Cell Tissue Bank DOI 10.1007/s10561-014-9447-8

ORIGINAL PAPER

The effect of supercritical carbon dioxide sterilization on the anisotropy of bovine cortical bone Nicholas Russell • Alain Rives • Matthew H. Pelletier • Tian Wang William R. Walsh



Received: 7 November 2013 / Accepted: 5 April 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract Bone allografts are used to replace bone that has been removed or to augment bone tissue in a number of clinical scenarios. In order to minimize the risk of infection and immune response, the bone is delipidated and terminally sterilized prior to implantation. The optimal method for bone graft sterilization has been the topic of considerable research and debate. Recently, supercritical carbon dioxide (SCCO2) treatments have been shown to terminally sterilize bone against a range of bacteria and viruses. This study aimed to evaluate the effect of these SCCO2 treatments on the anisotropic mechanical properties of cortical bone. Adult bovine cortical cubes were prepared and treated using SCCO2 and a range of common processing additives (ethanol, peracetic acid and hydrogen peroxide). The bone was mechanically tested in uniaxial compression in the axial, radial and tangential orientations. Ultimate stress, strain, elastic modulus, energy and stiffness were evaluated. This study found that SCCO2 treatment without additive

N. Russell  A. Rives  M. H. Pelletier  T. Wang  W. R. Walsh Surgical and Orthopaedic Research Laboratories, Prince of Wales Clinical School, University of New South Wales, Sydney, Australia W. R. Walsh (&) Surgical and Orthopaedic Research Laboratories, Prince of Wales Hospital, Level 1 Clinical Sciences Building, Avoca St Randwick, Sydney, NSW 2031, Australia e-mail: [email protected]; [email protected]

did not alter the ultimate stress, stiffness or energy to failure depreciably in any orientation. The addition of sterilants peracetic acid and hydrogen peroxide also preserved mechanical function, with no deleterious effect on stress or stiffness. This study highlights the expediency of SCCO2 treatment for bone allograft processing as terminal sterilization can be achieved while maintaining the intrinsic mechanical properties of the graft. Keywords Sterilization  Allograft  Bone  Mechanical  Anisotropy  Supercritical fluid

Introduction Bone grafts are used in a wide range of orthopaedic applications to replace or augment bone that has been removed due to injury or disease. Ideally the graft should exhibit mechanical properties comparable to the host bone, acting as a load bearing scaffold by mimicking the mechanical function of the bone that is replaced, while also actively participating in the healing process. Autograft bone is considered the ‘gold standard’ for the repair of bone defects due its superior osteogenic, osteoinductive and osteoconductive properties. However, associated risks such donor site morbidity, increased surgery time, increased risk of infection and a limited supply of donor bone limit its use (Vaccaro et al. 2002; Seiler and Johnson 2000; Ahlmann et al. 2002). Allograft bone is the logical

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alternative as it eliminates donor site morbidity, allows for anatomical matching and is readily available in a range of graft sizes and preparations. However, rigorous processing and terminal sterilization are required prior to use to minimize the possibility of an immune response or disease transmission. While effective at removing these infectious agents, some of these treatments have been shown to have a deleterious effect on the biological and/or mechanical properties of the graft (Currey et al. 1997; Cornu et al. 2000; Thoren and Aspenberg 1995; Akkus and Belaney 2005; Akkus and Rimnac 2001; DePaula et al. 2005; Anderson et al. 1992). A processing and sterilization method that preserves the mechanical and biological performance of allograft bone is necessary. Gamma irradiation is currently the gold standard for terminally sterilized allograft bone due to its efficacy against viral and bacterial disease transmission (Nguyen et al. 2007a, b; Campbell et al. 1994). However, studies have shown that gamma irradiation impairs the static (Hamer et al. 1999; Akkus and Rimnac 2001; Cornu et al. 2000) and dynamic (Mitchell et al. 2004; Akkus and Belaney 2005) mechanical properties of bone, with this damage occurring in a dose-dependent manner (Hamer et al. 1999; Currey et al. 1997). The gamma rays cause a dose dependent increase in the denaturation of bone collagen (Nguyen et al. 2012, 2013) and change the number and concentration of cross-links between collagen chains (Barth et al. 2011). This translates to a significant reduction in the post-yield (plastic) properties of cortical bone while the pre-yield (elastic) behaviour is unaffected (Currey et al. 1997; Akkus and Rimnac 2001; Hamer et al. 1996; Anderson et al. 1992). Clinically, these changes have been shown to affect the fracture toughness of allografts with fracture rates in patients treated with irradiated grafts almost double that of corresponding autograft patients (38 vs. 18 %) (Lietman et al. 2000). Considering most allograft fractures occur under dynamic loading conditions where forces are in the elastic region (Wang and Feng 2005), this suggests that there are other gamma irradiation induced factors involved which need to be investigated. A supercritical fluid is any substance above its critical temperature and pressure, resulting in unique physicochemical properties intermediate between a liquid and a gas (Gerd 2005). Supercritical carbon dioxide (SCCO2) has a critical temperature of 31.1 °C

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and critical pressure of 73.4 bar, which is attractive for allograft processing as it can be used to treat thermally labile biological tissues without deleterious effects on proteins and enzymes (White et al. 2006). Additionally, past its critical point SCCO2 has a gas-like diffusivity allowing it to easily penetrate microporous materials like bone, where its liquid-like density facilitates the extraction of organic components. Alloimmunogenicity is strongly correlated with the presence of human leukocyte antigen and major histocompatibility complexes which are found in bone components such as collagen, lipids and matrix proteins; and this is the rationale behind the processing and cleaning of allografts prior to sterilization. Some tissue banks currently process bone with solvents such as hydrogen peroxide and ethanol prior to sterilization which act doubly to remove these antigenic components while also reducing the bioburden in the graft. Fages et al. (1994) demonstrated the potential of SCCO2 treatment for allograft processing by effectively delipidating bone and thus removing some of these immunological concerns. Additionally, a number of other studies have reported terminal sterilization of a range of bacteria and viruses using SCCO2 at different experimental pressures and time (Spilimbergo et al. 2003; Watanabe et al. 2003; Dillow et al. 1999). While the mechanisms of inactivation are not clear, it has been speculated that the degree of inactivation is a function of the time, maximum pressure and temperature used; increasing as these parameters are increased (Dillow et al. 1999; Spilimbergo et al. 2003). The supplementation of these treatments with sporicidal and bactericidal additives such as hydrogen peroxide and ethanol has also been shown to improve the efficacy of treatment, allowing for milder treatment pressures and shorted experiment times (Zhang et al. 2006; Shieh et al. 2009; Qiu et al. 2009; Hemmer et al. 2007). The effect of SCCO2 treatment on the mechanical properties of allograft bone is limited. Nichols et al. (2009) found that SCCO2 treatment with the addition of active sterilant did not significantly affect the quasi static mechanical properties of human cortical bone when tested in three point bending. They also observed that the SCCO2 treated grafts appeared ‘cleaner’ compared to gamma irradiated bones, adding weight to the delipidation characteristics of supercritical fluid (SCF) treatments. Moreover, they found that under these same testing conditions terminal sterilization up

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to a sterility assurance level of 10-6 log reductions was achieved for B. atrophaeus inoculated on the bones. In the study conducted by our laboratory using the same SCCO2 conditions as Nichols et al. (2009), we reported that the mechanical properties of intact rabbit humeri were preserved compared to untreated controls in static three-point bending, four-point bending and torsion (Russell et al. 2013a). The aim of the present study was to examine the effect of different SCCO2 treatments on the anisotropy of cortical bone in compression; with the null hypothesis that SCCO2 treatment would preserve the mechanical properties of bone in all orientations compared to untreated controls. Cortical bone was treated using a number of SCCO2 treatments that incorporated common tissue banking processing additives, and were then mechanically tested in three anatomical orientations. In doing so, we hope to address the expediency of such treatments for the processing and sterilization of load bearing allograft bone. In addition, this work has aspects that are clinically relevant and has important implications regarding load transmission in bone. Understanding the effect of treatments which alter anisotropy of bone will provide insights into the relationship(s) between the bone constituents and overall mechanisms of bone functionality.

Materials and methods Preparation Six adult bovine femurs from different donors were obtained fresh from a local slaughter house. The diaphyses were dissected out and rough strips were cut in the longitudinal axis of the bone. From the rough strips, 270 cortical bone cubes (Fig. 1) (5 mm 9 5 mm 9 5 mm) were machined using a custom jig. The longitudinal plane of the beam was marked prior to machining to maintain orientation. During all stages of preparation the bone was kept well irrigated with Phosphate Buffered Saline (PBS) to avoid dehydration. The cubes were randomly assigned to one of six treatment groups (n = 45): Control and five SCCO2 groups with various treatment additives (Table 1). This method has previously been reported in testing the effect of gamma irradiation on the anisotropy of cortical bone (Russell et al. 2012).

Fig. 1 Schematic showing the orientation of the prepared cube specimens

Before treatment, each bone cube was measured in triplicate in the axial, radial and tangential planes using digital calipers. The weight was determined in both air and water using a precision vacuum scale (Sartorius BP110S), and using these weights apparent density was calculated by Archimedes principle. Treatment The control group was stored in phosphate buffered saline (PBS) at -20 °C following machining, and was thawed out to room temperature before mechanical testing. SCCO2 experiments were performed in-house using a custom supercritical fluid rig setup as previously described (Russell et al. 2013a, b). The treatment conditions used for each group are outlined in Table 1. For all SCCO2 experiments, the cubes were thawed out at room temperature prior to testing. The cubes were then loaded into the pressure vessel with or without an additive and thermally equilibrated at an operating temperature of 37 °C. In the SCF H2O2, SCF Peracetic Acid (PAA) and SCF Novakill groups, additive was introduced by pipetting it onto a cellulose pad that was placed inside the lid of the pressure vessel. The H2O2 and PAA additives were prepared in-house from stock solutions; while NovakillÒ additive was purchased from NovaSterilis (Lansing, NY, USA). In the SCF Ethanol group, the solution was simply poured into the vessel prior to adding the cubes. Once closed, the pressure vessel was then mounted onto the SCF rig inside the water bath and allowed to thermally equilibrate at the operating temperature of 37 °C.

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Cell Tissue Bank Table 1 Summary of the study sample size and treatment conditions for each group Group

Sample size (n)

Gas

Control

45

CO2

SCF control

45

CO2

SCF H2O2

45

CO2

SCF PAA

45

SCF ethanol SCF novakill

Treatment Pressure Bar

Temperature (°C)

Time h

Additive

Depressurization min

1

20







160

37

4



45

160

37

4

6 % H2O2 (2 mL)

45

CO2

160

37

4

14.7 % PAA (2 mL)

45

45

CO2

160

37

4

95 % EtOh (100 mL)

45

45

CO2

160

37

4

NovakillÒ (1.04 mL)

45

The carbon dioxide gas was then pressurized incrementally past its critical point (pc = 72.8 bar) and into the supercritical phase region where it was isolated at its operating pressure (either 100 or 160 bar) for the prescribed soaking time (either 1 or 4 h). Finally, the system was depressurized linearly at between 3 and 4 bar min-1 back to atmospheric pressure and the bones were extracted. The experimental parameters used in this study were adapted from the work of Nichols et al. (2009) who reported SAL 10-6 compliance under milder supercritical conditions. The additives used were chosen to represent common allograft processing agents, and as a means to examine any potential alteration in bone anisotropy following processing.

Elastic modulus: The slope of the linear elastic region, calculated as the stress divided by strain at the yield point (MPa). Stiffness: The specimen’s resistance to deformation (N mm-1) under the applied loading conditions, calculated as the gradient of the load–displacement curve in the linear elastic region. Ultimate stress: The maximum load divided by the cross-sectional area of the cube (MPa). Ultimate strain: The relative deformation of the cube at maximum load, calculated in relation to the original gauge length (%). Energy to failure: The total amount of energy absorbed (J) by the specimen, calculated as the area under the load–displacement curve to maximum load.

Mechanical testing

Scanning electron microscopy

Prior to the mechanical testing, dimensions were again taken in triplicate, and the cubes were weighed again for density calculations. Specimens were mechanically tested in unconfined uniaxial compression using a calibrated MTS 858 Bionix servo hydraulic testing machine (MTS, MN, USA). Fifteen cubes from each group were randomly assigned to axial, radial or tangential testing, respectively. Each cube was failed at 10 % strain min-1 in a PBS bath at room temperature. From the load–displacement output in each test, elastic and plastic mechanical properties were evaluated using a custom-written Matlab script (Matlab 7.11.0, The Mathworks, Inc.). The parameters measured in this study included: Yield stress: The maximal stress (MPa) at the yield point. Yield strain: The relative deformation (%) at the yield point.

Following mechanical testing, three randomly selected cubes from each group were imaged using a Hitachi TM-1000 Tabletop Microscope (Hitachi, Tokyo, Japan). The cubes were mounted onto adhesive carbon tabs fixed to a moveable stage and then placed into the vacuum chamber. No surface preparation was required, with the bone observed in its natural state. Low to moderate magnification (50–400 9 objectives) images were taken of each cube face in each orientation to observe fracture patterns and the mechanisms of failure.

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Statistics Statistical analyses were performed using IBM SPSS Statistics 20 (20.0, SPSS Inc) for windows. For the mechanical data, a Shapiro–Wilk test was first used to confirm the normality of the results. Then a one-way

Cell Tissue Bank Table 2 Mechanical results for yield strain showing the mean and standard deviation for each treatment group in each orientation Yield strain (%) Group

Fig. 2 Comparison of the yield stress in the control group versus treatment groups in the axial, radial and tangential orientations (results expressed as mean and standard deviation)

ANOVA followed by a Games Howell post hoc test was used to assess differences in each orientation and between groups for each mechanical parameter. Statistical changes in density were assessed using a student’s t test due to the paired nature of the data.

Results There were no changes to the physical appearance or dimensions of the samples following any of the SCF treatments. Yield stress Results for the control group showed distinctly greater yield stress in the axial orientation compared to the radial and tangential orientations, which appear transversely isotropic (Fig. 2). Following the SCF Control treatment, there was no significant effect on the yield stress in any orientation compared to the control, which was despite an 18 % increase in the radial yield stress (p = 0.130). The addition of hydrogen peroxide to the supercritical fluid treatment resulted in non-significant increases of 19, 9 and 14 % in the axial, radial and tangential orientations, respectively. However, this increase approached significance in the axial plane (p = 0.08). Under the same supercritical treatment conditions, the addition of peracetic acid and ethanol did not alter yield stress significantly in any direction. Under milder supercritical treatment conditions, the SCF Novakill treatment maintained yield properties compared to the control.

Axial (Z)

Radial (R)

Tangential (T)

Mean

STD

Mean

STD

Mean

STD

Control

0.039

0.006

0.037

0.006

0.034

0.006

SCF control

0.042

0.015

0.044

0.020

0.037

0.013

SCF H2O2

0.030*

0.002

0.031

0.006

0.031

0.005

SCF PAA

0.028*

0.002

0.036

0.005

0.029

0.004

SCF ethanol

0.033

0.007

0.037

0.012

0.033

0.005

SCF novakill

0.035

0.005

0.039

0.008

0.035

0.004

* Denotes significance difference as compared to the control at p \ 0.05

Yield strain Consistent with the yield stress results, supercritical fluid treatment alone had no significant effect on the yield strain of the bone in any orientation (Table 2). The addition of both hydrogen peroxide and peracetic acid additives resulted in a statistically significant drop of 23 % (p = 0.004) and 28 % (p \ 0.001) in the axial plane, respectively. However, the radial and tangential planes were relatively unaffected. As for the yield stress, both the SCF Ethanol and SCF Novakill groups preserved the yield strain properties of the samples in all orientations compared to the control. Elastic modulus The results for elastic modulus are shown in Fig. 3. It is evident from the graph that despite changes in magnitude, the characteristic relationship observed in the control group with the axial modulus substantially greater than the radial and tangential moduli which are similar, was maintained following all SCF treatments. Following SCF treatment alone, the axial elastic modulus was preserved. In the radial and tangential orientations there were increases of 74 and 51 %, respectively, though statistical significance was only reached in the radial orientation (p = 0.018). In both the SCF H2O2 and SCF PAA groups there were similar effects observed. The axial elastic modulus increased by 45 and 28 %, respectively, though this only reached significance in the SCF H2O2 group (p = 0.008). Correspondingly, in the radial and tangential planes

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Fig. 3 Comparison of the elastic modulus in the control group versus treatment groups in the axial, radial and tangential orientations (results expressed as mean and standard deviation); * denotes a significant difference as compared to the control at p \ 0.05, ** denotes a significant difference as compared to the control at p \ 0.001

Fig. 5 Comparison of the ultimate stress in the control group versus treatment groups in the axial, radial and tangential orientations (results expressed as mean and standard deviation)

Again, the stiffness properties of the bone samples were preserved following the SCF Control treatment. The SCF H2O2 treatment had a stiffening effect across all orthogonal planes. Most notably, there was a statistically significant (p \ 0.001), 30 % increase in the axial plane, and an almost significant (p = 0.052) climb of 36 % in the radial direction. Despite minor increases in axial stiffness, there was no significant change in stiffness properties following either of the SCF PAA, SCF Ethanol or SCF Novakill treatments. Ultimate stress

Fig. 4 Comparison of the stiffness in the control group versus treatment groups in the axial, radial and tangential orientations (results expressed as mean and standard deviation); ** denotes a significant difference as compared to the control at p \ 0.001

there were statistically significant rises of 110 and 68 %, and 79 and 67 %, respectively (all p \ 0.001). The addition of ethanol resulted in a similar effect to that of hydrogen peroxide and peracetic acid, albeit to a lesser extent. Elastic modulus was enhanced across all orientations, with 35 % (p = 0.08), 77 % (p = 0.036) and 52 % (p = 0.076) increases in the axial, radial and tangential planes, respectively. Contrary to these results, there was no significant change in elastic modulus following the SCF Novakill treatment. Stiffness The stiffness results revealed the same characteristic anisotropic behaviour seen for elastic modulus (Fig. 4).

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The results for the ultimate stress in the control and SCF treatment groups are shown in Fig. 5. In the control group, the axial and radial orientations had very similar ultimate stress values which were considerably greater than the tangential axes. The results of the SCF Control group were consistent with the control, despite a small 9 % decrease in axial stress; though this was not significant. There was no significant effect observed in any orientation in the SCF H2O2, SCF PAA, SCF Ethanol and SCF Novakill treatment groups. Ultimate strain The effect of SCF treatment alone on the ultimate strain was consistent with the ultimate stress findings, with no statistical change found for any orientation (Table 3). Moreover, similar behaviour was observed in the SCF H2O2, SCF PAA and SCF Novakill groups. In the SCF Ethanol group there were fairly uniform decreases in the ultimate strain of approximately 20 %

Cell Tissue Bank Table 3 Mechanical results for ultimate strain showing the mean and standard deviation for each treatment group in each orientation

Table 4 The effect of treatment on apparent density Apparent density (g cm-3)

Significance

Pre-treatment

Post-treatment

p value

Control

1.993 (0.061)

1.993 (0.061)



SCF control

2.018 (0.038)

1.969 (0.045)

[0.001

Group

Ultimate strain (%) Group

Axial (Z)

Radial (R)

Tangential (T)

Mean

STD

Mean

STD

Mean

STD

SCF H2O2

2.048 (0.034)

2.003 (0.038)

[0.001

Control

0.050

0.012

0.103

0.031

0.081

0.033

SCF PAA

2.036 (0.036)

2.007 (0.035)

[0.001

SCF control SCF H2O2

0.059 0.041

0.021 0.006

0.107 0.081

0.049 0.036

0.086 0.092

0.032 0.038

SCF ethanol

2.043 (0.032)

2.011 (0.038)

[0.001

SCF novakill

1.989 (0.051)

1.949 (0.050)

[0.001

SCF PAA

0.043

0.010

0.132

0.021

0.079

0.035

SCF ethanol

0.040

0.006

0.083

0.047

0.061

0.019

SCF novakill

0.047

0.008

0.107

0.030

0.069

0.024

Values are mean (std), statistical significance was reached following all SCF treatments

Density This study confirmed the extraction potential of SCF treatments with a significant 1–2 % (p \ 0.001) decrease in apparent density in all SCCO2 groups regardless of treatment conditions or additive (Table 4). Scanning electron microscopy

Fig. 6 Comparison of the energy to failure in the control group versus treatment groups in the axial, radial and tangential orientations (results expressed as mean and standard deviation); * denotes a significant difference as compared to the control at p \ 0.05

in all directions, though none of these were statistically significant. Energy to failure The mechanical results for energy to failure are displayed in Fig. 6. In the control group, there was considerable anisotropy observed with large differences in energy to failure reported for each orientation. This anisotropic behaviour was still present following the SCF Control treatment, with no significant change in any direction. Across all SCF additive groups, the most considerable changes were detected in the radial and tangential orientations, while there was no significant change in the energy to failure in the axial plane. The peracetic acid additive increased radial energy to failure by a statistically significant 63 % (p = 0.007).

Scanning electron microscopy images revealed no detectable difference between fracture behaviour in any of the treatment groups. There were, however, very distinct failure mechanisms in the axial orientation compared to the radial and tangential planes which supported the mechanical findings. In the axial orientation, microcracks accumulated on the loaded surface in no discernable pattern and eventually formed into larger macrocracks (Fig. 7a). In the tangential plane, multiple microcracks formed in parallel to the loading direction in a phenomenon called crack bridging. These cracks form along the weak cement lines between osteons and propagated into contiguous macrocracks. In some cases, fracture occurred through these macrocracks by micro-buckling (Fig. 7b). In the radial orientation, the fracture behaviour appeared to occur by more brittle mechanisms (Fig. 7c). Large shear planes perpendicular to the loading direction were present, with minimal microdamage evident on the surface. When loaded radially and tangentially, small macrocracks formed parallel to each other on the loaded cube face in the longitudinal direction (Fig. 7d). Presumably these cracks formed due to the weak cement line boundaries which surround osteons and

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Fig. 7 Scanning electron microscopy images showing the different fracture behaviour in the test orientations. Under axial loading conditions (a, b, c), microcracks accumulated on the loaded surface a, while larger macrocracks formed parallel to the loading direction in the tangential orientation b and large

shear planes were observed in the radial direction c. Under radial and tangential loading (d, e, f), small macrocracks formed perpendicular to the applied force on the loaded surface d and also on the remaining non-axial plane f. In the axial direction, a distinct fracture plane at 45°–60° was observed e

which are preferentially aligned in this direction. The location of the fracture plane varied across samples, though often formed on the edge of the cube at the radial vertex. In the axial orientation, there was a distinct fracture plane at approximately 45°–60° through the cube face in each sample which is an indication of compression-shear failure (Fig. 7e). The major fracture plane was jagged indicating crack

deflection, with significant microcracking also present suggesting the increased plastic properties in this orientation may be due to the microcrack toughening phenomenon. On the remaining surface there was considerable crack formation perpendicular to the applied load (Fig. 7f). There was also some crack deflection seen where the crack path was diverted to weak planes parallel to its original path.

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Discussion The development of optimal processing and sterilization techniques for bone allograft has been the focus of extensive investigation due to the limitations posed by autograft and the inability of synthetic bone graft materials to replicate the complex architecture and hierarchical arrangement of natural bone. Supercritical carbon dioxide treatment has unique solvent, extraction and diffusive properties which can be used to delipidate bone (Fages et al. 1994) and potentially minimize the use of harmful chemicals in allograft processing. Moreover, as long as the mild supercritical conditions of SCCO2 (T [31.1 °C, p [ 73.8 bar) are maintained, the treatment parameters can be easily manipulated to facilitate terminal sterilization (Spilimbergo et al. 2003; Watanabe et al. 2003; Dillow et al. 1999). Studies have shown that viral and bacterial inactivation can be enhanced by further processing with additives such as hydrogen peroxide and ethanol (Fages et al. 1998; Nichols et al. 2009; Qiu et al. 2009; Shieh et al. 2009; White et al. 2006; Zhang et al. 2006; Hemmer et al. 2007), which also allows for shorter treatment times and milder conditions. However, before this technology can be adopted for allograft processing, its effect on the mechanical properties of the bone must be established. Currently, very little research has been conducted examining the effect of these treatments on the mechanical properties of bone allograft, raising concerns over the use of this technique for load bearing grafts. This study aimed to evaluate the effect of various common allograft processing additives used in conjunction with SCF treatments on the anisotropic mechanical properties of bone, and, in doing so, gain some insights into the expediency of these treatments for allograft processing. The experiments were performed using conditions which have been found to terminally sterilize a range of bacteria and viruses (Nichols et al. 2009), and using common processing solutes, thus giving more weight to the mechanical outcomes. The anisotropic mechanical properties of the control group in this study are consistent with those reported by Novitskaya et al. (2011). In their study, 5 9 5 9 7.5 mm bovine cortical specimens prepared from the femur were compressed to failure at 1 9 10-3 s-1 in the axial, radial and tangential orientations. Under these similar experimental conditions, they reported ultimate stress values of 160, 165

and 120 MPa in the axial, radial and tangential orientations, respectively; which compare favorably to the corresponding values of 160, 165 and 130 MPa reported in the present study. These results conflict with previous assertions in literature that the axial orientation is significantly stronger than the radial and tangential planes, which are transversely isotropic (Reilly and Burstein 1974). However, Novitskaya et al. (2011) attributed this to the presence of circumferential lamellae in the radial test samples. Unlike cylindrical osteons, these lamellae are organized into a smooth mineral-collagen sheath which surrounds the bone in the longitudinal direction. This arrangement imparts greater mechanical strength in compression than cylindrical osteons which are more easily deformed due to the central Haversian canal. The scanning electron microscopy results in this study support this assumption. When loaded axially, very little microdamage was observed in the radial plane compared to the tangential plane where significant microcracking was present in the direction of osteon orientation. Furthermore, the fracture behaviour in the radial orientation where the large planes in radial face sheared off is consistent with failure due to the presence of weak cement lines between longitudinally orientated circumferential lamellae. The stiffness properties of this study also compare well with the Novitskaya et al. (2011) study which reported that axial stiffness was approximately 30 % greater than the radial and tangential planes. Our results showed that axial stiffness was 50 % greater than radial and tangential stiffness. However, these small differences could be attributed to the larger cross-sectional area of the radial and tangential planes used in the Novitskaya et al. (2011) study (5 9 7.5 mm, compared to 5 9 5 mm) where there was more bone to resist deformation. The first SCF experiment (SCF Control: 160 bar for 4 h at 37 °C with a 45 min depressurization) acted as the control group for the SCF treatments in order to elucidate the effect of the supercritical conditions alone on the mechanical properties of the bone. The results showed that both the plastic and elastic mechanical properties of the bone were preserved in the axial orientation following treatment. This was consistent in the other orientations, except for elastic modulus which was increased by 75 % in the radial direction and 51 % in the tangential direction; though statistical significance was only reached in the radial

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orientation (p = 0.018). Despite these increases, the preservation of the other elastic and plastic properties suggests that bone is resilient to SCCO2 treatment. This is especially noteworthy as the pressure and time conditions used in this experiment are more severe than what has been reported in the literature when SCF treatment has been used to terminally sterilize a range of bacteria (White et al. 2006; Checinska et al. 2011; Shieh et al. 2009) and viruses (Qiu et al. 2009). While not directly comparable, the results of this study are in agreement with Fages et al. (1994) regarding the nontoxic nature of SCCO2 treatment to collagenous proteins. The maintenance of the post-yield properties following treatment intimates that the structure and function of bone collagen is preserved as it is responsible for the toughness of bone in the plastic region. The second group of SCF experiments aimed to examine the effect of using different processing solutes and chemical sterilants in conjunction with SCCO2 on bone loading behaviour. Different solutions of hydrogen peroxide, peracetic acid and ethanol were chosen as these have been shown to improve the sterilization efficacy of SCF treatments (Fages et al. 1998; Hemmer et al. 2007; Nichols et al. 2009; Qiu et al. 2009; Shieh et al. 2009; White et al. 2006; Zhang et al. 2006) and are commonly used to defat allograft bone (Fages et al. 1994; Mitton et al. 2005) and minimize bioburden prior to terminal sterilization. In the first two treatments (SCF H2O2 and SCF PAA), 2 mL of either hydrogen peroxide or peracetic acid was introduced into the pressure vessel and treated under the same supercritical conditions as the SCF Control group. Both hydrogen peroxide and peracetic acid are oxidizing chemicals with proven disinfectant properties. Moreover, hydrogen peroxide is required in a reaction with acetic acid to maintain the equilibrium of a solution of peracetic acid in water (Dul’neva and Moskvin 2005). Consequently, both compounds are relatively unstable in water and would have a similar reactivity with bone. DePaula et al. (2005) investigated the effect of hydrogen peroxide soaking as a part of an allograft cleaning process on the mechanical properties of human cortical bone. In their study, human cortical cylinders were failed in the axial and tangential planes following a cleaning process which included soaking in 3 % hydrogen peroxide solution for 1 h. They reported no significant change in either ultimate stress or yield stress in either

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orientation, though as the hydrogen peroxide soaking was a part of a cleaning process it is difficult to isolate the effect of this treatment alone. In another study (Mitton et al. 2005), the ultimate stress, elastic modulus and energy to failure of cancellous bone was shown to be preserved after a SCF extraction procedure at 50 °C and 260 bar, followed by hydrogen peroxide and ethanol soaking. While not directly comparable, the results of the current study support these findings with no significant change in either yield stress or ultimate stress following SCF H2O2 or SCF PAA treatment in any orientation. We also found that both treatments increased the elastic modulus and stiffness in all orientations, with the most significant effect observed in the axial plane. However, despite these increases, the post-yield properties were not significantly altered. These findings suggest that the oxidative characteristics by which these compounds achieve inactivation of bacteria do not adversely affect the properties or relationship between bone constituents, and thus the anisotropic properties are preserved. Ethanol is another additive which is routinely used to process allograft bone due to its ability to dissolve medullary lipids and thus minimize the antigenic potential of allograft bone. As a result, a number of studies have investigated its effect on the mechanical properties of bone (Linde and Sørensen 1993; Unger et al. 2010; Beaupied et al. 2006). In one study, Linde et al. (1993) explored the effect of soaking in 70 % ethanol for 1, 10 and 100 days on the compression properties of human cancellous bone. While their results were not significant, they reported a trend towards increasing stiffness and elastic energy as the time in ethanol increased; and this tendency was also reported by Beaupied et al. (2006). This behaviour was also observed in our study, with an increase in stiffness in all orientations, though these changes were not statistically significant. This stiffening effect is likely due to the ethanol induced dehydration of bone which causes it to become more brittle, and consequently reduces its toughness (Evans 1969; Turner and Burr 1993). A reduction in post-yield properties was reported by Unger et al. (2010) in their study which examined the three-point bending properties of bovine cortical bone following 2 h of soaking in 95 % ethanol. They found that there was a significant increase in ultimate stress, while ultimate strain and plastic energy were reduced. Our results support these decreases in energy and ultimate strain, while we

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found no significant change in ultimate stress in any orientation. While these comparisons are useful, it is difficult to accurately relate the two studies given the difference in mechanical testing modalities, and the use of SCCO2 treatment in the present study. However, the results indicate that ethanol soaking with or without SCF treatment dehydrates bone, translating to an increased stiffness and a reduction in post yield toughness. Additionally, the uniform reductions observed across all orientations would suggest that the underlying mechanisms are more likely to be an alteration in the interaction between constituents as opposed to changes to the constituents themselves. The SCF Novakill treatment in this study replicated the experimental conditions used by Nichols et al. (2009) that achieved terminal sterilization of bacteria which were inoculated onto tendon samples. In their study, they also tested human cortical bone struts in three-point bending following sterilization and showed there was no significant change in ultimate bending stress, elastic modulus or toughness compared to untreated controls. Similarly, we have previously reported that the mechanical properties of intact rabbit humeri are preserved in static three-point bending, four-point bending and torsion (Russell et al. 2013b), and in dynamic three-point bending fatigue (Russell et al. 2013a) following the same treatment. The results of the current study support these findings in compression with no significant change in any mechanical parameter in any orientation following our SCF Novakill treatment. Taken together, these results highlight the considerable expediency of this treatment for load bearing allograft, facilitating terminal sterilization of the graft whilst maintaining the functional integrity of the graft. Finally, in all SCF treatment groups there was a statistically significant 1–2 % reduction in apparent density. This 1–2 % decrease is most likely due to the removal of medullary lipids from the samples, given that cortical bone is composed of approximately 1.5 % lipids by weight (Pietrzak and Woodell-May 2005). Fages et al. (1994) demonstrated the ability of SCCO2 treatment to delipidate bone in their study on cancellous bone. The unique diffusivity properties of SCCO2 allow it to penetrate microporous materials like bone easily, where its liquid-like density and viscosity properties enable it to selectively extract solutes. This is a very attractive property for allograft bone processing as medullary lipids are antigenic when

implanted. Additionally, lipid extraction improves integration of donor bone into the host by enhancing osteoconduction and providing channels for neovascularization (Thoren et al. 1995; Frayssinet et al. 1998). There are several limitations which need to be considered when interpreting the results of this study. First of all, this study investigated the mechanical properties of bone in compression which are not truly representative of physiological loading. It is, however, pertinent in providing some understanding of the anisotropy of bone since it is unusual for any tissue to be subjected to purely tension, compression, or shear loading. Moreover, the effect of these treatments on anisotropy in other mechanical testing methods may vary considerably due to the different role of each constituent under different loading conditions. This is reflected in studies which have shown that the anisotropy of bone is less pronounced in compression compared to tension (Reilly and Burstein 1975). Another limitation is that this study only investigated quasi static loading of the bone. The loads typically experienced by bone are cyclic in nature, and therefore fatigue loading may have more accurately represented physiological loading. However, the consistency of treatment, samples and testing method means that conclusions drawn are still valid for these conditions. Finally, the mechanical testing in this study was performed on small cuboidal bovine bone samples in order to evaluate any changes to the anisotropic properties of the bone in relation to its structural organization. Future investigation using larger bone samples of human tissue would be the logical progression of this research, allowing more clinically relevant conclusions to be drawn about the suitability of SCCO2 treatment for allograft preparation.

Conclusions The use of SCCO2 treatments for allograft processing is an exciting new area of research. The unique solvent, extraction and diffusive properties of the treatment offer many potentially beneficial outcomes for improved allograft performance in vivo. This coupled with the ability to terminally sterilize tissue and maintain the mechanical properties of the bone, underlines the expediency of these SCCO2 for allograft preparation.

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Cell Tissue Bank Acknowledgments The authors would like to acknowledge funding support for this research was provided by a grant from the Australian Orthopaedic Association. Conflict of interest The authors declare there are no conflicts of interest in the preparation of this manuscript.

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The effect of supercritical carbon dioxide sterilization on the anisotropy of bovine cortical bone.

Bone allografts are used to replace bone that has been removed or to augment bone tissue in a number of clinical scenarios. In order to minimize the r...
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