcium phosphate phases such as tricalcium phosphate(TCP) and tetra-calcium phosphate(TTCP) is the main reason for the loss of the mechanical properties of HA. Calcium phosphate phases are brittle and have weaker strength[9][14]. According to the results of the XRD analysis can be seen, When 10 wt% NiTi was added, the amount of calcium phosphate phase was less than the case of 15 wt% NiTi addition. This is another reason for the higher compressive strength of the HA-10%NiTi composite.

M AN U

SC

The density is directly related to the strength and by increasing the density strength increases[27]. As it is obvious from the results, strength is increased by adding Nitinol. The compressive strength of pure hydroxyapatite is 46 MPa that increased to 67.67 MPa by adding Nitinol. Maximum strength can be seen in the composite with 10% Nitinol that has the highest density and strength, by adding more Nitinol density and strength decreased. Elastic modulus with an addition of Nitinol decreased of 89.72GPa in pure HA to 25.76 GPa in HA-15 wt% NiTi composite. The reason for this decrease in the elastic modulus is the low level of elastic modulus in Nitinol. In this study, with the addition of nitinol to the hydroxyapatite, the elastic modulus has been reduced, which is more suitable for a bone substitute[7].

AC C

H HN5 HN10 HN15

elastic Total porosity Density(g/cm3) Compressive strength(MPa) modulus(GPa) (%) 32.97 2.118 46 89.72 29.16 2.295 58.34 42.29 26.18 2.438 67.67 36.59 28.95 2.409 35.67 25.76

EP

Sample code

TE D

Table. 1. characteristics of pure HA ceramic and HA-NiTi composite.

In this study, utilized the hydroxyapatite powder obtained from natural sources (calf bone). Values obtained for the strength of HA samples with values reported by Heidari[25] and Gheisari[24] matched. It was observed that the addition of Nitinol to 10 wt% compressive strength increased and better mechanical properties can be achieved. In order to investigate the mechanism of improving the mechanical properties of samples that were produced in this study, the images of fracture surface were prepared. Fig. 9 shows SEM images prepared from the surface. As

ACCEPTED MANUSCRIPT

TE D

M AN U

SC

RI PT

seen in the figure, there are many cracks due to poor mechanical properties of the matrix. By studying the path of cracks seem there are factors in the way that prevents the propagation of cracks directly. As can be seen in Fig. 10, cracks in some parts derailed or completely stopped and unable to continue its route. The reason for this can be attributed to particles of Nitinol. Nitinol has better mechanical properties compared to hydroxyapatite matrix. For this reason, when crack arrives they, cannot pass. As a result, these particles act as a barrier to crack and crack deflected or stopped. So Nitinol particles with crack deflection mechanism wasting crack energy and improve strength.

AC C

EP

Figure. 9. Scanning electron microscope image obtained from the fracture surface (100x magnification).

ACCEPTED MANUSCRIPT

Figure. 10. Scanning electron microscope image obtained from the fracture surface (a)1.80x magnification, b)750x magnification).

M AN U

SC

RI PT

By comparing the results obtained in this study with a report published about metal particles reinforcing, it was observed that also for them deviation crack by metal particles is a factor that improves the mechanical properties. For example, Chu et al. examined the effect of titanium on mechanical properties of hydroxyapatite. They showed that titanium metal phase promotes dehydration reactions of HA phase in the HA-20%Ti composite. These reactions reduce the manufacturing composite density and thus have a negative effect on the mechanical properties of the composite. However, Ti particles as agents of distorting crack cause improve mechanical properties. This report has been brought, when cracks arrive the Ti particles deviate into the matrix[7]. In another report, in order to improve the mechanical properties, silver oxide powders were used by different volume percentages. It was observed that with the addition of silver nanoparticles improves the mechanical properties of hydroxyapatite with crack bridging and also distract crack mechanisms[26].

EP

3.3. In vitro behavior

TE D

In conclusion, the present study also crack deflection mechanism has been improved the mechanical properties by adding Nitinol. But in HA-15%NiTi composite, Factors that cause the loss of the mechanical properties (decomposition phases of hydroxyapatite and low density), are greater than improvement factors of the mechanical properties. This is due to the low compressive strength of this composite compared to other samples.

3.3.1. Cell adhesion

AC C

Due to the fact that cell adhesion on the fine-grained surfaces is more than the other surfaces [28]. It is expected from nanocomposite samples that are generated in this study, to have good cell adhesion. SEM observation revealed the MG-63 cell attachment on different sample surfaces. Fig. 11 shows the morphologies of the cells on samples. As can be seen a large number of cells attached to the surface in all samples. The surface morphology of cells in contact with samples are plate shape. Which is indicative of early stages of bone formation.

ACCEPTED MANUSCRIPT

b

M AN U

SC

RI PT

a

AC C

EP

TE D

c

d

ACCEPTED MANUSCRIPT

f

M AN U

SC

RI PT

e

Figure. 11. SEM micrographs of osteoblasts in contact with samples, (a and b)H, (c and d)HN5, (e)HN10, (f)HN15. 3.3.2. MTT assay

AC C

EP

TE D

The MTT assay was used to quantitatively determine the proliferation of viable MG-63cells on the samples surfaces. Fig. 12 shows a comparison of ability to survive the cells in samples with negative control after 7 and 14 days of culture. All samples except HN15 composite showed good proliferation. In conjunction with nanohydroxyapatite a lot of research done that has shown great ability to growth and proliferation of this matter[29][30]. Also, there are reports suggesting that may be Nitinol show a small amount of toxicity in the early days which is related to the release of nickel in this matter. But over time its toxicity reduced and shows good cell growth and proliferation. In this study, it can be seen that the addition of Nitinol reduces cell proliferation. But generally up to 10 percent of Nitinol, no cell death was seen and nano biocomposites probably can be used in different medical applications.

SC

RI PT

ACCEPTED MANUSCRIPT

3.3.3. Alkaline phosphatase assay

M AN U

Figure. 12. MTT assay of cells on H, HN5, HN10 and HN15 samples after 7 and 14 days of incubation.

AC C

EP

TE D

The differentiated function of MG63 cells was assessed by monitoring their ALP activity. The ALP assay was studied after 7 and 14 days. Fig. 13 shows the ALP activity of MG63 cells cultured on the samples. Research carried out in relation to NiTi samples have shown that this matter improves osteoblast differentiation and cell growth[31]. But its ability to differentiate osteoblasts is lower than hydroxyapatite. This leads to the secretion of alkaline phosphatase reduced by increasing the percentage of Nitinol. However, the ALP activity in all cases is greater than the negative control.

Figure. 13. the ALP activity of MG63 cells cultured on H, HN5, HN10 and HN15 samples after 7 and 14 days.

ACCEPTED MANUSCRIPT

According to the results obtained from biological behavior, it can be said that HN10 nano biocomposites probably can be used in medical applications. 4. Conclusions

SC

RI PT

The purpose of this study was to develop nanocomposite for bone tissue engineering. An ideal replacement for bone tissue engineering should have proper mechanical and biological properties. accordingly, this study attempted that by adding NiTi improve the mechanical properties of hydroxyapatite. Natural HANiTi nanocomposites with 0, 5, 10 and 15 wt.% NiTi were fabricated by the powder metallurgy method. The results showed that the mechanical and biological properties of nanocomposites were influenced by adding NiTi. According to the results of this study can be said that:

M AN U

-Nitinol with the crack diversion mechanism was improved compressive strength in nanocomposites.

TE D

-XRD and FTIR curve prepared from sintered samples showed that the Nitinol promotes dehydration and decomposition reactions. New phases caused by decomposition reactions of HA phase and differences thermal expansion coefficient between the HA and NiTi prevents of appropriate sintering, thus reducing the mechanical properties. It was shown that in HN15 highest decomposition reactions happened. For this reason in HN15 composite mechanical properties decreased.

AC C

EP

- adding Nitinol, strength increases about one and a half times greater. by comparing the compressive strength results with the cancellous and cortical bone compressive strength, it was observed that although by using of Nitinol compressive strength improved, but still strength much less than bone strength. Due to the better mechanical properties of synthetic HA this study can also be performed on synthetic hydroxyapatite and compared the results with the present report. -Cell studies showed that up to 10 wt% NiTi no toxicity introduced into the system and show well growth, proliferation and cellular differentiation. In fact, compared to pure HA growth, proliferation and cellular differentiation of composite samples are less. But however, most of the sample control. As a general conclusion, we can say that the composite produced with 10wt% NiTi has good Mechanical and biological properties so that probably it can be used in

ACCEPTED MANUSCRIPT

medical applications. Yet this word( good biological properties) have required more experiments invivo and invitro to the confirmed truth.

RI PT

5. References

I. Ozden, M. Ipekoglu, N. Mahmutyazicioglu, and S. Altintas, “Effect of Al 2 O 3 , ZrO 2 , and TiO 2 Addition on the Mechanical Properties and the Microstructure of Natural Hydroxyapatite Obtained from Calf Femoral Bone,” vol. 494, pp. 199–204, 2012.

[2]

G. Silva, M. R. Baldissera, E. de S. Trichês, and K. R. Cardoso, “Preparation and characterization of stainless steel 316L/HA biocomposite,” Materials Research, vol. 16, no. 2, pp. 304–309, 2013.

[3]

C. Y. Tan, K. L. Aw, W. H. Yeo, S. Ramesh, M. Hamdi, and I. Sopyan, “Influence of Magnesium Doping in Hydroxyapatite Ceramics,” pp. 326– 329, 2008.

[4]

J. Venkatesan, B. Lowe, P. Manivasagan, K. H. Kang, E. P. Chalisserry, S. Anil, D. G. Kim, and S. K. Kim, “Isolation and characterization of nanohydroxyapatite from salmon fish bone,” Materials, vol. 8, no. 8, pp. 5426– 5439, 2015.

[5]

F. A. N. Xin, C. Jian, and Z. O. U. Jian-peng, “Bone-like apatite formation on HA / 316L stainless steel composite surface in simulated body fluid,” Transactions of Nonferrous Metals Society of China, vol. 19, no. 2, pp. 347– 352, 2008.

[6]

N. A. M. Barakat, M. S. Khil, A. M. Omran, F. A. Sheikh, and H. Y. Kim, “Extraction of pure natural hydroxyapatite from the bovine bones bio waste by three different methods,” Journal of Materials Processing Technology, vol. 209, no. 7, pp. 3408–3415, 2009.

[8]

M AN U

TE D

EP

AC C

[7]

SC

[1]

C. Chu, X. Xue, J. Zhu, and Z. Yin, “Fabrication and characterization of hydroxyapatite reinforced with 20 vol % Ti particles for use as hard tissue replacement,” Journal of Materials Science: Materials in Medicine, vol. 17, no. 3, pp. 985–992, 2002.

M. Atif, F. Afzal, P. Kesarwani, K. M. Reddy, S. Kalmodia, B. Basu, and K. Balani, “Functionally graded hydroxyapatite-alumina-zirconia biocomposite : Synergy of toughness and biocompatibility,” Materials Science & Engineering C, vol. 32, no. 5, pp. 1164–1173, 2012.

ACCEPTED MANUSCRIPT

[9]

M. Aminzare, A. Eskandari, M. H. Baroonian, A. Berenov, and Z. R. Hesabi, “Hydroxyapatite nanocomposites : Synthesis , sintering and mechanical properties,” vol. 39, pp. 2197–2206, 2013.

RI PT

[10] C. Kailasanathan, N. Selvakumar, and V. Naidu, “Structure and properties of titania reinforced nano-hydroxyapatite / gelatin bio-composites for bone graft materials,” Ceramics International, vol. 38, pp. 571–579, 2012. [11] R. Imani, “Characterization of a novel nanobiomaterial fabricated from HA , TiO 2 and Al 2 O 3 powders : an in vitro study,” prog biomater, 2014.

SC

[12] E. S. Ahn, N. J. Gleason, and J. Y. Ying, “The Effect of Zirconia Reinforcing Agents on the Microstructure and Mechanical Properties of HydroxyapatiteBased Nanocomposites,” vol. 3379, no. 20132, pp. 3374–3379, 2005.

M AN U

[13] H. Ghomi, M. H. Fathi, and H. Edris, “Effect of the composition of hydroxyapatite / bioactive glass nanocomposite foams on their bioactivity and mechanical properties,” Materials Research Bulletin, vol. 47, no. 11, pp. 3523–3532, 2012. [14] I. Mobasherpour, M. S. Hashjin, S. S. R. Toosi, and R. D. Kamachali, “Effect of the addition ZrO 2 – Al 2 O 3 on nanocrystalline hydroxyapatite bending strength and fracture toughness,” vol. 35, pp. 1569–1574, 2009.

TE D

[15] M. Tulinski and M. Jurczyk, “Applied Surface Science Nanostructured nickel-free austenitic stainless steel composites with different content of hydroxyapatite,” Applied Surface Science, vol. 260, pp. 80–83, 2012.

EP

[16] C. H. Fu, M. P. Sealy, Y. B. Guo, and X. T. Wei, “Austenite–martensite phase transformation of biomedical Nitinol by ball burnishing,” Journal of Materials Processing Technology, vol. 214, no. 12, pp. 3122–3130, 2014.

AC C

[17] T. Mousavi, F. Karimzadeh, and M. H. Abbasi, “Synthesis and characterization of nanocrystalline NiTi intermetallic by mechanical alloying,” vol. 487, pp. 46–51, 2008. [18] F. L. Nie, Y. F. Zheng, Y. Cheng, S. C. Wei, and R. Z. Valiev, “In vitro corrosion and cytotoxicity on microcrystalline , nanocrystalline and amorphous NiTi alloy fabricated by high pressure torsion,” Materials Letters, vol. 64, no. 8, pp. 983–986, 2010. [19] X. Liu, S. Wu, K. W. K. Yeung, Y. L. Chan, T. Hu, Z. Xu, X. Liu, J. C. Y. Chung, K. M. C. Cheung, and P. K. Chu, “Biomaterials Relationship between osseointegration and superelastic biomechanics in porous NiTi scaffolds,” vol. 32, pp. 330–338, 2011.

ACCEPTED MANUSCRIPT

[20] C. L. Chu, C. Y. Chung, P. H. Lin, and S. D. Wang, “Fabrication of porous NiTi shape memory alloy for hard tissue implants by combustion synthesis,” Materials Science and Engineering A, vol. 366, no. 1, pp. 114–119, 2004.

RI PT

[21] I. Gotman, D. Ben-david, R. E. Unger, T. Böse, E. Y. Gutmanas, and C. J. Kirkpatrick, “Acta Biomaterialia Mesenchymal stem cell proliferation and differentiation on load-bearing trabecular Nitinol scaffolds,” vol. 9, pp. 8440–8448, 2013.

SC

[22] K. Niespodziana, K. Jurczyk, and M. Jurczyk, “Synthesis of Niti Based Nanocomposites Reinforced by Ha Addition,” Archives of Metallurgy and Materials, vol. 61, no. 2, pp. 577–580, 2016.

M AN U

[23] M. Akmal, A. Raza, M. M. Khan, M. I. Khan, and M. A. Hussain, “Effect of nano-hydroxyapatite reinforcement in mechanically alloyed NiTi composites for biomedical implant,” Materials Science and Engineering C, vol. 68, no. May, pp. 30–36, 2016. [24] H. Gheisari, E. Karamian, and M. Abdellahi, “A novel hydroxyapatite – Hardystonite nanocomposite ceramic,” Ceramics International, vol. 41, no. 4, pp. 5967–5975, 2015.

TE D

[25] F. Heidari, M. Razavi, M. Ghaedi, and M. Forooghi, “Investigation of mechanical properties of natural hydroxyapatite samples prepared by cold isostatic pressing method,” Journal of Alloys and Compounds, vol. 693, pp. 1150–1156, 2017.

EP

[26] X. Zhang, G. H. M. Gubbels, R. A. Terpstra, and R. Metselaar, “Toughening of calcium hydroxyapatite with silver particles,” Journal of Materials Science, vol. 32, no. 1, pp. 235–243, 1997.

AC C

[27] I. Sopyan, M. Mel, S. Ramesh, and K. A. Khalid, “Porous hydroxyapatite for artificial bone applications,” vol. 6996, no. September, 2016. [28] I. O. Smith, L. R. McCabe, and M. J. Baumann, “MC3T3-E1 osteoblast attachment and proliferation on porous hydroxyapatite scaffolds fabricated with nanophase powder,” International Journal of Nanomedicine, vol. 1, no. 2, pp. 189–194, 2006. [29] G. Tetteh, A. S. Khan, G. C. Reilly, and I. U. Rehman, “Electrospun polyurethane / hydroxyapatite bioactive Scaffolds for bone tissue engineering : The role of solvent and hydroxyapatite particles $,” vol. 39, pp. 95–110, 2014. [30] Y. Kong, C. Bae, S. Lee, H. Kim, and H. Kim, “Improvement in

ACCEPTED MANUSCRIPT

biocompatibility of ZrO 2 – Al 2 O 3 nano-composite by addition of HA,” vol. 26, pp. 509–517, 2005.

AC C

EP

TE D

M AN U

SC

RI PT

[31] M. Li, T. Yin, Y. Wang, F. Du, X. Zou, H. Gregersen, and G. Wang, “Study of biocompatibility of medical grade high nitrogen nickel-free austenitic stainless steel in vitro,” Materials Science and Engineering C, vol. 43, pp. 641–648, 2014.

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

improve the mechanical properties of HA is proposed. Nitinol has been used in HA ceramic to improve its mechanical reliability. NHA- NiTi nanocomposites were fabricated by powder metallurgy. The structural stability of HA phase in HA- NiTi samples and mechanical strength of nanocomposites were studied.