Mechanical properties and biocompatibility of porous titanium scaffolds for bone tissue engineering

Author’s Accepted Manuscript Mechanical Properties and Biocompatibility of Porous Titanium Scaffolds for Bone Tissue Engineering Yunhui Chen, Jessica Ellen Frith, Ali DehghanManshadi, Hooyar Attar, Damon Kent, Nicolas Dominique Mathieu Soro, Michael J Bermingham, Matthew S Dargusch

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S1751-6161(17)30301-6 http://dx.doi.org/10.1016/j.jmbbm.2017.07.015 JMBBM2414

To appear in: Journal of the Mechanical Behavior of Biomedical Materials Received date: 16 May 2017 Revised date: 7 July 2017 Accepted date: 11 July 2017 Cite this article as: Yunhui Chen, Jessica Ellen Frith, Ali Dehghan-Manshadi, Hooyar Attar, Damon Kent, Nicolas Dominique Mathieu Soro, Michael J Bermingham and Matthew S Dargusch, Mechanical Properties and Biocompatibility of Porous Titanium Scaffolds for Bone Tissue Engineering, Journal of the Mechanical Behavior of Biomedical Materials, http://dx.doi.org/10.1016/j.jmbbm.2017.07.015 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 galley proof before it is published in its final citable 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.

Mechanical Properties and Biocompatibility of Porous Titanium Scaffolds for Bone Tissue Engineering Yunhui Chen*a, Jessica Ellen Frithc, Ali Dehghan-Manshadia, Hooyar Attara, Damon Kenta,b,d, Nicolas Dominique Mathieu Soroa, Michael J Berminghama,d, Matthew S Darguscha,d a. Queensland Centre for Advanced Materials Processing and Manufacturing (AMPAM), The University of Queensland, St. Lucia, 4072, Australia b. School of Science and Engineering, University of the Sunshine Coast, Maroochydore DC, QLD 4558, Australia c. Materials Science and Engineering, Monash University, 22 Alliance Lane, Clayton VIC 3800, Australia d. ARC Research Hub for Advanced Manufacturing of Medical Devices *Corresponding author: Yunhui Chen [email protected]

Abstract

Synthetic scaffolds are a highly promising new approach to replace both autografts and allografts to repair and remodel damaged bone tissue. Biocompatible porous titanium scaffold was manufactured through a powder metallurgy approach. Magnesium powder was used as space holder material which was compacted with titanium powder and removed during sintering. Evaluation of the porosity and mechanical properties showed a high level of

compatibility with human cortical bone. Interconnectivity between pores is higher than 95% for porosity as low as 30%. The elastic moduli are 44.2 GPa, 24.7 GPa and 15.4 GPa for 30%, 40% and 50% porosity samples which match well to that of natural bone (4-30 GPa). The yield strengths for 30% and 40% porosity samples of 221.7 MPa and 117 MPa are superior to that of human cortical bone (130-180 MPa). In-vitro cell culture tests on the scaffold samples using Human Mesenchymal Stem Cells (hMSCs) demonstrated their biocompatibility and indicated osseointegration potential. The scaffolds allowed cells to adhere and spread both on the surface and inside the pore structures. With increasing levels of porosity/interconnectivity, improved cell proliferation is obtained within the pores. It is concluded that samples with 30% porosity exhibit the best biocompatibility. The results suggest that porous titanium scaffolds generated using this manufacturing route have excellent potential for hard tissue engineering applications. Key words: Scaffold, Titanium, Powder Metallurgy, Space Holder, biocompatibility

1. Introduction

Bone tissue engineering aims to regenerate damaged bone tissues by combining cells with scaffolds, to act as templates for tissue regeneration and guide the growth of new tissue [1]. Engineered bone tissue has been viewed as a potential alternative to the conventional use of bone grafts, due to their limitless supply and no disease transmission [2]. Scaffolds are frameworks used to provide a three dimensional micro-environment where cells can proliferate, differentiate and generate the desired tissue [3]. A number of key considerations are important when designing or determining the suitability of a scaffold for use in bone tissue engineering. These include a relatively low modulus and high mechanical strength which match with that of bone, a high degree of biocompatibility and corrosion resistance, and adequate interconnectivity and porosity [3].

Porous titanium scaffold is regarded as a leading replacement for bone grafts [4][5]. Titanium and its alloys have been used successfully in the field of orthopaedic [6] and dental implants due to their excellent mechanical properties, corrosion resistance, and biocompatibility [7][8,9]. Meanwhile porous structures can facilitate cellular activities, such as the migration and proliferation of osteoblasts and mesenchymal cells as well as the transport of nutrients and oxygen required for vascularization during bone tissue development [10]. There are various methods to fabricate porous titanium scaffolds including powder metallurgy, foaming and additive manufacturing [11]. The advantage of powder metallurgy is its high production efficiency and low cost. Also, powder metallurgy is widely used in industry as the material blending and compaction processes allow for customization of material compositions, mechanical properties and shapes in order to mass-produce implants with desired characteristics [12]. Powder sintering can be coupled with techniques employing temporary space holders to form pores and can achieve high levels of porosity and good control over the porous structure of the scaffolds. The space holder particles are mixed with metallic powders, then compacted and removed either before or during sintering. Space holder materials such as sodium chloride [13], carbamide [14], sugar pellets [5], tapioca [15] and saccharose [16] have been used and are typically removed by evaporation or by dissolution. Magnesium has also been reported as a potential space holder material for fabricating porous scaffolds [17–21]. However, to the authors’ best knowledge, no work has thoroughly investigated the biocompatibility of porous scaffolds produced by this manufacturing route in respect to both their mechanical interaction with surrounding bone tissue and their biological interaction. In this work we focus on attaining porous scaffolds with optimized pore geometries and also investigate any potential contamination by the magnesium space holder. This is important as

magnesium residue left after the debinding process may negatively influence the ability of cells to adhere to the inert titanium scaffold.

In this work, porous titanium scaffolds are fabricated through powder metallurgy with magnesium powder as space holder material for tissue engineering applications. The purpose is to generate biomimetic scaffolds with high interconnectivity between pores and mechanical properties matching human cortical bone. In-vitro cell culture testing is used to demonstrate the biocompatibility and osseointegration potential. Cell viability and morphology is explored to indicate the potential of this manufacturing route for hard tissue engineering applications. 2. Materials & Methods

2.1 Preparation of scaffolds Porous Titanium scaffolds were produced using a powder metallurgy approach. The porosity was controlled by adjusting the volume ratio of Mg to Ti. Titanium hydride-dehydrate powder (>99% purity, provided by Wuyi Co., China) with particle sizes of around 45 µm and a normally distributed 40-50 µm size range was used as the primary matrix. Spherical magnesium powder (>99.5%, provided by Sigma Aldrich) with particle size of around 100 µm and normally distributed size range of 75-125 micrometers was used as the space holder. The magnesium powder size was chosen according to a review by Loh et al. [22] which reported that pore sizes around 100 μm facilitate cell migration, while high sphericity imparts optimal mechanical performance to the porous structures. The cell culture test aims to investigate the osseointegration potential of the scaffold, therefore 100 μm pore size was targeted to optimise their performance. Figure 1 is a scanning electron microscopy (SEM) image showing the size and morphology of Ti and Mg powders.

Titanium and Magnesium powders were mixed in a GlenMills Turbula T2F mixer for one hour with volumetric amounts of magnesium of 30, 40 and 50% corresponding to the desired levels of porosity. The powder mixtures were then uniaxially pressed at 600 MPa using a Carver 12 Tons Manual Hydraulic Press with a floating die to obtain cylindrical samples of 10 mm in diameter and 5 mm in height for material characterization. Long samples with height of 12 mm and diameter of 6 mm were also produced for compression tests in accordance with ASTM E9 - 09 which requires the height to diameter ratio of 2:1. The CP-Ti control group was manufactured by the same process using only titanium powders. Sintering was carried out in a Carbolite high temperature high vacuum tube furnace STF 15. Samples were sintered at 1300 °C for 2 hrs at a vacuum pressure of 10-5 Torr. Samples were placed on an Al2O3 ceramic crucible during sintering to prevent contamination. During sintering Mg powders start to evaporate at 400 °C [23] and consequently pores form. In order to prevent the influence of melting and boiling of Mg on the scaffold’s integrity, the furnace was slowly heated at 1ºC/min to 650 °C and held at this temperature for 20 minutes then heated at 4ºC/min to 1100 °C and held for a further 20 minutes before heating to the final sintering temperature at 4ºC/min. This process also facilitates maximal removal of the magnesium space holder prior to titanium sintering. 2.2 Density and porosity measurement The density and porosity of the sintered compacts was determined by the Archimedes method with oil impregnation. H-Galden ZT-180 was used instead of water to give more accurate results. The density of the sintered sample

and porosity

, were calculated using:

(1)

( (

) ) (2)

where

is the density of the H-Galden (1.69 g/mL at 21°C ),

used (KS7470, density 0.885 g/mL),

is the density of the oil

is the dry weight of the compact,

weight of the compact after oil infiltration, and

is the

is the weight of the oil infiltrated

compact measured while immersed in H-Galden. 2.3 Mechanical properties and microstructure analysis Mechanical properties of porous titanium scaffolds were investigated by compression testing. The tests were carried out with Instron 5584 test machine, using the cylindrical-shaped samples with height of 12 mm and diameter of 6 mm. Samples were prepared using precision CNC machining for geometric accuracy. The specimens were compressed at cross head velocity of 0.001 mm/s at room temperature. Each test coupon was subject to increasing load until fracture occured. The maximum compressive strength, yield strength and strain at fracture were obtained. Yield strengths and elastic modulus were determined using the 0.2%offset method. The composition of the specimen was determined with inductively coupled plasma optical emission spectrometry (ICP-OES), using Spectro-Arcos equipment. Specimens for the microstructure investigation were cut longitudinally, along the axis of the compact, and mounted in epoxy resin. Mounted specimens were ground on progressively finer SiC paper to 1200 grit and polished with a mixture of colloidal silica and H2O2 with a 9:1 volume ratio. Analysis of the pore morphologies was performed using a Tabletop Scanning Electron Microscope (SEM, TM3030).

2.4 Biocompatibility Analysis 2.4.1 Sample preparation Titanium disks (10 mm diameter; 1 mm thick) were fabricated from sintered samples using Struers Accutom Cut-off machine using diamond cutting disk (0.8mm thick). 2000 rpm cutting speed and 0.025 mm/s cutting rate were chosen to generate fine sectioned surface and intact pores open from the surface without further polishing. Disks were cleaned in an acetone bath using an ultrasonic cleaner for 20 mins. The disks were then rinsed with deionized water and dried under vacuum. The disks were individually wrapped in gauze to prevent damage and then sterilized by autoclaving. 2.4.2 Mesenchymal stem cell culture Human bone-marrow-derived MSC (hMSCs, Lonza) were cultured in DMEM-low glucose supplemented with 100 U/ml penicillin, 100 µg/mL streptomycin (DMEM/ps) and 10% fetal bovine serum (FBS) at 37 °C and 5% CO2. Upon reaching 80% confluence cells were passaged and replated at a density of 2500/cm2. Passage 6 cells were used in this study. 2.4.3 hMSC adhesion and proliferation The samples were divided into three groups with 3 samples within each group, namely, 30%, 40% and 50% porosity. Prior to seeding, all scaffolds were sterilised by immersion in 80% (w/v) ethanol, dried and rinsed in phosphate buffered saline (PBS). hMSCs were detached with TrypLE Select (Invitrogen), resuspended in DMEM/10% and seeded onto the scaffolds at a density of 5,000 cells/cm2. After 24 hrs of culture the samples were rinsed gently with PBS and fixed in 4% paraformaldehyde for 20min at room temperature (RT). After fixation the samples were rinsed with PBS and permeablised with 0.1% Triton-X-100 in PBS for 5 min at RT. Staining for the actin cytoskeleton was performed using ActinRed 555 Readyprobes (Invitrogen), according to the manufacturer’s instructions, and combined with

Hoechst 33342 (1:2000) for nuclear staining. After rinsing with PBS, the cells imaged using a Zeiss Axio Vert A.1 fluorescent microscope. 2.4.4 MTS Assay Cell viability was assessed using a MTS assay (CellTitre Aqueous One, Promega). hMSC viability was measured after 24hrs culture, using triplicates for each condition. To quantify cell viability, samples were moved to fresh culture plates containing 500ul culture medium supplemented with 100ul MTS solution. After 3 hrs, 100ul culture medium was removed and read on a spectophotomer (Multiskan Spectrum, Thermo Scientific) at 492 nm. 2.4.5 Statistical analysis All cellular experiments were run in triplicate. Data are presented as the mean value standard error of the mean and were analyzed with Student t-test. Statistical significance was considered at p < 0.05. 3. Results & Discussion

3.1 Density and porosity measurements Table 1 shows the measured average density and corresponding porosities for different magnesium fractions using triplicates. The error represents standard deviation. The difference among triplicates was less than 5%. Measured density is highly coherent with the designed value. When the porosity reaches 50 vol %, the density of the sample is 2.25 g/cm3, half of the value of pure titanium (4.506 g/cm3). Healthy human bone mineral density (BMD) on average is around 3.88 g/cm2 in males and 2.90 g/cm2 in females. Bone density plays an important role in implant outcome. The density ranges of porous samples produced in this study are consistent with the BMD range which can help to improve patient comfort and to keep rates of implant failure low [24]. It also should be noted that the present compacts with

the porosity greater than 30 vol % are promising for biomaterial applications, since the ideal porosity of implant materials is in the range of 30–90 vol %[25]. An indication of the pore interconnectivity can be obtained from the ratio of open porosity to the total porosity [26]. This suggests that the pores are mostly interconnected (above 95%) regardless of the levels of porosity and interconnectivity increases with the volume of magnesium powder. This is ascribed to the small geometry of magnesium powder with large powder particle contact area and its tendency to aggregate. Pore interconnectivity is also dependant on the degree of densification during sintering. 3.2 Structure observation Figure 2 shows the SEM images of polished cross-sections of the scaffolds revealing pore sizes, morphology and distribution. Clearly the magnesium space-holding particles have been removed. Inductively coupled plasma optical emission spectrometry (ICP-OES) results from the 50% porosity sample show that the residual Mg is only 0.01 wt%, as detailed in Table 2, which is at the lower limit of detection. The Mg residue is expected to be similar or even lower for the samples utilising lesser Mg additions. Pores are around 100 µm in diameter, which are coherent with the size distribution of the space holder. For low magnesium space holder additions, pores are generally round in shape and the pore walls have smooth curvatures. However, with increasing volume fractions of magnesium, increasing levels of aggregation lead to larger and more irregularly shaped pore clusters which greatly increased the interconnectivity. In addition to the larger macro pores formed by the Mg space holder, micro-pores are also visible as indicated in Figure 2(d). This is due to the incomplete sintering of the titanium powders and is a common feature in sintered titanium microstructures.

Computed tomography (Micro-CT) was used to measure not only the external and internal pore shapes but also 2D surface and 3D space parameters such as pore distribution and porosity related to bone histomorphometry. The cross sections of the scaffolds were recorded in three directions with step sizes of 0.01 mm. Mimics® was used to reconstruct the 3 dimensional structure from the hundreds of cross sections. Figure 3 corresponds to the centre of a sample designed with 40% porosity. The CT analysis showed a high level of correspondence between the designed porosity and the obtained one. Using Mimics®, the porosity was measured to be approximately 39.5%, which is very close to the results obtained from the Archimedes method. The voids inside the sample are uniformly distributed at around 100 µm with tight size distribution of around 10 µm size difference and high interconnectivity. The open pores are present from the surface through to the centre which is beneficial for osseointegration as it facilitates the transport of nutrients and oxygen required for vascularization during bone tissue development [10]. 3.3 Mechanical properties Figure 4 shows the compression stress-strain curves for scaffolds with different levels of porosity. As expected, the compressive Young’s modulus ( ) and the yield strengths (

) of

the samples decrease with increasing porosity. Table 3 summarizes the compression properties of the samples. As evident, the elastic modulus for CP-Ti sample is 92.4 GPa and is reduced to 44.2 GPa, 24.7 GPa and 15.4 GPa for 30%, 40% and 50% porous samples, respectively. It has been reported that elastic modulus of natural bone is between 4-30 GPa [27], therefore the elastic modulus values obtained for all porous samples are comparable with that of human cortical bone. Moreover, the yield strengths of the scaffolds, with the exception of the 50% designed porosity, are superior to that of natural bone (130-180 MPa) [27]. The scaffold design containing 40% porosity is particularly close to the stiffness and

strength of human cortical bone. These promising results indicate the suitability of the porous samples for implant applications since they are not only able to minimize the stress-shielding effect but they also show suitable yield strengths for the implant material to resist against permanent shape change under loading [28]. 3.4 hMSC adhesion and morphology The purpose of the test was to determine which porosity configuration offers the highest compatibility. The results showed that the 30% porosity scaffold shows better compatibility with higher cell adhesion and viability. This is due to a configuration of porosity, interconnectivity and surface area that is more suitable for the cells to adhere to and proliferate on. To assess the potential of the scaffolds structures (configuration of porosity, interconnectivity, surface area) to interact effectively with hMSCs, the scaffolds were seeded with cells and their viability and morphology assessed after 24 hrs. Fluorescent imaging showed adhesion of hMSCs to all scaffold types. The distribution of cells was heterogeneous, and cells appeared to cluster in the pores of the material, particularly in the 30% porosity scaffolds (Fig 5). The cells exhibited a partially-spread morphology with some filpodial protrusions suggestive of cells starting to interact with the underlying substrate. An MTS assay (Fig 6) showed viability of the cells on all scaffolds was equivalent to conventional tissue culture plates (TCP). There were no statistically significant differences between any of the samples based on Student t-test results. Together this indicates that the scaffolds were able to support the viability of hMSCs following attachment although future modifications may be of interest to further improve hMSC spreading and promote osteogenesis.

4. Conclusion This work demonstrates a biomimetic titanium scaffold manufactured using the powder metallurgy route with magnesium powder as the space holder material. The manufacturing process is flexible and it is straightforward to produce various designed porosity levels. In this work scaffolds with 30%, 40% and 50% porosity were developed. The porous scaffolds all show a very high level of interconnectivity which has been suggested to be beneficial for tissue ingrowth and osseintegration. The mechanical properties of the scaffolds closely match with human cortical bone and in-vitro cell cultures demonstrated the ability of the scaffolds to support hMSC adhesion and viability. Together our data suggest that the titanium scaffolds produced through this method may have excellent potential for use in bone tissue-engineering applications. Acknowledgment The authors acknowledge the support of Queensland Centre for Advanced Materials Processing and Manufacturing (AMPAM) and School of Materials Science and Engineering, Monash University. M. Dargusch acknowledges the support of the ARC Research Hub for Advanced Manufacturing of Medical Devices (IH150100024). J.E. Frith and M.J. Bermingham acknowledge the support of the ARC Discovery Early Career Researcher Awards. J.E. Frith is supported by DE13010098 and M.J. Bermingham is supported by DE160100260. References [1]

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Figures

Figure 1. Morphology of (a) Titanium (b) Magnesium powders used

Micro-pores

Figure 2. Pore sizes and distribution of (a) 30% (b) 40% (c) 50% porosity. And enlarged view of pore morphology of (d) 30% porosity sample

C Figure 3. Computed micro-tomography image of the porous titanium scaffold: (a) 3D view of the scaffold (b) one slice of the scan (c) enlarged view of the porous structure

Figure 4. Compression stress-strain curves of CP-Ti samples with different levels of porosity (a) sintered CP-Ti, porosity of (b) 30% (c) 40% (d) 50% and (e) the samples after compression testing.

Figure 5. Cell attachment and morphology on control and porous scaffold samples. Actin (green) and nucleus (blue). Scale bar = 50um.

Figure 6. MTS assay of hMSC viability after 24 hrs of culture. Data is presented as absorbance normalised to TCP controls with mean±SEM for n=3.

Tables Table 1. Density and porosity of sintered titanium samples Designed porosity, (vol %)

0

30

40

50

Density, (g/cm3)

4.29± 0.13

3.01± 0.1

2.64± 0.14

2.25± 0.11

Total porosity, (vol %)

4.7± 0.5

33.0± 0.23

41.3± 0.35

50.0± 0.28

Open porosity, (vol %)

0.7± 0.1

31.6± 0.15

39.9± 0.23

48.5± 0.17

15.3

95.9

96.6

96.9

Open to total porosity ratio, (vol %)

Table 2. Chemical analysis of the titanium powder and manufactured scaffold

Ti

Cb

Ob

Nb

Mga

Titanium powder (wt%)

Balanced

<0.01

0.12

0.02

<0.01

Scaffold with 50% porosity (wt%)

Balanced

0.01

0.27

0.30

0.01

ICP-OES/LECO

a

Analyzed by inductively coupled plasma optical emission spectroscopy (ICP-OES)

b

Analyzed by a carrier gas hot extraction gas analyzer

Table 3. Mechanical properties of the CP-Ti samples obtained from compression testing. E is Young’s modulus; σy denotes compressive yield strength; compressive strength and

Materials

(GPa)

(MPa)

92.4 ± 5

622 ± 14.5

indicates ultimate

denotes strain at fracture.

(MPa) 1506 ± 55

(%) 41 ± 6.5

Sintered CP-Ti

This work 44.2 ± 0.6

405 ± 15

524 ± 10.5

6.6 ± 1.6

30% Porosity

This work 24.7 ± 2.5

221.7 ± 17.5

301.7 ± 10

5.4 ± 2.1

40% Porosity

This work 15.4 ± 1.1

117 ± 6

120.3 ± 1.5

1.9 ± 0.6

50% Porosity

This work 4-30

Human bone

Reference

130-180 -

-

[29]