A Novel Ultra-porous Titanium Dioxide Ceramic with Excellent Biocompatibility ROYA SABETRASEKH, HANNA TIAINEN, S. PETTER LYNGSTADAAS, JANNE RESELAND AND HA˚VARD HAUGEN* Department for Biomaterials, Faculty for Dentistry, University of Oslo NO-0317 Oslo, Norway
ABSTRACT: The current study compares biocompatibility, cell growth and morphology, pore diameter distribution, and interconnectivity of a novel titanium dioxide (TiO2) bone graft substitute granules with three different commercially available bone graft granules NatixÕ , StraumannÕ BoneCeramic, and Bio-OssÕ . Human primary mesenchymal stem cells were cultured on the bone graft substitutes and cell viability and proliferation were evaluated after 1 and 3 days. The microstructural properties of the bone graft substitutes were evaluated by scanning electron microscopy, micro-computed tomography analysis, and mechanical testing. The cell viability and proliferation, porosity, interconnectivity, open pore size, and surface area-to-volume ratio of TiO2 granules were significantly higher than commercial bone granules (Bio-OssÕ and StraumannÕ BoneCeramic). KEY WORDS: bone graft substitute, titanium dioxide, microCT, human primary mesenchymal stem cells, porosity, cytotoxicity, bone graft.
INTRODUCTION
B
one grafts are used to repair and rebuild diseased bones in hips, knees, spine, and other bones and joints [1,2]. Bone grafting is used
*Author to whom correspondence should be addressed. E-mail:
[email protected]
JOURNAL OF BIOMATERIALS APPLICATIONS Vol. 25 — February 2011 0885-3282/11/06 0559–22 $10.00/0 DOI: 10.1177/0885328209354925 ß The Author(s), 2011. Reprints and permissions: http://www.sagepub.co.uk/journalsPermissions.nav
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for many types of orthopedic procedures that require bones to heal, e.g., bone loss caused by some types of fractures or even cancers [3–5]. The two main reasons to use bone grafting are to stimulate the bone to heal and to provide support to the skeleton by filling gaps between two bones. A bone graft substitute is used like ‘fertilizer’ to stimulate and speed the bone healing process [6]. To date, the dominant alternative for autologous grafting has been the use of donated allogenic bone grafts [7]. Harvesting tissue from donor source poses the risk of transmitting infectious diseases and causing immunogenic responses [8,9]. Allogenic bone grafts undergo a rigorous washing process that removes all of the living tissue to eliminate or greatly reduce this risk, which also decreases the osteoinduction properties of the graft [10]. There has been a huge shortage of supply in tissue donation each year [11]. Thus, there is an increasing need for alternative bone graft substitute materials. A variety of bone graft substitutes from natural and synthetic materials has been developed [12–15]. Regardless of their source, synthetic bone substitutes should ideally provide both immediate physical support and a biocompatible environment that enhances vascularization, and support osteogenesis and biomineralization [16,17]. In particular, interconnected porous structures are essential for cell penetration, cell in-growth, cell-to-cell interaction and vascularization, and also for allowing sufficient nutrient and waste exchange [18,19]. In association with osteoconductive properties, bone substitute should also offer structural support with mechanical integrity. Titanium oxide (TiO2) was chosen as bone graft substitute in the present study since this material has proven to fulfill many of the demands for a bone graft substitute material. TiO2 has shown to be biocompatible [20], enhance bone and vascular ingrowth [21] and to have a certain degree of bacteriostatic effect [22,23]. This study aims to compare this TiO2 bone graft substitute with three different commercially available bone graft substitute granules (NatixÕ from Tigran Technologies AB, BoneCeramic from Institut Straumann AG, and Bio-OssÕ from Geistlich Pharma AG). The cellular-bone graft substitute interactions on cytotoxicity and cell proliferation were examined with an in vitro assay using primary human mesenchymal stem cells (hMSCs). The bone graft substitute morphology, pore diameter distribution, interconnectivity were evaluated by scanning electron microscopy (SEM) and micro-computed tomography (microCT). The mechanical strength was investigated in a die-plunger compressive test.
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MATERIALS AND METHODS
TiO2 Bone Graft Substitute Material Fabrication Commercial TiO2 powder (Kronos 1171, Kronos Titan GmbH, Leverkusen, Germany) was used for the preparation of the ceramic slurry. The powder was cleaned in 1 M NaOH solution prior to use. The ceramic slurry was prepared by gradually adding 65 g of TiO2 powder to 25 mL of sterilized water and the pH of the dispersion was kept at 1.7 for the entire duration of the stirring with small additions of 1 M HCl. The TiO2 powder was stirred in the water in several steps at low rotation speed of 1000 rpm (Dispermat Ca-40, VMA-Getzmann GmbH, Reichshof, Germany). Temperature of the slurry was adjusted to room temperature during the low rotation speed stirring. When the slurry mixture was homogenous, the rotation speed was increased to 5000 rpm and the slurry was stirred for 5 h at this rotation speed. Temperature of the slurry was reduced to 158C during high speed stirring. Cylindrical polymer foam templates, 10 mm in both diameter and height, (60 ppi, Bulbren S, Eurofoam GmbH, Wiesbaden, Germany) were immersed in the ceramic slurry, and excess slurry was squeezed out of the foam templates between two polymer foam sheets to ensure that only a thin layer of slurry covered uniformly the entire surface area of the polymer template without blocking the pores (Figure 1).
Figure 1. Polyester-based polyurethane foams 60 ppi.
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The samples were then placed on a porous ceramic plate and allowed to dry at room temperature for at least 16 h before sintering. For the burnout of the polymer, the bone graft substitute materials were slowly heated to 4508C at a heating rate of 0.5 K/min. After 1 h holding time at 4508C, temperature was raised to 15008C at a rate of 3 K/min and the sintering time at this temperature was set to 40 h. The sintered bone graft substitute materials were then cooled back to room temperature at the cooling rate of 5 K/min (HTC-08/16, Nabertherm GmbH, Bremen, Germany). The sintered bone graft substitute materials were granulated and bone graft substitute material granules 0.9–2 mm in size were collected after sieving (Analysette 3, Fritsch GmbH, Idar-Oberstein, Germany). The TiO2 granules were sterilized prior to characterization and in vitro studies by autoclaving with saturated steam at temperature 1218C for 15 min. Commercial Bone Graft Substitute Material Samples In addition to the fabricated TiO2 bone graft substitute material granules, three different commercially available bone graft substitute materials were tested as listed in Table 1. All three commercial bone graft substitute materials were received as granules and used as received from the manufacturer. Cell Cultures hMSCs were obtained from Cambrex (Cambrex Bio Science Walkersville, MD, USA). The cells were cultured in MSC basal medium (Cambrex) supplemented with 10% fetal calf serum, 200 mM L-glutamine and 1% penicillin/streptomycin according to manufacturers’ instructions. 2 105 cells/mL were seeded on the various bone graft substitute material granules in 24-well cell culture plate. Table 1. List of commercial bone grafts used in the study. Abbreviation Natix
Õ
Product name Natix
Õ
Producer Tigran Tech AB
BoneCeramic StraumannÕ Institut BoneCeramic Straumann AG Bio-OssÕ Bio-OssÕ Geistlich Spongiosa granules Pharma AG *As reported by the manufacturer.
Granule size*
Material
0.7–1 mm
Commercially pure titanium Biphasic calcium phosphate Natural bone mineral of bovine origin
0.5–1 mm 1–2 mm
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Cell Viability Cell viability was evaluated by measuring of lactate dehydrogenase (LDH) activity in the cell culture medium. LDH activity was measured using the microplate-based Cytotoxicity Detection Kit (LDH) (Boehringer, Mannheim, Germany). Fifty microliters of cell culture media harvested at various time points was used according to the manufacturer’s protocol, and absorbance was measured at 492 nm using a ASYS Expert 96 (ASYS Hitech GmbH, Austria) analyzer. The absorbance of culture medium without cells for each bone graft substitute was used as individual background or negative control and subtracted to eliminate possible sample variations. LDH activities were calculated in percentage of the LDH activity from total cell death induced by 1% Triton X-100 treatments (positive control) on their respective granules. Cell Proliferation Assay Cell proliferation rate was determined by [3H]-Thymidine incorporation after 1 and 3 days of cell culturing. The cells were exposed to 1 mCi/ well [3H]-Thymidine (Amersham Biosciences, Buckinghamshire, UK) for 18 h prior to harvest of the cells. Cells were washed twice with icecold PBS and twice with ice-cold 5% trichloroacetic acid (TCA) to remove unincorporated [3H]-thymidine. Then cells were dissolved in 1 M NaOH (500 mL), and the cell solutions were transferred to 4 mL of Insta-gel II Plus liquid scintillation fluid and measured for 3 min in a Packard liquid scintillation counter (Packard, Zurich, Switzerland). Characterization The mechanical testing of the bone graft substitutes was performed closely adapted to the work of Long et al. [24]. Each sample (n ¼ 3 for each different material) consisted of a confined column of granules measuring 12.7 mm in diameter and 2.5 mL in volume. The samples were loaded at a displacement rate of 2.5 mm/min using a compressive mechanical testing machine (Zwicki, ZwickRoell, Ulm, Germany). The compressive strength was estimated from the load–displacement curves as a theoretical load corresponding to the transition from the initial linear region where the sample retains its porosity to the linear compaction region where porosity is eliminated. The initial visualization and optical observation of the microstructure of the bone graft substitutes were performed using a scanning electron microscope (TM-1000, Hitachi, Japan). The bone graft
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substitutes were mounted on aluminum stubs with conductive carbon tape and viewed with backscattered secondary ions at 15 kV accelerating voltage. Micro-computed tomography was used to determine nondestructively the 3D microstructure, porosity, and porous interconnectivity of the bone graft substitutes. The samples were mounted on a plastic sample holder and scanned with desktop 1172 micro-CT imaging system (Skyscan, Aartselaar, Belgium) at 8 mm voxel resolution using a source voltage of 100 kV and a current of 100 mA with 0.5 mm aluminum filter. Samples were rotated 1808 around their vertical axis and three absorption images were recorded every 0.4008 of rotation. These raw images of the samples were reconstructed with the standard SkyScan reconstruction software (NRecon) to serial coronal-oriented tomograms using 3D cone beam reconstruction algorithm. For the reconstruction, beam hardening was set to 20% and ring artifact reduction to 12. The image analysis of the reconstructed axial bitmap images was performed using the standard SkyScan software (CTan and CTvol). First, a thresholding analysis was performed to determine the threshold value for which the greyscale tomograms of bone graft substitutes were most accurately represented by their binarized counterparts in terms of porosity. Additional noise was removed by the ‘despeckling’ function. All objects smaller than 50 voxels and not connected to the 3D body were thus removed prior to further analysis. In order to eliminate potential edge effects, a cylindrical volume of interest (VOI) with a diameter of 5 mm and a height of 2.5 mm was selected in the center of the bone graft substitute. The porosity was then calculated as follows: Porosity ¼ 100% vol:% of binarized object ðbone graft substitute materialsÞ in VOI
ð1Þ
All images underwent 3D analysis, followed by the quantification of interconnectivity using the ‘shrink-wrap’ function, which allows measuring the fraction of pore volume in a bone graft substitute that is accessible from the outside through openings of a certain minimum size [25]. One can visualize this algorithm like placing a small sphere into the porous structure. If this sphere is able to reach the entire porous structure, the interconnectivity is 100%. The next step is to increase the diameter of the sphere and to verify how much of the porous structure is accessible for this particular sphere. In this study ‘spheres’ from 0 to 280 mm were used. The algorithm is based on a method by Camp et al.
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[26], and also similar to that of Lin et al. [27,28]. It first found voxels representing air that maintained connections to other air voxels and labeled regions of connected and isolated air with a connectedcomponents operation. It then performed a mathematical morphological operation known as shrink wrap or opening [29], which is a morphological erosion followed by a dilation of the air space in all three dimensions, essentially closing off small air connections. Void space was treated as 26-connected, and the solid phase therefore interpreted as 6-connected. Connected and isolated air regions were then labeled, and accessible void volume was computed as the number of air voxels maintaining connections with the outside air as a percentage of the total air voxels, as shown in Equation (2) [30]. A larger structuring element was chosen for the open operation and the process was performed again on the original segmented image. By performing iterations of this process using a successively larger structuring element for the open operation, bone graft substitute interconnectivity was assessed at successively larger minimum connection sizes. These sizes are multiples of the voxel size of the original images and correspond to the size of connections that are closed off. The accessible void fraction was computed for each iteration, so that sample interconnectivity was assessed over a range of minimum sizes that correspond to multiples of the voxel size [25]. The shrink-wrap process was performed between two 3D measurements to shrink the outside boundary of the VOI in a bone graft substitute through any openings the size of which is equal to or larger than a threshold value (0–280 mm were used in this study). Interconnectivity was calculated as follows: Interconnectivity ¼
V Vshrinkwrap 100% V Vm
ð2Þ
where V is the total volume of VOI, Vshrink-wrap is the VOI volume after shrink-wrap processing, and Vm is the volume of bone graft substitute material. The mean pore diameter distribution was found by measuring the material thickness on the inverse model. Additional noise was again removed using the ‘despeckling’ function, which removed all objects smaller than 50 voxels and not connected to the 3D body. Statistics Statistical comparison of the bone graft substitutes was performed using a nonparametrical test (ANOVA Analysis) (SigmaStat 3.5, Systat
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Figure 2. LDH activity in cell culture medium from cells cultured on NatixÕ , StraumannÕ BoneCeramic, Bio-OssÕ , and TiO2 granules. The LDH activity is calculated in % of total cell death induced by 1% Triton X-100. The data are presented as mean SEM. Figure (b) is normalized according to surface area (*p50.05 compared to TiO2, **p50.01 compared to TiO2).
Software Inc, San Jose, USA). Prior to the nonparametrical comparison test with the Holm–Sidak method, Normality and Equal Variance Test was performed. A probability of less than or equal to 0.05 was considered significant.
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RESULTS
Cytotoxicity
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At day 1 the LDH activity with Bio-OssÕ and NatixÕ were lower ( p50.01) than with TiO2, whereas there was no difference in LDH activity in the media with TiO2, Bio-OssÕ , and NatixÕ at day 3. The LDH activity with BoneCeramic, however, was higher ( p50.01) compared to TiO2 granules at day 3 (Figure 2(a)). When the LDH activities were adjusted for surface-to-volume areas the result for TiO2 was significantly lower ( p50.01) than NatixÕ and Bone Ceramic at day 1, and lower ( p50.01) than all the three commercially bone graft substitutes at day 3 (Figure 2(b)).
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Figure 3. Proliferation rates of hMSC cells cultured on NatixÕ , StraumannÕ BoneCeramic, Bio-OssÕ , and TiO2 bone graft substitutes. The proliferation rate was measured by the incorporation of [H3]-Thymidine (counts per minute). The data are presented as mean SEM. Figure (b) is normalized according to surface area (**p50.01 compared to TiO2, ##p50.01 compared to NatixÕ , $$p50.01 compared to BoneCeramic).
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Cell Proliferation Assay The cell growth on TiO2 granules were four times higher than on BioOssÕ and 6-fold higher than on BoneCeramic ( p50.01) at day 1, whereas there was no differences in cell growth on NatixÕ and TiO2. At day 3 the cell growth on TiO2 granules were approximately 3, 4, and 6 times higher than on NatixÕ , BoneCeramic, and Bio-OssÕ , respectively (Figure 3(a)). When the cell proliferation rate was adjusted for surfaceto-volume areas, the result with NatixÕ was significantly higher than BoneCeramic, Bio-OssÕ , and TiO2 granules at day 1, and significantly higher ( p50.01) compared to BoneCeramic and Bio-OssÕ for day 3. However, there was no significant difference between NatixÕ and TiO2 granules after day 3 ( p40.05). TiO2 granules and NatixÕ had higher proliferation rate than both BoneCeramic and Bio-OssÕ at day 1 and day 3. Bone-Ceramic had only higher proliferation rate than Bio-OssÕ at day 3 ( p50.001) (Figure 3(b))
Characterization of Bone Graft Substitutes TiO2 bone graft substitutes were compared with three different commercially available bone grafts, which are intended for similar applications as the prepared TiO2 bone graft substitutes. The SEM appearance of a TiO2 bone graft substitute and the three commercial bone grafts are shown in Figure 4 and Figure 5 presents the pore diameter distributions for all four bone graft substitutes. All bone graft substitutes displayed a rather wide pore size distribution but the majority of the pores in TiO2 granules were notably larger than in the three investigated commercial bone graft substitutes. Accordingly, the mean pore sizes for all three commercial bone graft substitutes were considerably smaller than for the TiO2 granules. Each of the three commercial bone graft substitutes also appeared considerably less porous when compared to the fabricated TiO2 bone graft substitutes (Table 2, Figure 4). In Table 2, selected structural parameters of the three commercial bone grafts are compared with those of the prepared TiO2 bone graft substitutes. This micro-CT data revealed the considerably higher porosity values of the TiO2 bone graft substitutes in comparison with all three commercial bone grafts. The higher porosity of the TiO2 bone graft substitute was also qualitatively observed from the SEM images presented in Figure 5. Also, the surface area-to-volume ratio is significantly higher for TiO2 bone graft substitutes than for any of the
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Figure 4. SEM images of four different bone graft substitutes showing the typical microscopic appearances of each bone graft substitute. (a) NatixÕ , (b) BoneCeramic, (c) Bio-OssÕ , and (d) TiO2 granules. 12 Natix® BoneCeramic
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Figure 5. Pore diameter distributions of the four different bone graft substitutes.
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commercial bone grafts. No significant difference was observed in the degree of anisotropy between the four bone graft substitute types. Table 2 also shows the compressive strength between the different bone graft substitutes. Again, there was no significance between the four groups. However, the metallic NatixÕ granules seemed more ductile in comparison to the ceramic bone grafts, which failed in brittle manner. NatixÕ granules retained their volume better during the compression test with losing only 37% of the original volume whereas the ceramic granules lost over 65% of the original volume of the granules (Table 2). This difference in packing volume was found statistically significant ( p50.01). An interconnectivity study was also performed on all commercial samples and the results were compared with the interconnectivity of the fabricated TiO2 bone graft substitutes. The results of the interconnectivity study are presented in Figure 6, which displays the interconnectivity of the pore volume as a function of the connection size. All bone graft substitutes were highly interconnected through openings up to 50 mm. However, with over 85% interconnectivity throughout the entire investigated minimum connection range, the interconnectivity of TiO2 bone graft substitutes far exceeded the interconnectivity of any of the other investigated bone graft substitutes, particularly at minimum connection sizes larger than 75 mm. The interconnectivity of NatixÕ granules decreased steadily from over 90% at minimum connection sizes below 75 mm to only 50% at above 200 mm BoneCeramic bone graft exhibited excellent interconnectivity (490%) through openings up to 75 mm but declined steeply to very poor interconnectivity values (530%) at minimum connection sizes above 250 mm. The interconnectivity of Bio-OssÕ bone graft dropped from 90% for
Table 2. Comparison of selected pore architectural characteristics and mechanical properties of four different bone graft granules. Presented numbers are mean values (¤p40.05). Parameter Porosity Intersection surface Surface area-to-volume ratio Pore size Strut thickness Degree of anisotropy Compressive strength Compact volume
Unit
NatixÕ
Bone Ceramic
Bio-OssÕ
TiO2
% mm2 1/mm mm mm – MPa %
55.8 20.9 18.3 241.6 191.1 1.54 0.59¤ 63.1
66.5 8.9 35.1 209.3 109.8 1.35 0.78¤ 24.9
41.6 22.5 21.2 129.9 188.1 1.66 0.55¤ 33.4
86.9 9.2 48.8 411.9 95.1 1.39 0.74¤ 35.5
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100
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80
60
40
Natix® BoneCeramic Bio-Oss® TIO2 granules
20 0
50
100
150
200
250
300
Minimum connection size (μm)
Figure 6. Interconnectivity of four different bone graft substitutes through openings smaller than 280 mm in diameter.
40 mm minimum connection size to mere 45% for 150 mm above which the interconnectivity remained virtually unchanged.
DISCUSSION
The present study showed that the high porosity, interconnectivity, and surface area-to-volume ratio properties of TiO2 bone graft substitute directly influenced the cellular responses and provided appropriate conditions for enhanced cell proliferation and viability of hMSCs. The high porosity values of the fabricated TiO2 bone graft substitutes in combination with the open pore structure achieved using the polymer sponge method led to a highly interconnected pore structure as has also seen with this processing method on other ceramics [31–35]. Consequently, the TiO2 bone graft substitutes outperformed all three commercial bone graft substitutes in respect of interconnectivity, particularly when the minimum connection size increased. The TiO2 bone graft substitutes had a large extent of open pore structure when compared to the other three bone graft substitutes. Interconnectivity of pores has
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been considered an important structural characteristic of tissue engineering scaffolds for quite some time [36,37]. Interconnectivity is one of the key scaffold parameters that can be measured quantitatively using micro-CT image processing algorithms such as the so-called ‘shrink-wrap function’ [25]. This has not been possible with the standard techniques such as mercury intrusion porosimetry, pycnometer, etc. [38]. These algorithms have evolved in the past years due to advances both in computer power, programming, and image resolution [28,39,40]. Computer-assisted image analysis and development of new algorithms provide further advancements and more sophisticated analyses will yield even more informative, quantitative parameters for describing scaffold architecture. These parameters may perhaps be used for correlation with and prediction of in vivo bone graft substitute performance [38]. The more open pore structure of TiO2 bone graft substitutes contributed to the significantly higher surface-to-volume ratio of these bone graft substitutes compared to the examined commercial bone graft substitutes. Surface-to-volume ratio is also an important characteristic for tissue engineering bone graft substitutes because a large surface area within the bone graft substitute is needed in order to achieve adequate cell attachment and growth for tissue regeneration [41]. Another factor that involves the surface-to-volume ratio is of particular importance to degradable bone graft substitutes [42], where more rapid dissolution rates were obtained with higher surface-to-volume ratio [43–45]. This would be particularly applicable for BoneCeramic that was used in this study. Since the surface area-to-volume ratio varied greatly for the different bone graft substitutes, one would therefore expect that cell density (the number of seeded cells in unit area of pore surface) affected the cell behavior such as cell adhesion and proliferation in the scaffolds. When the proliferation rate was normalized according to the surface area (Figure 3(b)), the significance levels between the groups increased tremendously in comparison to the nonnormalized (Figure 3(a)). The result showed that NatixÕ and TiO2 granules performed better than both BoneCeramic and Bio-OssÕ . Interestingly, there was no significant difference between NatixÕ and TiO2 at day 3, even though NatixÕ had almost four times lower surface area-to-volume ratio in comparison to TiO2 granules. The initial difference of the proliferation rate at day 1 may be due to variation in cell seeding density. Bio-OssÕ performed worst by all tested bone graft materials when normalized to surface area. Similar findings also seen by Wanschitz et al. [46]
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found that Bio-OssÕ did not generate increased proliferative responses of human PBMC from healthy donors. Even with IL-2 that induces proliferation of T lymphocytes, which encountered their specific antigen, the proliferation rate of PBMC from healthy donors was not increased after incubation with this bone grafting materials. Kubler et al. [47] showed that human osteoblasts had a different proliferation pattern according to the type of applied bone graft substitute, where PepGen P-15 showed the highest proliferation and differentiation rate followed by Osteograf, Algipore, and Bio-base. Bio-Oss showed the lowest. The surface area-to-volume ratio is assumed to play a major role in the viability and growth of cells. TiO2 granules had significantly lower LDH activity at all time points and showed the highest proliferation rate relative to area and volume at day 3. The higher cell viability and cell proliferation rate observed with TiO2 granules are likely due to higher surface area-tovolume ratio and larger pore size of TiO2 compared to the commercial graft materials tested. The larger pore size of TiO2 granules would allow for easier cell penetration, and nutrient and waste exchange. The higher surface area-to-volume ratio of TiO2 could provide more space for cell grow in longer culturing time. In general, scaffolds with higher porosities have been reported to display better bone in-growth over time [48–50]. The available space in the graft materials with lower total surface area may be occupied faster and consequently there is no more space available for further cell growth. Kasten et al. [51] showed that cells adhere more easily to mineral calcium deficient hydroxyapatite scaffold with high specific surface area (48 m2/g) as compared to b-tricalcium phosphate (b-TCP) with low surface area (0.5 m2/g). The higher porosity may be especially more beneficial with in vivo situation, which will result in higher tissue regeneration. Kruyt et al. [52] compared hydroxyapatite scaffolds with different porosities (70% porosity and 800 mm average pore size (70/800) versus 60% porosity and 400 mm average pore size (60/400)). More goat bone marrow stromal cells (gMSC) proliferated during a 6-day ex vivo culture in the 60/400 scaffolds. However, when scaffolds seeded with gMSC were implanted in bilateral paraspinal muscles in goats more bone formed in the 70/800 scaffolds. Furthermore, higher interconnectivity provided better cell–cell interaction, which enhanced cell proliferation and differentiation [53,54]. The average pore size of bone graft substitutes varied. However, the majority of pores were well above 100 mm in diameter for all analyzed bone graft substitutes excluding Bio-OssÕ , which had mean pore diameter of 130 mm with a considerable amount of pores smaller than 100 mm in diameter. This diameter is considered the minimum pore size for bone graft substitutes that are intended for bone tissue engineering
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applications and pore sizes above 300 mm are recommended for better bone regeneration [55,56]. The effective pore diameter is gradually reduced as cells attach and proliferate on bone graft substitute struts and pore walls, eventually forming new tissue within the bone graft substitute structure. All the morphological data from this study is gathered by micro-CT. The main advantage of using this machine is its ability to provide accurate 3D visualization and quantification of internal pore structures without any special sample preparation. Furthermore, micro-CT is the only current technique that can provide quantitative information on the interconnectivity of the pore spaces. Due to the nondestructive nature of micro-CT, the scanned scaffolds are left intact, allowing the same samples to be subjected to further testing. Micro-CT is a flexible analysis method that allows the evaluation of several different materials and scaffold types, including foams, textiles, and nanofiber scaffolds [38,40]. Despite the numerous advantages of micro-CT, there are some drawbacks associated with this technique. Micro-CT scanning and analysis processes can be time consuming, and are dependent on the computational capability of the hardware and software [38,57]. Image thresholding is a crucial step in micro-CT analysis and can be a major cause for erroneous results. Thresholding range is normally selected via histographics and visual estimation. Therefore, thresholding is a userdependent step, and incorrect selection of threshold range leads to incorrect analysis and visualization of the scanned scaffold. [38] There are also some imaging errors, such as ring artifacts and beam hardening, which are characteristic of computed tomography [58–60]. The quantification algorithms would benefit from further development in order to resolve such imaging errors closely related to this technique. Since micro-CT is still relatively new technology, improvements in these algorithms would be expected [38]. Owing to bone tissue’s key function of providing biomechanical support to the body, a certain degree of mechanical stability is required from a bone graft substitute. In the current study, no significant difference was found in the compressive strength between the different bone graft substitutes (Table 2). However, the method that was used for mechanical testing of the granules is better suited for assessing the mechanical properties of the ceramic granules than metallic NatixÕ granules due to the characteristically different behavior of these materials under compressive load. The ductility of NatixÕ allows more force to be used without breaking the granules and thus reducing the porosity and granule volume during the packing of the granules into a bone defect. This characteristic was seen as the considerably smaller change in the
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volume of the granule column during the compression test. All three commercial bone graft substitutes are currently being used in the filling and augmentation of intraoral and maxillofacial osteal defects, such as tooth extraction sites, bony defects of the alveolar ridge, expanded sinuses, and intrabony periodontal osteal defects and furcation [61–65]. The porous ceramic TiO2 bone graft substitutes are expected to perform well in these very same applications where bone regeneration is generally required. Moreover, TiO2 bone graft substitute blocks may be used for bone filling in some of the abovementioned applications instead of TiO2 granules, in which case the mechanical stability of the filled defect site would be greatly improved due to the higher compressive strength of the bone graft substitute. Compressive strength of 1.2 MPa has been reported for TiO2 bone graft substitute blocks [66].
CONCLUSION
All analyzed bone graft substitute materials were highly interconnected through openings up to 40 mm. However, the interconnectivity of TiO2 bone graft substitute far exceeded the interconnectivity of any of the other investigated bone graft substitute material, particularly at minimum connection sizes larger than 100 mm. Also the porosity and surface area-to-volume ration was higher for the TiO2 bone graft substitutes, where the porosity was 86.9%. This was more than twice as high as Bio-OssÕ (41.6%). Only the intersection surface area was higher for NatixÕ and Bio-OssÕ in comparison to the TiO2 bone graft substitutes, which was associated with the low porosity measured for NatixÕ and Bio-OssÕ . More importantly, the surface area-to-volume ratio was highest for the TiO2 bone graft substitutes. These results indicate that the TiO2 and NatixÕ bone graft substitutes have the adequate morphology properties; pore size and interconnectivity favored for tissue engineering and may therefore exceed the clinical performance seen by both BoneCeramic and Bio-OssÕ . No significant differences in mechanical strength were found when applying the plunger-die compression test; however, NatixÕ had the highest compact volume and BoneCeramic the lowest. The in vitro results from this study showed that the cytotoxicity of the TiO2 bone graft substitutes were significantly lower compared to the commercial bone graft substitutes. The proliferation of hMSC normalized to the surface area-to-volume ratio showed that NatixÕ and TiO2 outperformed Bio-OssÕ and BoneCeramic ( p50.01). NatixÕ had significant higher proliferation rate than TiO2 granules only at day 1.
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