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

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.


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 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 microorganisms surviving the treatment [21][22][23].
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 SCCO 2 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 SCCO 2 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], SCCO 2 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. SCCO 2 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 SCCO 2 sterilization protocol preserves the initial biomechanical properties of tendons; (b) histologically assess if SCCO 2 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 SCCO 2 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.

Gross Appearance and Dimensions
Compared to native tendons, gamma-irradiated tendons became shriveled and their surface granular; the SCCO 2 -NK-treated tendons became noticeably cleaner with effacement of the surface marks made on them prior to treatment; and SCCO 2 -SDS-treated tendons, especially the SCCO 2 -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 SCCO 2 -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 SCCO 2 -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). 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 SCCO 2 -NK in the respective column; e Significant difference from SCCO 2 -0.1%-SDS in the respective column; * Significant difference from pre-treatment measurements.

Mechanical Testing
In general, the mechanical properties of all treatment groups apart from SCCO 2 -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. SCCO 2 -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 SCCO 2 -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 SCCO 2 -0.1%-SDS was significantly lower than that of the standard SDS group (p = 0.035). The mean Young's modulus of the SCCO 2 -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 SCCO 2 -NK (p = 0.019) groups, while the mean Young's modulus of the SCCO 2 -NK group was also significantly greater than that of the standard SDS group (p = 0.003).

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 SCCO 2 -NK or SCCO 2 -0.1%-SDS-treated specimens (images g, h, I, and j, Figure 2), as evidenced by the quantitative results that SCCO 2 -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 SCCO 2 -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 SCCO 2 -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/mm 2 ). The cellularity of SCCO 2 -0.1%-SDS-treated tendons (682.32 ± 126.42 cells/mm 2 ) was significantly lower than that of the control tendons (818.91 ± 116.62 cells/mm 2 ) (p = 0.021), but this reduction is trivial when compared to the absolute number of cells left within the tendons (Table 3).

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 SCCO 2 -NK-, SCCO 2 -0.1%-SDS-, and SCCO 2 -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 SCCO 2 -1%-SDS-treated ones (images c and k, respectively, Figure 3). Specifically, for SCCO 2 -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).

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-NKand SCCO2-0.1%-SDS-treated samples, indicating no or minimal damage to the collagen contents. Also, the shapes of the spectra of native, SCCO2-NKand 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.

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 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 CH 2 group; and (b) 1742 cm −1 , which is associated with the carbonyl group [86]. SCCO 2 -1%-SDS treatment provoked a similar pattern of absorption peak changes.

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 SCCO 2 -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 SCCO 2 -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 SCCO 2 -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 SCCO 2 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 SCCO 2 without excessive additives generally retains the biomechanical integrity of soft tissues as assessed from most endpoints, the effect of SCCO 2 on the stiffness and Young's modulus of tissues remains indeterminate. Whilst the present study showed a statistically significant increase in the SCCO 2 -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 SCCO 2 , 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 SCCO 2 group. On the other hand, Balestrini et al. [72] observed significantly increased elastic modulus in SCCO 2 -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 SCCO 2 -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 SCCO 2 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 SCCO 2 was corroborated by the non-mechanical characterization of SCCO 2 -treated samples other than the SCCO 2 -1%-SDS group in the present study, reiterating the gentle character of SCCO 2 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 SCCO 2 -treated tendons and skins were generally comparable to those of the native tendons. Dillow et al. [57] have found that SCCO 2 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 CH 2 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 SCCO 2 -SDS hybrid decellularization protocols experimented in this study were formulated with the assumption that SCCO 2 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 NovaKill TM Gen2 for SCCO 2 -facilitated decellularization even more appealing. Nevertheless, even if the biomechanical properties remained intact, the SCCO 2 -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 SCCO 2 -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 NovaKill TM Gen2 (14.1% PAA) added to the SCCO 2 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 SCCO 2 -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 SCCO 2 -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 SCCO 2 -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 CO 2 -philic decellularizing agents are likely to be imperative for effective decellularization [94]. Indeed, CO 2 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 SCCO 2 with Dehypon ® LS-54, a common household and industrial surfactant, without affecting the mechanical integrity or biocompatibility.
Furthermore, dehydration of SCCO 2 -treated tissues observed in this study, as evidenced by the statistically significant decrease in CSA of SCCO 2 -0.1%-SDS-treated tendons, was also reported by Sawada et al. [95], where they successfully extracted cell nuclei from tissue with SCCO 2 -entrained ethanol. Casali et al. [81] were subsequently able to overcome this problem by pre-saturating SCCO 2 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 SCCO 2 -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., SCCO 2 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 NovaKill TM 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.

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].

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.

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].

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.

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 cobalt 60 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.

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 cobalt 60 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.

Standard SDS Decellularisation
The standard SDS treatment of the present study was strictly reproduced from the protocol of Pridgen et al. [82].

SCCO 2 -Based Treatment Protocols
An in-house custom-built supercritical fluid system was used to carry out SCCO 2 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 (SCCO 2 -NK) tendons were thawed, packaged into Tyvek pouches, loaded into the pressure vessel, and treated with SCCO 2 and 3.2 mL per 30 tendons of NovaKill TM Gen2 (~14.1% PAA and~4.9% H 2 O 2 ) (NovaSterilis, Lansing, NY, USA) under 39 • C and 102 bar to mirror the established SCCO 2 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 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% (

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 NovaKill TM 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 abovedescribed [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 NovaKill TM 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.  Figure 6. Schematic of the in-house supercritical fluid system [76].

Mechanical Testing
Group V (SCCO 2 -0.1%-SDS) and group VI (SCCO 2 -1%-SDS) tendons first received the above-described [82] 4-h pre-treatment with EDTA, followed by 2 h of SCCO 2 treatment under the identical conditions as group IV tendons. Also, in addition to NovaKill TM 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.

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 TestSuite TM 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. 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 cryoclamps 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 preload. The testing protocol was programmed in and executed by the MTS TestSuite TM 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.

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.

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, 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.

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, United Kingdom), 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.

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.

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.

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 underinvestigated 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 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.

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.

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.

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.

Conclusions
Development and modification of new allograft tendon and tissue processing protocols, in general, follow a multi-step progression. SCCO 2 represents an exceptionally promising but under-investigated agent, and the present study has demonstrated preservation of mechanical properties of SCCO 2 -treated tendons. The next advancement would be to identify the optimal CO 2 -philic detergents that complement SCCO 2 , and the treatment parameters to actualize sterilization and decellularization with minimal concentration and protocol run-time. The in vivo biocompatibility of SCCO 2 -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]. SCCO 2 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.

Conflicts of Interest:
The authors declare no conflicts of interest.