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Materials Chemistry and Physics 146 (2014) 99e104

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Materials Chemistry and Physics

journal homepage: www.elsevier .com/locate/matchemphys

Physicochemical characterization of InterOss� and Bio-Oss� anorganicbovine bone grafting material for oral surgery e A comparative study

David S.H. Lee*, Y. Pai, Steve ChangDepartment of Research and Development, SigmaGraft, 325 N Puente Street, Unit B, Brea, CA 92821, USA

h i g h l i g h t s

� The physicochemical characterization of InterOss� was substantially equivalent to that of Bio-Oss�.� The content of residual protein of InterOss� was relatively lower than that of Bio-Oss�.� The inner surface area of InterOss� was comparatively higher than that of Bio-Oss�.

a r t i c l e i n f o

Article history:Received 5 June 2013Received in revised form7 February 2014Accepted 1 March 2014

Keywords:BiomaterialsInorganic compoundsHeat treatmentMicrostructure

* Corresponding author. Tel.: þ1 714 525 0114; fax:E-mail address: wallee79@gmail.com (D.S.H. Lee).

http://dx.doi.org/10.1016/j.matchemphys.2014.03.0040254-0584/� 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

The anorganic bovine bone grafting materials have been widely used to fill bone defects in periodontaland maxillofacial surgery. The purpose of present study was to fully characterize our anorganic bone,InterOss�, by physical and chemical methods and to compare it with another anorganic bone, Bio-Oss�

that has been commercially distributed in dental bone graft substitute market since 1995. InterOss�

anorganic bone had been successfully prepared by chemical treatment (NaOH and H2O2) and lowtemperature (350 �C) annealing process with an extremely low heating rate (<0.3 �C min�1).Commercially available Bio-Oss� anorganic bone was chosen for comparison. The physical and chemicalanalysis indicated that the pore structure, microstructure, phase structure, and chemical composition ofInterOss� is substantially equivalent to that of Bio-Oss�. The BET analysis also showed that the innersurface area of InterOss� is comparatively higher than that of Bio-Oss�. Specially, the protein analysisshowed that the content of residual protein of InterOss� is relatively lower than that of Bio-Oss�. Basedon an equivalency to Bio-Oss� in terms of physical and chemical characterization with both higher innersurface area and lower residual protein content, the InterOss� can be a promising candidate as dentalbone grafting material in periodontal and maxillofacial surgery.

� 2014 Elsevier B.V. All rights reserved.

1. Introduction

The use of bone grafts in cranio-maxillofacial and dental sur-gery is growing increasingly [1,2]. The autografts and the allograftsare very effective as bone grafting substitute due to essentialphysicochemical and biological properties such as their immuneresponse, good osteoinductivity, and osteoconductivity [3e9].However, they have the disadvantages of limited supply andavailability [9e14]. Therefore, alternative biomaterials such asanimal source or synthetic bone grafts have been proposed andintensively studied. Specially, among these, the bovine origin bonegrafts have practically unlimited availability and good physico-chemical and structural similarity to human bone [15]. Therefore,

þ1 714 525 0116.

it has been attempted that the organic substances of bovine bonecan be removed without significantly modifying the nativecancellous structure of bone mineral [16e19]. Specially, in order toobtain the bone mineral with both high purity and nativecancellous structure (micropore, mesopore, and macroporestructure), the chemical treatment and the low temperature pro-cessing for a removal of the organic component of bovine bone arevery effective.

Therefore, in our present study, we developed the highly puri-fied InterOss� anorganic bone with the native structure by chem-ical treatment and low temperature processing with an extremelylow heating rate. This method can effectively achieve separated andpurified mineral from bovine bone, maintaining the native struc-ture of mineral as a similar techniquewith the extracting process ofBio-Oss� bone mineral. We thoroughly investigated our anorganicbone in terms of physicochemical characterization. We alsoconfirmed that the InterOss� anorganic bone has the

D.S.H. Lee et al. / Materials Chemistry and Physics 146 (2014) 99e104100

physicochemical and structural equivalency to the Bio-Oss� anor-ganic bone that has been mainly used as anorganic bovine bonegrafting material in dental surgery through a comparative studywith both two anorganic bones.

2. Experimental

2.1. Materials preparation

The InterOss� anorganic bone was extracted from bovine bone.All organic substances are removed by two-step chemical treat-ment (NaOH and H2O2) and low temperature annealing process.The residual organic substances after chemical treatment wereburned out at 350 �C, at a heating rate (<0.3 �C min�1). The Bio-Oss� anorganic bone was purchased from the manufacturer(Geistlich-Pharma, CH-6110 Wolhusen, Switzerland), as commer-cially available.

2.2. Physical characterization

The scanning electron microscopy (SEM, Hitachi Co., model S-4700, Japan) technique and image analyzer software program(PAX-it Image Analysis, MIS) were used to measure the size of thepores and particles of the bone mineral and to measure theiraverage size. The average size was observed and determined inquantity ranged from 100 to 150 numbers for size of particles andmicropores and in quantity within 5 numbers for size of meso-pores. The X-ray diffractometry (XRD, Rigaku, model D/MAX-2200/PC, Japan) using Cu Ka radiation and fourier transforminfrared spectroscopy (FT-IR, Thermo Scientific, model NICOLET-iS5, USA) techniques were used to analyze the microstructure ofbone mineral. The XRD pattern was performed for the identifica-tion of the constituent phase and the FT-IR pattern provides in-formation associated with the specific functional groups of thebone mineral. The inner surface area of the bone mineral wasmeasured by BrunauereEmmetteTeller (BET, BEL Japan Inc.,model BELSORP-max, Japan) surface area analysis and the areaunit was indicated as square meter per gram, m2 g�1. The indi-vidual experiment for the XRD, FT-IR, and BET analysis was

Fig. 1. SEM micrographs of both anorganic bones

performed three times and their mean and standard deviationwere calculated for each analysis.

2.3. Chemical characterization

The energy dispersive spectroscopy detector (SEM/EDS, HitachiCo., model S-4700, Japan) attached to an SEM was used to deter-mine the composition of chemical elements on the surface of thebone mineral. The Ca/P ratio of the bone mineral was measured byinductively coupled plasma mass spectrometer (ICP-MS, PerkineElmer, model ELAN 6000, USA). The individual experiment for theSEM/EDS and ICP-MS analysis was performed three times andtheir mean and standard deviation were calculated for eachanalysis.

2.4. Residual organic substance analysis

The protein analyzer (Vapodest, Gerhardt, Germany) usingKjeldahl method and thermogravimetric analysis (TGA, Mettler-Toledo, model TGA/DSC 1, USA) were used to confirm the contentof the residual organic substance of the bone mineral. The proteinanalysis and TGA curve provide information associated with thepotential crude protein remained in the bone mineral. The sampleof the TGAwas heated starting from room temperature (25 �C) andgoing up to 1000 �C, at a heating rate of 10 �C min�1, on analuminum support under a dynamic nitrogen atmosphere (N2),with a gas flow rate of the order of 50 ml min�1 [20]. The individualexperiment for the protein analyzer and TGA analysis was per-formed three times and their mean and standard deviation werecalculated for each analysis.

2.5. Statistical analysis

The SPSS for Windows version 21 statistical software (SPSS,Chicago, IL) was employed to perform the statistical analyses. Non-parametric ManneWhitney U test was performed having donethree individual experiments for comparison of physicochemicalcharacterization of two anorganic bone minerals. The statisticalsignificance was defined at P < 0.05.

: (a and c) InterOss� and (b and d) Bio-Oss�.

Fig. 3. FT-IR spectra of both anorganic bones: (a) InterOss� and (b) Bio-Oss�.

D.S.H. Lee et al. / Materials Chemistry and Physics 146 (2014) 99e104 101

3. Results

3.1. Physical characterization

Fig. 1 presents the SEM micrographs of InterOss� and Bio-Oss�.As shown in Fig. 1, it was confirmed that the mesopore andmicropore were obviously existed in both anorganic bones. Theaverage mesopore size (40e60 mm) of the InterOss� (Fig. 1(a)) wasslightly larger than that (30e40 mm) of the Bio-Oss� (Fig. 1(b)) butthe average micropore size (<1.5 mm) of the InterOss� (Fig. 1(c))was a little smaller than that (<3.0 mm) of the Bio-Oss� (Fig. 1(d)).Furthermore, the average particle size (1.5e2.5 mm) of the Inter-Oss� (Fig. 1(c)) was a little smaller than that (3.0e4.0 mm) of theBio-Oss� (Fig. 1(d)), and which had a similar tendency to averagemicropore size.

In the XRD patterns (Fig. 2) of both anorganic bones, the threedominant peaks such as (2q ¼ 32�), (2q ¼ 33�), and (2q ¼ 26�) wereassociated with synthetic hydroxyapatite (HAp), which are verifiedby comparing data obtained with the PDF (Powder Diffraction File)pattern 09-0432. From the XRD result, the HAp structure for theInterOss� (Fig. 2(a)) and Bio-Oss� (Fig. 2(b)) was low-crystallineapatite phase.

Fig. 3 presents the FT-IR spectra of both anorganic bones. Asindicated in Fig. 3, bands can be identified in the wavelength rangesof 1506e1570 cm�1, 1400e1477 cm�1, and 953e989 cm�1 corre-sponding to the functional group ðCO�2

3 Þ of the HAp for InterOss�

(Fig. 3(a)) and Bio-Oss� (Fig. 3(b)). Furthermore, the stretching vi-bration of the hydroxyl groups (OH�) was observed at around3572 cm�1 and 1638 cm�1 and the orthophosphates ðPO3�

4 Þ werealso observed at 960e1120 cm�1, 602 cm�1, and 570 cm�1.

Fig. 2. XRD patterns of both anorganic bones: (a) InterOss� and (b) Bio-Oss�.

The result of the inner surface area of both anorganic bonesmeasured by BET is listed in Table 1. As shown in Table 1, theaverage inner surface area 88.2 (�0.015; SD) m2 g�1 of the Inter-Oss� was comparatively higher than that 77.5 (�0.021; SD) m2 g�1

[21] of the Bio-Oss�, revealing no significant difference regardingthe P-value (P ¼ 0.33, P > 0.05) and dispersion value as standarddeviation in both anorganic boneminerals was very low because allvalues of inner surface area that were measured three times werevery close to its average value.

3.2. Chemical characterization

The chemical composition results of both anorganic bone min-erals obtained by EDS are presented in Fig. 4, Table 2, and Table 3.Fig. 4 shows spectra for the chemical element composition of theInterOss� (Fig. 4(a)) and Bio-Oss� (Fig. 4(b)) and the values ofcontent for an individual element were listed in Table 2 (InterOss�)and Table 3 (Bio-Oss�). As indicated in Fig. 4, Table 2, and Table 3,both anorganic bone minerals were mainly consisted of the Ca andP and an each average content value was above 63 wt.% (Ca) and33wt.% (P). The other elements except for Ca and Pwere Al, Mg, andNa and their average content value was very low below 1.7 wt.%.Meanwhile, Table 4 shows the P-values for comparison of content ofan individual element for the chemical composition of both anor-ganic boneminerals. ManneWhitney U test indicated no significantdifference regarding the P-values (P > 0.05) ranged from 0.06 to0.60 in content of all elements measured. This means that chemicalcomposition of both anorganic bone minerals is substantiallyequivalent each other.

The result of the Ca/P ratio of both anorganic bone mineralsmeasured by ICP-MS is listed in Table 1. The average Ca/P ratio of

Table 1The values of the inner surface area, Ca/P ratio, and crude protein of the InterOss�

and Bio-Oss� measured by BET, ICP-MS, and protein analyzer.

InterOss� Bio-Oss� P-value

Inner surface area (m2 g�1)(mean � SD)

88.2 � 0.015 77.5 � 0.021 0.33

Ca/P ratio mean � SD) 1.57 � 0.002 1.62 � 0.003 0.20Crude protein (%) mean � SD) 0.04 � 0.0015 0.118 � 0.0002 0.72

SD: standard deviation.

Fig. 4. EDS spectra for the chemical element composition of both bone minerals: (a) InterOss� and (b) Bio-Oss�.

D.S.H. Lee et al. / Materials Chemistry and Physics 146 (2014) 99e104102

InterOss� and Bio-Oss� was 1.57 (�0.002; SD) and 1.62 (�0.003;SD) [21], respectively. From the P-value (P¼ 0.20, P> 0.05), the Ca/Pratio of both anorganic bone minerals is substantially equivalenteach other with no significant difference. The dispersion value asstandard deviation in both anorganic bone minerals was very lowbecause all values of Ca and P element content and Ca/P ratio thatwere measured three times, respectively, were very close to theiraverage value.

3.3. Residual organic substance analysis

The crude protein content result of both anorganic bones ob-tained by protein analyzer is listed in Table 1. As shown in Table 1,the average crude protein content 0.04 (�0.0015; SD) % of theInterOss� was relatively lower than that 0.118 (�0.0002; SD) % [21]of the Bio-Oss�. Therewas no significant difference in crude proteincontent between both anorganic bone minerals through measure-ment of the P-value (P ¼ 0.72, P > 0.05).

Table 2The values of content of an individual element for the chemical composition of the Inter

Element App conc. (mean � SD) Intensity corrn. (mean � SD) Weig

Na K 0.13 � 0.002 1.0461 � 0.0002 1.65Mg K 0.11 � 0.002 0.9132 � 0.0001 1.56Al K 0.01 � 0.001 0.9883 � 0.0002 0.07P K 3.80 � 0.015 1.5103 � 0.0003 33.21Ca K 5.01 � 0.021 1.0408 � 0.0001 63.51Totals 100.00

SD: standard deviation.

Fig. 5 presents the TGA curves of both anorganic bones. Asshown in Fig. 5, the TGA curve of the InterOss� (Fig. 5(a)) showed asimilar pattern with that of the Bio-Oss� (Fig. 5(b)), indicating aplateau region (extremely low organic substances content) be-tween 280 �C and 550 �C. The average mass loss of the InterOss�

was about 3.26 (�0.002; SD) % (H2O content) and 3.00 (�0.011; SD)% (CO2 content) between 25 �Ce280 �C and 550 �Ce1000 �C,respectively. Also, the average mass loss of the Bio-Oss� was about2.54 (�0.002; SD) % (H2O content) and 2.68 (�0.002; SD) % (CO2content) at a same temperature range. The average loss content ofcarbonate corresponding to CO2 of InterOss� and Bio-Oss� wasabout 6.82 (�0.002; SD) % and 6.09 (�0.003; SD) %, respectively.Therefore, the average total residual mass at 1000 �C of bothanorganic bones was about 93.4 (�0.020; SD) % for InterOss�

(Fig. 5(a)) and 94.2 (�0.015; SD) % for Bio-Oss� (Fig. 5(b)). The P-values for comparison of loss content of H2O, CO2, and carbonate inboth anorganic bone minerals were 0.18 (P > 0.05), 0.14 (P > 0.05),and 0.07 (P > 0.05), respectively. This suggests that the content of

Oss� obtained by EDS.

ht% (mean � SD) Weight% sigma (mean � SD) Atomic% (mean � SD)

� 0.006 0.59 � 0.002 2.56 � 0.002� 0.004 0.47 � 0.003 2.29 � 0.002� 0.002 0.39 � 0.002 0.10 � 0.005� 0.026 1.03 � 0.002 38.36 � 0.003� 0.015 1.13 � 0.003 56.69 � 0.010

Table 3The values of content of an individual element for the chemical composition of the Bio-Oss� obtained by EDS.

Element App conc. (mean � SD) Intensity corrn. (mean � SD) Weight% (mean � SD) Weight% sigma (mean � SD) Atomic% (mean � SD)

Na K 0.08 � 0.001 1.0412 � 0.0001 1.07 � 0.002 0.62 � 0.002 1.67 � 0.002Mg K 0.09 � 0.002 0.9169 � 0.0002 1.35 � 0.007 0.48 � 0.001 2.00 � 0.015Al K 0.01 � 0.001 0.9938 � 0.0001 0.08 � 0.002 0.39 � 0.003 0.11 � 0.002P K 3.57 � 0.002 1.5151 � 0.0002 33.40 � 0.007 1.08 � 0.001 38.75 � 0.002Ca K 4.70 � 0.019 1.0412 � 0.0003 64.10 � 0.010 1.18 � 0.002 57.47 � 0.002Totals 100.00

SD: Standard Deviation.

D.S.H. Lee et al. / Materials Chemistry and Physics 146 (2014) 99e104 103

residual components such as H2O, CO2, and carbonate afterextracting of both anorganic bone minerals is substantially equiv-alent each other. The dispersion value as standard deviation in bothanorganic bone minerals was very low because all values of crudeprotein content and H2O, CO2, carbonate content, and total residualmass in TGA analysis that were measured three times, respectively,were very close to their average value.

4. Discussion

The Bio-Oss� is grouped as anorganic bone grafts (xenografts)extracted from bovine bone, and is indicated as bone defect fillingmaterials [22] due to its osteoconductive property. Specially, theBio-Oss� is a widely studied as natural apatites consisting of thehighly purified and native cancellous mineral after the removal ofthe organic substances of bovine bone during an annealing at lowtemperature (300 �Ce350 �C) with chemical treatment [23]. Thephysicochemical properties of the anorganic bone fabricated by thisprocessing method favorably affect the biological responses such asthe degradation rate associated with the bone repairing [24e26],osteogenesis [27], and osseous growth [24,28]. Thus, we comparedand analyzed the physicochemical characterizations of our anor-ganic bone (InterOss�) derived by chemical treatment and lowtemperature processing with an extremely low heating rate tothose of the Bio-Oss�.

The both anorganic bones presented the mesopore and micro-pore on their surface through SEM and BET analysis. Generally, thesize, shape, and distribution aspect of the mesopore and microporein the pore structure of the InterOss� were similar with those ofBio-Oss�. The existence of these pores in the granule form increasesthe inner surface area of the anorganic bones, supporting osteo-conduction, encouraging bone growth inside the pores [24]. Fromthe BET result, the inner surface area of InterOss� and Bio-Oss�

were a higher level within the ranges reported for the humancancellous bone (50e100m2 g�1). Therefore, it is supposed that thehighly inner surface area of both anorganic bones was closelyassociated with increasing density of micropore. Specially, themicropore allows the penetration of body fluids into the implant[29] and can be a strategy to manipulate resorption and dissolutionrate [30e34].

The XRD and FT-IR analysis indicated that both anorganic boneshave a typical HAp phase structure. The XRD curve of the InterOss�

Table 4The P-values for comparison of content of an individual element for the chemicalcomposition of the InterOss� and Bio-Oss�.

Element App conc. Intensity corrn. Weight% Weight%sigma

Atomic%

Na K 0.57 0.25 0.35 0.33 0.29Mg K 0.50 0.26 0.24 0.34 0.19Al K 0.50 0.25 0.50 0.36 0.43P K 0.09 0.19 0.21 0.26 0.20Ca K 0.07 0.24 0.14 0.24 0.16

had a similar tendency with the Bio-Oss�, suggesting a low degreeof crystalline apatite resulting from the low temperature process-ing. This crystalline degree is associated with the solubility orbiodegradation of the biomaterials, in other words, the lowercrystalline HAp materials are more soluble [35]. Therefore, it isconsidered that both anorganic bones are more prone to degrada-tion. From the FT-IR result, the bands in both anorganic bones aresimilar with each other, and no bands associated with other com-pounds excluding chemical groups corresponding to the HAp areobserved. Specially, the low intensity and absence of peak for OeHstretching band that is the norm for majority of the crystalline HApat around 3572 cm�1 and 632 cm�1, respectively confirmed that thecrystalline degree of HAp material for both anorganic bones wasreduced.

The EDS and ICP analysis verified that the main component ofboth bone minerals was Ca and P that are main element of thehuman bone. The values of the Ca/P ratio of both bone mineralscarried out by the ICP-MS techniquewere closely approached to thetheoretical value of 1.67 for synthetic HAp structure. This Ca/P ratioas well as the crystalline degree is associated with the dissolutionrate or bioabsorption rate of the HAp materials [35].

Fig. 5. TGA curves of both anorganic bones: (a) InterOss� and (b) Bio-Oss�.

D.S.H. Lee et al. / Materials Chemistry and Physics 146 (2014) 99e104104

The crude protein content of the InterOss� through the proteinanalyzer was relatively reduced comparing to that of the Bio-Oss�.It is supposed that the InterOss� bone mineral was more highlypurified resulting from the long time annealing process with anextremely low heating rate. The loss of mass observed in the TGAcurve of both bone minerals suggests the presence of residualorganic substances but it is suggested that their content wasextremely low, showing a plateau region. The effect of processingtemperature is associated with a removal of carbonate content inthe bonemineral, which occurs at temperature above 400 �C [9,35].Therefore, the residual carbonate in both boneminerals was existeddue to the low temperature processing. This carbonate is ther-moanalytically determined by the ratio of the molar mass of CaCO3and CO2: CaCO3 ¼ 2.273 � CO2 [36].

5. Conclusions

This paper summarizes comparative physicochemical charac-terization studies of InterOss� and Bio-Oss�, a cancellous bovinebone mineral for bone void filler in the dental surgeries. Specially,the Bio-Oss� has been successfully used clinically as a bone graftsubstitute during the past decade. The physical and chemicalproperties of the InterOss� were very similar with those of the Bio-Oss�. According to the results in this paper, it is expected that theInterOss� will perform equivalently as effective as Bio-Oss� forbone void filler in the oral surgical applications.

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