Mechanical properties of animal ligaments: a review and comparative study for the identification of the most suitable human ligament surrogates

The interest in the properties of animal soft tissues is often related to the desire to find an animal model to replace human counterparts due to the unsteady availability of human tissues for experimental purposes. Once the most appropriate animal model is identified, it is possible to carry out ex-vivo and in-vivo studies for the repair of ligamentous tissues and performance testing of replacement and support healing devices. This work aims to present a systematic review of the mechanical properties of ligaments reported in the scientific literature by considering different anatomical regions in humans and several animal species. This study was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) method. Moreover, considering the lack of a standard protocol for preconditioning of tissues, this aspect is also addressed. Ninety-six studies were selected for the systematic review and analysed. The mechanical properties of different animal species are reported and summarised in tables. Only results from studies reporting the strain rate parameter were considered for comparison with human ligaments, as they were deemed more reliable. Elastic modulus, ultimate tensile stress, and ultimate strain properties are graphically reported identifying the range of values for each animal species and to facilitate comparison between values reported in the scientific literature in animal and human ligaments. Useful similarities between the mechanical properties of swine, cow, and rat and human ligaments have been found. Supplementary Information The online version contains supplementary material available at 10.1007/s10237-023-01718-1.


Introduction
The interest in the mechanical properties of animal ligaments is often correlated with finding a useful model for human ones.Since ethical reasons make difficult to find human ligaments to run in vitro and in vivo tests, animal specimens are commonly employed.In fact, animal models are preferred in preclinical studies for two main types of research purposes: (i) evaluation of tissue healing through different strategies (for example, after growth factors and stem cell injection) and (ii) the evaluation of mechanical properties of suture pattern under validation and testing of innovative repair technologies.Surgical repair techniques commonly employed in human's and animal's traumatology (DeLong and Waterman 2015; Dabbene et al. 2018) rely on the results of mechanical studies, based on reported properties of the original and intact anatomical structures.
Nevertheless, not all animal ligaments are biomechanically comparable to their humans' anatomical counterparts.Therefore, it is needed to discuss the differences between these latter and animal ligaments, even if few studies in the literature made a direct comparison between human and animal ligaments (Baah-Dwomoh et al. 2018;Noyes and Grood 1976).
This review aims to provide a more detailed analysis of similarities and differences between human and various animal species to find the most suitable human ligament surrogate.Uniaxial tensile tests performed on equine, bovine, ovine, caprine, swine, canine, rodents, leporidae, and human ligaments were considered.This work is closely related to a similar comparison between animal and human tendons previously conducted by this research group (Burgio et al. 2022).

Human and animal ligaments
Like tendons, ligaments are characterised by a hierarchical structure and are made of mesenchymal cells inside a supporting matrix and an extracellular matrix containing a high amount of collagen fibres (type I and type III collagen are the most abundant), water and to a lesser extent of elastin, glycoproteins, and proteoglycans (Rumian et al. 2007).
Despite the similar composition, in tendons collagen fibrils are placed in parallel to each other and along the whole length of the tendon.On the contrary, the collagen fibrils of the ligaments are not uniformly orientated, and this organisation is fundamental to withstand multidirectional loads (Rumian et al. 2007).
Even localisation and function, as well as the different arrangement of the components, contribute to defining differences in the biomechanical characteristics of tendons and ligaments: both of these structures must be able to withstand tensile loads, but while the tendons are subjected mostly to uniaxial forces, the ligaments are subjected to multiaxial loads (the force components directions depend on the directions of movement allowed to the joint) (Rumian et al. 2007).

Common applications of animal surrogates
Concerning biomechanics, it is important to consider that, unlike humans, almost all animals are quadrupeds and often have different and more limited ranges of motion in the corresponding joints (Bascuñán et al. 2019).However, there are many instances where they are extensively used.In this section, will be discussed different animal models encountered in the research.Considering human biomechanics, the main subject of investigation is the knee joint; therefore, over the years several studies with different animal models have been done to better understand its anatomy and biomechanics.On the other hand, only a few articles dealing with other anatomical sites were found, and these will be discussed in a specific subsection.

Animal models for knee joint
Knee joint ligaments injuries are one of the most widespread lesions; for this reason, several animal models have been widely employed to better understand the anatomy and biomechanics.Numerous studies dealing with knee ligament reconstruction via suture patterns, graft, or Ligament Advanced Reinforcement System (LARS) used animal specimens to perform tests, especially bovine (Eleswarapu et al. 2011), rabbit (Woo et al. 1992), rat (Yiannakopoulos et al. 2005), sheep (Weiler et al. 2001;Viateau et al. 2013), swine (Kim et al. 2014), and monkey (Noyes and Grood 1976).To the best of our knowledge, the study carried out by Noyes and Grood (Noyes and Grood 1976) is the only one in the literature that deals with a nonquadruped animal model, and the authors reported similar results with respect to the canine model.
The anterior cruciate ligament (ACL) is critical for knee joint stability in humans and animals, and its injury results in joint instability rapidly causing osteoarthritis (Comerford et al. 2005).The canine knee model is largely used to make studies on knee ligaments and tendons due to its similarity with its human counterpart (Beynnon et al. 1994).
The sheep stifle joint has often been used as an animal model for human ACL reconstruction.However, Radford et al. (1996), showed that the ovine stifle is not suitable for testing full-size human clinical ACL implants.The reason for this statement is that when compared to human joints the overall shape of the distal femur is narrower, and the femoral condyles do not have extensive articular surfaces distally.Thus, the range of motion of the stifle is not adapted for taking loads in full extension and cannot attain a straight-leg posture (Radford et al. 1996).
Moreover, it was concluded that the stifle joint of the sheep is both morphologically and biomechanically similar to the human knee, but there are detailed differences relating to ligament's fibres geometry.In conclusion, the authors reported that the ovine stifle is a valid animal model for experimental work on menisci and cruciate ligaments (Radford et al. 1996).
The rabbit knee has often been used as an animal model for the study of cruciate ligaments (posterior cruciate ligament (PCL) and ACL) and collateral ligaments (medial collateral ligament (MCL) and the lateral collateral ligament (LCL)).It is well accepted in the orthopaedic community that unrepaired injuries to either cruciate ligament will eventually result in chronic secondary degenerative joint changes, most notably in the menisci and in the articular cartilage.Few studies have been proposed to analyse the pathological consequences of cruciate ligament ruptures in the medial and collateral ligaments.Among them, Tozilli and Arnoczky (Tozilli and Arnoczky 1988) have not found significant changes in the biomechanical properties of rabbit LCL after a complete section of the anterior and posterior cruciate ligaments.
Another knee ligament involved in common trauma is the MCL; therefore, it is of great importance to find suitable animal surrogates.A relevant case study was conducted by Germscheid et al. (2011), in which was reported that porcine MCL is comparable in shape and size and in its failure mechanism to the adult human MCL.

Other animal models
Animal models are often used also to investigate causes and consequences of human diseases on the related ligaments.For example, a frequent trauma highly explored is the chronic neck pain caused by whiplash; in this context, several tensile failure studies (Lee et al. 2006;Quinn and Winkelstein 2007) of the C6/C7 rat cervical facet capsular ligament have been conducted to better understand the whiplash-related pain.Other studies were also conducted to better understand pelvic floor disorders that often result on permanent compromission of pelvic ligaments, affecting millions of women every year.The pelvic anatomy of the Macaca species is approximately identical to that of the human, providing a unique opportunity to study pelvic supportive ligaments (Vardy et al. 2005) and related mechanical and structural changes after injuries.Studied on nonquadruped animals which have a certain relevance, since they have a posture and joint range of motion more similar to that of humans.Unfortunately, in our research work, only one study on non-quadrupeds animals met the eligibility criteria and therefore was considered worthy of being reviewed.The results obtained are interesting, and comparisons with human ligaments have been performed in paragraph 4.1.1.

Effects of experimental setup parameters
First of all, it is necessary to specify that to characterise the ligaments and evaluate the integrity of the tissues after surgical repair, uniaxial tensile tests are generally carried out on the bone-ligament-bone (blb) complexes rather than on the single, isolated ligament.This procedure is preferred due to the limited sizes of the single ligament and its slipperiness at the anchor points with the clamps.The bone provides a secure hold on clamps during in-vitro testing.In contrast, the blb complex has one drawback: often the break occurs near the insertions (avulsion) instead of the expected "midsubstance failure" (Sample 2017;Martin et al. 2015).
Due to the variability in the ligament's mechanical properties introduced by the animal species, age, sex, testing conditions, tensile testing device and orientation of the ligaments or blb complexes in relation to the imposed stress, it is crucial to standardise a protocol to obtain data easily comparable with each other (Beynnon and Amis 1998).In this systematic review, wherever available, these parameters are always reported for completeness and proper comparison of the results.Nevertheless, this investigation of the existing scientific literature highlighted the lack of a commonly accepted standard.This point will be addressed in a dedicated section.
For example, there has been much discussion on the influence that the storage of the samples could have on the mechanical properties of the specimens.The debate is still open, but it seems that freezing up to three months does not significantly modify the structural and mechanical properties of the samples, as proven by Woo and colleagues (Woo et al. 1986), studying the influence of conservation on rabbit MCL ligaments (Martin et al. 2015;Beynnon and Amis 1998).In fact, in the main part of the experimental studies reported in this review, the specimens were kept at low temperature (freezing) and defrosted shortly before the actual test.Generally, specimens were maintained hydrated in solution during tests.For the conservation of the specimens, a physiological solution is commonly used, but also the phosphate buffered saline and Ringer's solution are usable (Martin et al. 2015).
The aim of this study is to analyse the setup parameters used during the experimental tests.In particular, two main factors influence the mechanical response: (i) the strain rate and displacement rate values set during the test and (ii) the preconditioning before the test.These aspects will be discussed in detail in the rest of the paper.

Difference between human and animals knee biomechanics
The substantial impact of knee ligaments injury, such as ACL, PCL, and collateral ligaments, has generated a big research field, thus allowing to explore their mechanisms of injury and the development of new treatment strategies.
In fact, several large animal models are commonly used to study knee ligaments repair mechanisms, but no species is currently considered as the gold standard.However, each animal model has limitations, which should be carefully considered.Regarding the human ACL, it is well known that is anatomically divided into three bundles: the anteromedial (AM), intermediate (IM), and posterolateral (PL), each of them performing different functions within the knee joint.
Other animal species as dog and goat ACL have only two bundles, rabbit ACL has not bundles, and only pig and goat ACL have three bundles (Bascuñán et al. 2019).Furthermore, biomechanical studies on the human ACL have shown that different bundles of ligaments have opposite behaviour during knee joint extension and flexion.Nevertheless, no animal ACL presents that mechanical behaviour in different portions (Bascuñán et al. 2019).Goat and swine appear to be a valid surrogate of ACL, since they present the greatest similarities with human ones (Bascuñán et al. 2019).
Another aspect to consider when experimental studies on knee animal models are designed is the difference in the mechanical properties of the knee ligaments at different angles of work.Wingfield et al. (2000) analysed the influence of two different knee angles in the mechanical properties of dog CraCl.However, no significant difference in the mechanical properties was found, but it is well known that cruciate ligaments in humans are influenced by the knee angle.Further studies need to evaluate more precisely this aspect.

Eligibility criteria
The primary aim of this review is a systematic revision of the scientific literature reporting tensile-testing mechanical properties of healthy ligaments in different animal species (bovine, dog, equine, monkey, mouse, ovine, rabbit, rat, swine).The mechanical properties were collected to compare the mechanical behaviour and identify the most suitable animal model.
In the cases where the data were expressed in units of measures that did not belong to SI units, they were converted into the corresponding SI units.Furthermore, to improve data accuracy, the expression of these properties as mean value ± standard deviation (SD) was required.All articles that presented the following characteristics were excluded: (i) results of the tensile test represented only in a graphic form, expressed only as mean without standard deviation, percentage, or range of values; (ii) studies on pathological or damaged ligaments only; (iii) study conducted on ligaments harvested from paediatric or elderly patients; (iv) studies evaluating the healing process of injured ligaments through the insertion of allografts or autografts or that included the use of different kinds of scaffolds or growth factors; (v) studies that report only compression and shear stress values and viscoelastic properties of the specimens; (vi) studies with data derived from finite element models; (vii) studies that perform biaxial test.

Information sources and search
The main databases were PubMed, Google Scholar, Science Direct, Springer, Taylor and Francis, Wiley-Blackwell, and PicoPolito (Politecnico di Torino search engine).The keywords used to find the articles in the primary research were: "ligaments", "animal ligaments", "human ligaments", "biomechanics", "mechanical characterisation", "mechanical properties", "structural properties", "stress-strain", "tensile test", "failure test", "strain rate", "Young's modulus", "ultimate tensile stress", and "ultimate strain".All the collected data were exported to Microsoft Excel and analysed.The research was conducted by four authors (S.C., M.M., F.S., and G.S.) working independently, each of them investigating one-quarter of the number of articles analysed and then reviewing them together one by one over three months.This study was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) method.

Data items
Specifically, the following mechanical properties were considered: elastic modulus or Young's modulus (MPa), stiffness ( N mm −1 ), maximal load (N), ultimate tensile stress (MPa), ultimate strain (%), and energy absorbed at failure ( N mm ).Additionally, regarding the experimental setup of the tensile tests, the preconditioning application, the strain rate ( % min −1 ), and the displacement rate ( mm min −1 ) values set for the tests were reported.

Additional analysis
In order to evaluate all the aspects related to the experimental tensile tests, the two methodologies that are employed to perform the tests were considered: "strain-controlled mode" and "displacement-controlled mode".The information about the control mode adopted by various authors during tensile tests was reported with the relative values of strain rate, where "SCM" stands for "strain controlled mode" and "DCM" stands for "displacement-controlled mode".
Additionally, the type of preconditioning used for the tests was reported and evaluated to give some guidelines in the results section.

Study selection
The initial research of peer-reviewed articles published in the selected databases using the mentioned keywords includes more than 2000 manuscripts.Then, the title and abstracts were analysed to include the papers and 263 manuscripts for the full-text evaluation were selected.Following the eligibility criteria, 95 articles were evaluated to obtain values of the mechanical properties (Fig. 1).

Comparison between the mechanical properties of animal and human ligaments
All the collected data reported in the previous tables were organised in different bar graphs.Each bar in the graphs represents the range of values assumed by a specific mechanical property analysed; the bar is delimited by the standard deviation (STD) values centred on the mean value of the data considered.In certain cases, the same reference provides several bars with different values because, in the same article, animals of different breeds, different sexes, different ages, or right/left limbs were studied.As a result, different values were obtained in the same article, although the type of sample preparation and strain/displacement rate were the same.
All the data reported in the previous tables were organised in different bar graphs.The elastic modulus, the ultimate tensile stress, and the ultimate strain report the strain rate in mm/min (Figs. 2, 3 and 4) and in %/min (Figs. 5, 6 and 7).For standardisation, values reported in mm/min and in cm/ min have been modified to obtain values in mm/s.Data that did not report the strain rate values were not used for graphing and analysis.During the evaluation of all the articles related to rabbit ligaments, different MCL elastic modulus values were found.In particular, the article of Xie et al. (Xie et al. 2021) shows an MCl elastic modulus equal to 3 GPa, a greater value compared to the other articles.The high variability in the results may be due to the experimental setup, since they used a tension-torsion combined testing machine.Given that the elastic modulus value obtained by Xie et al. appears to be an outlier, this study was removed from our evaluation.
For better data visualisation and comparison of the mechanical properties of the ligaments between different animal species and the human, each species was associated with a specific colour: bovine (blue), dog (light blue), equine (green), monkey (light green), goat (yellow), sheep (orange), rabbit (red), rat (fuchsia).Regarding the mechanical properties of the human ligaments, grey was chosen.

Results of mechanical property evaluation in mm/min
See Figs. 2, 3 and 4.
New Zealand (male, 3.  na measured as the slope of the stress-strain curve between 4.0 and 6.5% strain; z Young's modulus measured as the slope of the stress-strain curve between 2 and 4% strain; aa Young's modulus measured as the slope of the stress-strain curve between 3 and 5% strain; ab Young's modulus obtained from the unloading stage; ac Young's modulus calculated at 10-20% strain of the stress-strain curve; ad Young's modulus calculated at 2-6% strain of the stress-strain curve; ae Young's modulus calculated at 4-8% strain of the stress-strain curve; af Young's modulus is the slope of the linear regression between 15 and 65% of the failure stress on the failure stress-strain curve, ag Young's modulus at a strain range of 0.04-0.10;ah Young's modulus referred to DIC, at 20 MPa stress; ai Young's modulus referred to grip-to-grip at 20 MPa stress; aj Young's modulus referred to DIC, at 3% strain; ak Young's modulus referred to grip-to-grip at 3% strain; al Young's modulus calculated by considering only the stress-strain data in the interval (: strain at the Ultimate Tensile Strength (UTS))

Results of mechanical property evaluation in %/ min
See Figs. 5, 6 and 7.

Results of additional analysis-type of preconditioning
Table 5 reports the preconditioning that has been performed for different animal species and human.

Discussion
The mechanical properties evaluation of animal's and human's ligaments obtained from literature was performed in this review, considering the strain rate with two different units (mm/min and %/min).The analysis only dealt with the comparison between human and animal ligaments; thus, no comparison was performed among the mechanical properties of animal ligaments.From the analysis of the bar graphs, it was observed that generally, for each species, the values of the mechanical properties are included in a specific range.
In particular, there is evidence that the value of strain rate has an effect on the mechanical properties of the ligaments (Pioletti et al. 1999).Differences in specimen behaviour at high and low strain rate values were shown in several papers.For instance, (Woo et al. 1990a) showed that the  and 7).In other cases, for the same strain rate values, some mechanical properties show very different value as data obtained for rabbit MCL, v = 10 mm/min (Weiss et al. 1991) for elastic modulus (Fig. 2).Before the evaluation of the similarity between human ligaments and animal ligaments, it is important to specify that two different types of overlapping were found.The partial similarity means an overlapping between data, but the animal ligament shows a range of values that exceed human ligament values range.On the other hand, total similarity means that the animal ligaments show a range of values that is within the human ligament values range.The partial and total similarity between human and animal ligaments is reported in Appendix 1 and 2 in Supplementary material.Only the total similarity for all the parameters evaluated in this work is discussed in the following subsection, additionally, the percentage of overlap between the animal species and human ligament range was reported (%, of overlap between the distributions considered as the overlap with respect to the human values range).

Evaluation of mechanical property in mm/min
Analysing the mechanical parameters obtained with a strain rate in mm/min (as reported in Figs. 2, 3 and 4), it can be observed that: • Human AL (Zens et al. 2015) has a partial similarity for each animal ligament in terms of elastic modulus and ultimate stress.It has a verified total similarity of 38,7% in terms of ultimate strain with dog CraCL (Wingfield et al. 2000).

Evaluation of mechanical property in %/min
Analysing the mechanical parameters obtained with a strain rate in %/min in Figs. 5, 6 and 7, it can be observed that: • Human ACL (Noyes and Grood 1976) has no similarities for elastic modulus.For the ultimate stress, there are only partial similarities with calf CauCL, LCL and MCL (Eleswarapu et al. 2011).• Human ACL (Chandrashekar et al. 2006) has only partial similarities for elastic modulus and ultimate stress.Instead, for the ultimate strain, there are total similarities with goat ACL (Jackson et al. 1991) between 25% and 33.33%.• Human anterolater PCL (Race and Amis 1994) presents total similarities in terms of elastic modulus with monkey ACL (Noyes and Grood 1976) of 21.82% and goat ACL (Jackson et al. 1993) of 31.09%.For the ultimate stress, there is total similarities with swine LCL (Bonner et al. 2015) of 72.36%.For the ultimate strain, there is a total similarity with swine LCL (Bonner et al. 2015) of 56.60%.(Quapp and Weiss 1997) presents only partial similarities for elastic modulus and ultimate strain.• Human MCL (transverse) (Quapp and Weiss 1997) presents total similarities in terms of elastic modulus with cow PL (Oskui et al. 2016) at different strain rate values, 0.28% (600%/min), 7.56% (6000%/min), and 9.80% (60,000%/min).• Human MPFL (Criscenti et al. 2016) presents total similarity in terms of ultimate stress with calf MCL (Eleswarapu et al. 2011).For ultimate strain, there is a total similarity with goat ACL (Jackson et al. 1991) of 29.48%.• Human ALL (strain rate of 3000%/min) (Mattucci et al. 2012) presents a total similarity in terms of ultimate strain with cow PL (Oskui et al. 2016) at strain rate of 6000%/min and 60,000%/min of 6.12%.• Human ALL (strain rate of 12,000%/min) (Mattucci et al. 2012) presents only partial similarities for ultimate stress.• Human ALL (strain rate of 900,000%/min) (Mattucci et al. 2012) presents only partial similarities for ultimate stress.

Type of preconditioning
The preconditioning consists typically of 10/20 cycles of loading/unloading until a certain value or inside an interval of tension or deformation.As can be seen in Table 5, in the majority of the reviewed articles, the specimens underwent preconditioning by 10 cycles of approximately 0-5% strain (Shetye et al. 2009, Woo et al. 1990b, Ng et al. 1995, Woo et al. 1992, Danto and Woo 1993, Murao et al. 1997, Ma et al. 2009, Moon et al. 2006, Kim et al. 2014, Wilson et al. 2012, Quapp and Weiss 1997, Criscenti et al. 2016, Kusayama et al. 1994, Hewitt et al. 2002, Schleifenbaum et al. 2016, Moore et al. 2004and Moore et al. 2005) or around 50 N (Becker et al. 1994, Viateau et al. 2013, Wijdicks et al. 2010and Robinson et al. 2005).It is also possible to observe that in many cases (Diotalevi et al. 2018, Woo et al. 1990b, Abramowitch et al. 2003, Weiler et al. 2004, Woo et al. 1992, Panjabi et al. 1996, Woo et al. 1986, Weiss et al. 1991, Xie et al. 2021, Kim et al. 2014, Tan et al. 2015and Schleifenbaum et al. 2016) the loading/unloading cycles are performed at the same strain rate used during the tensile tests.Lastly, it can be said that the type of preconditioning varies with different ligaments in various animal species and human specimens.In fact, it is important to point out that in general there is no standardisation in terms of the number of cycles and the value of deformation or tension at which the preconditioning is performed.

Limitations
The individuation from the existing scientific literature of the most suitable surrogate to imitate the behaviour of human ligaments is hampered by several inhomogeneities in the experimental test protocol.This study also did not consider parameters such as animal age, sex, and lifetime activity.These parameters may influence the biomechanical characteristics of soft tissues.Additionally, the comparison of ligaments should be conducted by evaluating their composition.Future studies should compare the influence of these parameters on the mechanical properties of animal and human tendons, which would lead to a more accurate assessment of the ligament to be used for ex vivo testing.Moreover, here the mechanical properties of knee animals and human ligaments were reported evaluating only a uniaxial tensile test condition.Further studies will be needed to analyse their mechanical behaviour at different angles.

Conclusions
This systematic review aimed at defining the most suitable surrogates for mimicking the behaviour of human ligaments when subjected to uniaxial tensile tests.For this reason, the scientific literature was reviewed, evaluating the experimental studies involving the mechanical properties of animal ligaments.Differences and similarities between human and animal ligaments were highlighted and commented upon and the best candidates were determined and discussed.The comparison between the mechanical properties of animal ligaments highlighted how they cannot always be compared with their human counterparts; on the other hand, there are many similarities between different anatomical parts.In general, no specific animal ligaments can provide a suitable model for its respective human counterpart concerning all the three primary mechanical properties (Young modulus, ultimate tensile stress, and ultimate tensile strain) at the same strain rate.It is interesting to note that in the current scientific literature, different animal models (bovine, dog, rabbit, and swine) were adopted to evaluate the knee repair technologies; nevertheless, despite this wide use, no clear similarities were found in their mechanical properties.Further studies will be needed to further compare the mechanical properties of these ligaments and ensure that the scientific evidence derived from such experimental studies can be considered reliable.Several similarities were observed in some properties between animal and human ligaments.These similarities were found despite the ligaments having been analysed at different strain rates.The results showed similarities between animal and human ligaments that should be considered in the evaluation of scaffolds and sutures.
Considering the results reported for tests performed in mm/min: • Swine CL with a displacement rate of 45 mm/min is comparable (total similarity in terms of elastic modulus, ultimate tensile stress and ultimate strain) with human AB-IGHL with a displacement rate of 10 mm/min; It's important mentioning that monkey RL with a displacement rate of 6 mm/min has a partial similarity with human RL with a displacement rate of 5 mm/min for elastic modulus and ultimate tensile stress.This result should be further analysed in future works.
Considering the results reported for tests performed in %/min: • Swine LCL with a strain rate of 60%/min is comparable (total similarity in terms of ultimate stress and ultimate strain but not for elastic modulus) with human anterolateral PLL with a strain rate of 12,000%/min; • Swine LCL with a strain rate of 6o %/min and 600%/ min are comparable (total similarity in terms of ultimate stress and ultimate strain but not for elastic modulus) with human anterolateral PCL with a strain rate of 3000%/min; • Swine LCL with a strain rate of 6o %/min is comparable (total similarity in terms of ultimate stress and ultimate strain but not for elastic modulus) with human PLL with a strain rate of 12,000%/min; • Cow PL with a strain rate of 6o %/min and 600% is comparable (total similarity in terms of elastic modulus and ultimate stress but not for ultimate strain) with human CL with a strain rate of 3000%/min.Moreover, the cow PL at different strain rate shows some partial similarities with human CL with a strain rate of 900,000%/min; • Cow PL with a different strain rate is comparable (total similarity in terms of elastic modulus and ultimate stress but not for ultimate strain) with human ISL with strain rates of 3000%/min and 12,000%/min.The human ISL (3000%/min and 12,000%/min) shows some partial similarities with calf CauCL for elastic modulus and ultimate stress.Moreover, increasing the strain rate, some partial similarities with cow PL remain.
In our previous review, similarities between human, swine, equine, rabbit, rat, and goat tendons were found and discussed in detail.Here, the analysis of the mechanical properties for human and animal ligaments reported similarities between human and swine, cow, and rat ones.Comparing these two reviews, it can be stated that there are similarities between the mechanical properties of human and animals' tendons and ligaments.In particular, the species with most similarities for both tendons and ligaments are swine and rat.These results may pave the way for future works.
As a concluding remark, it seems highly probable that the choice of parameter setting significantly affects the results of the experimental studies reviewed and discussed here.Unfortunately, different authors reported their results with different settings.The lack of standard test settings (strain rate, pre-conditioning) for the experiments should be considered when interpreting the results reported in the scientific literature.Future studies will be needed to evaluate ligaments from different animals and anatomical regions with the same test conditions and strain rate, in a fully comparable way.Based on the evaluation of mechanical characterisation of ligaments analysed in this work, the authors thought the following suggestions for best practices.After the tendon extraction from the anatomical site, it is important to use the same protocol for each of them.It is advisable to not perform the test on frozen samples.However, in case of frozen samples, the defrosting process should be done at least 24 h before the tests.Furthermore, before the test, the specimens' thickness and width should be measured.These measurements can be done either in a normal condition or with a preload.The preload value should be evaluated based on the literature information; if no data are available, the preload should not exceed 10 Newton.Of course, all the parameters used for the test should be fully reported in the article and defined after an evaluation of the literature on the specific tissue.Based on this review, the standard preconditioning for ligaments should be 20 cycles at 1%/s of strain rate (starting from the preload force).Finally, the range where Young's modulus was calculated should be reported in the article.

Fig. 1
Fig. 1 Workflow followed to identify, exclude and select the articles

Fig. 7
Fig. 7 Ultimate strain for the considered animal species (%/min) 0.0 and 0.3 mm extension (approximately 0 and 3% strain of the mid-substance of the ligament) at a rate of 10 mm • min −1 Woo et al. (1992) ACL 10 cycles from 0.0 to 0.3 mm elongation (approximately 0-3% strain of the mid-substance of the ligament), at a rate of 0.2 mm • s −1 Danto and Woo (1993) ACL 21 cycles of stretching between 0 and 0.5 mm (approximately 5% strain) at 1 mm • s −1 and on the 22nd cycle stretched until failure Panjabi et al. (1996) PCL 10 cycles between 0 and 0.5 mm deformation at a rate of 10 mm • min −1 Murao et al. (1997) PCL 10 cycles between 0 and 0.5 mm deformation at a rate of 10 mm • min −1 Ma et al. (2009) LCL 3 cycles were performed by slowly cycling the ligament from its unloaded state just into the linear portion of its load-deformation response and then back to zero load Tozilli and Arnoczky (1988) MCL 10 cycles of loading-unloading to 1 mm of elongation at a rate of 1 c m • min −1 Woo et al. (1986) MCL 10 cycles of loading-unloading to 1 mm of elongation Woo et al. (1990a) MCL stretching the FMTC 10 times to the in situ strain level previously determined for each MCL specimen, at an elongation rate of 1 cm/ min Weiss et al. (1991) MCL 10 cycles between 0.0 and 0.5 mm extension (approximately 0 and 3% strain of the MCL substance, respectively) at an extension rate of 10 mm • min −1 Woo et al. (1992) MCL 10 cycles of between 0 and 1.5 mm of elongation Moon et al. (2006) MCL 5 min of a static preload of 0.5 N and then the maximum load was loaded and unloaded at a rate of 5 mm • min −1 at 0.5% of the maximum load 20 times Xie et al. (2021) Rat MCL 5 cycles of load as low as the cyclic stretching and then stretched to failure immediately Su et al. (2008) Thoracic FCL 30 cycles to 0.1 mm at 0.05 mm • s −1 Freedman et al. (2012)Cervical FCL 30 cycles to 0.2 mm (approximately 5% of load at gross failure)Quinn and Winkelstein (2007) 1 and 10 N at 10 mm • min −1 , and repeated five times, then held at 0 N for 10 s Bonner et al. (2015) MCL 2 cycles from − 20 N to + 8 N at 1 mm • min −1 Germscheid et al. (2011) MPFL 10 cycles of cyclic tension between 0 and 2 mm at an extension rate of 10 mm • min −1 Kim et al. (2014) CL 5 cycles from 0.25 to 1.0 N at 0.75 mm • s −1 Tan et al. (2015) Human ACL, PCL, LCL, MCL 5 cycles to an intermediate load (approx.147 N) at a strain rate of 5cm • min −1 Trent et al. (1976) ACL 20 cycles between 25 and 150 N tension at 0.25 Hz Chandrashekar et al. (2006) LCL, PFL Several cycles by slowly cycling the specimens from an unloaded state to the linear portion of their load deformation curve and back to zero load LaPrade et al. (2005) LCL 5 loading cycles to a maximum load of 35 to 50 N tension at 0.5 Hz Ciccone et al. (2006) LCL, MCL 10 cycles to a nominal 2 N and then to 3.5% strain at 1 Hz Wilson et al. (2012) MCL 10 cycles to a maximum amplitude of 0.5 mm at a rate of 10 mm • min −1 Quapp and Weiss (1997) MCL, POL 10 cycles of 10 N to 50 N tension at 0.1 Hz Wijdicks et al. (2010) MCL 10 cycles between 1 and 40 N tension at a crosshead speed of 10 mm • min −1 Robinson et al. (2005) MPFL 10 cycles to 3% of strain at a strain rate of 0.1%•s −1 Criscenti et al. (2016) MFL 10 cycles of 0-2 mm extension at a crosshead speed of 20 mm • min −1 Kusayama et al. (1994) MFL 10 load cycles resulting in 2 mm of extension at 20 mm • min −1 Gupte et al. (2002a) SHIL, IHIL, IS, FAL 10 cycles of loading to 5% strain Hewitt et al. (2002) IL, IS, PF crosshead displacement of 20 mm • min −1 and a maximum strain of 5% Schleifenbaum et al. (2016) ALL, PLL, CL, LF, ISL 20 cycles of loading to 10% strain at a frequency of approximately 1 Hz Mattucci et al. 2012) LF 5 load cycles were applied (from the unloaded condition) up to 9.8 N and subsequently to 19.6 N Nachemson and Evans (1968) IGHL 10 cycles 1-2 mm at 50 mm • min −1 Lee et al. (1999) AB-IGHL 10 cycles between elongation limits of 0-0.3 mm at a rate of 10 mm • min −1 Moore et al. (2004) PB-IGHL 10 cycles between elongation limits of 0-0.3 mm at a rate of 10 mm • min −1 Moore et al. (2005)Scapholunate Ligament 25 times to 15% of their initial lengths at a rate of 66% of the initial lengths at a rate of 200 HzJohnston et al. (2004)

Table 1
All the selected articles are grouped by animal species and human

Table 2
Acronyms list to indicate the ligaments quoted in this review

Table 3
Mechanical properties of animal ligaments, grouped by species.'na' indicates unavailable data

Table 4
Mechanical properties of human ligaments.'na' indicates unavailable data

Table 5
Type of preconditioning divided by animal species and human.'na' indicates unavailable data

Table 5
• Swine USL with a displacement rate of 45 mm/min is comparable (total similarity in terms of elastic modulus and ultimate strain but not for ultimate stress) with human PB-IGHL with a displacement rate of 10 mm/ min; • Swine ACL and posterolateral ACL with a displacement rate of 19.8 mm/min are comparable (total similarity in terms of elastic modulus and ultimate strain but not for ultimate stress) with human posteromedial PCL with a displacement rate of 1000 mm/min; • Rat MCL with a displacement rate of 30 mm/min is comparable (total similarity in terms of elastic modulus and ultimate stress but not for ultimate strain) with human posteromedial PCL with a displacement rate of 1000 mm/min; • Swine PCL with a displacement rate of 19.8 mm/min is comparable (total similarity in terms of elastic modulus and ultimate stress but not for ultimate strain) with human anterolateral PCL with a displacement rate of 1000 mm/min;