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A thorough physicochemical characterisation of 14 calcium phosphate-based bone substitution materials in comparison to natural bone
ARTICLE IN PRESS
Biomaterials 25 (2004) 987–994
A thorough physicochemical characterisation of 14 calcium phosphate-based bone substitution materials in comparison to natural bone D. Tadic, M. Epple* Solid State Chemistry, Faculty of Chemistry, University of Bochum, Universitatsstr. 150, D-44780 Bochum, Germany Received 10 March 2003; accepted 22 July 2003
Abstract Fourteen different synthetic or biological bone substitution materials were characterised by high-resolution X-ray diffractometry, infrared spectroscopy, thermogravimetry, and scanning electron microscopy. Thus, the main parameters chemical composition, crystallinity, and morphology were determined. The results are compared with natural bone samples. The materials fall into different classes: Chemically treated bone, calcined bovine bone, algae-derived hydroxyapatite, synthetic hydroxyapatite, peptideloaded hydroxyapatite, and synthetic b-TCP ceramics. r 2003 Elsevier Ltd. All rights reserved. Keywords: Bone graft materials; Chemical analysis; Calcium phosphates
1. Introduction Filling of bone defects is a significant question in every day clinical work. Autogeneous bone is still the most effective bone graft substitution material (‘‘gold standard’’), fulfilling all essential physicochemical and biological properties, despite its inherent limitations (availability, post-operative pain) [1–5]. The most common alternative to the autograft material are (human) allografts or (animal, e.g. bovine) xenografts. Allografts have the disadvantages of limited supply and potential infectivity (e.g. HIV, Hepatitis). With xenografts there are the questions of unfavourable immune response and also of infectivity. Autogenous bone is osteogenic (the cells within a donor graft synthesise new bone at the implantation site), osteoinductive (new bone is formed by the active recruitment of host mesenchymal stem cells from the surrounding tissue, which differentiate into bone-forming osteoblasts), osteoconductive (vascularisation and new bone formation into the transplant) and highly biocompatible [6]. This process is facilitated by the *Corresponding author. Tel.: +49-234-3224-151; fax: +49-2343214-558. E-mail address: [email protected] (M. Epple). 0142-9612/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0142-9612(03)00621-5
presence of growth factors within the autogenous bone material (mainly bone morphogenetic proteins [7]). These characteristics should be present in an ideal substitute and all bone graft substitution materials can be described by these characteristics [8]. Synthetic calcium phosphate ceramics [9] with their excellent biocompatibility are common alternatives to autogeneous bone, xenograft or allograft materials. They have gained acceptance for various dental or medical applications which include, e.g., fillers for periodontal defects, alveolar ridge augmentation, maxillofacial reconstruction, ear implants, spine fusion, and coatings for metallic implants [10–15]. Bone grafts and synthetic calcium phosphates (such as b-tricalcium phosphate; b-TCP, and hydroxyapatite; HAP) are commonly used as blocks, cements, pastes, powders or granules. The aim of this article is to describe the chemical and physical properties of these bone graft materials and to compare them to natural bone. As model for autologous spongiosa, natural bone samples were analysed. The biological performance of a synthetic material depends on fundamental parameters: chemical composition, morphology, and biodegradability. A wide range of analytical methods (IR, XRD, TG, REM) was used to investigate these properties. As each of these methods has its
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limitations, it is necessary to combine all results to obtain a comprehensive view.
2. Materials Fourteen different bone substitution materials were used. The following data were taken from the manufacturer’s specification. All samples were obtained directly from the manufacturers in sealed vials and used without further treatment, except for grinding or cutting to a size appropriate for analysis. Bioresorbs, Chronoss, Ceross, Cerasorbs, and Vitosss are synthetic b-tricalcium phosphates from high-temperature calcination processes. Bioresorbs is available as porous granulate (particle size: 0.5–2 mm) mainly for dental application. Chronoss and Ceross are also granular materials with a particle size of 0.5–1.4 mm and pore sizes of 100–500 mm (60% pore volume), also mainly for dental application. Cerasorbs is available as porous granulate (pore size >5 mm) in particle sizes of 0.05–2 mm for dental application and as machined macroporous blocks for orthopaedic applications. Vitosss is a porous granulate (pore size 10–1000 mm; porosity approx. 90%; particle size 3–5 mm) for dental application. PepGen P-15s is a calcined bovine bone (1100 C; hydroxyapatite) coated with a pentadecapeptide (P-15, a part of the sequence of collagen). It is available as granulate with a particle size of 0.25–0.42 mm and used in dental applications. Endobons and Cerabones are high-temperature sintered bovine bone materials (>1200 C), containing the sintered inorganic part of bone (hydroxyapatite). They are usually administered as highly porous blocks (pore size typically 1 mm; porosity typically 50%) with dimensions in the centimeter-range. Cerabones is also available as granulate (not studied here). These materials are used in orthopaedic surgery. Algipores is a algae-derived hydroxyapatite. It is prepared by the hydrothermal conversion of the original calcium carbonate of the algae in the presence of ammonium phosphate at about 700 C. This process preserves the porosity of the algae. It is available as granulate with particle sizes of 0.3–2 mm and pores in the range of 5–10 mm and used for dental application. Ostims is a nanocrystalline precipitated hydroxyapatite that still contains about 40% of water. It has a viscous, fluid-like consistence and can therefore be directly injected into a defect. Note the difference to self-hardening bone cements as this is a fluid dispersion. It can be used in dental and orthopaedic surgery. BioOsss is the inorganic component of bovine bone (i.e., the mineral). All organic material is removed by a stepwise annealing process (up to 300 C), followed by a chemical treatment (NaOH) that leaves a porous
hydroxyapatite bone chip material. The particle size of the granulate is 0.25–2 mm. It is used mainly in dental surgery. Kiel bone is a bovine bone graft material that was treated chemically with chloroform/methanol and H2O2 to remove all organic components except of the collagen. It is not in clinical use anymore. It was available as centimeter-sized block. Tutoplasts is obtained as either human or bovine graft material after a multi-step chemical treatment (osmolysis, NaOH, H2O2, acetone). It is available as granulate (particle size 0.25–2 mm) and as centimetersized block or cylinder. The interconnecting porosity of the bone is still present in both cases (pore sizes of some hundred mm). The data for synthetic hydroxyapatite synthesised by precipitation (purchased from Merck Chemical Division, Darmstadt, Germany) and human callus bone and human tumor bone were taken from our earlier study on the structure of bone and bone substitution materials [16].
3. Methods High-resolution X-ray powder diffractometry (XRD) was carried out in transmission geometry (ground samples on Kapton foil) at beamline B2 at HASYLAB/DESY, Hamburg, Germany, with wavelengths of ( (depending on the individual experiabout l ¼ 1:2 A ment) [16]. For Ceross, Chronoss, Bioresorbs, Vitosss and PepGen P-15s, diffraction data were measured on a Bruker AXS D8 Advance laboratory ( Infrared spectroinstrument (Cu Ka radiation, 1.54 A). scopy (IR) was carried out with a Perkin-Elmer 1720 instrument (KBr pellet, transmission mode, 400– 4000 cm1, resolution 2 cm1, 10 scans). Thermogravimetric analysis (TGA) was carried out with a TG/DTAS II, Seiko Exstar 6000 instrument (5–15 mg; 25– 1000 C; 10 K min1; dynamic oxygen atmosphere; 300 ml min1; Al2O3 crucibles). Scanning electron microscopy (SEM) was carried out with a LEO 1530 instrument on gold-sputtered samples.
4. Results and discussion Fig. 1 shows the macroscopic morphologies of the different materials. Bioresorbs, Chronoss, Ceross, Cerasorbs, Vitosss, PepGens, Algipores, BioOsss, and Tutoplasts are available as granulate with typical particle sizes of a few hundred mm to a few millimeters. Ostims is a fluid paste with nanoscopic apatite particles in aqueous dispersion. Cerabones, Endobons, and Tutoplasts have the bone-like structure with interconnecting porosity as they are all derived from natural
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Bioresorb®
Chronos®
β-TCP ceramics
Ceros®
Cerasorb®
Vitoss®
Hydroxyapatite-based materials
PepGen® P-15
Cerabone®
Ostim®
CO2m’’; peak at 37.3 2Y) [17]. Traces of CaO are also seen in Cerabones at the same position in 2Y: The inorganic phase of PepGens is also a highly crystalline hydroxyapatite (with no traces of CaO). Algipores is a moderately crystalline hydroxyapatite phase with no detectable foreign phases. Ostims is prepared by rapid precipitation, keeping the crystals within the nanometer range. This is indicated by the broad diffraction peaks in Fig. 2c that correspond to synthetic hydroxyapatite. No foreign phases are visible. Interestingly, the crystallinity is close to that of BioOsss that is prepared from bovine bone. Even smaller crystals lead to even broader diffraction peaks. All bone-like samples fall into this category (Fig. 2d). They all contain hydroxyapatite-like mineral and there are no distinct differences. The only exception is a content of octacalcium phosphate (OCP; Ca8H2(PO4) 5H2O) in Tutoplasts (bovine) as indicated by asterisks in Fig. 2d (peaks between 21 and 24 2Y). The diffraction peak broadening by small crystallites can be semi-quantitatively estimated by the Scherrer equation [18] (Table 1): b1=2 ¼ ðKl57:3Þ=ðD cos YÞ:
BioOss®
Tutoplast®
Fig. 1. Macromorphology of the different bone graft materials.
bone by either thermal or chemical treatment. In contrast, blocks of Cerasorbs are prepared by coldisostatic pressing, followed by mechanical drilling of millimeter-sized holes. The results of the X-ray diffraction experiments that are indicative for the chemical composition (presence of crystalline phases) are shown in Fig. 2. Four of the five b-TCP ceramics show small amounts of impurities besides the major phase (peaks marked with asterisks in Fig. 2a). They all exhibit a high crystallinity as indicated by the narrow diffraction peaks. Bioresorbs contains some b-Ca2P2O7 (calcium pyrophosphate; peak at 30.8 2Y) and a-TCP (peak at 22.9 2Y). Chronoss and Ceross contain some a-TCP and some hydroxyapatite (peak at 31.6 2Y). Vitosss contains some b-Ca2P2O7 (peaks at 29.0 and 30.2 2Y). In all cases, the amount of foreign phases is very small. Cerabones and Endobons are prepared by hightemperature calcination from bovine bone, and consequently the hydroxyapatite is highly crystalline (very narrow diffraction peaks). Endobons also contains small amounts of calcium oxide (CaO) that results from decomposition of the carbonate content of the original bone mineral (carbonated apatite; ‘‘CaCO3-CaO+
989
ð1Þ
Here, b1=2 is the peak width (as full-width at half maximum) in 2Y; K is a constant that we set to 1 (as ( D is the often done), l is the X-ray wavelength in A, average domain size (roughly the crystallite size) and Y is the diffraction angle of the corresponding reflex. This equation gives an estimate of the crystallite size. It should be noted, however, that structural disorder and strain phenomena, e.g. caused by carbonate substitution, can also lead to a peak broadening effect [18]. Therefore, the given values should be mainly used for comparison among the samples. All bone samples have essentially the same anisotropic crystal size, i.e. about 25 nm in c-direction [(0 0 2) and (0 0 4)] and about 9 nm in a-direction [(2 1 0)/(1 2 0) and (1 3 0)/(3 1 0)]. The Tutoplasts process does not change the mineral particle size. BioOsss and Ostims have slightly larger particles (about double as much in each direction). In the case of BioOsss, this may be due to the heating during the preparation as Rogers et al. reported first structural changes in the mineral phase between 200 C and 400 C [17]. Synthetic hydroxyapatite and Algipores show almost isotropic particles about three times larger than bone mineral particles in each direction. For the highly crystalline samples Cerabones, Endobons and PepGens, the diffraction peak width is at the minimum given by the experimental setup, therefore only a lower limit for the crystallite size can be given (but see below for SEM pictures). The infrared spectra are shown in Fig. 3. All b-TCP ceramics are identical and show only the expected calcium phosphate bands (Fig. 3a). The hydroxyapatitebased ceramics in Fig. 3b show only calcium phosphate
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990
Bioresorb
Chronos
*
*
(R)
(R )
*
Ceros
Cerasorb
* 20
25
(a)
Vitoss
* 30
35
PepGen (R) P15
intensity
* intensity
(R )
*
*
(R)
*
Endobon
*
Cerabone
(R)
(R)
(R)
Algipore
40
20
25
(b)
diffraction angle / ˚2 θ
(R)
30 diffraction angle / ˚2 θ
35
40
Kiel Bone
(synthetic) Ostim
(R)
Tumor Bone
intensity
intensity
hydroxyapatite
Callus Bone (R)
* * ** *
Tutoplast (bovine)
(R)
Tutoplast (human)
(R)
BioOss
20
(c)
25 30 diffraction angle / °2θ
35
40
(d)
20
25 30 diffraction angle / ˚2 θ
35
40
( The Fig. 2. X-ray diffraction data for all investigated samples. All data were either measured at or converted to the Cu Ka wavelength (1.54 A). displayed range in 2y was chosen to optimally represent the relevant features.
Table 1 Estimation of the domain size from diffraction peak broadening of all investigated materials in nanometers Diffraction line index ( 2y [ ] at Cu Ka (l ¼ 1:54 A) PepGens Endobons Cerabones Algipores Synthetic hydroxyapatite Ostims BioOsss Kiel bone Tumor bone Callus bone Tutoplast (bovine)s Tutoplast (human)s
(1 1 1)
(0 0 2)
(2 1 0) (1 2 0)
(1 3 0) (3 1 0)
(1 1 3)
(2 2 2)
(2 1 3) (1 2 3)
(0 0 4)
22.9 >64 >71 >64 59 30 21 29 19 24 20 21 14
25.9 >54 >64 >80 65 38 36 36 24 22 21 27 27
29.0 >54 >67 >65 45 25 22 23 10 12 10 17 18
39.8 >56 >63 >84 40 29 21 17 8 9 9 8 9
43.8 >42 >64 >56 29 40 24 21 23 20 24 21 13
46.7 >57 >62 >57 44 26 19 21 19 18 18 12 16
49.5 >43 >64 >58 38 33 25 25 15 14 14 17 19
53.1 >44 >64 >88 36 42 35 29 25 22 22 20 22
bands in a sharp and split way, as indicative for the high crystallinity. Algipores shows some carbonate bands (approx. 1400 cm1) that are probably due to remnants of the production process (from calcium carbonate algae). Although PepGens contains a bioactive peptide, there are no bands of organic material (as seen below). This is due to the small amount present. In Fig. 3c,
nanocrystalline hydroxyapatite ceramics are shown. The phosphate bands are generally broader because of the small crystallite size. In addition, there are bands of water, and, except for Ostims, of carbonate. This shows that synthetic hydroxyapatite as well as BioOsss contain small amounts of incorporated carbonate. The bone samples that are shown in Fig. 3d all contain
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991
(R)
Chronos Ceros
(R)
(R) (R)
Cerasorb Vitoss
(R)
(R)
PepGen
absorbance / a.u.
absorbance / a.u.
Bioresorb
(R)
Endobon
(R)
Cerabone
O-H
P-O
4000
3500
3000
(a)
2500
2000
1500
wave number / cm
1000
(R)
Algipore
C-O
P-O
P-O
P-O
500
4000
-1
3500
3000
(b)
2500 2000 1500 1000 -1 wave number / cm
500
Bone (Kiel)
Ostim
(R)
BioOss
absorbance / a.u.
absorbance / a.u.
Hydroxyapatite synthetic
Bone (tumor) Bone (callus)
(R)
O-H
O-H
P-O
C-O
H-P-O
N-H O-H
P-O
4000
3500
3000
2500
2000
1500
1000
500
4000
-1
wave number / cm
(c)
(d)
3500
C-H
3000
2500
2000
C-O P-O O-H
1500
Tutoplast (bovine)
(R)
Tutoplast P-O (human) H-P-O
(R)
1000
500
-1
wave number / cm
Fig. 3. Infrared spectroscopy on the bone graft materials with the bands assigned to structural features. 100 ~9 % water
95
sample mass / %
90 85 80
~ 26 % organic material
75 70 65
~ 3,6 % CO2
60
100
200
300 400 500 temperature / ˚C
600
700
800
900
Fig. 4. Typical thermogravimetric curve of Tutoplasts (bovine), showing the three regions of mass loss that can be used to derive the chemical composition.
collagen and organic tissue in variable amounts. In addition to the bands of calcium phosphate, we can see a multitude of bands that are related to the organic material and incorporated water. All samples were subjected to thermogravimetric analysis [19] to determine the content of water, organic material (like collagen), and mineral (calcium phos-
phate). A typical curve is shown in Fig. 4 (Tutoplasts human). As derived from earlier experiments with mass spectrometric analysis of the released gases, three ranges of mass loss can be assigned to specific processes [16]. From room temperature to about 200 C, incorporated water is lost. Above about 300 C, organic material like collagen, fat tissue, proteins start to burn. At about 400 C, only the mineral phase (calcium phosphate) is left. If the mineral contains some carbonate in the form of carbonated apatite, there is a mass loss between about 400 C and 900 C [16,20]. Therefore, it is possible to determine the mineral content and its carbonate content from such TG experiments. Note that all biological apatites are carbonated apatites [15]. Table 2 shows all compositional data as determined from TG experiments (average of two experiments). All b-TCP phases, except for Vitosss, show no mass loss, indicating the absence of any volatile or combustible material. In the case of Vitosss, a small mass loss was registered between 200 C and 400 C. This may be due to an organic binder used for granulation. All calcined hydroxyapatite samples (PepGens, Cerabones, and Endobons) show no mass loss, as expected due to the preparation of these materials by high-temperature calcination. The amount of peptide in PepGens is too small to result in a detectable mass loss.
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Table 2 Chemical composition, as derived from thermogravimetric experiments. The nature of the mineral phase was derived from previous diffraction experiments
Bioresorbs Chronoss Ceross Cerasorbs Vitosss PepGens Endobons Cerabones Algipores Ostims BioOsss Kiel bone Tumor bone Callus bone Tutoplasts (bovine) Tutoplasts (human)
H2O (wt%)
Soft tissue+organic bone matrix (wt%)
Mineral phase (wt%)
Formal content of CaCO3 (wt%)
Content of TCP
Formal content of HAP (wt%)
Formal ratio apatite: CaCO3 (w:w)
0 0 0 0 0 0 0 0 0.3 40.4 3 7.8 5.7 6.9 9 9.5
0 0 0 0 1.2 0 0 0 2.4 0 0 28.7 21.2 47.7 26 34
100 100 100 100 98.8 100 100 100 97.3 \quad 59.6 97 63.5 73.1 45.4 65 56.5
0 0 0 0 0 0 0 0 2.3 0 3.4 3.7 5.2 1.4 8 7.5
100 100 100 100 98.8 — — — — — — — — — — —
— — — — — 100 100 100 95 59.6 93.6 59.8 67.9 44 57 49
— — — — — — — — 41 — 28 16 13 31 7 6.5
Note the traces of impurities in some cases (Fig. 2).
Algipores contains small amounts of water, probably a small amount of organic material and some carbonate, as indicated by the weight loss at high temperature (decomposition of carbonated hydroxyapatite to hydroxyapatite and calcium oxide). Ostims contains about 40 wt% of water; the remainder is a carbonate-free hydroxyapatite. BioOsss contains a small amount of water but no detectable combustible material. The inorganic phase is a carbonated hydroxyapatite. The materials that still contain all or most of the organic bone matrix (Kiel bone, natural bone, Tutoplasts) have a similar composition with some water content (6– 10 wt%), some organic material (20–50 wt%) and carbonated hydroxyapatite as mineral phase. It is interesting to see that the carbonate content in the bone-like materials (BioOsss, Kiel bone, Tutoplasts) is highly variable. If we formally compute a weight ratio of Ca5(PO4)3OH to CaCO3 in these samples, we obtain values between 6 and 30. As the range in natural bone samples is also highly variable (we found ratios from 13 to 37 in four bone samples [16]), we can conclude that the identity with bone mineral is still present, even after extensive chemical and moderate thermal treatment. Fig. 5 shows representative SEM pictures that illustrate the typical morphology of these classes of materials. In Fig. 5a, Cerasorbs shows the granular appearance of a sintered material with visible micropores at high magnification. In Fig. 5b, the calcined bovine bone (Cerabones) has the interconnecting porous structure of the original bone. In higher magnification, primary crystallites of sintered hydroxyapatite are visible with particle sizes of a few micrometers. In Fig. 5c, the chemically converted algae structure of Algipores can be seen. The graded porosity (resembling cortical and cancellous bone, but on a much
(a)
(b)
(c)
(d)
Fig. 5. SEM pictures of four representative bone graft materials. (a) Cerasorbs, (b) Cerabones, (c) Algipores and (d) Tutoplasts (bovine).
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Table 3 Summary of all data obtained for the different materials. The mechanical stability of granulates refers to their ability to retain a cm-sized threedimensional shape Sample
Chemical composition
Crystallinity Morphology
Expected biodegradability
Mechanical stability
BioResorbs
b-TCP, traces of calcium pyrophosphate and a-TCP b-TCP, traces of a-TCP and HAP b-TCP, traces of a-TCP and HAP b-TCP
High
Porous granulate
Moderate
Low
High
Porous granulate
Moderate
Low
High
Porous granulate
Moderate
Low
High
Porous granulate; drilled porous blocks Porous granulate
Moderate Moderate
Low (granulate) to high (blocks) Low
Porous Porous Porous Porous
Slow Slow Slow Moderate
Low High High Low
Paste Porous granulate Porous block (bone-like)
Fast Fast Fast
None Low High
Porous block (bone-like)
Fast
High
Porous block (bone-like)
Fast
High
Porous block (bone-like)
Fast
High
Porous block (bone-like)
Fast
High
ChronOSs Ceross Cerasorbs Vitosss
PepGens Endobons Cerabones Algipores Ostims BioOsss Kiel bone Callus bone Tumor bone Tutoplasts (bovine) Tutoplasts (human)
b-TCP, traces of calcium High pyrophosphate and possibly organic binder HAP High HAP, traces of calcium oxide High HAP, traces of calcium oxide High Carbonated HAP, traces of Moderate organic binder (?) HAP dispersed in water Nano Carbonated HAP, water Nano Carbonated HAP, water, Nano organic bone matrix Carbonated HAP, water, Nano organic bone matrix Carbonated HAP, water, Nano organic bone matrix Carbonated HAP, traces of Nano OCP, water, organic bone matrix Carbonated HAP, water, Nano organic bone matrix
smaller dimension) is due to the biological requirements of the algae. At high magnification, we can see the primary particles of a micrometer or less. In Fig. 5d, the chemically treated bovine bone material Tutoplasts is shown. As in Fig. 5b, we can see the interconnecting macroporosity of bone; however, as this material was not sintered, it still contains the collagen matrix. At high magnification, we do not see sintered hydroxyapatite but the fibrous structure of the original bone. Table 3 summarises all structural and morphological information in a concise way. It can be seen that these materials strongly differ in their composition. It is also clear the mere denomination ‘‘calcium phosphate ceramics’’ is by no means sufficient to fully characterise a material. With respect to biodegradability, it is possible to make some reasonable predictions, based on literature data. b-TCP ceramics are faster degradable than HAP ceramics [21–23]. In addition, there is a difference between sintered HAP ceramics and precipitated HAP ceramics, the former showing a very slow (if any) biodegradation. If the crystallite size of the HAP ceramics is very small (like in bone) and/or if there is carbonate incorporated, the biodegradation is strongly enhanced due to a higher solubility [22–26]. Even more strongly, this applies to bone grafts that still contain the collagen matrix. In these cases, usually a fast biode-
granulate block (bone-like) block (bone-like) granulate
gradation is observed and a biological potency of the incorporated bone matrix is postulated [7,27].
5. Conclusions 14 different bone graft materials were investigated, and the results were compared to synthetic hydroxyapatite and natural bone samples (as reference for autologous bone). Their composition and morphology is strongly different, therefore the materials cover a wide range of applications, ranging from permanent implants to rapidly degradable implants with osteogenic potency.
Acknowledgements This project was supported by the Fonds der Chemischen Industrie (Frankfurt am Main, Germany).
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[16] Peters F, Schwarz K, Epple M. The structure of bone studied with synchrotron X-ray diffraction, X-ray absorption spectroscopy and thermal analysis. Thermochim Acta 2000;361:131–8. [17] Rogers KD, Daniels P. An X-ray diffraction study of the effects of heat treatment on bone mineral microstructure. Biomaterials 2002;23:2577–85. [18] Klug HP, Alexander LE. X-ray diffraction procedures for polycrystalline and amorphous materials. New York: WileyInterscience; 1974. [19] Cammenga HK, Epple M. Basic principles of thermoanalytical techniques and their applications in preparative chemistry. Angew Chem Int Ed Engl 1995;34:1171–87; Cammenga HK, Epple M. Angew Chem 1995;1107:1284–301. [20] Suchanek W, Shuk P, Byrappa K, Riman RE, TenHuisen KS, Janas VF. Mechanochemical-hydrothermal synthesis of carbonated apatite powders at room temperature. Biomaterials 2002;23:699–710. [21] Cao W, Hench LL. Bioactive materials. Ceramics Int 1996;22: 493–507. [22] Doi Y, Iwanaga H, Shibutani T, Morikawa Y, Iwayama Y. Osteoclastic responses to various calcium phosphates in cell cultures. J Biomed Mat Res 1999;47:424–33. [23] Lu J, Descamps M, Dejou J, Koubi G, Hardouin P, Lemaitre J, Proust JP. The biodegradation mechanism of calcium phosphate biomaterials in bone. J Biomed Mat Res Appl Biomater 2002; 63:408–12. [24] Leeuwenburgh A, Layrolle P, Barr"ere F, de Bruijn JD, Schoonman J, van Blitterswijk CA, de Groot K. Osteoclastic resorption of biomimetic calcium phosphate coatings in vitro. J Biomed Mat Res 2001;56:208–15. [25] Tadic D, Peters F, Epple M. Continuous synthesis of amorphous carbonated apatites. Biomaterials 2002;23:2553–9. [26] Fulmer MT, Ison IC, Hankermayer CR, Constantz BR, Ross J. Measurements of the solubilities and dissolution rates of several hydroxyapatites. Biomaterials 2002;23:751–5. [27] Du C, Cui FZ, Feng QL, Zhu XD, de Groot K. Tissue response to nano-hydroxyapatite/collagen composite implants in marrow cavity. J Biomed Mater Res 1998;42:540–8.
Biomaterials 25 (2004) 987–994
A thorough physicochemical characterisation of 14 calcium phosphate-based bone substitution materials in comparison to natural bone D. Tadic, M. Epple* Solid State Chemistry, Faculty of Chemistry, University of Bochum, Universitatsstr. 150, D-44780 Bochum, Germany Received 10 March 2003; accepted 22 July 2003
Abstract Fourteen different synthetic or biological bone substitution materials were characterised by high-resolution X-ray diffractometry, infrared spectroscopy, thermogravimetry, and scanning electron microscopy. Thus, the main parameters chemical composition, crystallinity, and morphology were determined. The results are compared with natural bone samples. The materials fall into different classes: Chemically treated bone, calcined bovine bone, algae-derived hydroxyapatite, synthetic hydroxyapatite, peptideloaded hydroxyapatite, and synthetic b-TCP ceramics. r 2003 Elsevier Ltd. All rights reserved. Keywords: Bone graft materials; Chemical analysis; Calcium phosphates
1. Introduction Filling of bone defects is a significant question in every day clinical work. Autogeneous bone is still the most effective bone graft substitution material (‘‘gold standard’’), fulfilling all essential physicochemical and biological properties, despite its inherent limitations (availability, post-operative pain) [1–5]. The most common alternative to the autograft material are (human) allografts or (animal, e.g. bovine) xenografts. Allografts have the disadvantages of limited supply and potential infectivity (e.g. HIV, Hepatitis). With xenografts there are the questions of unfavourable immune response and also of infectivity. Autogenous bone is osteogenic (the cells within a donor graft synthesise new bone at the implantation site), osteoinductive (new bone is formed by the active recruitment of host mesenchymal stem cells from the surrounding tissue, which differentiate into bone-forming osteoblasts), osteoconductive (vascularisation and new bone formation into the transplant) and highly biocompatible [6]. This process is facilitated by the *Corresponding author. Tel.: +49-234-3224-151; fax: +49-2343214-558. E-mail address: [email protected] (M. Epple). 0142-9612/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0142-9612(03)00621-5
presence of growth factors within the autogenous bone material (mainly bone morphogenetic proteins [7]). These characteristics should be present in an ideal substitute and all bone graft substitution materials can be described by these characteristics [8]. Synthetic calcium phosphate ceramics [9] with their excellent biocompatibility are common alternatives to autogeneous bone, xenograft or allograft materials. They have gained acceptance for various dental or medical applications which include, e.g., fillers for periodontal defects, alveolar ridge augmentation, maxillofacial reconstruction, ear implants, spine fusion, and coatings for metallic implants [10–15]. Bone grafts and synthetic calcium phosphates (such as b-tricalcium phosphate; b-TCP, and hydroxyapatite; HAP) are commonly used as blocks, cements, pastes, powders or granules. The aim of this article is to describe the chemical and physical properties of these bone graft materials and to compare them to natural bone. As model for autologous spongiosa, natural bone samples were analysed. The biological performance of a synthetic material depends on fundamental parameters: chemical composition, morphology, and biodegradability. A wide range of analytical methods (IR, XRD, TG, REM) was used to investigate these properties. As each of these methods has its
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limitations, it is necessary to combine all results to obtain a comprehensive view.
2. Materials Fourteen different bone substitution materials were used. The following data were taken from the manufacturer’s specification. All samples were obtained directly from the manufacturers in sealed vials and used without further treatment, except for grinding or cutting to a size appropriate for analysis. Bioresorbs, Chronoss, Ceross, Cerasorbs, and Vitosss are synthetic b-tricalcium phosphates from high-temperature calcination processes. Bioresorbs is available as porous granulate (particle size: 0.5–2 mm) mainly for dental application. Chronoss and Ceross are also granular materials with a particle size of 0.5–1.4 mm and pore sizes of 100–500 mm (60% pore volume), also mainly for dental application. Cerasorbs is available as porous granulate (pore size >5 mm) in particle sizes of 0.05–2 mm for dental application and as machined macroporous blocks for orthopaedic applications. Vitosss is a porous granulate (pore size 10–1000 mm; porosity approx. 90%; particle size 3–5 mm) for dental application. PepGen P-15s is a calcined bovine bone (1100 C; hydroxyapatite) coated with a pentadecapeptide (P-15, a part of the sequence of collagen). It is available as granulate with a particle size of 0.25–0.42 mm and used in dental applications. Endobons and Cerabones are high-temperature sintered bovine bone materials (>1200 C), containing the sintered inorganic part of bone (hydroxyapatite). They are usually administered as highly porous blocks (pore size typically 1 mm; porosity typically 50%) with dimensions in the centimeter-range. Cerabones is also available as granulate (not studied here). These materials are used in orthopaedic surgery. Algipores is a algae-derived hydroxyapatite. It is prepared by the hydrothermal conversion of the original calcium carbonate of the algae in the presence of ammonium phosphate at about 700 C. This process preserves the porosity of the algae. It is available as granulate with particle sizes of 0.3–2 mm and pores in the range of 5–10 mm and used for dental application. Ostims is a nanocrystalline precipitated hydroxyapatite that still contains about 40% of water. It has a viscous, fluid-like consistence and can therefore be directly injected into a defect. Note the difference to self-hardening bone cements as this is a fluid dispersion. It can be used in dental and orthopaedic surgery. BioOsss is the inorganic component of bovine bone (i.e., the mineral). All organic material is removed by a stepwise annealing process (up to 300 C), followed by a chemical treatment (NaOH) that leaves a porous
hydroxyapatite bone chip material. The particle size of the granulate is 0.25–2 mm. It is used mainly in dental surgery. Kiel bone is a bovine bone graft material that was treated chemically with chloroform/methanol and H2O2 to remove all organic components except of the collagen. It is not in clinical use anymore. It was available as centimeter-sized block. Tutoplasts is obtained as either human or bovine graft material after a multi-step chemical treatment (osmolysis, NaOH, H2O2, acetone). It is available as granulate (particle size 0.25–2 mm) and as centimetersized block or cylinder. The interconnecting porosity of the bone is still present in both cases (pore sizes of some hundred mm). The data for synthetic hydroxyapatite synthesised by precipitation (purchased from Merck Chemical Division, Darmstadt, Germany) and human callus bone and human tumor bone were taken from our earlier study on the structure of bone and bone substitution materials [16].
3. Methods High-resolution X-ray powder diffractometry (XRD) was carried out in transmission geometry (ground samples on Kapton foil) at beamline B2 at HASYLAB/DESY, Hamburg, Germany, with wavelengths of ( (depending on the individual experiabout l ¼ 1:2 A ment) [16]. For Ceross, Chronoss, Bioresorbs, Vitosss and PepGen P-15s, diffraction data were measured on a Bruker AXS D8 Advance laboratory ( Infrared spectroinstrument (Cu Ka radiation, 1.54 A). scopy (IR) was carried out with a Perkin-Elmer 1720 instrument (KBr pellet, transmission mode, 400– 4000 cm1, resolution 2 cm1, 10 scans). Thermogravimetric analysis (TGA) was carried out with a TG/DTAS II, Seiko Exstar 6000 instrument (5–15 mg; 25– 1000 C; 10 K min1; dynamic oxygen atmosphere; 300 ml min1; Al2O3 crucibles). Scanning electron microscopy (SEM) was carried out with a LEO 1530 instrument on gold-sputtered samples.
4. Results and discussion Fig. 1 shows the macroscopic morphologies of the different materials. Bioresorbs, Chronoss, Ceross, Cerasorbs, Vitosss, PepGens, Algipores, BioOsss, and Tutoplasts are available as granulate with typical particle sizes of a few hundred mm to a few millimeters. Ostims is a fluid paste with nanoscopic apatite particles in aqueous dispersion. Cerabones, Endobons, and Tutoplasts have the bone-like structure with interconnecting porosity as they are all derived from natural
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Bioresorb®
Chronos®
β-TCP ceramics
Ceros®
Cerasorb®
Vitoss®
Hydroxyapatite-based materials
PepGen® P-15
Cerabone®
Ostim®
CO2m’’; peak at 37.3 2Y) [17]. Traces of CaO are also seen in Cerabones at the same position in 2Y: The inorganic phase of PepGens is also a highly crystalline hydroxyapatite (with no traces of CaO). Algipores is a moderately crystalline hydroxyapatite phase with no detectable foreign phases. Ostims is prepared by rapid precipitation, keeping the crystals within the nanometer range. This is indicated by the broad diffraction peaks in Fig. 2c that correspond to synthetic hydroxyapatite. No foreign phases are visible. Interestingly, the crystallinity is close to that of BioOsss that is prepared from bovine bone. Even smaller crystals lead to even broader diffraction peaks. All bone-like samples fall into this category (Fig. 2d). They all contain hydroxyapatite-like mineral and there are no distinct differences. The only exception is a content of octacalcium phosphate (OCP; Ca8H2(PO4) 5H2O) in Tutoplasts (bovine) as indicated by asterisks in Fig. 2d (peaks between 21 and 24 2Y). The diffraction peak broadening by small crystallites can be semi-quantitatively estimated by the Scherrer equation [18] (Table 1): b1=2 ¼ ðKl57:3Þ=ðD cos YÞ:
BioOss®
Tutoplast®
Fig. 1. Macromorphology of the different bone graft materials.
bone by either thermal or chemical treatment. In contrast, blocks of Cerasorbs are prepared by coldisostatic pressing, followed by mechanical drilling of millimeter-sized holes. The results of the X-ray diffraction experiments that are indicative for the chemical composition (presence of crystalline phases) are shown in Fig. 2. Four of the five b-TCP ceramics show small amounts of impurities besides the major phase (peaks marked with asterisks in Fig. 2a). They all exhibit a high crystallinity as indicated by the narrow diffraction peaks. Bioresorbs contains some b-Ca2P2O7 (calcium pyrophosphate; peak at 30.8 2Y) and a-TCP (peak at 22.9 2Y). Chronoss and Ceross contain some a-TCP and some hydroxyapatite (peak at 31.6 2Y). Vitosss contains some b-Ca2P2O7 (peaks at 29.0 and 30.2 2Y). In all cases, the amount of foreign phases is very small. Cerabones and Endobons are prepared by hightemperature calcination from bovine bone, and consequently the hydroxyapatite is highly crystalline (very narrow diffraction peaks). Endobons also contains small amounts of calcium oxide (CaO) that results from decomposition of the carbonate content of the original bone mineral (carbonated apatite; ‘‘CaCO3-CaO+
989
ð1Þ
Here, b1=2 is the peak width (as full-width at half maximum) in 2Y; K is a constant that we set to 1 (as ( D is the often done), l is the X-ray wavelength in A, average domain size (roughly the crystallite size) and Y is the diffraction angle of the corresponding reflex. This equation gives an estimate of the crystallite size. It should be noted, however, that structural disorder and strain phenomena, e.g. caused by carbonate substitution, can also lead to a peak broadening effect [18]. Therefore, the given values should be mainly used for comparison among the samples. All bone samples have essentially the same anisotropic crystal size, i.e. about 25 nm in c-direction [(0 0 2) and (0 0 4)] and about 9 nm in a-direction [(2 1 0)/(1 2 0) and (1 3 0)/(3 1 0)]. The Tutoplasts process does not change the mineral particle size. BioOsss and Ostims have slightly larger particles (about double as much in each direction). In the case of BioOsss, this may be due to the heating during the preparation as Rogers et al. reported first structural changes in the mineral phase between 200 C and 400 C [17]. Synthetic hydroxyapatite and Algipores show almost isotropic particles about three times larger than bone mineral particles in each direction. For the highly crystalline samples Cerabones, Endobons and PepGens, the diffraction peak width is at the minimum given by the experimental setup, therefore only a lower limit for the crystallite size can be given (but see below for SEM pictures). The infrared spectra are shown in Fig. 3. All b-TCP ceramics are identical and show only the expected calcium phosphate bands (Fig. 3a). The hydroxyapatitebased ceramics in Fig. 3b show only calcium phosphate
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990
Bioresorb
Chronos
*
*
(R)
(R )
*
Ceros
Cerasorb
* 20
25
(a)
Vitoss
* 30
35
PepGen (R) P15
intensity
* intensity
(R )
*
*
(R)
*
Endobon
*
Cerabone
(R)
(R)
(R)
Algipore
40
20
25
(b)
diffraction angle / ˚2 θ
(R)
30 diffraction angle / ˚2 θ
35
40
Kiel Bone
(synthetic) Ostim
(R)
Tumor Bone
intensity
intensity
hydroxyapatite
Callus Bone (R)
* * ** *
Tutoplast (bovine)
(R)
Tutoplast (human)
(R)
BioOss
20
(c)
25 30 diffraction angle / °2θ
35
40
(d)
20
25 30 diffraction angle / ˚2 θ
35
40
( The Fig. 2. X-ray diffraction data for all investigated samples. All data were either measured at or converted to the Cu Ka wavelength (1.54 A). displayed range in 2y was chosen to optimally represent the relevant features.
Table 1 Estimation of the domain size from diffraction peak broadening of all investigated materials in nanometers Diffraction line index ( 2y [ ] at Cu Ka (l ¼ 1:54 A) PepGens Endobons Cerabones Algipores Synthetic hydroxyapatite Ostims BioOsss Kiel bone Tumor bone Callus bone Tutoplast (bovine)s Tutoplast (human)s
(1 1 1)
(0 0 2)
(2 1 0) (1 2 0)
(1 3 0) (3 1 0)
(1 1 3)
(2 2 2)
(2 1 3) (1 2 3)
(0 0 4)
22.9 >64 >71 >64 59 30 21 29 19 24 20 21 14
25.9 >54 >64 >80 65 38 36 36 24 22 21 27 27
29.0 >54 >67 >65 45 25 22 23 10 12 10 17 18
39.8 >56 >63 >84 40 29 21 17 8 9 9 8 9
43.8 >42 >64 >56 29 40 24 21 23 20 24 21 13
46.7 >57 >62 >57 44 26 19 21 19 18 18 12 16
49.5 >43 >64 >58 38 33 25 25 15 14 14 17 19
53.1 >44 >64 >88 36 42 35 29 25 22 22 20 22
bands in a sharp and split way, as indicative for the high crystallinity. Algipores shows some carbonate bands (approx. 1400 cm1) that are probably due to remnants of the production process (from calcium carbonate algae). Although PepGens contains a bioactive peptide, there are no bands of organic material (as seen below). This is due to the small amount present. In Fig. 3c,
nanocrystalline hydroxyapatite ceramics are shown. The phosphate bands are generally broader because of the small crystallite size. In addition, there are bands of water, and, except for Ostims, of carbonate. This shows that synthetic hydroxyapatite as well as BioOsss contain small amounts of incorporated carbonate. The bone samples that are shown in Fig. 3d all contain
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991
(R)
Chronos Ceros
(R)
(R) (R)
Cerasorb Vitoss
(R)
(R)
PepGen
absorbance / a.u.
absorbance / a.u.
Bioresorb
(R)
Endobon
(R)
Cerabone
O-H
P-O
4000
3500
3000
(a)
2500
2000
1500
wave number / cm
1000
(R)
Algipore
C-O
P-O
P-O
P-O
500
4000
-1
3500
3000
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2500 2000 1500 1000 -1 wave number / cm
500
Bone (Kiel)
Ostim
(R)
BioOss
absorbance / a.u.
absorbance / a.u.
Hydroxyapatite synthetic
Bone (tumor) Bone (callus)
(R)
O-H
O-H
P-O
C-O
H-P-O
N-H O-H
P-O
4000
3500
3000
2500
2000
1500
1000
500
4000
-1
wave number / cm
(c)
(d)
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C-H
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1500
Tutoplast (bovine)
(R)
Tutoplast P-O (human) H-P-O
(R)
1000
500
-1
wave number / cm
Fig. 3. Infrared spectroscopy on the bone graft materials with the bands assigned to structural features. 100 ~9 % water
95
sample mass / %
90 85 80
~ 26 % organic material
75 70 65
~ 3,6 % CO2
60
100
200
300 400 500 temperature / ˚C
600
700
800
900
Fig. 4. Typical thermogravimetric curve of Tutoplasts (bovine), showing the three regions of mass loss that can be used to derive the chemical composition.
collagen and organic tissue in variable amounts. In addition to the bands of calcium phosphate, we can see a multitude of bands that are related to the organic material and incorporated water. All samples were subjected to thermogravimetric analysis [19] to determine the content of water, organic material (like collagen), and mineral (calcium phos-
phate). A typical curve is shown in Fig. 4 (Tutoplasts human). As derived from earlier experiments with mass spectrometric analysis of the released gases, three ranges of mass loss can be assigned to specific processes [16]. From room temperature to about 200 C, incorporated water is lost. Above about 300 C, organic material like collagen, fat tissue, proteins start to burn. At about 400 C, only the mineral phase (calcium phosphate) is left. If the mineral contains some carbonate in the form of carbonated apatite, there is a mass loss between about 400 C and 900 C [16,20]. Therefore, it is possible to determine the mineral content and its carbonate content from such TG experiments. Note that all biological apatites are carbonated apatites [15]. Table 2 shows all compositional data as determined from TG experiments (average of two experiments). All b-TCP phases, except for Vitosss, show no mass loss, indicating the absence of any volatile or combustible material. In the case of Vitosss, a small mass loss was registered between 200 C and 400 C. This may be due to an organic binder used for granulation. All calcined hydroxyapatite samples (PepGens, Cerabones, and Endobons) show no mass loss, as expected due to the preparation of these materials by high-temperature calcination. The amount of peptide in PepGens is too small to result in a detectable mass loss.
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Table 2 Chemical composition, as derived from thermogravimetric experiments. The nature of the mineral phase was derived from previous diffraction experiments
Bioresorbs Chronoss Ceross Cerasorbs Vitosss PepGens Endobons Cerabones Algipores Ostims BioOsss Kiel bone Tumor bone Callus bone Tutoplasts (bovine) Tutoplasts (human)
H2O (wt%)
Soft tissue+organic bone matrix (wt%)
Mineral phase (wt%)
Formal content of CaCO3 (wt%)
Content of TCP
Formal content of HAP (wt%)
Formal ratio apatite: CaCO3 (w:w)
0 0 0 0 0 0 0 0 0.3 40.4 3 7.8 5.7 6.9 9 9.5
0 0 0 0 1.2 0 0 0 2.4 0 0 28.7 21.2 47.7 26 34
100 100 100 100 98.8 100 100 100 97.3 \quad 59.6 97 63.5 73.1 45.4 65 56.5
0 0 0 0 0 0 0 0 2.3 0 3.4 3.7 5.2 1.4 8 7.5
100 100 100 100 98.8 — — — — — — — — — — —
— — — — — 100 100 100 95 59.6 93.6 59.8 67.9 44 57 49
— — — — — — — — 41 — 28 16 13 31 7 6.5
Note the traces of impurities in some cases (Fig. 2).
Algipores contains small amounts of water, probably a small amount of organic material and some carbonate, as indicated by the weight loss at high temperature (decomposition of carbonated hydroxyapatite to hydroxyapatite and calcium oxide). Ostims contains about 40 wt% of water; the remainder is a carbonate-free hydroxyapatite. BioOsss contains a small amount of water but no detectable combustible material. The inorganic phase is a carbonated hydroxyapatite. The materials that still contain all or most of the organic bone matrix (Kiel bone, natural bone, Tutoplasts) have a similar composition with some water content (6– 10 wt%), some organic material (20–50 wt%) and carbonated hydroxyapatite as mineral phase. It is interesting to see that the carbonate content in the bone-like materials (BioOsss, Kiel bone, Tutoplasts) is highly variable. If we formally compute a weight ratio of Ca5(PO4)3OH to CaCO3 in these samples, we obtain values between 6 and 30. As the range in natural bone samples is also highly variable (we found ratios from 13 to 37 in four bone samples [16]), we can conclude that the identity with bone mineral is still present, even after extensive chemical and moderate thermal treatment. Fig. 5 shows representative SEM pictures that illustrate the typical morphology of these classes of materials. In Fig. 5a, Cerasorbs shows the granular appearance of a sintered material with visible micropores at high magnification. In Fig. 5b, the calcined bovine bone (Cerabones) has the interconnecting porous structure of the original bone. In higher magnification, primary crystallites of sintered hydroxyapatite are visible with particle sizes of a few micrometers. In Fig. 5c, the chemically converted algae structure of Algipores can be seen. The graded porosity (resembling cortical and cancellous bone, but on a much
(a)
(b)
(c)
(d)
Fig. 5. SEM pictures of four representative bone graft materials. (a) Cerasorbs, (b) Cerabones, (c) Algipores and (d) Tutoplasts (bovine).
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Table 3 Summary of all data obtained for the different materials. The mechanical stability of granulates refers to their ability to retain a cm-sized threedimensional shape Sample
Chemical composition
Crystallinity Morphology
Expected biodegradability
Mechanical stability
BioResorbs
b-TCP, traces of calcium pyrophosphate and a-TCP b-TCP, traces of a-TCP and HAP b-TCP, traces of a-TCP and HAP b-TCP
High
Porous granulate
Moderate
Low
High
Porous granulate
Moderate
Low
High
Porous granulate
Moderate
Low
High
Porous granulate; drilled porous blocks Porous granulate
Moderate Moderate
Low (granulate) to high (blocks) Low
Porous Porous Porous Porous
Slow Slow Slow Moderate
Low High High Low
Paste Porous granulate Porous block (bone-like)
Fast Fast Fast
None Low High
Porous block (bone-like)
Fast
High
Porous block (bone-like)
Fast
High
Porous block (bone-like)
Fast
High
Porous block (bone-like)
Fast
High
ChronOSs Ceross Cerasorbs Vitosss
PepGens Endobons Cerabones Algipores Ostims BioOsss Kiel bone Callus bone Tumor bone Tutoplasts (bovine) Tutoplasts (human)
b-TCP, traces of calcium High pyrophosphate and possibly organic binder HAP High HAP, traces of calcium oxide High HAP, traces of calcium oxide High Carbonated HAP, traces of Moderate organic binder (?) HAP dispersed in water Nano Carbonated HAP, water Nano Carbonated HAP, water, Nano organic bone matrix Carbonated HAP, water, Nano organic bone matrix Carbonated HAP, water, Nano organic bone matrix Carbonated HAP, traces of Nano OCP, water, organic bone matrix Carbonated HAP, water, Nano organic bone matrix
smaller dimension) is due to the biological requirements of the algae. At high magnification, we can see the primary particles of a micrometer or less. In Fig. 5d, the chemically treated bovine bone material Tutoplasts is shown. As in Fig. 5b, we can see the interconnecting macroporosity of bone; however, as this material was not sintered, it still contains the collagen matrix. At high magnification, we do not see sintered hydroxyapatite but the fibrous structure of the original bone. Table 3 summarises all structural and morphological information in a concise way. It can be seen that these materials strongly differ in their composition. It is also clear the mere denomination ‘‘calcium phosphate ceramics’’ is by no means sufficient to fully characterise a material. With respect to biodegradability, it is possible to make some reasonable predictions, based on literature data. b-TCP ceramics are faster degradable than HAP ceramics [21–23]. In addition, there is a difference between sintered HAP ceramics and precipitated HAP ceramics, the former showing a very slow (if any) biodegradation. If the crystallite size of the HAP ceramics is very small (like in bone) and/or if there is carbonate incorporated, the biodegradation is strongly enhanced due to a higher solubility [22–26]. Even more strongly, this applies to bone grafts that still contain the collagen matrix. In these cases, usually a fast biode-
granulate block (bone-like) block (bone-like) granulate
gradation is observed and a biological potency of the incorporated bone matrix is postulated [7,27].
5. Conclusions 14 different bone graft materials were investigated, and the results were compared to synthetic hydroxyapatite and natural bone samples (as reference for autologous bone). Their composition and morphology is strongly different, therefore the materials cover a wide range of applications, ranging from permanent implants to rapidly degradable implants with osteogenic potency.
Acknowledgements This project was supported by the Fonds der Chemischen Industrie (Frankfurt am Main, Germany).
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