Bioceramics in Simulated Body Fluid

1 23

Journal of Materials Science:Materials in MedicineOfficial Journal of the European Societyfor Biomaterials ISSN 0957-4530 J Mater Sci: Mater MedDOI 10.1007/s10856-014-5229-x

Investigating the surface reactivity of SiO2–TiO2–CaO–Na2O/SrO bioceramics as afunction of structure and incubation timein simulated body fluid

Y. Li, A. Coughlan & Anthony. W. Wren

1 23

Your article is protected by copyright and all

rights are held exclusively by Springer Science

+Business Media New York. This e-offprint is

for personal use only and shall not be self-

archived in electronic repositories. If you wish

to self-archive your article, please use the

accepted manuscript version for posting on

your own website. You may further deposit

the accepted manuscript version in any

repository, provided it is only made publicly

available 12 months after official publication

or later and provided acknowledgement is

given to the original source of publication

and a link is inserted to the published article

on Springer's website. The link must be

accompanied by the following text: "The final

publication is available at link.springer.com”.

Investigating the surface reactivity of SiO2–TiO2–CaO–Na2O/SrObioceramics as a function of structure and incubation timein simulated body fluid

Y. Li • A. Coughlan • Anthony. W. Wren

Received: 21 January 2014 / Accepted: 21 April 2014

� Springer Science+Business Media New York 2014

Abstract This study focuses on evaluating the biocom-

patibility of a SiO2–TiO2–CaO–Na2O/SrO glass and glass–

ceramic series. Glass and ceramic samples were synthe-

sized and characterized using X-ray diffraction. Each

material was subject to maturation in simulated body fluid

over 1, 7 and 30 days to describe any changes in surface

morphology. Calcium phosphate (CaP) deposition was

observed predominantly on the Na? containing amorphous

and crystalline materials, with plate-like morphology. The

precipitated surface layer was also observed to crystallize

with respect to maturation, which was most evident in the

amorphous Na? containing glasses, Ly-N and Ly-C. The

addition of Sr2? greatly reduced the solubility of all sam-

ples, with limited CaP precipitation on the amorphous

samples and no deposition on the crystalline materials. The

morphology of the samples was also different, presenting

irregular plate-like structures (Ly-N), needle-like deposits

(Ly-C) and globular-like structures (Ly-S). Cell culture

analysis presented a significant increase in cell viability

with the Na? materials, 134 %, while the Sr2? containing

glasses, 60–80 % and ceramics, 60–85 % presented a

general reduction in cell viability, however these reduc-

tions were not significant.

1 Introduction

In recent years bioactive glasses and ceramics have stim-

ulated interest as materials that can stimulate the regener-

ation of bone tissue [1]. A common and widely studied

characteristic of bioactive glasses and glass–ceramics if the

formation of a biologically active apatite (A) layer which

supports bone bonding which can be evaluated using

simulated body fluid (SBF), which is a solution that con-

tains an ionic composition similar to that of human blood

plasma [2, 3]. Prior to the 1970s, artificial materials that

were implanted into the human body, specifically bone

defects, resulted in the materials being encapsulated by

fibrous tissue which resulted in the materials isolation for

the surrounding bone. In the early 1970s, Hench [4, 5]

produced glass in the Na2O–CaO–SiO2–P2O5 system that

spontaneously bonds to living bone without the formation

of surrounding fibrous tissue. Since this development,

many different types of ceramics such as sintered

hydroxyapatite, sintered b-tricalcium phosphate, A/b-tri-

calcium phosphate biphasic ceramics and glass/ceramic A–

W (wollastonite) have been shown to bond to living bone

[2].

Additionally, many different materials have been pro-

duced from bioactive glass and ceramics that can be used

for numerous medical applications. These materials include

glass–ceramic scaffolds [6–8] for bone repair, glass

microspheres for cancer treatment [9–11], composite

materials for drug release [12, 13] and also composite

materials where bioactive glasses are used to improve

bioactivity or mechanical strength [14–17]. Bioactive glass

and ceramics are a valuable addition to medical materials

as they can incorporate biological compatibility with

mechanical strength and bone adhesion, vital components

for skeletal repair [18]. However, uncertainties still exists

Y. Li � Anthony. W. Wren (&)

Inamori School of Engineering, Alfred University, Alfred,

NY 14802, USA

e-mail: [email protected]

A. Coughlan

School of Materials Engineering, Purdue University,

West Lafayette, IN, USA

123

J Mater Sci: Mater Med

DOI 10.1007/s10856-014-5229-x

Author's personal copy

relating to the mechanical durability due to their interac-

tions within the biological environment, and also the effect

of mechanical strain and the dissolution rate of materials,

and as such, altering the mechanical properties of a bio-

active glass without compromising its bioactivity is of key

interest [18]. This concern has previously been highlighted

when producing glass–ceramic scaffolds from 45S5 Bio-

glass�, where it has been suggested that crystallization of

45S5 Bioglass� can reduce its bioactivity as, post sintering,

crystallization turns the material from being a bioactive

material into an inert ceramic [19]. However, there have

since been studies that suggest the predominant crystal

phase of 45S5 Bioglass� (Na2Ca2Si3O9), can significantly

improve the mechanical properties of the material, and that

crystallization does not inhibit the bioactivity as precipi-

tation of an amorphous calcium phosphate (CaP) occurs in

biological fluids [20–22].

This study was conducted to determine any differences

in bioactivity and subsequent changes in surface mor-

phology, specifically in SBF and cell culture, as a function

of material composition (Na?/Sr2? concentration), incu-

bation time, and structure (amorphous/crystalline). The

starting glass composition, SiO2–TiO2–CaO–Na2O/SrO,

alters Na? and Sr2? concentration which will affect the

dissolution rate of the glasses. A previous study by the

authors on the solubility of these materials determined that

(1) crystallization greatly reduces the ion release rate, (2)

pH is also reduced with crystallization, (3) the mechanical

durability of the materials is greatly enhanced by crystal-

lization where no significant changes are observed after

30 days in aqueous media and (4) the Na? containing

materials produces higher ion release rates than the Sr2?

analogues. The glass composition utilized in this study was

selected as Na? is critical in promoting dissolution of the

glass as it acts as a network modifier within the glass

network [22, 23]. Sr2? also acts as a network modifier

within the glass, however, it shares atomic similarities to

Ca2? and has been previously investigated and applied to

treating postmenopausal women with osteoporosis [24, 25].

Titanium (Ti) has been incorporated as numerous studies

cite that Ti containing materials result in the deposition of

CaP surface layer when incubated in SBF [26]. That can be

attributed to the formation of Ti–OH groups providing

favourable conditions for Ca precipitation [26].

2 Materials and methods

2.1 Glass synthesis

Three glass compositions (Ly-N, Ly-C, Ly-S) were formu-

lated for this study with the principal aim being to inves-

tigate any property changes with the modification of

sodium (Na?) and strontium (Sr2?) in the glass. A control

glass (Ly-C) was also formulated which contained equal

quantities of Na? and Sr2?. Glasses were prepared by

weighing out appropriate amounts of analytical grade

reagents and ball milling (1 h, Table 1).

2.1.1 Glass powder production

The powdered mixes were oven dried (100 �C, 1 h) and

fired (1,500 �C, 1 h) in platinum crucibles and shock

quenched into water. The resulting frits were dried, ground

and sieved to retrieve glass powders with a maximum

particle size of 45 lm.

2.1.2 Disc sample preparation

Disc samples (Ly-N, Ly-C, Ly-S) were prepared by

weighing approximately 0.5 g powder into a stainless steel

die (sample dimensions 1.5 9 6/ mm) which was pressed

under 3 tonnes of pressure. Disc samples were kept

amorphous for Ly-N, Ly-C, Ly-S by heat treating the

pressed discs below the glass transition temperature for

24 h, and crystalline analogues were heat treated to the

sintering temperature for Ly-N (653 �C), Ly-C (713 �C)

and Ly-S (825 �C) in order to determine any differences in

bioactivity as a result of structure. Disc samples were then

used for SBF testing and while 100 ll extracts were used

for indirect cell culture analysis.

2.2 Glass characterization

2.2.1 X-ray diffraction (XRD)

Diffraction patterns were collected using a Siemens D5000

XRD Unit (Bruker AXS, Inc., WI, USA). Glass powder

samples were packed into standard stainless steel sample

holders. A generator voltage of 40 kV and a tube current of

30 mA was employed. Diffractograms were collected in

the range 10� \ 2h\ 80�, at a scan step size 0.02� and a

step time of 10 s. Any crystalline phases present were

identified using JCPDS (Joint Committee for Powder Dif-

fraction Studies) standard diffraction patterns (data repro-

duced from previously published manuscript [27], Fig. 1;

Table 3).

Table 1 Glass composition

(mol. fr.)Ly-N Ly-C Ly-S

SiO2 0.55 0.55 0.55

TiO2 0.05 0.05 0.05

CaO 0.22 0.22 0.22

Na2O 0.18 0.09 0.00

SrO 0.00 0.09 0.18


123


2.2.2 Hot stage microscopy (HSM)

A MISURA side view HSM, Expert Systems (Modena,

Italy), with image analysis system and electrical furnace,

with max temperature of 1,600 �C and max rate of 80 �C/

min. The parameters for this experiment were a heat rate of

20 �C/min from 20 to 500 �C and 5 �C/min from 500 to

1,200 �C. The computerized image analysis system auto-

matically records and analyses the sample geometry during

heating.

2.2.3 Scanning electron microscopy and energy dispersive

X-ray analysis (SEM–EDS)

Backscattered electron imaging was carried out with an

FEI Co. Quanta 200F Environmental SEM. Additional

compositional analysis was performed with an EDAX

Genesis Energy-Dispersive Spectrometer. All EDS spectra

were collected at 20 kV using a beam current of 26 nA.

Quantitative EDS spectra were subsequently converted into

relative concentration data.

Fig. 1 Scanning electron

microscopy of a Ly-N, bLy-C and c Ly-S including

corresponding EDX and

quantitative composition in

mol%


123


2.3 Biocompatibility testing

2.3.1 Sample preparation

Discs of each glass were autoclaved prior to use and sterile

de-ionized water was used as the solvent to prepare

extracts. The volume of extract was determined using

Eq. 1. Vs is the volume of extract used, Sa is the exposed

surface area of the disc.

Vs ¼ Sa

10: ð1Þ

Samples (n = 3 amorphous, and n = 3 crystalline) were

aseptically immersed in appropriate volumes of sterile de-

ionized water and agitated at (37 ± 2 �C) for 1, 7 and

30 days after which 100 ll of fluid was used for cytotox-

icity testing.

2.3.2 SBF trial

SBF was produced in accordance with the procedure out-

lined by Kokubo and Takadama [2]. The composition of

SBF is outlined in Table 2. The reagents were dissolved in

order, from reagent 1 to 9, in 500 ml of purified water

using a magnetic stirrer. The solution was maintained at

36.5 �C. 1 M-HCl was titrated to adjust the pH of the SBF

to 7.4. Purified water was then used to adjust the volume of

the solution up to 1 l. Glass/ceramic discs (n = 2) were

immersed in concentrations of SBF as determined by Eq. 1

and were subsequently stored in for 1, 7 and 30 days in an

incubator at 37 �C. A JEOL JSM-840 SEM equipped with

a Princeton Gamma Tech energy dispersive X-ray (EDX)

system was used to obtain secondary electron images and

carry out chemical analysis of the surface of glass and

ceramic discs. All EDX spectra were collected at 20 kV,

using a beam current of 0.26 nA. Quantitative EDX con-

verted the collected spectra into concentration data by

using standard reference spectra obtained from pure ele-

ments under similar operating parameters.

2.3.3 Cell culture analysis

The established cell line L929 (American Type Culture

Collection CCL 1 fibroblast, NCTC clone 929) was used in

this study as required by ISO10993 part 5. Cells were

maintained on a regular feeding regime in a cell culture

incubator at 37 �C/5 % CO2/95 % air atmosphere. Cells

were seeded into 24 well plates at a density of 10,000 cells

per well and incubated for 24 h prior to testing with both

extracts and cement discs. The culture media used was

M199 media supplemented with 10 % fetal bovine serum

(Sigma Aldrich, Ireland) and 1 % (2 mM) L-glutamine

(Sigma Aldrich, Ireland). The cytotoxicity of cement

extracts was evaluated using the methyl tetrazolium (MTT)

assay in 24 well plates. Aliquots (100 ll) of undiluted

sample were added into wells containing L929 cells in

culture medium (1 ml) in triplicate over 1, 7 and 30 days.

Cement discs (n = 3 amorphous, and n = 3 crystalline)

were placed in the plate wells and were tested after 24 h.

Each of the prepared plates was incubated for 24 h at

37 �C/5 % CO2. The MTT assay was then added in an

amount equal to 10 % of the culture medium volume/well.

The cultures were then re-incubated for a further 2 h

(37 �C/5 % CO2). Next, the cultures were removed from

the incubator and the resultant formazan crystals were

dissolved by adding an amount of MTT Solubilization

solution [10 % Triton X-100 in acidic isopropanol (0.1 N

HCI)] equal to the original culture medium volume. Once

the crystals were fully dissolved, the absorbance was

measured at a wavelength of 570 nm. Aliquots (100 ll) of

tissue culture water were used as controls, and cells were

assumed to have metabolic activities of 100 %.

2.4 Statistical analysis

One-way analysis of variance was employed to compare

the difference in cell viability as a function of maturation

(1, 7 and 30 days) for each material tested. Comparison of

relevant means was performed using the post hoc Bonfer-

roni test. Differences between groups was deemed signifi-

cant when P B 0.05.

3 Results

3.1 Material characterization

Characterization techniques utilized for this study includes

scanning electron microscopy (SEM/EDX), XRD and

HSM. SEM and the corresponding EDX are presented in

Fig. 1 for Ly-N, Ly-C and Ly-S. Figure 1a presents Ly-

N which confirms the composition contains Si4?, Ca2?,

Ti4? and Na?, which has a composition similar to batch

Table 2 Ionic composition of SBF [2]

Orders Reagents Amount

1 NaCl 7.996 g

2 NaHCO3 0.350 g

3 KCl 0.224 g

4 K2HPO4�3H2O 0.228 g

5 MgCl2�6H2O 0.305 g

6 1 M-HCl 40 ml

7 CaCl2 0.278 g

8 Na2SO4 0.071 g

9 NH2C(CH2OH)3 6.057 g


123


calculations. Minor differences include Si4? slightly higher

at 60 % and Na? at 11 %. Figure 1b presents Ly-C which

has a composition similar to the batch calculations, addi-

tionally, minor difference include Si4? at 57 % and Ca2?

slightly lower than batch calculations at 19 %. Both Na?

and Sr2? levels were determined to be 9 %, similar to batch

calculations. Figure 1c presents Ly-S which also has a

composition close to the original batch calculation. Very

slight compositional differences include Si4? being 56 %

and Ca2? being slightly lower than original calculations at

20 %. XRD patterns for each material are presented in

Fig. 2 with the complete list of crystal phases and crystal

size for Ly-N, Ly-C and Ly-S. Ly-N presented in Table 3.

The crystal phases for Ly-C contain sodium calcium sili-

cate phases (combeite) in addition to SiO2. Ly-S was found

to contain multiple crystal phases and each is listed in

Table 3, however as there is no Na? in the starting com-

position, no sodium calcium silicate phases (combeite)

exist. Additionally, the crystal size for each phase was

calculated and for Ly-N the mean crystal size was 601 A,

for Ly-C the crystal size exceeded 1,000 A and for Ly-S the

mean crystal size was 348 A. HSM data is presented in

Fig. 3 and presents the sintering (Ts), softening (Tf) and

melting (Tm) temperature of Ly-N, Ly-S and Ly-C. From

Fig. 3 it is evident the thermal properties of each material

change as the Na?/Sr2? concentration differs. Regarding

Ly-N the Ts was found to be 653 �C, however as Sr2? is

substituted the Ts increases to 713 �C (Ly-C) and 825 �C

(Ly-S). Regarding the Tf it was found to decrease from

866 �C (Ly-N) to 745 �C (Ly-C) and to increase to

1,243 �C (Ly-S) as the Sr2? concentration increased. The

Tm for Ly-N and Ly-C were similar at 1,068 and 1,115 �C,

respectively, whereas Ly-S was found to be 1,244 �C.

3.2 Evaluation of surface dissolution and reactivity

SBF testing was conducted on glass and ceramic disc

samples with respect to (1) composition, (2) amorphous

and crystalline structure and (3) incubation time, over 1, 7

and 30 days. With respect to Figs. 4, 6 and 8, images are

presented at 1, 7 and 30 days for both the amorphous and

crystalline analogues, and the corresponding EDX of the

30 day samples. Figure 4 presents the Ly-N SBF results

where after 1 day incubation there was no CaP deposition

present. It is evident from Fig. 4 that after 7 days

Fig. 2 X-ray diffraction of a amorphous materials and b glass–

ceramic materials

Table 3 Crystal phases identified for Ly-N, Ly-C and Ly-S (see

Fig. 1)

Phase ID Reference

codes

Crystal size

(A)

Ly-N

Combeite - Na2.2Ca1.9Si3O9 04-04–2757 612

Sodium: Na2Ca3Si6O16 04-012–8681 591

Ly-C

Combeite: Na4.8Ca3Si6O18 04-007–5453 [1,000

Silicon dioxide: SiO2 04-007–5453 [1,000

Ly-S

Strontium silicon: Sr2Si3 01-089–2593 348

Titanium oxide: Ti8O15 04-007–0444 138

Calcium silicon: CaSi2 04-007–0647 229

Strontium silicide: SrSi 01-076–7303 317

Strontium titanium silicate:

Sr2TiSi2O8

04-006–7366 261

Silicon oxide: SiO2 00-029–0085 [1,000

Perovskite: CaTiO3 04-015–4851 229

Fig. 3 Hot stage microscopy testing of Ly-N, Ly-C and Ly-S present-

ing sintering, softening and melting temperature for each material


123


incubation, the amorphous Ly-N surface was completely

covered in CaP. The crystalline counterpart did experience

CaP deposition, however it was minimal compared to the

amorphous Ly-N. However, after 30 days the surfaces of

the amorphous and crystalline analogues of Ly-N were

fully covered in CaP. The corresponding EDX detected the

presence of phosphate at *9 and *4 wt% for the amor-

phous and crystalline Ly-N, respectively. Figure 5 presents

the 30 days SEM images of the amorphous and crystalline

Ly-N at 1, 5 and 50k magnification. With respect to

Ly-C (Fig. 6) the surface of the amorphous Ly-C is similar

to Ly-N amorphous; however the crystalline counterpart

lacks the porous surface presented by Ly-N crystalline. CaP

deposition can be seen on the 7 days Ly-C amorphous and

the surface is fully covered after 30 days. Corresponding

EDX from 30 day samples presents high P levels in the Ly-

C amorphous, however a relatively low P signal was

present for Ly-C crystalline.

In order to investigate any changes in the surface of the

materials with respect to incubation time, XRD was con-

ducted on the samples that presented complete coverage by

CaP both after 30 days incubation in SBF. Figure 7 pre-

sents diffraction patterns of Ly-N (amorphous and crystal-

line) and Ly-C (amorphous) before and after 30 days

immersion in SBF. From Fig. 7a is evident that after

30 days crystal peaks are forming from the initially glassy

structure, which can be directly attributed to the deposition

of CaP as the starting material is amorphous. Crystal peaks

were identified as CaP (PDF 04-014-2292, Ca3(PO4)2)

which suggests that the crystal phase is an immature form

of hydroxyapatite. Figure 7b presents the crystalline ana-

logue of Ly-N which is initially predominantly crystalline.

However, after 30 days in SBF, the region corresponding

to 5–30� 2h exhibit a relatively minor amorphous region in

addition to the loss of a number of peaks in the 5–30� 2h,

and between 50 and 75� 2h. This is indicative of a poorly

Fig. 4 SEM images of amorphous and crystalline Ly-N after 1, 7 and 30 days in SBF and 30 day EDX of the amorphous and crystalline surfaces


123


crystalline CaP surface layer which is predominantly

amorphous with minor crystal formation. Attempts to

identify any newly formed crystal regions were inconclu-

sive. Figure 7c presents Ly-C amorphous which also pre-

sents a number of minor crystal peaks which are present at

55 and 60� 2h. However, this was less pronounced than

Ly-N amorphous which made phase identification difficult.

Regarding Ly-S, SEM imaging and the corresponding

30 days EDX are presented in Fig. 8. With respect to the

amorphous samples, CaP deposition can be observed at

each time period (1, 7 and 30 days), however to a much

lesser degree than the Na? containing glasses. It is evident

from the EDX that after 30 days CaP is localized, and is

not as prevalent in density as Ly-N amorphous and

Fig. 5 Low and high magnification SEM images of Ly-N amorphous and crystalline samples after 30 days in SBF


123


crystalline and Ly-C amorphous. With respect to the

crystalline analogue of Ly-S, there was relatively minor

detection of P at less than 0.5 wt% for each time period.

When comparing the CaP structures formed with respect to

each material (Fig. 9), it is evident that different mor-

phologies exist for both Na? and Sr2? containing materials.

Figure 9a presents Ly-N at 7 days displaying full surface

coverage with highly irregular, plate-like crystal formation.

Figure 9a presents the intermediate glass (Ly-C) at 7 days

which predominantly displays needle-like crystals that

seem to be growing epitaxially, in addition, it is evident

that precipitation is beginning on the particles on the left of

the same image. This may be the beginning the formation

of plate-like crystal similar to the Ly-N as after 30 days the

final morphology is similar to that of Ly-N at 30 days. With

respect to Ly-S, CaP deposition presented spherical glob-

ular like projections that were far less abundant that the

plate-like projections presented on Ly-N and Ly-C.

3.3 Evaluation of cytocompatibility

Regarding cell culture analysis, Fig. 10a presents the

cytocompatibility of the amorphous samples while

Fig. 10b presents the crystalline analogues. It is evident

from Fig. 10a that Ly-N presented a steady increase in cell

viability. The increase was not deemed significant at 1 day

(94 %, P = 1.000) or 7 day (111 %, P = 0.900), however

at 30 days a significant increase was observed (134 %,

P = 0.0001). Ly-C amorphous remained relatively con-

stant over each time period with no significant change

(P = 0.037–0.197) when compared to the growing cell

population. The Sr2? containing series, Ly-S, produced

significantly lower cell viability at 1 day (70 %,

P = 0.007) and 7 day (59 %, P = 0.0001) however, no

significant change in viability (68 %) was observed at

30 days. With respect to the crystalline analogues there

was no significant increase in cell viability for any of the

Fig. 6 SEM images of amorphous and crystalline Ly-C after 1, 7 and 30 days in SBF and 30 day EDX of the amorphous and crystalline surfaces


123


Fig. 7 XRD of samples with surface changes in SBF before and after 30 days incubation

Fig. 8 SEM images of amorphous and crystalline Ly-S after 1, 7 and 30 days in SBF and 30 day EDX of the amorphous and crystalline surfaces


123


materials. There was a significant decrease in viability for

Ly-C at 7 days (59 %, P = 0.000), and 30 days (71 %,

P = 0.001). Ly-S also presented an overall reduction in cell

viability; however this change did not reach significance.

4 Discussion

4.1 Material characterization

SEM coupled with EDX was used to image the glass

particles and estimate the glass composition. The mean

particle size was previously determined for each glass,

which were similar at 4.6 lm for Ly-N, 3.9 lm for

Ly-C and 4.6 lm for Ly-S [27]. Particle size distribution

can significantly influence biocompatibility through chan-

ges in exposed surface area and the subsequent particle

dissolution rate. It can also be observed that the Na?

containing glasses (Ly-N, Ly-C) were found to have smaller

particles agglomerated to larger particles, in particular with

Ly-N. This agglomeration of the Na? containing glasses

may be due to electrostatic charge build up during pro-

cessing. With respect to glass composition, the batch

calculation closely resembled the data acquired by EDX.

XRD was conducted on each material to confirm the

amorphous/crystalline state and to determine the phases

present in the crystalline materials. Sodium calcium silicate

phases were found to exist in Ly-N and Ly-C, whereas

numerous phases were detected in Ly-S (Table 3). Previous

studies suggest that crystallization converts glass from

being a bioactive to an inert material and, in the case of

glass–ceramic scaffolds, the mechanical integrity is also

compromised. However additional studies by Chen et al.

[28] suggest that crystalline 45S5 Bioglass� forms Na2-

Ca2Si3O9 phases that can significantly improve the

mechanical properties of the material, that crystallization

does not inhibit bioactivity with respect to bone bonding

ability and when immersed in body fluids the crystalline

Na2Ca2Si3O9 decomposes and transits to amorphous CaP.

Studies by Clupper and Hench [20] determined that the

predominant crystal phase associated with Bioglass�,

Na2Ca2Si3O9 slightly decreases the formation kinetics of

an A layer on Bioglass� surface but did not totally suppress

its formation. Further studies by Filho et al. [21] found that

there is no compromise in bioactivity for the 45S5 glass–

ceramic system even when 100 % crystalline. Their study

Fig. 9 SEM images of CaP deposition on a Ly-N, b Ly-C and c Ly-S

Fig. 10 Cell viability of

amorphous and crystalline

samples


123


in particular focused on determining the surface precipi-

tation reactions in SBF and determined that by inducing

crystallinity (ranging from 8 to 100 %), the materials

maintained their bioactivity in SBF [21]. HSM determined

that differences in the thermal properties of the materials

existed where the sintering temperature increases with an

increase in Sr2? concentration, i.e. Ly-N \ Ly-C \Ly-S. This may be due to the divalent nature of Sr2?,

charge neutralizing intermittent non bridging oxygen sites

in the glass, where two Na? atoms are required to fulfill the

same vacancy. This would likely result in higher temper-

atures to decompose the Ly-S glass particles to the extent

required for sintering, as compared to Ly-N.

4.2 Evaluation of surface characteristics and reactivity

With respect to the amorphous surface for each material,

differences in surface morphology exist where the particles

are fused together resulting in a ‘cobblestone’ like surface.

The crystalline analogues for each material formed a por-

ous/dense or irregular surface morphology relatively spe-

cific to the materials composition. CaP deposition on the

Ly-N supports earlier findings by Clupper and Hench,

where the formation of an A layer was decreased but not

totally suppressed. The higher resolution images (Fig. 5)

confirm the complete coverage of both Ly-N amorphous

and crystalline surfaces with CaP. Higher resolution SEM

images (50k) present a floral-plate like CaP present for

both materials, which is similar in structure to A deposition

on A–W glass–ceramic [2]. This suggests that the CaP

deposited is similar irrespective of amorphous/crystalline

structure, with the only difference being the time required

for this surface layer to deposit. Ly-C amorphous presented

CaP deposition the covered the surface after 30 days. The

lack of surface precipitation on Ly-C crystalline may be

due to a number of factors. The lower Na? concentration in

the starting glass results in reduced solubility, which

reduces the rate of ion release from the material. Previous

ion release studies on these materials by the authors

resulted in the Na? containing materials being more solu-

ble than the Sr2? (i.e. Ly-N [ Ly-C [ Ly-S) [27]. In

addition, the amorphous materials were found to be far

more soluble than the crystalline analogues [27]. XRD

conducted on samples with full CaP surface coverage

determined that the evolution of crystal structures was

identified predominantly in samples containing Na?.

Although the amorphous samples Ly-N and Ly-C induced

complete surface coverage after 30 days, the crystalline

analogue of Ly-N produced the same surface morphology

which took longer to produce as there was no significant

coverage after 7 days, suggesting that a solubility limit

exists. The precipitation of the CaP crystals support this

where the Na? containing materials produced plate-like

crystals (Fig. 9) after 30 days, whereas Ly-S produced

globular-like crystals which were only sporadically dis-

tributed on the surface after 30 days. Also, it seems the

reduced solubility in addition to the inclusion of Sr2?,

results in an overall reduction in CaP deposition.

4.3 Evaluation of cytocompatibility

A general trend that can be observed with the cell viability

data is that as Sr2? is incorporated into the glass, there is a

general reduction in cell viability. Ly-N produced the

highest cell viability after 30 days at 134 %, which is

likely attributed to the solubility [27] and the release of

Na?. Na? is well known to be critical to cellular metabo-

lism including providing the required ion concentration

gradients between the interstitial fluids and the cytoplasm.

As such, cells have the ability to tightly control the influx/

efflux of ions like Na?, K? and Ca2?. The solubility of the

Ly-C and Ly-S is lower [27] than Ly-N which may be a

contributing factor to the reduction/insignificant change in

cell viability. In particular with Ly-S, it is also possible that

Sr2? hinders the metabolic process in this particular cell

line, as positive reports have been established regarding

Sr2? use in osteoblasts [24]. With respect to the crystalline

analogues, which were much less soluble than the amor-

phous counterparts [27], there were relatively insignificant

changes in cell viability. This may be due to a combination

of the reduced ion release rate, in addition to these cells not

requiring Sr2? for metabolism and ion homeostasis.

5 Conclusion

The work determined that the Na? containing glasses and

ceramics consistently produced CaP surface layers more

readily than the Sr2? containing materials. However, dif-

ferent morphologies of CaP was observed with differences

in glass composition. Additionally, the CaP surface layer

was observed to crystallize after 30 days with the Na?

containing materials. Testing of the liquid extracts in cell

culture was observed to significantly increase cell viability

in the Na? containing glasses, while no significant toxicity

was experienced with the Sr2? and crystalline analogues of

each material. This study concludes that the inclusion of

Na? significantly enhances the surface reactivity of

bioceramics, and that the addition of ions with different

electronic states can significantly influence the ion release

rate, atomic arrangement and morphology of the precipi-

tated surface A layer, and the associated cellular response.


123


References

1. Jones JR. Review of bioactive glass: from Hench to hybrids. Acta

Biomater. 2013;9:4457–86.

2. Kokubo T, Takadama H. How useful is SBF in predicting in vivo

bone bioactivity. Biomaterials. 2006;27:2907–15.

3. Kokubo T, Kim H-M, Kawashita M. Novel bioactive materials

with different mechanical properties. Biomaterials. 2003;24:

2161–75.

4. Hench LL. The story of Bioglass. J Mater Sci Mater Med. 2006;

17:967–78.

5. Hench LL. Genetic design of bioactive glass. J Eur Ceram Soc.

2009;29(7):1257–65.

6. Haimi S, Gorianc G, Moimas L, Lindroos B, Huhtala H, Raty S,

Kuokkanen H, Sandor GK, Schmid C, Miettinen S, Suuronen R.

Characterization of zinc-releasing three-dimensional bioactive

glass scaffolds and their effect on human adipose stem cell pro-

liferation and osteogenic differentiation. Acta Biomater.

2009;5(8):3122–31.

7. Vargas GE, Mesones RV, Bretcanu O, Lopez JMP, Boccaccini

AR, Gorustovich A. Biocompatibility and bone mineralization

potential of 45S5 Bioglass�-derived glass–ceramic scaffolds in

chick embryos. Acta Biomater. 2009;5(1):374–80.

8. Chen QZ, Rezwan K, Francon V, Armitage D, Nazhat SN, Jones

FH, Boccaccini AR. Surface functionalization of Bioglass�-

derived porous scaffolds. Acta Biomater. 2007;3(4):551–62.

9. Anderson JH, Goldberg JA, Bessent RG, Kerr DJ, McKillop JH,

Stewart I, Cooke TG, McArdle CS. Glass yttrium-90 micro-

spheres for patients with colorectal liver metastases. Radiother

Oncol. 1992;25(2):137–9.

10. Bortot MB, Prastalo S, Prado M. Production and characterization

of glass microspheres for hepatic cancer treatment. Proc Mater

Sci. 2009;1:351–8.

11. da Costa Guimaraes C, Moralles McRoberto Martinelli J. Monte

Carlo simulation of liver cancer treatment with 166Ho-loaded

glass microspheres. Radiat Phys Chem. 2014;95:185–7.

12. Ladron de Guevara-Fernandez S, Ragel CV, Vallet-Regi M.

Bioactive glass-polymer materials for controlled release of ibu-

profen. Biomaterials. 2003;24(22):4037–43.

13. Zhao L, Yan X, Zhou X, Zhou L, Wang H, Tang J, Yu C.

Mesoporous bioactive glasses for controlled drug release.

Microporous Mesoporous Mater. 2008;109:210–5.

14. Ravarian R, Moztarzadeh F, Hashjin MS, Rabiee SM, Khoshakh-

lagh P, Tahriri M. Synthesis, characterization and bioactivity

investigation of bioglass/hydroxyapatite composite. Ceram Int.

2010;36(1):291–7.

15. Goller G. The effect of bond coat on mechanical properties of

plasma sprayed bioglass–titanium coatings. Ceram Int.

2004;30(3):351–5.

16. Goller G, Demirkiran H, Oktar FN, Demirkesen E. Processing

and characterization of bioglass reinforced hydroxyapatite com-

posites. Ceram Int. 2003;29(6):721–4.

17. Habibe AF, Maeda LD, Souza RC, Barboza MJR, Daguano

JKMF, Rogero SO, Santos C. Effect of bioglass additions on the

sintering of Y-TZP bioceramics. Mater Sci Eng C. 2009;29(6):

1959–64.

18. Kashyap S, Griep K, Nychka JA. Crystallization kinetics, min-

eralization and crack propagation in partially crystallized bioac-

tive glass 45S5. Mater Sci Eng C. 2011;31(4):762–9.

19. Chen QZ, Thompson ID, Boccaccini AR. 45S5 Bioglass�-

derived glass–ceramic scaffolds for bone tissue engineering.

Biomaterials. 2006;27(11):2414–25.

20. Clupper DC, Hench LL. Crystallization kinetics of tape cast

bioactive glass 45S5. J Non-Cryst Solids. 2003;318:43–8.

21. Filho OP, LaTorre GP, Hench LL. Effect of crystallization on

apatite-layer formation of bioactive glass 45S5. J Biomed Mater

Res. 1996;30:509–14.

22. Chen X, Meng Y, Li Y, Zhao N. Investigation on bio-minerali-

zation of melt and solgel derived bioactive glasses. Appl Surf Sci.

2008;255(2):562–4.

23. Serra J, Gonzalez P, Liste S, Chiussi S, Leon B, Perez-Amor M,

Ylanen HO, Hupa M. Influence of the non-bridging oxygen

groups on the bioactivity of silicate glasses. J Mater Sci Mater

Med. 2002;13:1221–5.

24. Marie PJ. Strontium ranelate; a novel mode of action optimizing

bone formation and resorption. Osteoporos Int. 2005;16:S7–10.

25. Marie PJ. Strontium ranelate: new insights into its dual mode of

action. Bone. 2007;40:S5–8.

26. Takadama H, Kim HM, Kokubo T, Nakamura T. XPS study of

the process of apatite formation on bioactive Ti–6Al–4V alloy in

simulated body fluid. Sci Technol Adv Mater. 2001;2:389–96.

27. Li Y, Coughlan A, Laffir FR, Pradhan D, Mellott NP, Wren AW.

Investigating the mechanical durability of bioactive glasses as a

function of structure, solubility and incubation time. J Non-Cryst

Solids. 2013;380:25–34.

28. Chen QZ, Li Y, Jin LY, Quinn JMW, Komesaroff PA. A new

sol–gel process for producing Na2O-containing bioactive glass

ceramics. Acta Biomater. 2010;6:4143–53.


123


Bioceramics in Simulated Body Fluid

Documents

Transcript of Bioceramics in Simulated Body Fluid