Different sterilization methods used for human tendons – A systematic review

Abstract


Background
In orthopedic reconstruction, the use of tendon allografts has become more widely accepted in recent years, especially in the anterior cruciate ligament reconstruction. There have been multiple studies conducted which back the use of tendon allografts. The bene ts include reduced operation time, reduced donor site morbidity, and unaltered mechanics secondary to harvesting. Additionally, studies conducted on animals and humans have demonstrated that soft tissue allografts are statistically comparable to autografts on an anatomical and biomechanical basis. [1]- [10] Anterior cruciate ligament (ACL) reconstruction is a common procedure in orthopedic practice. A critical decision to be made is the choice of graft. Despite autografts proving capable and displaying good outcomes, graft harvest can cause continual pain at the site of harvest and limit the range of motion. For this reason, a signi cant increase in allograft use can be seen in the last decade, and despite the higher costs, it remains a feasible choice, speci cally in revision cases. In young patients, an increased rate of failure of allografts has been recorded. This mostly seems to be caused by the sterilization process, as some studies also reported increased failure rates when comparing fresh-frozen allografts and autografts. A couple of studies displayed more inferior results in sterilization compared with fresh-frozen allografts. A major risk of using allografts is disease transmission. Septic arthritis after ACL reconstruction has been reported as a risk factor for unsterilized allografts. Also, higher rates of postoperative infections have been reported when allografts were aseptically processed rather than irradiated allografts or autografts. [11]- [21] A potential complication of using allografts is the risk of disease transmission. Possible diseases include both infections from HIV, Hepatitis C, and bacterial infection from organisms. Whilst the prevalence of the transmission of the disease via allograft tissue is minimal; the potential impact is high. After multiple documented cases of sepsis and infection following ACL reconstruction using allografts, the bacterial transmission of diseases has come to the forefront. These cases were associated with non-irradiated tissue grafts. For this reason, an effort has been made to reduce the scarce but possibly disastrous occurrences of bacterial infection by using enhanced sterilization techniques. [10], [22]- [25] 1.1. Aim of the study The present systematic literature review aims to identify and to categorize existing sterilization methods and their effects on the mechanical properties (failure load/ultimate tensile strength and Young's modulus of elasticity) of the tendons.
Combining the collected materials should allow us to investigate how different methods can differentiate between participant groups and which methods are more encouraging in research or clinical practice. included: tendon, allograft tendon, tendon sterilization, biomechanical testing, mechanical properties, and synonyms of these terms. The expressions were matched to each database. Other sources included reference lists of formerly cited articles containing our published works on similar topics. (Table 1) For example, a search strategy used for the Science Direct database is as follows. In the Advanced search, the following phrases were added in All elds: (allograft tendon OR tendon) AND (tendon sterilization OR ligament sterilization OR sterilization AND biomechanical properties OR mechanical properties OR modulus). The search was re ned to journal publications. Publication dates were set from 2008 to the Present, with the search performed on March 31st, 2020. This search in the Science Direct database yielded 82 records. Key search terms were identi ed and agreed upon by DF and RMK; electronic search and downloading of results were conducted by DF. Screening, eligibility check of materials, and data extraction were executed by DF, BK, and BS.

Screening materials
The associated materials were screened based on title and abstract, removing duplicate entries. The material of unrelated topics, aims, or completely theoretical work was excluded. Proof of concept articles was not omitted.

Eligibility check of materials
Inclusion and exclusion criteria were determined to check for eligibility. Studies had to meet all the inclusion criteria to be included in the nal synthesis ( Table 1). Studies that either met an exclusion criterion or otherwise failed to meet inclusion criteria were excluded. These criteria were created to provide a quality assessment to a certain extent, i.e., the methods applied had to be well communicated, and the evaluation of the measurement results had to be objective. No additional quality assessment was carried out on the materials included.

Data extraction
In compliance with the objective of this research, the nal overview of the types of sterilization was to extract relevant information on the assessment of mechanical properties. The data collected from the articles included a) author and date, b) type of tendon, c) type of sterilization, d) sterilization dose, and g) measured and calculated parameters such as failure load/ultimate tensile strength and Young's modulus of elasticity. Studies with a detailed explanation of the tendon irradiation and mechanical test and the experimental procedure that was completed.
Studies without detail or incomplete descriptions of the tendon irradiation and mechanical test and the experimental procedures that were completed.

Assessment of results
Studies with unbiased results based on measurable parameters.
Studies with biased scoring/assessments of results, not (fully) based on measurable parameters.

Results
The database search and additional sources resulted in 284 records ( The following were the reasons for exclusion: several studies applied criteria outside of the scope of our de nition of tendon irradiation and mechanical properties, e.g., effects of gamma radiation if the tendon was infected with HIV. If multiple studies described similar tendon irradiation with an equal measurement method and similar instrumentation for assessment, the newest publication was used (30 pieces). Several theoretical articles did not detail a speci c measurement setup and were therefore excluded (32 pieces). Some articles used a biased scoring assessment (6 pieces), and a few articles had an unrelated aim of the study, e.g., clinical case (7 pieces).

Type of sterilization
These studies evaluated the effect of sterilization's type: non-sterilized and non-frozen tendons (fresh tendons) [27, 28, 29, 30; Table 9.] and chlorhexidine [52; Table 10.]. The structure of the tendons may be destroyed by sterilization, so the method used is important. The following subsections detailed these sterilization methods, and the section of conclusion compared the failure load and Young's modulus of elasticity results found in the literature.

Non-sterilized tendon (fresh/control tendon)
Four articles mention the fresh grafts, which did not receive any sterilization [8, 27-30, 43-45, 50, 52; Table 2.]. After they were procured from human cadavers, they were placed in a saline solution and cooled until the measurement. In comparative studies, non-sterilized tendons are measured as a standard to be able to tell to what extent the sterilization method and dose change its properties. Baldini et al. [27] analyzed three groups of anterior and posterior tibialis tendons. The results showed that the failure load and Young's modulus of elasticity of the fresh group are weaker than the gamma-irradiated and SCCO2 sterilized groups [27;  The failure load of the irradiated tendons deteriorated relative to the control group. With an increase in dose, the mechanical properties of the tendon improved to 35 kGy, and after that, they deteriorated [34]. In the article by Hangody et al. [40], the target doses were 21 and 42 kGy, and ve different types of tendons were tested. In the case of the 21 kGy dose, the failure load and Young's modulus of elasticity of tendons improved versus the frozen group. In the case of the 42 kGy dose, the mechanical properties of some grafts improved, and others deteriorated [40].s In the second group, the effect of gamma irradiation was examined compared to the control group. The values between 14.6-40 kGy radiation dose were examined. In two cases [33,36] [32] compared three age groups ("young", "middle", "old"). Deterioration can be observed in the "middle" group and the "old" group. There are values of failure load, and Young's modulus of elasticity, no noticeable change in the "young" group [32].

BioCleanse
Following the BioCleanse protocol, all tendons were closed in a chamber and were exposed to a solvent at differing pressures and vacuum cycles [8, 43-45, 54; Table 6.]. The treatment group was exposed to the chemical solutions for twice as long as the standard time of exposure. This was designed to portray the worst outcome for degrading the structural and mechanical characteristics of the donor material [54]. Based on the results, the failure load and Young's modulus of elasticity showed no signi cant differences between gamma irradiation and BioCleanse groups [8, 43-45, 54; Table 6.].  Table 7.] compared to the method with gamma irradiation, but the differences were not signi cant. were then closed and subjected to gamma irradiation at an average dose of 15 kGy. The sterilized muscles were stowed at -80°C [50], and they showed better mechanical results than the control group.
This hybrid procedure has not been compared with other sterilization methods [50; Table 8.].

Chlorhexidine
In this chemical process, the experimental group is immersed in a 4% chlorhexidine gluconate solution for 30 minutes, while the control group was kept moist in saline gauze. The results of patellar tendons were not compared with other sterilization procedures [52]. The experimental group had better mechanical properties than the control group [52; Table 10].

Discussion
The two factors that can affect the mechanical properties of a tendon are the type of tendon and the sterilization methods. Tendon grafts show to be promising for transplants. It is important to sterilize the tendons to ensure no bacteria is transmitted that could cause infections. We reviewed peroneus longus, peroneus brevis, tibialis anterior, tibialis posterior, patellar tendon, human exor digitorum super cial, Achilles, bone-patellar tendon-bone, semitendinosus, semitendinosus + gracilis, fascia latta types of tendon and gamma irradiation, SCCO2, ethylene oxide, BioCleanse, E-Beam, PE-R, hydrogen peroxide, chlorhexidine sterilization methods.
The supercritical carbon dioxide (SCCO2) sterilization method has been discussed only by Baldini et al.   [42].
The BioCleanse wash poses the same issues as the hydrogen peroxide and chlorhexidine method [43][44][45]54; Table 6.]. Since this is a wash, only the outside of the tendon is sterilized. Dangerous bacteria might still be found in the tendon. The BioCleanse solution is not as effective as gamma irradiation. The sterilization method is addressed in three studies [  [43][44][45].
The effect of sterilization by Electron Beam (E-beam) was analyzed in three articles [30, 46-47,  consisting of immersing the tendon in a chemical solution and then using gamma irradiation. This method yielded a worse failure load than the Electron beam sterilization and gamma sterilization. [50].
The hydrogen peroxide method is just a wash; the inside of the tendon is not properly sterilized, which can lead to infections. Gardner et al. [51] examined the effect of hydrogen peroxide on several different types of tendons. The ultimate tensile strength was not compared to other sterilization methods or nonsterilized tendons [ Table 9.]. Comparing the results of the ultimate tensile strength of previous studies with the results, hydrogen peroxide is not a suitable method for sterilizing tendons [51].
The Chlorhexidine chemical solution is also a wash, the same as the hydrogen peroxide method [52].
While it does pose better results of failure load for the tendon, the inside of the tendon is not properly sterilized. According to the method of Sobel et al. [52] showed that the value of the failure load increases as a result of sterilization (non-sterilized: 1876 N, Chlorhexidine: 2219 N) [ Table 10.].

Limitation
This review analyzed and classi ed multiple existing sterilization methods used in determining the biomechanical properties of tendons. Since this review looked at different techniques, a meta-analysis could not be performed. Limitations can also surface because only three major scienti c databases were utilized in the search for papers. Because of the full range of this research, it only provides an overview of multiple sterilization processes. It does not discuss the advancement of any single process.

Conclusion
The purpose of this literature review was to systematically evaluate existing literature to compare the biomechanical effects of different sterilization methods on commonly used tendon allografts. The biomechanical properties of tendons that were determined included failure load and Young's modulus of elasticity.
Based on a systematic literature review, it can be said that the most common method of sterilization is gamma radiation. However, after comparing the different literature, it is established that mechanical properties are improved, compared to non-sterilized tendons, with the E-beam sterilization method. The method is not widespread, due to the lack of laboratories and instruments suitable for conducting this treatment. The procedure using Chlorhexidine has yielded similar results to the E-beam. Only a few studies is available on the subject, so further research is needed.
Based on the above-mentioned data, we recommend freezing and gamma irradiation or electron beam at 14.8-28.5 kGy [