Processing, characterization and biological testing of poroustitanium obtained by space-holder technique

Y. Torres • J. A. Rodrıguez • S. Arias •

M. Echeverry • S. Robledo • V. Amigo •

J. J. Pavon

Received: 27 January 2012 / Accepted: 16 May 2012 / Published online: 30 May 2012

� Springer Science+Business Media, LLC 2012

Abstract The high Young’s modulus of titanium with

respect to that one of the bone is the main cause of the stress-

shielding phenomenon, which promotes bone resorption

around implants. Development of implants with a low

Young’s modulus has gained increased importance during

the last decade, and the manufacturing of porous titanium

is one of the routes to reduce this problem. In this work,

porous samples of commercially pure titanium grade IV

obtained by powder metallurgy with ammonium bicarbonate

(NH4HCO3) as space-holder were studied. Evaluations of

porosity and mechanical properties were used to determine

the influence of compaction pressure for a fixed NH4HCO3

content. Measurements by ultrasound tests gave Young’s

modulus results that were low enough to reduce stress

shielding, whilst retaining suitable mechanical strength.

Biological tests on porous cp Ti showed good adhesion of

osteoblasts inside the pores, which is an indicator of poten-

tial improvement of osteointegration.

Introduction

Among all metallic biomaterials used for bone replace-

ment, titanium and some of its alloys are recognized as the

most successful materials for this purpose [1, 2]. This

success is due to their highly biocompatible behaviour and

their fulfilment of the mechanical requirements of the

application [3–6]. However, their high Young’s modulus

values mean that implants and prostheses do not transfer

the entire applied load to the bone generating a stress-

shielding phenomenon [7–9]. As bone is a dynamic tissue

the structure and density of which are modulated by

applied load, mismatch between the Young’s modulus of

titanium and that of bone generates a loss of bone density,

termed bone resorption [10, 11]. Other factors involved in

stress shielding are implant design, biomechanics and the

biological environment. However, the Young’s modulus of

the implant material is probably the most accessible

parameter to be modified to reduce the problem [1]. Many

failures of titanium implants are associated with fractures

of the surrounding bone and subsequent loosening of the

load-bearing component of the prosthesis. Therefore, it is

desirable to design new implants and prostheses with a

lower stiffness than those currently used, which would

allow the stress-shielding problem to be solved or reduced

without any important detrimental effect on mechanical

strength. Within this context, many research works can

currently be found dedicated to the development of new

implant materials with a bone-matching modulus, such as

metastable b-titanium alloys [12], magnesium and its

alloys [13] and porous materials [14, 15]. There are several

manufacturing processes for the latter, among which are

highlighted: the electron beam melting process [16], creep

expansion of argon-filled pores [17], directional aqueous

freeze casting [18], rapid prototyping techniques [19],

Y. Torres (&) � J. A. Rodrıguez

Department of Mechanical and Materials Engineering,

E.T.S. de Ingenierıa, Universidad de Sevilla, Avda.

Camino de los Descubrimientos, s/n, 41092 Sevilla, Spain

e-mail: ytorres@us.es

S. Arias � M. Echeverry � J. J. Pavon

Biomaterials Research Group, Bioengineering Program,

University of Antioquia, Medellın, Colombia

S. Robledo

PECET, University of Antioquia, Medellın, Colombia

V. Amigo

Departamento de Ingenierıa Mecanica y de Materiales,

Universidad Politecnica de Valencia, Camino de Vera s/n,

46022 Valencia, Spain

123

J Mater Sci (2012) 47:6565–6576

DOI 10.1007/s10853-012-6586-9

laser-engineered net shaping [20], electric current acti-

vated/assisted sintering techniques [21, 22], conventional

powder metallurgy [23] and space-holder techniques [24].

In this last process, most of the space-holders (ammonium

bicarbonate [25–28], carbamide [29] and PVA [30]) are

completely evaporated at low temperatures, whilst others

(e.g., sodium chloride [31–35]) are removed by a dissolu-

tion process, generally in water.

Space-holder techniques offer a route to controlling

porosity parameters such as pore morphology and per-

centage. Important efforts and advances within this field

are widely recognized [36]. Most of these works have

focused on the influence of processing conditions on

porosity parameters [24, 29, 37, 38], as well as on the

mechanical response to uniaxial compression [26, 29, 39].

A few of them have studied other interesting properties

such as fracture [27], fatigue [28] and biological testing

[40–42]. These previous manuscripts have not assessed

the overall relationship between the above properties:

processing, microstructure, mechanical and biological

behaviour.

The aim of this work is to evaluate the influence of

compaction pressure on both mechanical properties and the

results of in vitro biological testing of porous cp Ti samples

manufactured by space-holder technique. Samples were

fabricated by a non-conventional powder metallurgy (PM)

process using NH4HCO3 as space-holder. Some process

conditions were fixed according to previous works by the

authors [43]: space-holder content and the procedure for its

elimination, as well as sintering time and temperature. The

influence of different compaction pressures was evaluated

through changes in microstructural and mechanical behav-

iour (yield strength, conventional and dynamic Young’s

modulus) and, finally, the samples were biologically tested

to determine their osteointegration potential.

Materials and methods

Processing of samples

The commercially pure Titanium (CP Ti) powder

(SE-JONG Materials Co. Ltd., Korea) used for the blends

was manufactured by a hydrogenation/dehydrogenation pro-

cess. The particle size distribution corresponded to 10, 50

and 90 % passing percentages, of 9.7, 23.3 and 48.4 lm,

respectively. The chemical composition of the powder used

was equivalent to CP Ti Grade IV according to the ASTM

F67-00 Standard [44]. CP Ti has an apparent density

of 1.30 ± 0.01 g/cm3 (28.8 ± 0.1 %) and a tap density

of 1.77 ± 0.04 g/cm3 (39.2 ± 0.8 %). Its morphology is

approximately equiaxial and its surface is very rough

(Fig. 1). The ammonium bicarbonate particles (NH4HCO3,

Cymit Quımica S.L, Spain) employed as space-holder, has

a particle size corresponding to 10, 50 and 90 % passing

percentages of 73, 233 and 497 lm, respectively, with a

purity of 99.9 %. The initial NH4HCO3 was sieved up to a

range between 100 and 200 lm to obtain a homogeneous

pore size. NH4HCO3 powder has an apparent density of

0.738 ± 0.01 g/cm3 (46.5 ± 0.1 %) and a tap density of

0.87 ± 0.04 g/cm3 (54.83 ± 0.8 %) (Fig. 1). The blends

of CP Ti powder and NH4HCO3 granules [CP Ti ?

NH4HCO3], with a NH4HCO3 concentration of 50 vol.%,

were prepared using a Turbula� T2C blender for 40 min to

ensure good homogenization. This NH4HCO3 content was

chosen according to the previous works by the authors with

conventional PM [35] and space-holder results with NaCl

[45] based on the optimum porosity to match the Young’s

modulus of cortical bone. The compacting step was carried

out using an Instron 5505 universal machine to apply the

pressures used (200, 400, 600 and 800 MPa). The com-

pacting loading rate was 6 kN/s, dwelling time was 2 min

and unloading time was 15 s for decreasing load up to

150 N. Elimination of NH4HCO3 from green samples

was performed thermally by placing the samples in an

oven at 100 �C for 10 h. The sintering process was per-

formed in a Carbolyte� STF 15/75/450 ceramic furnace

with a horizontal tube at 1250 �C for 2 h using high vac-

uum (*5�10-5 mbar). Diameter of compaction die (8 mm)

and powder mass were selected to obtain samples in which

the effect of compaction pressure was minimized [35].

These parameters were kept constant for compression tests

(height/diameter = 0.8 [46]).

Density and porosity characterization

Density measurement was carried out by the Archimedes’

method with distilled water impregnation. This method was

chosen for its experimental simplicity and reasonable

reliability (ASTM C373-88 [47]). This method basically

consisted of: (1) weighing the dry sample, (2) warming the

sample in distilled water for 5 h, (3) leaving the sample for

24 h (to ensure that water penetrates through the porosity),

(4) removing the sample and letting the outer water drain

off, (5) weighing the sample again (mass of saturated

sample), (6) once again, weighing the sample immersed in

water. The total and interconnected porosity were calcu-

lated from density measurements. Three specimens were

employed for each material and three measurements were

made per each sample (9 for each material). The porosity

evaluation by image analysis (IA) was performed using a

Nikon Epiphot optical microscope coupled with a Jenoptik

Progres C3 camera and suitable analysis software (Image-

Pro Plus 6.2). Before the image analysis, the sectioned

parts were prepared by a sequence of conventional steps

(resin mounting, grinding and polishing) followed by a

6566 J Mater Sci (2012) 47:6565–6576

123

mechanochemical polishing with magnesium oxide and

hydrogen peroxide. Image analysis was assessed using 10

pictures of 59 for each sample (one for each kind of

material). The main porosity characteristics estimated by

this method were: (i) the pore shape factor (Ff = 4pA/PE2,

where A is the pore area and PE is the experimental

perimeter of the pore) [48]; (ii) the mean free path between

the pores (k, measure of the mean size of titanium matrix.

This parameter is calculated in the same way as cemented

carbide [49]); (iii) the equivalent diameter (Deq) [48]; and

(iv) the porosity itself.

Mechanical testing

For mechanical compression testing, the specimen dimen-

sions were fixed according to those recommended (height/

diameter = 0.8) in Standard ASTM E9-89a [46]. The tests

were carried out using a universal electromechanical Instron

machine 5505 by applying a strain rate of 0.005 mm/

mm min. All tests were run up to a strain of 50 % and,

afterwards, both Young modulus, E, and yield strength, ry,

were estimated. The Young’s modulus estimation from the

compression stress–strain curves was corrected by including

testing machine stiffness (87.9 kN/mm). Three specimens

were employed for each space-holder percentage. These

tests were performed on the samples in which porosity

(density) and dynamic Young’s modulus were already

measured. Measurement of dynamic Young’s modulus was

performed by an ultrasound technique using a Krautkramer

USM 35� flaw detector. This method allowed both the lon-

gitudinal and transverse propagation velocities of acoustic

waves to be determined. To evaluate the longitudinal waves,

a Panametric S-NDT� 4 MHz ultrasonic transducer was

used with an ultrasonic couplant (Sonotrace grade 30�). For

transverse waves, a Panametric S-V153� 1.5 MHz shear

wave transducer was used using a shear wave couplant

(Panametrics-NDT(TM)). The Metals Handbook section on

non-destructive evaluation and quality control [50] was used

as a reference and includes the results of measurements on

nonporous CP Ti samples corresponding to velocities of

longitudinal and transverse waves of 6.1 and 3.12 km/s,

respectively. Wave velocities through porous samples were

measured by minimizing delay times of transducers fol-

lowing an iterative measurement protocol. Mathematical

Fig. 1 Raw powder materials. a, b Ti; c, d NH4HCO3. a, c represent a general view and b, d are detailed images from the surfaces. It is

remarkable to notice the superficial roughness of both Ti and NH4HCO3 powder

J Mater Sci (2012) 47:6565–6576 6567

123

expression was employed to calculate the dynamic Young’s

modulus once the acoustic wave velocities were measured

(see Eq. 1, [51]). This equation has been used by other

authors [52] and their experimental results were consistent

with poro-micromechanical model predictions based on the

stiffness and strength properties of pure titanium and on the

specific porosity of the sample.

Ed ¼ qv2

T 3v2L � 4v2

T

� �

v2L � v2

T

ð1Þ

where q is the density (g/cm3) and VL and VT are the

longitudinal and transverse velocities, respectively.

In vitro biological testing

The samples were sterilized in water steam at a pressure of

2300 mbar at 121 �C before cytotoxicity and cell mor-

phology tests. These were performed using an osteoblast

cell line SAOS-2 (ATCC, USA). Before testing, the cells

were cultured in culture bottles and grown for 72 h in an

incubator at 37 �C with 5 % CO2. The culture media was

prepared with 10 % foetal bovine serum (FBS) and 90 %

McCoy’s medium (Sigma-Aldrich). Cytotoxicity and cell

morphology tests were both based on the protocol descri-

bed by Denizot et al. [53] and adapted by Echeverry [54].

Porous Ti samples were placed in a culture plate of 48

wells, two of which were used for cytotoxicity-positive

controls and which would contain only culture media and

cells. Osteoblasts were then harvested by adding 0.05 %

trypsin (Sigma) to culture bottles with suitable cell densi-

ties (for 10 min at 37 �C). The cell suspensions were then

decanted into centrifuge tubes and spun at 1300 rpm, for

10 min, to obtain a precipitate. Each Ti sample was then

seeded in a cell suspension (500000 cells/mL) and incu-

bated at 37 �C for 72 h. Cell viability was determined by

the yellow tetrazolium MTT (Sigma) method. The stop

solution consisted of 10 % sodium dodecyl sulphate

(Fischer, USA) and 50 % isopropanol. Production of for-

mazan was determined using a microplate reader (Bench-

mark, Biorad) at 570 nm. Results were expressed as the

cell death percentage. Regarding the cell morphology, the

cell suspension for seeding was obtained by following a

similar protocol as for cytotoxicity; however, in this case, a

concentration of 10000 cells/cm2 was used for each sam-

ple. The seeded samples were incubated at 37 �C for 72 h.

Osteoblasts were then fixed in the material with 2 % glu-

taraldehyde for 12 h at 4 �C. Cell dehydration was

achieved with increasing concentrations of ethanol (30, 50,

70, 80, 90 and 100 %) in phosphate buffered saline (PBS).

Finally, the samples were subjected to critical point drying

and coated with gold to be observed by scanning electron

microscopy (SEM).

Results and discussion

Density and porosity characterization

Porous samples were structurally stable during the whole

process for all the compaction pressures used. Table 1

shows that both total and interconnected porosity for the

whole compaction pressure range were always lower than

the nominal space-holder content (50 vol.%) and those

porosities were slightly higher as the compaction pressure

was increased. This difference with respect to nominal

space-holder content is assumed to be a consequence of

several facts: a higher compaction pressure implies a more

severe plastic deformation of the space-holder, which

allows the coalescence between pores, and therefore, a

higher interconnected porosity; this makes space-holder

elimination easier. Besides this, there is a normal porosity

reduction after sintering, with respect to the nominal one,

which is a well-known phenomenon in both PM and space-

holder practice; it is assumed to be a consequence of

metallic framework shrinkage during sintering, which gen-

erates a reduction in surface energy of particles [37, 55, 56].

This same phenomenon has been observed and discussed

by the authors in a previous work with NaCl as space-

holder [45]. Water content of the space-holder can also

have a small influence on porosity reduction with respect

to initial NH4HCO3 percentage. The slight increment

in the interconnected porosity (36.3–39.9 vol.%) as the

compaction pressure is increased confirms the above

mentioned mechanism because it promotes the coalescence

of pores.

Mechanical testing

The evaluation of Young’s modulus in porous materials is

controversial. Young’s modulus measurements from uni-

axial compression tests are significantly lower than

dynamic measurements. Greiner et al. [57] associated this

discrepancy to super-elastic deformation within the linear-

elastic range of NiTi materials; stiffness measured by

Table 1 Total and interconnected porosity as a function of com-

paction pressure

Powder

metallurgy

process

Compaction

pressure (MPa)

Porosity (%)

Total Interconnected

Space-holder 200 42.8 ± 2.6 36.3 ± 0.4

400 44.1 ± 2.5 37.6 ± 1.3

600 44.7 ± 2.5 38.5 ± 0.3

800 45.0 ± 2.5 39.9 ± 0.2

Conventional 0 41.5 ± 2.5 40.0 ± 0.3

211.5 7.2 ± 2.5 1.9 ± 0.1

6568 J Mater Sci (2012) 47:6565–6576

123

ultrasonic technique decreases with increasing porosity

in agreement with Eshelby’s elasticity-based theory for

closed, spherical porosity. We reported a similar trend for

C.P. Ti obtained by a conventional powder-metallurgy

process [35], as well as in recent work [45] developed with

space-holder (NaCl). These authors related this difference

to the testing machine stiffness in which the mechanical

system and sample were considered as two springs in

series. Moreover, it must be remembered that Ti matrix is

different at each cross section of the cylindrical sample

during a compression test; the material collapse starts at the

section with the lowest Ti content. In works such as those

mentioned above, the reliability and certainty of ultrasound

measurements were validated by comparison with a well

known and accepted pore-elasticity model, such as that of

Nielsen [58].

Initially, influence of testing machine stiffness on the

Young’s modulus evaluations by uniaxial compression

tests was determined. From stress–strain curves (see Fig. 2)

and Young’s modulus measurements with ultrasound tech-

nique (Table 2), it can be observed that increments of

compaction pressure reduce both Young’s modulus and

yield strength with a reduction in the latter. The Young’s

modulus reduction by ultrasound technique is less marked

and even it can be assumed to keep approximately con-

stant (24.63 ± 1.4–24.11 ± 1.5 GPa) because such a small

porosity reduction does not have a strong influence on the

total stiffness of the sample. However, the influence of

compaction pressure on yield strength is clearly greater

(215 ± 11–174.5 ± 6.6 MPa) because this mechanical prop-

erty is sensitive to other parameters besides porosity: neck

strength, pore shape factor, equivalent diameter of pores

and mean separation between pores. With respect to the

influence of intrinsic neck strength, it is normally assumed

that this parameter is greater as the compaction pressure is

increased; this is associated with a breakdown of the oxide

layer and the consequent promotion of a cold welding

effect between particles and subsequent better diffusion

during the sintering. However, from Table 2, it seems that

this is not the case. From Table 2, it is observed that

Young’s modulus and yield strength of space-holder sam-

ples are between those from loose sintering and conven-

tional PM with 211.5 MPa of compaction pressure. This

comparison confirms role of porosity obtained by space-

holder technique.

The pore shape factor, Ff, is more related to influences

on both mechanical strength and fatigue life, as well as in

the case of mean separation between pores, k. Results in

Table 3 are consistent with the expected influence of Ff on

yield strength (greater yield strength values for greater Ff

values). However, k behaviour does not show a significant

role on yield strength. On the other hand, evaluation of the

equivalent diameter of pores, Deq, as compaction pressure

was increased (200–800 MPa) for NH4HCO3 contents

between 30 and 70 vol.% showed increments between 30

and 40 % (Table 3). This latter trend is clearly consistent

with the fact that a smaller pore size indicates greater yield

strength. In summary, despite all the above, the mentioned

parameters are expected to influence mechanical strength;

in this case, dominant role of pore shape factor and

equivalent diameter (pore size) explain the observed behav-

iour of yield strength for increased compaction pressure.

Therefore, the influence of pore equivalent diameter and

pore shape factor can be considered important enough to

affect yield strength in addition to the evident diminishing

effect due to the total and interconnected porosity. It should

be pointed out that this whole line of reasoning was devel-

oped by considering that time and sintering temperatures

were fixed variables, which are well known to have a clear

effect on the mechanical strength of the final PM samples.

Concerning the cortical bone replacement (E * 20

[59, 60]), the best stiffness result measured by ultrasound

technique (24.09 ± 1.5–24.11 ± 1.51 GPa) corresponded

to a space-holder content of 50 % (45 % porosity) for

compaction pressures between 600 and 800 MPa. How-

ever, once the match with Young’s modulus of cortical

bone is considered, it is also important to highlight the

sample which offers a better mechanical balance between

stiffness and strength. In that sense, the best sample was the

one with 50 vol.% of NH4HCO3 (42.8 % porosity) for

200 MPa of compaction pressure: E = 24.63 ± 1.46 GPa

and ry = 215 ± 11 MPa (Cortical bone, ry = 150 MPa).

Despite this optimum value of yield strength being rela-

tively close to that of cortical bone, bigger pores (larger

than 50 lm [61], 100 lm [62–65] and even 150 lm [66]),

Fig. 2 Compression stress–strain curves and yield strength values for

different compaction pressures. Every curve corresponds to a single

run test between carried out for every compaction pressure. The

concerning mean and dispersion values for every batch are shown in

Table 2

J Mater Sci (2012) 47:6565–6576 6569

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will help bone ingrowth into the porous sample, which will

partially reduce the risk of fatigue failure.

In vitro biological testing

Cytotoxicity results were expressed as cell death percent-

ages, according to a simple calculation from the different

absorbance signals from a Biorad reader [54]. Most of the

samples presented cell death percentages lower than 25 %,

which indicates that they are not potentially toxic. How-

ever, some other samples presented a higher value of cell

mortality because of their interconnected porosity, which

allowed cells to settle at the bottom of the recipient; this

fact partially prevents a complete cell recovery to be

accomplished, as will be discussed later in this paper (see

Fig. 3).

Figure 4 summarizes the morphology and cell adhesion

behaviour in samples with increased compaction pressure.

It should be noted that all samples exhibit good osteoblast

adhesion inside the pores, indicating an important osteo-

integration potential.

Morphological details of the cells inside the pores are

diverse and they depend on the topographical features of

that inner surface (see Fig. 5). Osteoblasts on all samples

seem to be well attached to peaks inside the pores or

between depressions, which are separated by a length

similar to osteoblast diameter (*10 lm); this feature is

more clearly observed with the lowest compaction pres-

sure. For some of these inner surfaces, cells presented a

high dorsal surface activity with small diameter cytoplas-

mic extensions, which appear to spread up to the highest

peaks of the surface. The cell morphology becomes wide-

spread with fewer small diameter extensions, as the rough-

ness peaks become more separated and smaller, which

correspond to increasing compaction pressure. Increments

in compaction pressure reduce cell adhesion because the

space-holder becomes more flat as compaction pressure is

Table 2 Mechanical properties estimated from uniaxial compression tests and ultrasound technique

Powder metallurgy process Compaction pressure (MPa) Sintering

(�C, h)

Young’s modulus (GPa) Yield strength

(MPa)Uniaxial compression test Dynamic (ultrasound

technique)Uncorrected Corrected

Space-holder 200 1250; 2 4.5 10.6 ± 2.2 24.6 ± 1.4 215 ± 11

400 5.1 2.7 ± 1.6 24.8 ± 1.6 188 ± 10

600 3.1 8.5 ± 1.8 24.1 ± 1.5 192 ± 8

800 3.1 9.3 ± 1.8 24.1 ± 1.5 174.5 ± 6.6

Conventional 0 1000; 2 3.4 9.6 ± 1.6 23.9 ± 1.8 68 ± 6

211.5 1300; 2 8.8 59.5 ± 2.1 105.4 ± 1.1 620

Measurements errors correspond to standard deviation. Note: yield strength and Young’s modulus for bulk Ti are 650 MPa and 110 GPa,

respectively

Table 3 Microstructural

parameters as a function of

space-holder content and

compaction pressure

Powder metallurgy process Compaction

pressure (MPa)

Deq (lm) Ff k (lm)

Space-holder vol.% 30 200 14 ± 29 0.94 ± 0.15 46.3 ± 85.9

800 18 ± 38 0.93 ± 0.16 56.5 ± 101.9

70 200 38 ± 71 0.81 ± 0.23 48.5 ± 88.0

800 53 ± 97 0.75 ± 0.24 58.7 ± 101.6

Conventional 0 16 ± 17 0.74 ± 0.30 32.8 ± 28.7

211.5 13 ± 15 0.84 ± 0.21 161.7 ± 131.2

Fig. 3 Cytotoxicity results presented as cell death in terms of

compaction pressure

6570 J Mater Sci (2012) 47:6565–6576

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increased. Therefore, once the space-holder is removed, the

inner surface of pores will be smoother. This mechanical

response of the space-holder to increasing compaction

pressures is a consequence of its very low fracture tough-

ness related to its high water content associated with its

hygroscopic behaviour; these facts are responsible for its

plastic-like behaviour under pressure. These results are

consistent with the work of Laptev et al. [37] in which they

found that 50 % NH4HCO3 was the critical content at

which it started to respond in a highly plastic manner as a

lubricant was not necessary for the ejection of green

samples. For comparison purposes, it can be seen that the

inner surfaces of pores of a loose-sintering sample (no

compaction pressure, no space-holder) appear very smooth

(Figs. 6a, 7a).

It is also interesting to note that cell death behaviour in

Fig. 3 can be considered consistent with the better cell

adhesion mentioned above: samples obtained with the

Fig. 4 SEM micrographs of osteoblast morphologies inside the pores for different compaction pressures: a Image of an osteoblast on a simply

machined monolithic Ti sample; b 200 MPa; c 400 MPa; d 600 MPa; e 800 MPa

J Mater Sci (2012) 47:6565–6576 6571

123

lowest compaction pressure show the highest cell death

percentage despite them having less interconnected porosity

(Table 1).

The better cell morphologies observed suggest that

parameters associated with roughness inside the pores are

close to those recognized as improving cell adhesion: for

Boyan and Swartchz [67], cells sense the roughness when

the peak height is higher than 2 lm and lower than the cell

diameter (10–20 lm), as well as for a separation between

peaks no longer than the cell diameter. In a later work,

Aparicio et al. [68] showed more restricted values of these

parameters for optimal osteoblast adhesion for a roughness

obtained by sandblasting with alumina particles: mean

height of peaks (vertical parameter), Ra = 4–5 lm, and

mean separation between peaks (horizontal parameter),

Sm = 70–80/cm. It must be noted that osteoblast mor-

phology on a simply machined monolithic Ti sample pre-

sents a typical rounded shape, indicating a poor cell

adhesion to the substrate (Fig. 4a).

Results of cell adhesion observed in Figs. 4 and 5 are

partly consistent with those desired values of surface

roughness: Ra parameter seems to be within the optimum

values; however, it is appreciated that separation between

peaks is similar to cell diameter (a higher Sm parameter)

which is different to what was reported by the above

authors. The good adhesion results, despite a longer sepa-

ration between peaks, could be explained in terms of sur-

face curvature inside the pores: this factor can play an

important role in cell adhesion and it is not present when

the tested surface is flat, as was the case in the works of the

above authors. However, the real influence of surface

curvature on cell adhesion must be the subject of a detailed

evaluation in a separate study and this will be a subject of

future works.

Cell behaviour observed in Figs. 4 and 5, indicates

that, as compaction pressure is increased, inner roughness

of pores deviates with respect to desired values and cells

become more flat, exhibiting a fibroblast-like behaviour

(less osteoblastic) with a clearly less adhesive force. The

high dorsal activity of cells attached to rough surfaces is

also an indicator of the active state of cell phenotype

development that is, at the same time, an indicator of a

better differentiation response of osteoblasts. Interestingly,

the better cell adhesion results obtained in this work

Fig. 5 SEM micrographs showing details of osteoblast adhesion inside the pores for different compaction pressures: a 200 MPa; b 400 MPa;

c 600 MPa; d 800 MPa

6572 J Mater Sci (2012) 47:6565–6576

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(compaction pressure of 200 MPa) are coincident with

samples of the best mechanical balance for cortical bone

replacement: E = 24.6 GPa and ry = 215 MPa. How-

ever, it must be mentioned that reaching a complete

mechanical balance will require a detailed fatigue evalu-

ation in which case inner roughness of pores for samples

compacted with 200 MPa could have some influence.

This is part of current research that the authors will be

submitting soon.

Conclusions

Once the influence of compaction pressure for a fixed

space-holder content (NH4HCO3) designed for porous

titanium implants was evaluated, the following conclusions

were drawn:

• Total and interconnected porosity of samples for the

whole compaction pressure range were always lower

Fig. 6 SEM micrographs of porosity behaviour for different compaction pressures: a porosity of a conventional PM sample, loose sintering at

1300 �C for 2 h; b 200 MPa; c 400 MPa; d 600 MPa; e 800 MPa

J Mater Sci (2012) 47:6565–6576 6573

123

than nominal space-holder content; both kinds of

porosities were higher as the compaction pressure was

increased. This porosity behaviour is assumed to be a

consequence of severe plastic deformation of space-

holder as compaction pressure is increased.

• Mechanical testing showed that increments of compac-

tion pressure slightly reduce Young’s modulus because

such a small porosity reduction does not have a strong

influence on it. Significant reduction of yield strength

with compaction pressure can be explained because this

mechanical property is especially sensitive to the

equivalent diameter of pores. This parameter presents

increments between 30 and 40 %, which is large

enough to influence yield strength, in addition to an

evident diminishing effect on total and interconnected

porosity.

• The optimum balance of mechanical properties with

respect to cortical bone was achieved in a sample with

42.8 % porosity and 200 MPa of compaction pressure:

E = 24.6 GPa and ry = 215 MPa. Both the percentage

Fig. 7 SEM micrographs showing roughness inside the pores for different compaction pressures: a porosity of a conventional PM sample, loose

sintering at 1300 �C for 2 h; b 200 MPa; c 400 MPa; d 600 MPa; e 800 MPa

6574 J Mater Sci (2012) 47:6565–6576

123

of big pores (larger than 50, 100 lm and even 150 lm)

and the interconnected porosity achieved are factors

that can improve the ingrowth of cortical bone through

the porous implants, partially reducing the risk of

fatigue failure.

• Cytotoxicity tests showed that all porous samples were

non-toxic, which validates the biological viability of all

processing conditions. Biological tests also showed

osteoblast behaviour inside residual pores from a space-

holder technique. The osteoblast morphologies at all

compaction pressures were considered to be promising

for improving osteointegration of titanium implants.

The attachment force of cells is better for rougher inner

surfaces of the pores, i.e., increments in compaction

pressure reduce cell adhesion because the space-holder

becomes more flat as compaction pressure is increased.

In that sense, the better cell adhesion results obtained in

this work (compaction pressure of 200 MPa) are

coincident with samples with the best mechanical

balance for cortical bone replacement: E = 24.6 GPa

and ry = 215 MPa.

• The optimum behaviour of samples obtained in this

work with lowest compaction pressure (200 MPa), with

50 vol.% NH4HCO3, indicates that it is a promising

route for developing titanium implants with a high

cellular adhesion (improved osteointegration), suitable

bone ingrowth and a clearly reduced stress shielding

with a reasonably good mechanical strength.

Acknowledgments This work was supported by the Ministerio de

Ciencia y Tecnologıa, MICINN (Spain) through the project Ref.

MAT2010-20855. Furthermore, the authors want to thank laboratory

technicians J. Pinto and M. Sanchez, and the undergraduate student I.

Nieto for their assistance in microstructure characterization and

mechanical testing.

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