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Article

Effects of SCCO2, Gamma Irradiation, and Sodium Dodecyl Sulfate Treatments on the Initial Properties of Tendon Allografts

by
Yikan Sun
,
Vedran Lovric
,
Tian Wang
,
Rema A. Oliver
and
William R. Walsh
*
Surgical and Orthopedic Research Laboratories, Prince of Wales Clinical School, Prince of Wales Hospital, University of New South Wales, Randwick, New South Wales 2031, Australia
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2020, 21(5), 1565; https://doi.org/10.3390/ijms21051565
Submission received: 28 November 2019 / Revised: 7 February 2020 / Accepted: 15 February 2020 / Published: 25 February 2020
(This article belongs to the Special Issue Tendon/Ligament Reconstruction by Tissue Engineering)
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Abstract

:
Sterile and decellularized allograft tendons are viable biomaterials used in reconstructive surgeries for dense connective tissue injuries. Established allograft processing techniques including gamma irradiation and sodium dodecyl sulfate (SDS) can affect tissue integrity. Supercritical carbon dioxide (SCCO2) represents a novel alternative that has the potential to decellularize and sterilize tendons with minimized exposure to denaturants, shortened treatment time, lack of toxic residues, and superior tissue penetration, and thus efficacy. This study attempted to develop a single-step hybrid decellularization and sterilization protocol for tendons that involved SCCO2 treatment with various chemical additives. The processed tendons were evaluated with mechanical testing, histology, scanning electron microscopy (SEM), and Fourier-transform infrared (FTIR) spectroscopy. Uniaxial mechanical testing showed that tendons treated with SCCO2 and additive NovaKillTM Gen2 and 0.1% SDS had significantly higher (p < 0.05) ultimate tensile stress (UTS) and Young’s modulus compared to gamma-irradiated and standard-SDS-treated tendons. This was corroborated by the ultrastructural intactness of SCCO2-treated tendons as examined by SEM and FTIR spectroscopy, which was not preserved in gamma-irradiated and standard SDS-treated tendons. However, complete decellularization was not achieved by the experimented SCCO2-SDS protocols used in this study. The present study therefore serves as a concrete starting point for development of an SCCO2-based combined sterilization and decellularization protocol for allograft tendons, where additive choice is to be optimized.

1. Introduction

Tendinous and ligamentous injuries are extremely common, especially among at-risk population subgroups such as military recruits, recreational and elite runners, and professional athletes, and contribute to much morbidity and functional loss [1,2,3,4]. The reported incidence rates of anterior cruciate ligament (ACL) injuries that require surgical reconstruction, for instance, are up to 0.52 cases per 1000 person-years in Australia and climb to as high as 1.52–37.62 cases per 1000 person-years for professional athletes [5]. Autogenous tendons are currently considered the gold standard for grafting of large defects [6]. However, they are associated with limited availability, prolonged surgical and rehabilitation time, donor site morbidity, and potential functional deficits [7,8,9,10,11,12]. Use of allografts, on the other hand, circumvents the above-mentioned drawbacks associated with autograft harvesting and implantation, but is discouraged by higher costs, the immunogenicity of the donor cellular materials [13], and most notably, the much-dreaded risk of disease transmission [14,15,16,17,18]. Consequently, in addition to routine donor screening and adherence to aseptic harvesting and handling techniques [19], allograft tendons are recommended to be terminally sterilized to achieve a sterility assurance level (SAL) of 10−6 [20], which denotes a 1 in 100,000 probability of any micro-organisms surviving the treatment [21,22,23].
Currently, gamma irradiation stands as the mainstay for musculoskeletal allograft sterilization [24,25,26], which inactivates pathogens by damaging the nucleic acids through direct energy deposition and generation of free radicals [27,28]. Nevertheless, it undermines the biomechanical and biological properties of allograft tendons in a dose-dependent manner [29,30,31,32], which is thought to be mediated by the irradiation-induced free radicals cross-linking collagen [33]. Consequently, controversy persists regarding whether the routinely used moderate-dose 25 kGy gamma irradiation to achieve acceptably low risk of transmission markedly compromises the initial mechanical properties of allograft tendons [31,34,35,36,37,38,39,40,41,42,43,44,45,46,47].
Contrarily, supercritical carbon dioxide (SCCO2), which has many applications in the food and pharmaceutical industries [48,49,50], emerges as a promising alternative that possesses many properties of an ideal sterilant for biomaterials. As CO2 enters its supercritical state at 31.1 °C and 1099 psi (Figure 1), its gas-like viscosity and diffusability allow it to readily permeate the tissue matrices, while its liquid-like density and solvation power [51,52,53] enable synergy with modifier solutions to improve sterilization efficacy [54,55,56,57]. The low critical temperature of CO2 makes it suitable for treating heat-sensitive biomaterials by not denaturing the protein content of the tissue [57,58,59,60]. SCCO2 can be easily withdrawn without toxic residues from treated biomaterials simply through depressurization and out-gassing [55,61]. Besides, CO2 is non-toxic, non-inflammable, and chemically inert, and thus does not constitute an environmental or occupational hazard [62,63]. Meanwhile, its natural abundance and recyclability guarantee the economic feasibility of industrial-scale CO2-based sterilization [49,64].
SCCO2 has been demonstrated to inactivate viruses [65,66,67], vegetative bacteria [57,62,68], and, working synergistically with additives such as peracetic acid (PAA) and hydrogen peroxide (H2O2), bacterial spores [69,70] to achieve SAL 10−6 [54,61], though the exact mechanism remains obscure [71]. Pioneering studies have found it to be a gentle agent that appears to preserve the mechanical properties of a variety of tissues such as heart valves [59], lungs matrices [72], amniotic membrane [73], bones [66,74,75,76,77], acellular dermal matrices [69], and menisci [78], while there is still a paucity of literature on tendons [40,46,79].
Donor cells and residual DNA have been shown to induce prolonged adverse host inflammatory reactions against allograft tendons in vivo that can lead to clinical inferiority to autografts [13]. Therefore, decellularization, the process of removing resident cells and nuclear material, has been increasingly recognized as a crucial step in the tissue engineering process to mitigate their antigenicity. Decellularization usually entails agitated wash in protein-denaturing detergents followed by prolonged washing with phosphate-buffered saline (PBS) or deionized water to unload toxic residues introduced [80]. It is proposed that SCCO2 may be equally, if not more, effective at removing chemical residues while drastically expediating the washing process owing to its solvent properties, diffusibility, and ease of withdrawal from tissue, as described above. Indeed, Casali et al. [81] have recently successfully purged cytotoxic detergent from porcine aortas using SCCO2 following solution-based decellularization treatment, shortening the final cleansing step to merely one hour, which typically lasts more than 24 h with saline wash. Additionally, similar to how it drastically improves the sterilization efficacy of PAA while minimizing the chemical concentration required [54], SCCO2 has the potential to directly synergize with additive decellularizing detergents such as sodium dodecyl sulfate (SDS) to remove the immunogenic cellular materials, but to our knowledge, such a possibility has not yet been extensively explored by published studies.
SCCO2 can potentially engender a single-step protocol with shortened treatment time that produces sterile and decellularized tendon allografts without compromising their biomechanical properties. The aims of the present study was therefore to (a) test if the established SCCO2 sterilization protocol preserves the initial biomechanical properties of tendons; (b) histologically assess if SCCO2 is able to preserve the morphology of, and decellularize tendons with appropriate additive chemicals; and, in addition, (c) microscopically and molecularly characterize tendons undergoing different treatments through field-emission scanning electron microscopy (FE-SEM) and Fourier-transform infrared (FTIR) spectroscopy, respectively. It was hypothesized that SCCO2 treatment would not affect the initial biomechanical properties, ultrastructure, and chemical signatures of tendon grafts and would be able to decellularize tendons with appropriate additives.
A total of 6 groups were proposed for the study: (I) Untreated control; (II) Moderate-dose gamma irradiation (~25 kGy); (III) Standard SDS [82]; (IV) SCCO2 treatment with the sterilising NovaKillTM Gen2 additive (SCCO2-NK); (V) SCCO2 with NovaKillTM Gen2 and 0.1% SDS (SCCO2-0.1%-SDS); and (VI) SCCO2 with NovaKillTM Gen2 and 1% SDS (SCCO2-1%-SDS).

2. Results

2.1. Gross Appearance and Dimensions

Compared to native tendons, gamma-irradiated tendons became shriveled and their surface granular; the SCCO2-NK-treated tendons became noticeably cleaner with effacement of the surface marks made on them prior to treatment; and SCCO2-SDS-treated tendons, especially the SCCO2-0.1%-SDS treated ones, turned out to be visibly dehydrated, to the extent that they were appreciably transparent, and only regained the typical white and glossy texture of tendons after immersing in PBS for at least 15 min. No perceptible change in the outward appearance was observed for the standard-SDS-treated tendons.
The mean pre- and post-treatment cross-sectional area (CSA) and thickness of the standard SDS group were significantly greater than those of the control group (p < 0.05), while the mean post-treatment CSA and thickness of SCCO2-0.1%-SDS group were significantly smaller than those of the Standard SDS group (p < 0.05). No significant differences were found between the pre- and post-treatment dimensions of tendons in all groups except the SCCO2-0.1%-SDS group, where there was a statistically significant decrease in CSA (p = 0.020) and thickness (p = 0.008), which was in concordance with their dehydrated appearance post-treatment (Table 1).

2.2. Mechanical Testing

In general, the mechanical properties of all treatment groups apart from SCCO2-1%-SDS were greater than the control group. This may reflect a potential drying artefact that may have accounted for this increase. All tendons failed at the mid-substance and were included in the statistical analysis. SCCO2-0.1%-SDS-treated tendons were found to be significantly stiffer than the native tendons (p = 0.014) and the standard-SDS-treated tendons (p = 0.003) (Table 2a). The mean ultimate tensile stress (UTS) of the standard SDS group was significantly lower than that of the gamma group (p = 0.006), and reduced compared to that of the control, although statistical significance was not reached (p = 0.062); whereas the mean UTS of the SCCO2-0.1%-SDS group was significantly greater than those of the control (p = 0.011), gamma (p = 0.011), and standard SDS (p < 0.001) groups. The mean failure strain of the SCCO2-0.1%-SDS was significantly lower than that of the standard SDS group (p = 0.035). The mean Young’s modulus of the SCCO2-0.1%-SDS group was significantly higher than those of the control (p < 0.001), gamma (p < 0.001), standard SDS (p < 0.001), and SCCO2-NK (p = 0.019) groups, while the mean Young’s modulus of the SCCO2-NK group was also significantly greater than that of the standard SDS group (p = 0.003).
However, with a 10-fold increase in concentration of additive SDS from 0.1% to 1%, the SCCO2-1%-SDS-treated group had significantly decreased stiffness than the SCCO2-0.1%-SDS group (p = 0.004), significantly decreased UTS than the control (p < 0.001), SCCO2-NK (p = 0.001), gamma (p < 0.001), and SCCO2-0.1%-SDS (p < 0.001) groups, and significantly reduced Young’s modulus than the SCCO2-NK (p = 0.033) and SCCO2-0.1%-SDS (p < 0.001) groups (Table 2b).
Notably, there were no significant differences detected between the control and gamma irradiation groups in regards to the mechanical endpoints analyzed.

2.3. Histomorphology

The morphology of the extracellular matrix (ECM) was examined and cellularity assessed (Figure 2).
Gamma irradiation and standard SDS treatment both inflicted substantial damage to the ECM of tendons (images c, d, e, and f, Figure 2), as evidenced by perceptible differences in histomorphological appearances in comparison to control (images a and b, Figure 2). Specifically, irregularly-shaped dead space devoid of pink-staining ECM and separation of the collagen fibers were notable in the transverse and longitudinal sections of the gamma-irradiated tendons (images c and d, Figure 2). The increased gaps between collagen fibers in the longitudinal sections were also observed in the standard SDS group (image f, Figure 2), albeit to a lesser extent. These findings on microscopic observation were in keeping with the quantitative results, where there was a significantly increased percentage of void (area not occupied by ECM) in gamma-irradiated (41.81 ± 4.50%) and standard-SDS-treated (35.21 ± 4.48%) tendons than in native tendons (29.08 ± 4.96%) (p < 0.001 and p = 0.015, respectively) (Table 3).
Conversely, no remarkable morphological changes of collagen fibers were noted for the SCCO2-NK or SCCO2-0.1%-SDS-treated specimens (images g, h, I, and j, Figure 2), as evidenced by the quantitative results that SCCO2-NK-treated tendons have significantly less percentage of void (27.61 ± 6.49%) compared to gamma-irradiated (41.81 ± 4.50%) and standard-SDS-treated (35.21 ± 4.48%) ones (p < 0.001 and p = 0.001, respectively), and so do SCCO2-0.1%-SDS-treated tendons (27.35 ± 7.09%) (p < 0.001 and p = 0.001 when comparisons made to gamma-irradiated and standard-SDS-treated tendons, respectively). Interestingly however, the spaces between the collagen fibers as observed in the transverse and longitudinal sections of the native tendons appeared to be nearly obliterated by the SCCO2-1%-SDS treatment (images k and l, Figure 2), which was corroborated by drastically decreased empty space on quantitative analysis (7.48 ± 5.18%) (Table 3).
Only the standard SDS treatment achieved thorough decellularization with near absence of distinguishable cell nuclei histologically (3.17 cells/mm2). The cellularity of SCCO2-0.1%-SDS-treated tendons (682.32 ± 126.42 cells/mm2) was significantly lower than that of the control tendons (818.91 ± 116.62 cells/mm2) (p = 0.021), but this reduction is trivial when compared to the absolute number of cells left within the tendons (Table 3).

2.4. Scanning Electron Microscopy

Tendon surfaces and the collagen triple helices running in the longitudinal direction were observed under high magnification (Figure 3).
Under approximately 10,000× magnification, the surfaces of SCCO2-NK-, SCCO2-0.1%-SDS-, and SCCO2-1%-SDS-treated tendons (images h, j and l, respectively, Figure 3) resembled that of the native tendons (image b, Figure 3), whereas gamma-irradiated and standard-SDS-treated tendons (images d and f, respectively, Figure 3) exhibited numerous fissures on their surface, exposing the collagen fibers underneath.
Under ultrahigh (~100,000×) magnification, disruption to the collagen banding pattern was appreciable in all treated tendons, especially the standard-SDS- and SCCO2-1%-SDS-treated ones (images c and k, respectively, Figure 3). Specifically, for SCCO2-1%-SDS treated tendons, the surface of the collagen fibers appeared granular and occasional breaks in fibers were readily identifiable. Meanwhile, standard SDS treatment caused destructive changes to the characteristic collagen triple helices, where innumerable bulges annihilated the smooth cylindrical contour seen in the pristine fibers of control tendons (image a, Figure 3).

2.5. Fourier-Transform Infra-Red Spectroscopy

The FTIR spectra of short tendon segments yielded valuable insights into their spatial molecular composition (Figure 4). No significant shifts or absolute reductions in absorption peaks, including primarily the amide I (1634 cm−1), II (1548 cm−1), and III (1250 cm−1) peaks associated with collagen [83,84], were detected in the SCCO2-NK- and SCCO2-0.1%-SDS-treated samples, indicating no or minimal damage to the collagen contents. Also, the shapes of the spectra of native, SCCO2-NK- and SCCO2-0.1%-SDS-treated tendons between 1125 and 970 cm−1 are strikingly similar, implying preservation of various other ECM components such as proteoglycans [85].
Conversely, standard SDS and gamma irradiation appeared to have obliterated the peaks at around (a) 2920 cm−1 and 2850 cm−1, which correspond to, respectively, the asymmetric and symmetric stretching of the collagen-associated CH2 group; and (b) 1742 cm−1, which is associated with the carbonyl group [86]. SCCO2-1%-SDS treatment provoked a similar pattern of absorption peak changes.

3. Discussion

Decellularization and terminal sterilization are crucial steps in soft tissue allograft manufacturing. In principle, any viable constituents should be removed, or at least deactivated, in the end-product, and the ECM ideally conserved in its intact three-dimensional microarchitecture, which contains naturally occurring collagen, and is responsible for most of its mechanical robustness [87,88]. Tissues are processed in this manner so that the risk of disease transmission is minimized with the removal of pathogens, adverse host response avoided in the absence of cellular materials, and graft incorporation promoted owing to the capability of intact ECM to induce healing and remodeling [80]. However, concerns have been raised regarding the harmful effect of the currently used physical and chemical processing techniques on the biomechanical properties on allograft tendons, which might have been implicated in their suboptimal clinical performance and propensity to fail.
Our results suggest that the SCCO2-based NovaKill sterilization protocol, even with the addition of 0.1% SDS to the antimicrobial cocktail, was able to preserve the mechanical integrity of the tendons, as they showed similar if not greater level of UTS and Young’s modulus compared to the untreated tendons. This finding largely coincides with the results of the few previously published studies on SCCO2-treated tendons and collagenous soft tissues, where they were consistently found to have comparable or increased UTS to the untreated tendons [40,46,59,79]. It should also be noted that SCCO2-0.1%-SDS-treated tendons were biomechanically superior to the standard-SDS-treated tendons with respect to UTS and Young’s modulus, even though the standard SDS protocol re-performed in the present study also employed the same concentration of SDS (0.1%) and was the least destructive one to accomplish decellularization among the trialed protocols in the original study conducted by Pridgen et al. [82]. Admittedly, although it is capable of lysing cell and nuclear membranes, SDS along with some other commonly used decellularizing chemicals, such as tri(n-butyl)phosphate (TnBP) and Triton X-100, is notoriously harmful to the components of ECM [89]. This is where SCCO2 may be able to facilitate infiltration of and potentially synergize with modifier solutions with minimized concentration required to precipitate the same level of decellularization and sterilization efficacy with much less exposure time to such denaturants [54], thereby preserving the ultrastructure and thus the biomechanical properties to the greatest extent.
While SCCO2 without excessive additives generally retains the biomechanical integrity of soft tissues as assessed from most endpoints, the effect of SCCO2 on the stiffness and Young’s modulus of tissues remains indeterminate. Whilst the present study showed a statistically significant increase in the SCCO2-0.1%-SDS group compared to control, Baldini et al. [46], Mazyar et al. [79], and Antons et al. [90] all found reduced stiffness or elastic modulus in tendons treated with SCCO2, although the statistically significant finding in the first study might have been partly due to the smaller mean CSA of the tendons allocated to the SCCO2 group. On the other hand, Balestrini et al. [72] observed significantly increased elastic modulus in SCCO2-treated lung matrices, similar to what we discovered in tendons in this study. Cross-linking of ECM induced by high pressure was proposed as the underlying mechanism, but is confronted by the negative differential scanning calorimetry (DSC) results reported by Hennessey et al. [59] in SCCO2-treated porcine aortic valves, even though it was admitted that transient cross-linking could have possibly happened and went undetected by the analyses. The effect of SCCO2 on the stiffness of soft tissues is therefore controversial and warrants further investigation.
Moreover, the present study found that gamma-irradiated tendons at 25 kGy showed similar mechanical properties to those of the control tendons. This agrees well with the report of Balsly et al. [39], where patella tendon grafts were tested at 18.3–21.8 kGy as well as 24–28.5 kGy, while others report damage to the collagen and deleterious effects on the mechanical properties [31,35,37,41,42,43,44,47], especially if gamma irradiation treatment is conducted under room temperature instead of with dry ice [36,91]. This highlights the ongoing debate regarding whether the conventional 25 kGy gamma irradiation used for terminal sterilization significantly undermines the in vitro mechanical properties of musculoskeletal allografts [31,35,36,37,38,39,40,41,42,43,44,45,46,47].
The preservation of biomechanical properties by SCCO2 was corroborated by the non-mechanical characterization of SCCO2-treated samples other than the SCCO2-1%-SDS group in the present study, reiterating the gentle character of SCCO2 found by many pilot studies [40,46,59,69,72,73,74,75,76,77,78,79]. Previously, Antons et al. [90] also resorted to SEM for ultrastructural examination and found that the orientation, size, and density of collagen fibers in SCCO2-treated tendons and skins were generally comparable to those of the native tendons. Dillow et al. [57] have found that SCCO2 treatment did not alter the characteristic IR peaks of poly(lactic-co-glycolic) acid, a type of thermally and hydrolytically labile biomaterial analogous to collagenous tissues. On the contrary, both gamma-irradiated and standard-SDS treated tendons sustained disruptions to their ultrastructure and molecular composition to certain extents, as evidenced by alterations in multiple FTIR absorption peaks associated with collagen. Interestingly, Tilley et al. [86] detected a very similar pattern of changes in collagenous scaffolds dissolved in pH 3.3 aqueous solution regarding the reduction in peaks associated with CH2 and carbonyl groups, which were attributed to hydroxylation of these groups. Moreover, the present study’s findings of statistically significant increased area of void and separation of collagen fibers histologically in gamma-irradiated specimens echo with the findings of Irani et al. [79] and Bui et al. [78], reaffirming the destructive nature of ionizing irradiation to ECM [32].
The SCCO2-SDS hybrid decellularization protocols experimented in this study were formulated with the assumption that SCCO2 would demonstrate a comparable degree of synergy with SDS as with PAA [54]. Additionally, PAA has been reported to enhance detergent penetration into tissue [92,93], and while being a potent sterilant, is included in a number of decellularization protocols [80], which made the outlook of combining SDS and PAA-containing NovaKillTM Gen2 for SCCO2-facilitated decellularization even more appealing. Nevertheless, even if the biomechanical properties remained intact, the SCCO2-0.1%-SDS protocol was not able to thoroughly decellularize tendons under the conditions utilized in this study (i.e., 2 h, 39 °C and 102 bar without agitation by stirrer). Although the reduction in cellularity in SCCO2-0.1%-SDS-treated tendons was found to be statistically significant, it is dwarfed by the sheer number of cells that remained in the treated tendons. We initially believed that this ineffective decellularization could be due to the very low concentration and volume of the SDS modifier solution used. It was previously found that 2 mL of NovaKillTM Gen2 (14.1% PAA) added to the SCCO2 treatment system only translated into 0.018% effluent PAA concentration, which was 10 times lower than the typical level of PAA tissues are exposed to in conventional treatment [72]. Thus, although a quantitative measure was not used in the present study, the concentration of SDS specimens that were exposed to the SCCO2-0.1%-SDS protocol of this study was likely to be low, for the SEM and FTIR changes in the standard-SDS-treated tendons were not manifested in the SCCO2-0.1%-SDS-treated ones. Consequently, we hypothesized that a higher concentration of additives or changes to the treatment conditions such as longer treatment time and higher pressure could potentially result in better decellularization outcomes. However, a 10-fold increase in SDS concentration implemented in the SCCO2-1%-SDS protocol did not achieve any decrease in cell count while the mechanical integrity and microscopic features of the tendons were demolished, presumably suggesting overexposure to SDS. Therefore, we propose that the use of CO2-philic decellularizing agents are likely to be imperative for effective decellularization [94]. Indeed, CO2 is non-polar and requires a compatible cosolvent to remove polar molecules such as phospholipids (abundant in cell membranes) and DNA, and Antons et al. [90] did successfully reduce DNA content of the tendons by combining SCCO2 with Dehypon® LS-54, a common household and industrial surfactant, without affecting the mechanical integrity or biocompatibility.
Furthermore, dehydration of SCCO2-treated tissues observed in this study, as evidenced by the statistically significant decrease in CSA of SCCO2-0.1%-SDS-treated tendons, was also reported by Sawada et al. [95], where they successfully extracted cell nuclei from tissue with SCCO2-entrained ethanol. Casali et al. [81] were subsequently able to overcome this problem by pre-saturating SCCO2 with water.
The present study does suffer from many limitations. The choice of ovine hindlimb digital tendons ensured the availability as each sheep was able to yield a total of 4 viable tendons for mechanical testing. However, the discrepancy in dimensions (Table 1) rendered failure load and stiffness data less interpretable, making a direct comparison of these results with other studies’ inappropriate. Meanwhile, the apparent decrease in CSA due to dehydration might have caused an overestimation of the Young’s modulus and the UTS of the SCCO2-0.1%-SDS-treated tendons [81]. Also, the present study primarily focused on failure testing, whereas cyclic testing and suture pull-out strength testing might have better depicted the mode of loading tendons that are subject to in vivo and therefore would have been more clinically relevant [45,96]. Furthermore, sterility verification of the treated samples was not performed in the present study, although the samples were treated using the identical methods (i.e., SCCO2 conditions and additives) that have been proven to achieve SAL 10−6 in a previous study [54]. It is unlikely, but still possible, that SDS added to the treatment concoction might interact with NovaKillTM Gen2 and downgrade its sterilization efficacy, making it necessary to have two separate treatment steps. As for the assessment of decellularization thoroughness, Crapo et al. [89] proposed an acclaimed three-item standard that entails quantitative measurements of DNA content and fragment length, which however, were not incorporated into the present study.

4. Materials and Methods

4.1. Sample Procurement and Preparation

120 extensor digital tendons were harvested from the hindlimbs of 20 skeletally mature sheep aged more than 2 years immediately following euthanasia for other ethically approved studies conducted at our institution. Harvested tendons were soaked in PBS and frozen at −60 °C until further treatment and/or testing. All tendons were stored for a maximum of 6 months and underwent no more than 3 freeze–thaw cycles to minimize the effect of repetitive freeze–thaw cycles on their mechanical strength [97,98].

4.2. Study Design

6 groups were proposed as previously discussed in the introduction section. Each group was randomly allocated with 16 full-length tendons (approximately 10 mm in length) for mechanical testing and 4-cm tendon segments produced from 4 full-length tendons for non-mechanical assessments (Figure 5). Samples underwent different treatments accordingly, and if not otherwise specified, were soaked in PBS, and either frozen until mechanical testing or fixed in 10% neutral buffered formalin to be prepared for histology, FE-SEM, and FTIR spectroscopy.

4.3. Gamma Irradiation Treatment

Samples were packaged in Tyvek sterilization pouches (Livingstone International, Mascot, Australia), sealed in a Styrofoam box filled with dry ice to maintain a temperature below −20 °C throughout treatment, and then transferred to an external treatment facility, where they were irradiated with a cobalt60 source at the dose of 25 kGy under standardized operating procedures (Steritech, Wetherill Park, Australia). Treated tendons were then returned to the −60 °C freezer, or thawed and fixed in formalin.

4.4. Standard SDS Decellularisation

The standard SDS treatment of the present study was strictly reproduced from the protocol of Pridgen et al. [82]. Briefly, samples were thawed and washed at room temperature on an orbital shaker at 20 rpm with 0.1% (w/v) ethylenediaminetetracetic acid (EDTA) (Sigma-Aldrich, St. Louis, MO, USA) in de-ionized water (Merck Millipore, Burlington, MA, USA) for 4 h with a single change of solution at the 2-h mark. The pre-treated samples were then washed in 0.1% (w/v) SDS (Sigma-Aldrich, St. Louis, MO, USA) prepared in 0.1% (w/v) EDTA for 24 h with a single change at 12 h, followed by PBS wash for 1 h with a single change at 30 min.

4.5. SCCO2-Based Treatment Protocols

An in-house custom-built supercritical fluid system was used to carry out SCCO2 treatment protocols (Figure 6) [76]. A cellulose pad secured with a stainless-steel basket at one end of the 660-mL pressure vessel was soaked in and hence served as the vehicle for additive chemicals. Group IV (SCCO2-NK) tendons were thawed, packaged into Tyvek pouches, loaded into the pressure vessel, and treated with SCCO2 and 3.2 mL per 30 tendons of NovaKillTM Gen2 (~14.1% PAA and ~4.9% H2O2) (NovaSterilis, Lansing, NY, USA) under 39 °C and 102 bar to mirror the established SCCO2 sterilization protocol that has been proven to achieve SAL 10−6 [54]. The treatment vessel was slowly depressurized over 30 min, and the tendons retrieved and either frozen or formalin-fixed until further analysis
Group V (SCCO2-0.1%-SDS) and group VI (SCCO2-1%-SDS) tendons first received the above-described [82] 4-h pre-treatment with EDTA, followed by 2 h of SCCO2 treatment under the identical conditions as group IV tendons. Also, in addition to NovaKillTM Gen2, the additive chemical cocktail was reinforced with 0.8 mL per gram of tissue wet mass of the 0.1% SDS and 0.1% ETDA solution for group V, and 1% SDS and 0.1% ETDA for group VI, with an attempt to decellularize.

4.6. Mechanical Testing

The pre- and post-treatment width (w) and thickness (t) of the tendons were measured with a digital caliper at the center of the mid-substance section intended to be tested and the two points 10 mm away towards each end (Figure 7). The cross-sectional area (CSA) was assumed elliptical and calculated with the formula CSA = π × t × w/4 [42]. The mean CSA of the tested section is then estimated by averaging the calculated CSA at the three equidistant points. Uniaxial tensile testing was conducted by the MTS 858 Mini Bionix® axial servohydraulic tabletop test system (MTS, Eden Prairie, MN, USA) with a 1 kN load cell. Thawed tendons were rigidly griped at both ends by cryo-clamps with interlocking rectangular teeth. The gauge length was set at 25 mm. Testing commenced after a freeze-line was observed at the gripping edge of each clamp, and tendons taut with a 2 N pre-load. The testing protocol was programmed in and executed by the MTS TestSuiteTM Multipurpose Elite Software (MTS, Eden Prairie, MN, USA), with force and displacement data collected at 10 Hz. Each tendon was subjected to 10 pretensioned cycles between 10 and 20 N at 1 Hz, stretched to and held at 8% strain for 10 s, ramped back to 0% strain to fully relax, and then ramped at a rate of 100% strain/min to failure, which was defined as a plummet in force profile succeeding the maximum force reached. Testing data were processed by an in-house MATLAB (Mathworks, Natick, MA, USA) script, where the force-displacement and stress-strain graphs were plotted to derive both the mechanical properties, namely failure load and stiffness, and the CSA-normalized material properties, namely ultimate tensile stress (UTS), maximum strain, and Young’s modulus, of the tested tendons for data analysis.

4.7. Histology

Three transverse and two longitudinal sections were obtained along the length of each treated and formalin-fixed 4 cm tendon segment (Figure 8), which were automatically processed with Shandon™ Excelsior™ ES tissue processor (Thermo Fisher Scientific, Kalamazoo, MI, USA) and embedded in paraffin wax with standard procedures. Five-micron slices were then obtained with a hand-operated Leica RM 2165 Microtome (Leica Instrument GmbH, Nussloch, Germany) from each section and stained with haematoxylin and eosin (H&E). An Olympus BX43 microscope was employed in conjunction with a DP72 digital camera (Olympus, Shinjuku, Japan) to thoroughly examine the histological appearance of tendons at both low and high magnifications. The cellularity and percentage of empty space (void) within each snapshot were quantitatively determined with an in-house MATLAB (Mathworks, Natick, MA, USA) script. For each group, the average results of a total of 18 images taken under 400× magnification at random locations of transverse H&E-stained cross-sections (2 per cross-section, 3 cross-sections per tendon segment, and 3 tendon segments per group) were deemed representative of the overall picture.

4.8. Scanning Electron Microscopy

Formalin-fixed samples were decanted in deionized water for 15 min, dehydrated with increasing grades of ethanol, critical-point-dried with an Autosamdri-815 Critical Point Dryer (Tousimis, Rockville, MD, USA), mounted onto 12.5-mm sample stubs, sputter-coated with platinum (40 mA, 2 × 2.5 min) using an Emitech K575X Sputter Coater (Quorum Technologies, Lewes, UK), and then examined under the FEI Nova NanoSEM 230 field-emission scanning electron microscope (Thermo Fisher Scientific, Waltham, MA, USA). Snapshots of the longitudinal collagen fibres and tendon surfaces were obtained from 100× to 120,000× magnifications.

4.9. Fourier-Transform Infrared Spectroscopy

Following critical point drying, samples from each group were analyzed with a Spectrum Two FT-IR Spectrometer (PerkinElmer, Waltham, MA, USA). The attenuated total reflectance (ATR) mode was used to compensate for the discrepancy in the thickness of the samples. For each specimen, a constant pressure was applied to obtain a consistent absolute absorbance and the spectra were generated from 4000 to 400 cm−1 based on 64 background-subtracted scans, and then ATR corrected and smoothed with the PerkinElmer Spectrum software for analysis.

4.10. Statistical Analysis

Statistical analysis was performed using IBM SPSS statistics 25 (SPSS Inc., Chicago, IL, USA) for all quantitative endpoints using a one-way analysis of variance (ANOVA) if not otherwise specified. A statistically significant effect in ANOVA was followed by a Tukey’s post hoc test if the homogeneity of variances assumption was met, or if not, Games–Howell post hoc test. A p value of less than 0.05 is considered statistically significant. All figures are reported as mean ± standard deviation.

5. Conclusions

Development and modification of new allograft tendon and tissue processing protocols, in general, follow a multi-step progression. SCCO2 represents an exceptionally promising but under-investigated agent, and the present study has demonstrated preservation of mechanical properties of SCCO2-treated tendons. The next advancement would be to identify the optimal CO2-philic detergents that complement SCCO2, and the treatment parameters to actualize sterilization and decellularization with minimal concentration and protocol run-time. The in vivo biocompatibility of SCCO2-treated tendons should then be studied as biomechanical integrity is only one of the many determinants for clinical success. Host cell infiltration and repopulation of implanted tendon allografts, for example, are indispensable for graft incorporation and maintenance against progressive attenuation of mechanical strength with everyday use [99,100]. The capacity of tendon allografts to promote and accommodate cellular ingrowth is affected by treatment and usually limited due to the compact nature of the collagen structure [101,102]. SCCO2 has the potential of creating porous scaffolds that are advantageous to cellular attachment [78,103,104], and validating such a possibility is certainly a worthwhile undertaking for future in vivo studies.

Author Contributions

Conceptualization, W.R.W., V.L., and Y.S.; methodology, V.L. and Y.S.; software, T.W.; validation, T.W., V.L., and W.R.W.; formal analysis, Y.S.; investigation, Y.S. and T.W. and R.A.O.; resources, W.R.W.; data curation, Y.S.; writing—original draft preparation, Y.S.; writing—review and editing, V.L.; visualization, Y.S.; supervision, V.L.; project administration, W.R.W.; funding acquisition, n/a. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Acknowledgments

The authors would like to thank Yin Yao and Isabelle Morrow at the Mark Wainwright Analytical Centre for training on sample preparation for and operation of the SEM; and Hayley Franklin for cutting and staining the histology samples.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

ANOVAAnalysis of variance
ATRAttenuated total reflectance
CSACross-sectional area
ECMExtracellular matrix
EDTAEthylenediaminetetracetic acid
(FE-)SEM(Field-emission) scanning electron microscopy
FTIRFourier-transform infrared (spectroscopy)
H&EHaematoxylin and eosin
PAAPeracetic acid
PBSPhosphate-buffered saline
SALSterility assurance level
SCCO2Supercritical carbon dioxide
SDSSodium dodecyl sulfate
UTSUltimate tensile stress

References

  1. Clayton, R.A.; Court-Brown, C.M. The epidemiology of musculoskeletal tendinous and ligamentous injuries. Injury 2008, 39, 1338–1344. [Google Scholar] [CrossRef] [PubMed]
  2. Hootman, J.M.; Macera, C.A.; Ainsworth, B.E.; Addy, C.L.; Martin, M.; Blair, S.N. Epidemiology of musculoskeletal injuries among sedentary and physically active adults. Med. Sci. Sports Exerc. 2002, 34, 838–844. [Google Scholar] [CrossRef]
  3. Leanderson, C.; Leanderson, J.; Wykman, A.; Strender, L.-E.; Johansson, S.-E.; Sundquist, K. Musculoskeletal injuries in young ballet dancers. Knee Surg. Sports Traumatol. Arthrosc. 2011, 19, 1531–1535. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Gianotti, S.M.; Marshall, S.W.; Hume, P.A.; Bunt, L. Incidence of anterior cruciate ligament injury and other knee ligament injuries: A national population-based study. J. Sci. Med. Sport 2009, 12, 622–627. [Google Scholar] [CrossRef] [PubMed]
  5. Moses, B.; Orchard, J.; Orchard, J. Systematic review: Annual incidence of ACL injury and surgery in various populations. Res. Sports Med. 2012, 20, 157–179. [Google Scholar] [CrossRef] [PubMed]
  6. Robertson, A.; Nutton, R.W.; Keating, J.F. Current trends in the use of tendon allografts in orthopaedic surgery. J. Bone Jt. Surg. Br. Vol. 2006, 88, 988–992. [Google Scholar] [CrossRef] [Green Version]
  7. Engelman, G.H.; Carry, P.M.; Hitt, K.G.; Polousky, J.D.; Vidal, A.F. Comparison of Allograft Versus Autograft Anterior Cruciate Ligament Reconstruction Graft Survival in an Active Adolescent Cohort. Am. J. Sports Med. 2014, 42, 2311–2318. [Google Scholar] [CrossRef]
  8. Cole, D.W.; Ginn, T.A.; Chen, G.J.; Smith, B.P.; Curl, W.W.; Martin, D.F.; Poehling, G.G. Cost Comparison of Anterior Cruciate Ligament Reconstruction: Autograft Versus Allograft. Arthrosc. J. Arthrosc. Relat. Surg. 2005, 21, 786–790. [Google Scholar] [CrossRef]
  9. Jost, P.W.; Dy, C.J.; Robertson, C.M.; Kelly, A.M. Allograft Use in Anterior Cruciate Ligament Reconstruction. HSS J. 2011, 7, 251–256. [Google Scholar] [CrossRef] [Green Version]
  10. Bottoni, C.R.; Smith, E.L.; Shaha, J.; Shaha, S.S.; Raybin, S.G.; Tokish, J.M.; Rowles, D.J. Autograft Versus Allograft Anterior Cruciate Ligament Reconstruction: A Prospective, Randomized Clinical Study With a Minimum 10-Year Follow-up. Am. J. Sports Med. 2015, 43, 2501–2509. [Google Scholar] [CrossRef]
  11. Paschos, N.; Vasiliadis, H.S. Allograft versus autograft for reconstruction of anterior cruciate ligament rupture in adults. Cochrane Database Syst. Rev. 2013. [Google Scholar] [CrossRef]
  12. Wei, J.; Yang, H.B.; Qin, J.B.; Yang, T.B. A meta-analysis of anterior cruciate ligament reconstruction with autograft compared with nonirradiated allograft. Knee 2015, 22, 372–379. [Google Scholar] [CrossRef] [PubMed]
  13. Keane, T.J.; Londono, R.; Turner, N.J.; Badylak, S.F. Consequences of ineffective decellularization of biologic scaffolds on the host response. Biomaterials 2012, 33, 1771–1781. [Google Scholar] [CrossRef] [PubMed]
  14. Ireland, L.; Spelman, D. Bacterial Contamination of Tissue Allografts – Experiences of the Donor Tissue Bank of Victoria. Cell Tissue Bank. 2005, 6, 181–189. [Google Scholar] [CrossRef] [PubMed]
  15. Conrad, E.U.; Gretch, D.R.; Obermeyer, K.R.; Moogk, M.S.; Sayers, M.; Wilson, J.J.; Strong, D.M. Transmission of the hepatitis-C virus by tissue transplantation. J. Bone Jt. Surg. Am. 1995, 77, 214–224. [Google Scholar] [CrossRef] [PubMed]
  16. Sanzén, L.; Carlsson, Å. Transmission of human T-cell lymphotrophic virus type 1 by a deep-frozen bone allograft. Acta Orthop. Scand. 1997, 68, 72–74. [Google Scholar] [CrossRef]
  17. Centers for Disease Control and Prevention. Update: Unexplained deaths following knee surgery—Minnesota, 2001. MMWR Morb. Mortal. Wkly. Rep. 2001, 50, 1080. [Google Scholar]
  18. Centers for Disease Control and Prevention. Update: Allograft-Associated Bacterial Infections―United States, 2002. Morb. Mortal. Wkly. Rep. 2002, 51, 207–210. [Google Scholar]
  19. Fishman, J.A.; Greenwald, M.A.; Grossi, P.A. Transmission of Infection With Human Allografts: Essential Considerations in Donor Screening. Clin. Infect. Dis. 2012, 55, 720–727. [Google Scholar] [CrossRef] [Green Version]
  20. Bettin, D.; Harms, C.; Polster, J.; Niemeyer, T. High incidence of pathogenic microorganisms in bone allografts explanted in the morgue. Acta Orthop. Scand. 1998, 69, 311–314. [Google Scholar] [CrossRef]
  21. Lambert, B.J.; Mendelson, T.A.; Craven, M.D. Irradiation and ethylene oxide terminal sterilization experiences with drug eluting stent products. AAPS PharmSciTech 2011, 12, 1116–1126. [Google Scholar] [CrossRef] [PubMed]
  22. Brubaker, S.; Lotherington, K.; Zhao, J.; Hamilton, B.; Rockl, G.; Duong, A.; Garibaldi, A.; Simunovic, N.; Alsop, D.; Dao, D.; et al. Tissue recovery practices and bioburden: A systematic review. Cell Tissue Bank. 2016, 17, 561–571. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  23. von Woedtke, T.; Kramer, A. The limits of sterility assurance. GMS Krankenh. Interdiszip. 2008, 3, Doc19. [Google Scholar]
  24. Dziedzic-Goclawska, A.; Kaminski, A.; Uhrynowska-Tyszkiewicz, I.; Stachowicz, W. Irirradiation as a Safety Procedure in Tissue Banking. Cell Tissue Bank. 2005, 6, 201–219. [Google Scholar] [CrossRef]
  25. Campbell, D.G.; Li, P. Sterilization of HIV with irirradiation: Relevance to infected bone allografts. Aust. N. Z. J. Surg. 1999, 69, 517–521. [Google Scholar] [CrossRef]
  26. Fideler, B.M.; Vangsness, T.C.; Moore, T.; Li, Z.; Rasheed, S. Effects of Gamma Irirradiation on the Human Immunodeficiency Virus. A Study in Frozen Human Bone-Patellar Ligament-Bone Grafts Obtained from Infected Cadavera. J. Bone Jt. Surg. Am. Vol. 1994, 76, 1032–1035. [Google Scholar] [CrossRef]
  27. Buchalla, R.; Schüttler, C.; Bögl, K.W. Irradiation sterilization of medical devices. Effects of ionizing irradiation on ultra-high molecular-weight polyethylene. Irradiat. Phys. Chem. 1995, 46, 579–585. [Google Scholar] [CrossRef]
  28. Silindir, M.; Özer, A.Y. Sterilization Methods and the Comparison of E-Beam Sterilization with Gamma Irradiation Sterilization. FABAD J. Pharm. Sci. 2009, 34, 43–53. [Google Scholar]
  29. DiBartola, A.C.; Everhart, J.S.; Kaeding, C.C.; Magnussen, R.A.; Flanigan, D.C. Maximum load to failure of high dose versus low dose gamma irirradiation of anterior cruciate ligament allografts: A meta-analysis. Knee 2016, 23, 755–762. [Google Scholar] [CrossRef]
  30. Salehpour, A.; Butler, D.L.; Proch, F.S.; Schwartz, H.E.; Feder, S.M.; Doxey, C.M.; Ratcliffe, A. Dose-dependent response of gamma irirradiation on mechanical properties and related biochemical composition of goat bone-patellar tendon-bone allografts. J. Orthop. Res. Off. Publ. Orthop. Res. Soc. 1995, 13, 898–906. [Google Scholar] [CrossRef] [PubMed]
  31. Fideler, B.M.; Vangsness, C.T.J.; Lu, B.; Orlando, C.; Moore, T. Gamma Irirradiation: Effects on Biomechanical Properties of Human Bone-Patellar Tendon-Bone Allografts. Am. J. Sports Med. 1995, 23, 643–646. [Google Scholar] [CrossRef] [PubMed]
  32. Somers, P.; Cuvelier, C.A.; Somer, F.D.; Cornelissen, M.; Cox, E.; Verloo, M.; Chiers, K.; van Nooten, G. Gamma irradiation alters the ultrastructure in tissue-engineered heart valve scaffolds. Tissue Eng. Part A 2009, 15, 3597–3604. [Google Scholar] [CrossRef] [PubMed]
  33. Nguyen, H.; Morgan, D.A.F.; Forwood, M.R. Sterilization of allograft bone: Effects of gamma irirradiation on allograft biology and biomechanics. Cell Tissue Bank. 2007, 8, 93–105. [Google Scholar] [CrossRef] [PubMed]
  34. Nguyen, H.; Morgan, D.A.; Forwood, M.R. Sterilization of allograft bone: Is 25 kGy the gold standard for gamma irirradiation? Cell Tissue Bank. 2007, 8, 81–91. [Google Scholar] [CrossRef]
  35. Gibbons, M.J.; Butler, D.L.; Grood, E.S.; Bylski-Austrow, D.I.; Levy, M.S.; Noyes, F.R. Effects of gamma irirradiation on the initial mechanical and material properties of goat bone-patellar tendon-bone allografts. J. Orthop. Res. 1991, 9, 209–218. [Google Scholar] [CrossRef]
  36. Smith, C.W.; Young, I.S.; Kearney, J.N. Mechanical Properties of Tendons: Changes with Sterilization and Preservation. J. Biomech. Eng. 1996, 118, 56–61. [Google Scholar] [CrossRef]
  37. Curran, A.R.; Adams, D.J.; Gill, J.L.; Steiner, M.E.; Scheller, A.D. The Biomechanical Effects of Low-Dose Irirradiation on Bone-Patellar Tendon-Bone Allografts. Am. J. Sports Med. 2004, 32, 1131–1135. [Google Scholar] [CrossRef]
  38. Schimizzi, A.; Wedemeyer, M.; Odell, T.; Thomas, W.; Mahar, A.T.; Pedowitz, R. Effects of a Novel Sterilization Process on Soft Tissue Mechanical Properties for Anterior Cruciate Ligament Allografts. Am. J. Sports Med. 2007, 35, 612–616. [Google Scholar] [CrossRef]
  39. Balsly, C.R.; Cotter, A.T.; Williams, L.A.; Gaskins, B.D.; Moore, M.A.; Wolfinbarger, L., Jr. Effect of low dose and moderate dose gamma irirradiation on the mechanical properties of bone and soft tissue allografts. Cell Tissue Bank. 2008, 9, 289–298. [Google Scholar] [CrossRef]
  40. Nichols, A.; Burns, D.C.; Christopher, R. Studies on the Sterilization of Human Bone and Tendon Musculoskeletal Allograft Tissue Using Supercritical Carbon Dioxide. J. Orthop. 2009, 6, e9. [Google Scholar]
  41. McGilvray, K.C.; Santoni, B.G.; Turner, A.S.; Bogdansky, S.; Wheeler, D.L.; Puttlitz, C.M. Effects of (60)Co gamma irradiation dose on initial structural biomechanical properties of ovine bone--patellar tendon--bone allografts. Cell Tissue Bank. 2011, 12, 89–98. [Google Scholar] [CrossRef] [PubMed]
  42. Ren, D.; Sun, K.; Tian, S.; Yang, X.; Zhang, C.; Wang, W.; Huang, H.; Zhang, J.; Deng, Y. Effects of gamma irirradiation and repetitive freeze–thaw cycles on the biomechanical properties of human flexor digitorum superficialis tendons. J. Biomech. 2012, 45, 252–256. [Google Scholar] [CrossRef] [PubMed]
  43. Conrad, B.P.; Rappe, M.; Horodyski, M.; Farmer, K.W.; Indelicato, P.A. The effect of sterilization on mechanical properties of soft tissue allografts. Cell Tissue Bank. 2013, 14, 359–366. [Google Scholar] [CrossRef] [PubMed]
  44. Yanke, A.B.; Bell, R.; Lee, A.; Kang, R.W.; Richard, C.; Mather, I.; Shewman, E.F.; Wang, V.M.; Bernard, R.; Bach, J. The Biomechanical Effects of 1.0 to 1.2 Mrad of Gamma Irirradiation on Human Bone–Patellar Tendon–Bone Allografts. Am. J. Sports Med. 2013, 41, 835–840. [Google Scholar] [CrossRef]
  45. Elenes, E.Y.; Hunter, S.A. Soft-tissue allografts terminally sterilized with an electron beam are biomechanically equivalent to aseptic, nonsterilized tendons. J. Bone Jt. Surg. Am. 2014, 96, 1321–1326. [Google Scholar] [CrossRef] [Green Version]
  46. Baldini, T.; Caperton, K.; Hawkins, M.; McCarty, E. Effect of a novel sterilization method on biomechanical properties of soft tissue allografts. Knee Surg. Sports Traumatol. Arthrosc. 2016, 24, 3971–3975. [Google Scholar] [CrossRef]
  47. Edwards, J.H.; Herbert, A.; Jones, G.L.; Manfield, I.W.; Fisher, J.; Ingham, E. The effects of irirradiation on the biological and biomechanical properties of an acellular porcine superflexor tendon graft for cruciate ligament repair. J. Biomed. Mater. Res. Part B Appl. Biomater. 2017, 105, 2477–2486. [Google Scholar] [CrossRef] [Green Version]
  48. Brunner, G. Applications of Supercritical Fluids. Annu. Rev. Chem. Biomol. Eng. 2010, 1, 321–342. [Google Scholar] [CrossRef]
  49. Tarafa, P.J.; Jimenez, A.; Zhang, J.; Matthews, M.A. Compressed carbon dioxide (CO2) for decontamination of biomaterials and tissue scaffolds. J. Supercrit. Fluids 2010, 53, 192–199. [Google Scholar] [CrossRef]
  50. Duarte, A.R.C.; Mano, J.F.; Reis, R.L. Supercritical fluids in biomedical and tissue engineering applications: A review. Int. Mater. Rev. 2009, 54, 214–222. [Google Scholar] [CrossRef] [Green Version]
  51. Hyatt, J.A. Liquid and supercritical carbon dioxide as organic solvents. J. Org. Chem. 1984, 49, 5097–5101. [Google Scholar] [CrossRef]
  52. Knez, Ž.; Markočič, E.; Leitgeb, M.; Primožič, M.; Knez Hrnčič, M.; Škerget, M. Industrial applications of supercritical fluids: A review. Energy 2014, 77, 235–243. [Google Scholar] [CrossRef]
  53. Pasquali, I.; Bettini, R.; Giordano, F. Supercritical fluid technologies: An innovative approach for manipulating the solid-state of pharmaceuticals. Adv. Drug Deliv. Rev. 2008, 60, 399–410. [Google Scholar] [CrossRef] [PubMed]
  54. White, A.; Burns, D.; Christensen, T.W. Effective terminal sterilization using supercritical carbon dioxide. J. Biotechnol. 2006, 123, 504–515. [Google Scholar] [CrossRef]
  55. Zhang, J.; Davis, T.A.; Matthews, M.A.; Drews, M.J.; LaBerge, M.; An, Y.H. Sterilization using high-pressure carbon dioxide. J. Supercrit. Fluids 2006, 38, 354–372. [Google Scholar] [CrossRef]
  56. Shieh, E.; Paszczynski, A.; Wai, C.M.; Lang, Q.; Crawford, R.L. Sterilization of Bacillus pumilus spores using supercritical fluid carbon dioxide containing various modifier solutions. J. Microbiol. Methods 2009, 76, 247–252. [Google Scholar] [CrossRef]
  57. Dillow, A.K.; Dehghani, F.; Hrkach, J.S.; Foster, N.R.; Langer, R. Bacterial inactivation by using near- and supercritical carbon dioxide. Proc. Natl. Acad. Sci. USA 1999, 96, 10344. [Google Scholar] [CrossRef] [Green Version]
  58. Enomoto, A.; Nakamura, K.; Nagai, K.; Hashimoto, T.; Hakoda, M. Inactivation of Food Microorganisms by High-pressure Carbon Dioxide Treatment with or without Explosive Decompression. Biosci. Biotechnol. Biochem. 1997, 61, 1133–1137. [Google Scholar] [CrossRef]
  59. Hennessy, R.S.; Jana, S.; Tefft, B.J.; Helder, M.R.; Young, M.D.; Hennessy, R.R.; Stoyles, N.J.; Lerman, A. Supercritical Carbon Dioxide–Based Sterilization of Decellularized Heart Valves. JACC Basic Transl. Sci. 2017, 2, 71–84. [Google Scholar] [CrossRef]
  60. Hemmer, J.D.; Drews, M.J.; LaBerge, M.; Matthews, M.A. Sterilization of bacterial spores by using supercritical carbon dioxide and hydrogen peroxide. J. Biomed. Mater. Res. Part B Appl. Biomater. 2007, 80B, 511–518. [Google Scholar] [CrossRef]
  61. Bernhardt, A.; Wehrl, M.; Paul, B.; Hochmuth, T.; Schumacher, M.; Schütz, K.; Gelinsky, M. Improved Sterilization of Sensitive Biomaterials with Supercritical Carbon Dioxide at Low Temperature. PLoS ONE 2015, 10, e0129205. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  62. Cinquemani, C.; Boyle, C.; Bach, E.; Schollmeyer, E. Inactivation of microbes using compressed carbon dioxide―An environmentally sound disinfection process for medical fabrics. J. Supercrit. Fluids 2007, 42, 392–397. [Google Scholar] [CrossRef]
  63. Beckman, E.J. Supercritical and near-critical CO2 in green chemical synthesis and processing. J. Supercrit. Fluids 2004, 28, 121–191. [Google Scholar] [CrossRef]
  64. Matthews, M.A.; Warner, L.S.; Kaiser, H. Exploring the Feasibility of Using Dense-Phase Carbon Dioxide for Sterilization. Available online: https://www.mddionline.com/exploring-feasibility-using-dense-phase-carbon-dioxide-sterilization (accessed on 8 April 2018).
  65. Fages, J.; Poirier, B.; Barbier, Y.; Frayssinet, P.; Joffret, M.L.; Majewski, W.; Bonel, G.; Larzul, D. Viral inactivation of human bone tissue using supercritical fluid extraction. ASAIO J. 1998, 44, 289–293. [Google Scholar] [PubMed]
  66. Fages, J.; Jean, E.; Frayssinet, P.; Mathon, D.; Poirier, B.; Autefage, A.; Larzul, D. Bone allografts and supercritical processing: Effects on osteointegration and viral safety. J. Supercrit. Fluids 1998, 13, 351–356. [Google Scholar] [CrossRef] [Green Version]
  67. Perrut, M. Sterilization and virus inactivation by supercritical fluids (a review). J. Supercrit. Fluids 2012, 66, 359–371. [Google Scholar] [CrossRef]
  68. Mun, S.; Hahn, J.-S.; Lee, Y.-W.; Yoon, J. Inactivation behavior of Pseudomonas aeruginosa by supercritical N2O compared to supercritical CO2. Int. J. Food Microbiol. 2011, 144, 372–378. [Google Scholar] [CrossRef]
  69. Qiu, Q.-Q.; Leamy, P.; Brittingham, J.; Pomerleau, J.; Kabaria, N.; Connor, J. Inactivation of Bacterial Spores and Viruses in Biological Material Using Supercritical Carbon Dioxide With Sterilant. J. Biomed. Mater. Res. Part B-Appl. Biomater. 2009, 91B, 572–578. [Google Scholar] [CrossRef]
  70. Checinska, A.; Fruth, I.A.; Green, T.L.; Crawford, R.L.; Paszczynski, A.J. Sterilization of biological pathogens using supercritical fluid carbon dioxide containing water and hydrogen peroxide. J. Microbiol. Methods 2011, 87, 70–75. [Google Scholar] [CrossRef]
  71. Zhang, J.; Dalal, N.; Gleason, C.; Matthews, M.A.; Waller, L.N.; Fox, K.F.; Fox, A.; Drews, M.J.; LaBerge, M.; An, Y.H. On the mechanisms of deactivation of Bacillus atrophaeus spores using supercritical carbon dioxide. J. Supercrit. Fluids 2006, 38, 268–273. [Google Scholar] [CrossRef]
  72. Balestrini, J.L.; Liu, A.; Gard, A.L.; Huie, J.; Blatt, K.M.; Schwan, J.; Zhao, L.; Broekelmann, T.J.; Mecham, R.P.; Wilcox, E.C.; et al. Sterilization of Lung Matrices by Supercritical Carbon Dioxide. Tissue Eng. Part C Methods 2016, 22, 260–269. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  73. Wehmeyer, J.L.; Natesan, S.; Christy, R.J. Development of a Sterile Amniotic Membrane Tissue Graft Using Supercritical Carbon Dioxide. Tissue Eng. Part C Methods 2015, 21, 649–659. [Google Scholar] [CrossRef] [PubMed]
  74. Frayssinet, P.; Asimus, E.; Autefage, A.; Fages, J. Histological evaluation of xenogeneic bone treated by supercritical CO2 implanted into sheep. J. Mater. Sci. Mater. Med. 1995, 6, 473–478. [Google Scholar] [CrossRef]
  75. Frayssinet, P.; Rouquet, N.; Mathon, D.; Autefage, A.; Fages, J. Histological integration of allogeneic cancellous bone tissue treated by supercritical CO2 implanted in sheep bones. Biomaterials 1998, 19, 2247–2253. [Google Scholar] [CrossRef] [Green Version]
  76. Russell, N.A.; Rives, A.; Pelletier, M.H.; Bruce, W.J.; Walsh, W.R. The effect of sterilization on the mechanical properties of intact rabbit humeri in three-point bending, four-point bending and torsion. Cell Tissue Bank. 2013, 14, 231–242. [Google Scholar] [CrossRef]
  77. Russell, N.; Rives, A.; Bertollo, N.; Pelletier, M.H.; Walsh, W.R. The effect of sterilization on the dynamic mechanical properties of paired rabbit cortical bone. J. Biomech. 2013, 46, 1670–1675. [Google Scholar] [CrossRef]
  78. Bui, D.; Lovric, V.; Oliver, R.; Bertollo, N.; Broe, D.; Walsh, W.R. Meniscal allograft sterilisation: Effect on biomechanical and histological properties. Cell Tissue Bank. 2015, 16, 467–475. [Google Scholar] [CrossRef]
  79. Irani, M.; Lovric, V.; Walsh, W.R.J.C.; Banking, T. Effects of supercritical fluid CO2 and 25 kGy gamma irirradiation on the initial mechanical properties and histological appearance of tendon allograft. Cell Tissue Bank. 2018. [Google Scholar] [CrossRef]
  80. Wang, S.; Wang, Y.; Song, L.; Chen, J.; Ma, Y.; Chen, Y.; Fan, S.; Su, M.; Lin, X. Decellularized tendon as a prospective scaffold for tendon repair. Mater. Sci. Eng. C 2017, 77, 1290–1301. [Google Scholar] [CrossRef]
  81. Casali, D.M.; Handleton, R.M.; Shazly, T.; Matthews, M.A. A novel supercritical CO2-based decellularization method for maintaining scaffold hydration and mechanical properties. J. Supercrit. Fluids 2018, 131, 72–81. [Google Scholar] [CrossRef] [Green Version]
  82. Pridgen, B.C.; Woon, C.Y.; Kim, M.; Thorfinn, J.; Lindsey, D.; Pham, H.; Chang, J. Flexor tendon tissue engineering: Acellularization of human flexor tendons with preservation of biomechanical properties and biocompatibility. Tissue Eng. Part C Methods 2011, 17, 819–828. [Google Scholar] [CrossRef] [PubMed]
  83. de Campos Vidal, B.; Mello, M.L.S. Collagen type I amide I band infrared spectroscopy. Micron 2011, 42, 283–289. [Google Scholar] [CrossRef] [PubMed]
  84. Figueiredo, M.M.; Gamelas, J.A.F.; Martins, A.G. Characterization of Bone and Bone-Based Graft Materials Using FTIR Spectroscopy; Intech Europe: Rijeka, Croatia, 2012; pp. 315–338. [Google Scholar]
  85. Khayyeri, H.; Blomgran, P.; Hammerman, M.; Turunen, M.J.; Löwgren, A.; Guizar-Sicairos, M.; Aspenberg, P.; Isaksson, H. Achilles tendon compositional and structural properties are altered after unloading by botox. Sci. Rep. 2017, 7, 13067. [Google Scholar] [CrossRef] [PubMed]
  86. Tilley, J.M.; Chaudhury, S.; Hakimi, O.; Carr, A.J.; Czernuszka, J.T. Tenocyte proliferation on collagen scaffolds protects against degradation and improves scaffold properties. J. Mater. Sci. Mater. Med. 2012, 23, 823–833. [Google Scholar] [CrossRef]
  87. Chen, J.; Xu, J.; Wang, A.; Zheng, M. Scaffolds for tendon and ligament repair: Review of the efficacy of commercial products. Expert Rev. Med Devices 2009, 6, 61–73. [Google Scholar] [CrossRef]
  88. Berthiaume, F.; Maguire, T.J.; Yarmush, M.L. Tissue Engineering and Regenerative Medicine: History, Progress, and Challenges. Annu. Rev. Chem. Biomol. Eng. 2011, 2, 403–430. [Google Scholar] [CrossRef]
  89. Crapo, P.M.; Gilbert, T.W.; Badylak, S.F. An overview of tissue and whole organ decellularization processes. Biomaterials 2011, 32, 3233–3243. [Google Scholar] [CrossRef] [Green Version]
  90. Antons, J.; Marascio, M.G.; Aeberhard, P.; Weissenberger, G.; Hirt-Burri, N.; Applegate, L.A.; Bourban, P.E.; Pioletti, D.P. Decellularised tissues obtained by a CO2-philic detergent and supercritical CO2. Eur. Cells Mater. 2018, 36, 81–95. [Google Scholar] [CrossRef]
  91. Hamer, A.J.; Stockley, I.; Elson, R.A. Changes in allograft bone irradiated at different temperatures. J. Bone Jt. Surg. Br. Vol. 1999, 81, 342–344. [Google Scholar] [CrossRef]
  92. Bottagisio, M.; Pellegata, A.F.; Boschetti, F.; Ferroni, M.; Moretti, M.; Lovati, A.B. A new strategy for the decellularisation of large equine tendons as biocompatible tendon substitutes. Eur. Cells Mater. 2016, 32, 58–73. [Google Scholar] [CrossRef] [Green Version]
  93. Whitlock, P.W.; Smith, T.L.; Poehling, G.G.; Shilt, J.S.; Van Dyke, M. A naturally derived, cytocompatible, and architecturally optimized scaffold for tendon and ligament regeneration. Biomaterials 2007, 28, 4321–4329. [Google Scholar] [CrossRef] [PubMed]
  94. Selcan, G.; Bahar, A.; Pezhman, H.; Murat, A.H. Supercritical Carbon Dioxide-Assisted Decellularization of Aorta and Cornea. Tissue Eng. Part C Methods 2017, 23, 540–547. [Google Scholar] [CrossRef]
  95. Sawada, K.; Terada, D.; Yamaoka, T.; Kitamura, S.; Fujisato, T. Cell removal with supercritical carbon dioxide for acellular artificial tissue. J. Chem. Technol. Biotechnol. 2008, 83, 943–949. [Google Scholar] [CrossRef]
  96. Hahn, J.M.; İnceoğlu, S.; Wongworawat, M.D. Biomechanical Comparison of Krackow Locking Stitch Versus Nonlocking Loop Stitch With Varying Number of Throws. Am. J. Sports Med. 2014, 42, 3003–3008. [Google Scholar] [CrossRef] [PubMed]
  97. Huang, H.; Zhang, J.; Sun, K.; Zhang, X.; Tian, S. Effects of repetitive multiple freeze–thaw cycles on the biomechanical properties of human flexor digitorum superficialis and flexor pollicis longus tendons. Clin. Biomech. 2011, 26, 419–423. [Google Scholar] [CrossRef]
  98. Chang, E.Y.; Bae, W.C.; Statum, S.; Du, J.; Chung, C.B. Effects of repetitive freeze–thawing cycles on T2 and T2* of the Achilles tendon. Eur. J. Radiol. 2014, 83, 349–353. [Google Scholar] [CrossRef]
  99. Woods, T.; Gratzer, P.F. Effectiveness of three extraction techniques in the development of a decellularized bone-anterior cruciate ligament-bone graft. Biomaterials 2005, 26, 7339–7349. [Google Scholar] [CrossRef]
  100. Chen, C.-H. Graft healing in anterior cruciate ligament reconstruction. Sports Med. Arthrosc. Rehabil. 2009, 1, 21. [Google Scholar] [CrossRef] [Green Version]
  101. Lovati, A.B.; Bottagisio, M.; Moretti, M. Decellularized and Engineered Tendons as Biological Substitutes: A Critical Review. Stem Cells Int. 2016. [Google Scholar] [CrossRef] [Green Version]
  102. Reverchon, E.; Baldino, L.; Cardea, S.; De Marco, I. Biodegradable synthetic scaffolds for tendon regeneration. Muscles Ligaments Tendons J. 2012, 2, 181–186. [Google Scholar]
  103. Kim, S.H.; Jung, Y.; Kim, S.H. A biocompatible tissue scaffold produced by supercritical fluid processing for cartilage tissue engineering. Tissue Eng. Part C Methods 2013, 19, 181–188. [Google Scholar] [CrossRef] [PubMed]
  104. Aydin, H.M.; Turk, M.; Calimli, A.; Piskin, E. Attachment and growth of fibroblasts on poly(L-lactide/epsilon-caprolactone) scaffolds prepared in supercritical CO2 and modified by polyethylene imine grafting with ethylene diamine-plasma in a glow-discharge apparatus. Int. J. Artif. Organs 2006, 29, 873–880. [Google Scholar] [CrossRef] [PubMed]
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Figure 1. Phase diagram of CO2 showing the critical point at 31.1 °C and 1099 psi [54].
Figure 1. Phase diagram of CO2 showing the critical point at 31.1 °C and 1099 psi [54].
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Figure 2. Representative high-resolution histological images of transverse and longitudinal sections. In haematoxylin and eosin (H&E) sections, collagen was stained pale pink with interspersed dark blue nuclei conspicuous in all groups except the standard SDS group. The histological dead space in the transverse sections are indicated with *. Separation of collagen bundles are indicated with black arrowheads. Scale bar at the bottom right represents 20 μm.
Figure 2. Representative high-resolution histological images of transverse and longitudinal sections. In haematoxylin and eosin (H&E) sections, collagen was stained pale pink with interspersed dark blue nuclei conspicuous in all groups except the standard SDS group. The histological dead space in the transverse sections are indicated with *. Separation of collagen bundles are indicated with black arrowheads. Scale bar at the bottom right represents 20 μm.
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Figure 3. The typical appearances of the collagen banding patterns as observed under ~100,000× magnification (left column, a,c,e,g,i,k) and the tendon surfaces as observed under ~10,000× magnification (right column, b,d,f,h,j,l). Black arrowheads in the left column indicate breaks in collagen fibers and those in the right column indicate sites of surface breakage and exposure of underlying collagen fibrils.
Figure 3. The typical appearances of the collagen banding patterns as observed under ~100,000× magnification (left column, a,c,e,g,i,k) and the tendon surfaces as observed under ~10,000× magnification (right column, b,d,f,h,j,l). Black arrowheads in the left column indicate breaks in collagen fibers and those in the right column indicate sites of surface breakage and exposure of underlying collagen fibrils.
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Figure 4. Representative FTIR spectra of the control, gamma-irradiated, standard-SDS-treated, SCCO2-NK-treated, SCCO2-0.1%-SDS-treated, and SCCO2-1%-SDS-treated tendons. The red and black boxes highlight the reductions of absorption peaks associated with CH2 and carbonyl groups, respectively.
Figure 4. Representative FTIR spectra of the control, gamma-irradiated, standard-SDS-treated, SCCO2-NK-treated, SCCO2-0.1%-SDS-treated, and SCCO2-1%-SDS-treated tendons. The red and black boxes highlight the reductions of absorption peaks associated with CH2 and carbonyl groups, respectively.
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Figure 5. A study design flowchart showing the sample size for each group and endpoint, and processing and storage steps. Abbreviations: SDS, sodium dodecyl sulfate; SCCO2, supercritical carbon dioxide; PBS, phosphate-buffered saline; FE-SEM, field-emission scanning electron microscopy; FTIR, Fourier-transform infrared.
Figure 5. A study design flowchart showing the sample size for each group and endpoint, and processing and storage steps. Abbreviations: SDS, sodium dodecyl sulfate; SCCO2, supercritical carbon dioxide; PBS, phosphate-buffered saline; FE-SEM, field-emission scanning electron microscopy; FTIR, Fourier-transform infrared.
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Figure 6. Schematic of the in-house supercritical fluid system [76].
Figure 6. Schematic of the in-house supercritical fluid system [76].
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Figure 7. The locations of width and thickness measurements. The bold black frame represents a full-length tendon. The central mid-substance encircled in the red frame was designated as the gauge length for mechanical testing. The dashed lines pointed by black arrows delineate where the width and thickness measurements were taken.
Figure 7. The locations of width and thickness measurements. The bold black frame represents a full-length tendon. The central mid-substance encircled in the red frame was designated as the gauge length for mechanical testing. The dashed lines pointed by black arrows delineate where the width and thickness measurements were taken.
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Figure 8. Sample preparation for histology. The bold frame represents a 4-cm tendon segment and dash lines indicate where cuts were made with a scalpel to 3 transverse and 2 longitudinal sections. From each of these sections, 5-micron slices were acquired for staining with haematoxylin and eosin (H&E). Two 40× magnification images were taken for each cross-section, culminating in 18 images per group as there were 3 transverse cross-sections per tendon segment, and 3 tendon segment per group.
Figure 8. Sample preparation for histology. The bold frame represents a 4-cm tendon segment and dash lines indicate where cuts were made with a scalpel to 3 transverse and 2 longitudinal sections. From each of these sections, 5-micron slices were acquired for staining with haematoxylin and eosin (H&E). Two 40× magnification images were taken for each cross-section, culminating in 18 images per group as there were 3 transverse cross-sections per tendon segment, and 3 tendon segment per group.
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Table
Table 1. Pre- and post-treatment dimensions.
Table 1. Pre- and post-treatment dimensions.
GroupCross-Sectional Area (CSA)
(mm2)		Thickness (mm)Width (mm)
Pre-TreatmentPost-TreatmentPre-TreatmentPost-TreatmentPre-TreatmentPost-Treatment
Control3.75 ± 0.961.64 ± 0.212.90 ± 0.56
25 kGy Gamma4.61 ± 1.32 a 4.34 ± 1.001.84 ± 0.291.69 ± 0.213.15 ± 0.533.26 ± 0.62
Std SDS4.89 ± 0.82 a4.78 ± 0.89 a1.85 ± 0.14 a1.85 ± 0.17 ab3.39 ± 0.63 a3.32 ± 0.67 a
SCCO2-NK4.33 ± 0.844.14 ± 0.73 c1.70 ± 0.191.70 ± 0.18 c3.25 ± 0.593.06 ± 0.60
SCCO2-0.1%-SDS4.31 ± 0.953.53 ± 0.84 bcd*1.73 ± 0.121.62 ± 0.11 c*3.16 ± 0.662.78 ± 0.65 bc
SCCO2-1%-SDS4.05 ± 0.81 c4.17 ± 0.79 ce1.73 ± 0.161.76 ± 0.22 e2.97 ± 0.51 c3.02 ± 0.42
Although the measurements were conducted on the same tendons for each group, the pre- and post-treatment measurements could not be matched. Thus, independent samples t-tests were run to compare the pre- and post-treatment dimensions for each group. a Significant difference from control in the respective column; b Significant difference from 25 kGy Gamma in the respective column; c Significant difference from Std SDS in the respective column; d Significant difference from SCCO2-NK in the respective column; e Significant difference from SCCO2-0.1%-SDS in the respective column; * Significant difference from pre-treatment measurements.
Table
Table 2. The mean values of mechanical properties (failure load and stiffness) (a) and material properties (UTS, failure strain, and Young’s modulus) (b) of all groups.
Table 2. The mean values of mechanical properties (failure load and stiffness) (a) and material properties (UTS, failure strain, and Young’s modulus) (b) of all groups.
(a)Failure Load (N)Stiffness (N/mm)
Control330.34 ± 79.09 77.03 ± 31.83
25 kGy Gamma408.27 ± 121.86 92.58 ± 23.95
Std SDS 350.55 ± 128.77 72.95 ± 29.80
SCCO2-NK376.79 ± 93.19 98.54 ± 26.22
SCCO2-0.1%-SDS 400.31 ± 123.79 108.65 ± 21.86 ac
SCCO2-1%-SDS 203.07 ± 103.14 abcde 73.09 ± 24.16 e
(b)Ultimate Tensile Stress (MPa)Failure Strain (%)Young’s Modulus (MPa)
Control89.76 ± 15.40 40.01 ± 10.12 574.20 ± 253.81
25 kGy Gamma93.88 ± 14.02 40.73 ± 6.90 557.87 ± 135.27
Std SDS 72.68 ± 17.35 b 42.70 ± 9.68 405.43 ± 101.82
SCCO2-NK92.57 ± 23.43 36.68 ± 7.20 648.40 ± 181.71 c
SCCO2-0.1%-SDS 112.84 ± 19.84 abc 34.44 ± 5.32 c 852.63 ± 193.67 abcd
SCCO2-1%-SDS 50.75 ± 26.27 abde 28.93 ± 5.35 abc 456.55 ± 157.34 de
a Significant difference from control in the respective column; b Significant difference from 25 kGy Gamma in the respective column; c Significant difference from Std SDS in the respective column; d Significant difference from SCCO2-NK in the respective column; e Significant difference from SCCO2-0.1%-SDS in the respective column.
Table
Table 3. Void percentage and cellularity across groups.
Table 3. Void percentage and cellularity across groups.
GroupVoid (%)Cellularity (Cells/mm2)
Control29.08 ± 4.96818.91 ± 116.62
25 kGy Gamma41.81 ± 4.50 a750.61 ± 176.45
Std SDS35.21 ± 4.48 ab3.17 ± 9.49 ab
SCCO2-NK27.61 ± 6.49 bc764.65 ± 127.28 c
SCCO2-0.1%-SDS27.35 ± 7.09 bc 682.32 ± 126.42 ac
SCCO2-1%-SDS7.48 ± 5.18 abcde824.65 ± 252.15 c
a Significant difference from control in the respective column; b Significant difference from 25 kGy Gamma in the respective column; c Significant difference from Std SDS in the respective column; d Significant difference from SCCO2-NK in the respective column; e Significant difference from SCCO2-0.1%-SDS in the respective column.

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MDPI and ACS Style

Sun, Y.; Lovric, V.; Wang, T.; Oliver, R.A.; Walsh, W.R. Effects of SCCO2, Gamma Irradiation, and Sodium Dodecyl Sulfate Treatments on the Initial Properties of Tendon Allografts. Int. J. Mol. Sci. 2020, 21, 1565. https://doi.org/10.3390/ijms21051565

AMA Style

Sun Y, Lovric V, Wang T, Oliver RA, Walsh WR. Effects of SCCO2, Gamma Irradiation, and Sodium Dodecyl Sulfate Treatments on the Initial Properties of Tendon Allografts. International Journal of Molecular Sciences. 2020; 21(5):1565. https://doi.org/10.3390/ijms21051565

Chicago/Turabian Style

Sun, Yikan, Vedran Lovric, Tian Wang, Rema A. Oliver, and William R. Walsh. 2020. "Effects of SCCO2, Gamma Irradiation, and Sodium Dodecyl Sulfate Treatments on the Initial Properties of Tendon Allografts" International Journal of Molecular Sciences 21, no. 5: 1565. https://doi.org/10.3390/ijms21051565

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