ISO 23317:2012
(Main)Implants for surgery — In vitro evaluation for apatite-forming ability of implant materials
Implants for surgery — In vitro evaluation for apatite-forming ability of implant materials
ISO 23317:2012 specifies a method for detecting apatite formed on a surface of a material in simulated body fluid (SBF). It is applicable to implant surfaces intended to come into direct bone contact.
Implants chirurgicaux — Évaluation in vitro de la capacité de formation d'apatite des matériaux d'implants
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Standards Content (Sample)
INTERNATIONAL ISO
STANDARD 23317
Second edition
2012-10-01
Implants for surgery — In vitro
evaluation for apatite-forming ability
of implant materials
Implants chirurgicaux — Évaluation in vitro de la capacité de
formation d’apatite des matériaux d’implants
Reference number
ISO 23317:2012(E)
©
ISO 2012
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ISO 23317:2012(E)
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ii © ISO 2012 – All rights reserved
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ISO 23317:2012(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Apparatus . 2
5 Test specimen . 2
5.1 Specimen configuration and size . 2
5.2 Specimen preparation . 3
6 Simulated body fluid . 3
6.1 General . 3
6.2 Reagents for SBF . 3
6.3 Preparation of SBF . 4
6.4 Confirmation of ion concentration of SBF . 6
6.5 Preservation of SBF . 6
7 Procedure. 6
8 Test report . 8
Annex A (informative) Apparatus for preparing SBF . 9
Annex B (informative) Preparation of standard glasses for evaluating apatite-forming ability .10
Bibliography .12
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ISO 23317:2012(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International
Standards adopted by the technical committees are circulated to the member bodies for voting.
Publication as an International Standard requires approval by at least 75 % of the member bodies
casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 23317 was prepared by Technical Committee ISO/TC 150, Implants for surgery, Subcommittee
SC 1, Materials.
This second edition cancels and replaces the first edition (ISO 23317:2007), which has been
technically revised.
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ISO 23317:2012(E)
Introduction
It has been revealed that materials of various kinds bond to living bone through a layer of apatite. It has
been shown that this apatite layer can be reproduced on their surfaces in an acellular and protein-free
simulated body fluid (SBF) with ion concentrations nearly equal to those of human blood plasma, and
that apatite thus formed is similar to the bone mineral in its composition and structure.
This evaluation of apatite-forming ability on implant material in SBF is useful for evaluating its in vivo
bone-bonding ability preliminary to animal experiments. When a bioactive material is implanted in a
living body, a thin layer rich in Ca and P forms on its surface. The material then connects to the living
tissue through this apatite layer without a distinct boundary. It has been shown that this apatite layer
can be reproduced on the surfaces of materials in SBF as well, and that apatite thus formed is similar to
bone mineral in its composition and structure. As bioactivity increases, apatite forms on the material
surface in a shorter time in proportion to this increase. The formation of apatite layers can be detected
by thin film X-ray diffraction spectrometry and/or scanning electron microscopy.
The apatite formed in the SBF is, however, similar to bone apatite in the following ways.
— Ca-deficient type apatite.
— Lower Ca/P atomic ratio than stoichiometric apatite.
2+ + - -
— Containing some impurities such as Mg , Na , Cl , HCO .
3
— Low crystallinity.
NOTE 1 The material which forms apatite on its surface in vivo can bond to living bone, since this apatite is
biologically active. Their in vivo apatite deposition can be reproduced on their surfaces even in vitro in SBF. For
1)
1
example, in vivo calcification on surfaces of Bioglass® , CaO-SiO glasses, Na O-CaO-SiO glasses, Cerabone® A-W,
2 2 2
1
Ceravital® -type glass-ceramic, sintered hydroxyapatite and alkali-heat-treated titanium metal, are correlated
with in vitro calcification in SBF. However, this does not exclude the possibility that materials, which do not form
apatite on their surfaces in vivo, bond to living bone. For example, it is reported that such resorbable materials
as beta-tricalcium phosphate [Ca (PO ) ] and calcium carbonate bond to living bone without forming an apatite
3 4 2
layer on their surfaces.
NOTE 2 It has been reported that glasses with different compositions in the system Na O-CaO-SiO show
2 2
a correlation between bone-forming ability of materials implanted into a bone defect of a rabbit and apatite-
forming ability on its surface in SBF.
1) Trade names of the products are examples of suitable products available commercially. This information
is given for the convenience of the users of this document and does not constitute an endorsement by ISO of these
products.
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INTERNATIONAL STANDARD ISO 23317:2012(E)
Implants for surgery — In vitro evaluation for apatite-
forming ability of implant materials
1 Scope
This International Standard specifies a method for detecting apatite formed on a surface of a material in
simulated body fluid (SBF). It is applicable to implant surfaces intended to come into direct bone contact.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
ISO 3696:1987, Water for analytical laboratory use — Specification and test methods
ISO 14630, Non-active surgical implants — General requirements
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 14630 and the following apply.
3.1
apatite
group of calcium-phosphates including bone mineral and the main inorganic constituent of bones and
teeth similar to hydroxyapatite, which has the composition Ca (PO ) (OH)
10 4 6 2
2− − + 2+
Note.1.to.entry: Bone mineral also contains ions such as C0 , F , Na and Mg .
3
3.2
apatite-forming ability
capability to develop apatite on the surface
3.3
bioactivity
property that elicits a specific biological response at the interface of the material, which results in the
formation of a bond between tissue and material
3.4
induction period
time to detect apatite formation on a surface of a specimen after soaking the specimen in simulated
body fluid
3.5
simulated body fluid
SBF
inorganic solution having a similar composition to human blood plasma without organic components
3.6
standard glass for evaluating apatite-forming ability
class of standard glasses with certain chemical compositions as shown in Annex B showing given apatite-
forming abilities in SBF and when implanted in an animal body
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ISO 23317:2012(E)
3.7
thin film X-ray diffraction spectrometry
TF-XRD
method for detecting minerals in a thin layer at the surface of a material from a diffraction pattern
obtained by X-ray with small glancing angle against the surface of the sample
4 Apparatus
4.1 Electric balance, capable of measuring a mass with an accuracy of ± 1 mg.
4.2 Water bath equipped with magnetic stirrer, to maintain the temperature of the solution within
the range of (36,5 ± 2) °C with an accuracy of ± 0,2 °C.
4.3 pH meter, capable of measuring the pH of a solution with an accuracy of ± 0,01.
4.4 Thermometer, capable of measuring the temperature of a solution with an accuracy of ± 0,1°C.
4.5 Thin film X-ray diffraction spectrometer (TF-XRD), capable of detecting apatite formed in a thin
layer at the surface of a material.
4.6 Scanning electron microscope (SEM), capable of observing apatite grains and/or layers formed
on a plain surface of a material with a magnification up to × 10 000.
5 Test specimen
5.1 Specimen configuration and size
This International Standard allows specimens of any configuration and size derived from implant parts
and devices to be used. However, a disc or rectangular plate specimen is highly recommended because
bioactivity of a material is evaluated by confirmation of apatite formed on the surface of the material
using TF-XRD and/or SEM. Recommended specimen dimensions are shown in Figure 1.
Dimensions in millimetres
a) Disc specimen
b) Rectangular specimen
Figure 1 — Recommended specimen dimensions
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ISO 23317:2012(E)
5.2 Specimen preparation
5.2.1 General
This International Standard allows several options for specimen preparation. The specimens should be
machined, if necessary, to alter the configurations of original implants.
5.2.2 Basic machining procedure
In the case of a rectangular thin plate specimen, as shown in Figure 1 b), the following procedure shall
be used. Specimens shall be ground using a diamond wheel of grit size between 120 and 400. Conditions
such as depth of cut per pass, wheel speed and others depend on the ground material. Water soluble
materials, such as bioactive standard glasses, shall be machined under non-aqueous conditions.
Where a customary machining procedure has been developed that is completely satisfactory for apatite-
forming ability testing, this customary procedure can be used.
6 Simulated body fluid
6.1 General
Simulated body fluid (SBF) as defined in Table 1 shall be used.
Table 1 — Ion concentrations of SBF and human blood plasma
−3
Concentration (10 mol) in
Ion
SBF Blood plasma
(pH 7,40) (pH 7,2 to 7,4)
+
Na 142,0 142,0
+
K 5,0 5,0
2+
Mg 1,5 1,5
2+
Ca 2,5 2,5
−
Cl 147,8 103,0
HCO 4,2 27,0
3−
2−
HPO 1,0 1,0
4
2−
SO 0,5 0,5
4
NOTE 1 For SBF as defined in Table 1, a correlation was observed between in vivo bone ingrowth and in vitro
apatite-forming ability.
NOTE 2 Other kinds of SBFs have been proposed in the literature, some of which have shown a correlation
between in vivo bone ingrowth and in vitro apatite-forming ability.
6.2 Reagents for SBF
For the preparation of SBF, only reagents of the following recognized analytical grade chemicals and
only water in accordance with ISO 3696:1987, grade 2, shall be used.
6.2.1 Sodium chloride (NaCl).
6.2.2 Sodium hydrogen carbonate (NaHCO ).
3
6.2.3 Potassium chloride (KCl).
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ISO 23317:2012(E)
6.2.4 Di-potassium hydrogen phosphate trihydrate (K HPO · 3H O).
2 4 2
6.2.5 Magnesium chloride hexahydrate (MgCl · 6H O).
2 2
6.2.6 Hydrochloric acid solution, c(HCl) = 1 mol/l.
6.2.7 Calcium chloride (CaCl ) or calcium chloride dihydrate (CaCl · 2H O).
2 2 2
6.2.8 Sodium sulfate (Na SO ).
2 4
6.2.9 Tris-hydroxymethyl aminomethane (TRIS): ((HOCH2) CNH ).
3 2
6.3 Preparation of SBF
6.3.1 General
Since SBF is supersaturated with respect to apatite, an inappropriate preparation method can lead to
the homogeneous precipitation of apatite in the solution.
During its preparation the solution shall remain colourless, transparent and without deposit on the
surface of the bottle. If any precipitation occurs, stop preparing SBF, abandon the solution and restart
by washing the apparatus.
In Table 2, the reagents for the preparation of 1 l of SBF are given in the required order of dissolution.
Table 2 — Ion concentrations of SBF and human blood plasma
a b
Order Reagent Amount Container Purity Formula weight
1 6.2.1 8,035 g weighing paper 99,5 % 58,443 0
2 6.2.2 0,355 g weighing paper 99,5 % 84,006 8
3 6.2.3 0,225 g weighing bottle 99,5 % 74,551 5
4 6.2.4 0,231 g weighing bottle 99,0 % 228,222 0
5 6.2.5 0,311 g weighing bottle 98,0 % 203,303 4
6 6.2.6 39 ml graduated cylinder — —
c
7 6.2.7 0,292 g weighing bottle 95,0 % 110,984 8
8 6.2.8 0,072 g weighing bottle 99,0 % 142,042 8
9 6.2.9 6,118 g weighing paper 99,0 % 121,135 6
10 6.2.6 0 ml to 5 ml syringe dropper — —
a
The amounts of the reagents are changed depending upon their purities.
b
The purity given in this table is a typical purity for reagent available in most countries.
c
If calcium chloride dihydrate (CaCl ) · H O is used, attention shall be given to the different molar weight:
2 2 2
— amount0,371 g;
— purity 99,0 %;
— formula weight 147,015 2.
6.3.2 Step 1
Put 700 ml of ion-exchanged and distilled water, with a stirring bar, into a 1 litre plastic beaker. Set
it in the water bath (4.2) on the magnetic stirrer and cover it with a watch glass or plastic wrap. Heat
the water in the beaker to (36,5 ± 1,5)°C while stirring. Annex A shows an example of an apparatus for
preparing the SBF.
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ISO 23317:2012(E)
6.3.3 Step 2
Dissolve the 1st to 8th reagents in the required order given in Table 2 in the distilled water at
(36,5 ± 1,5)°C, while considering the following.
a) Glass containers should be avoided. A plastic containe
...
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