Paired versus two-group experimental design for rheological studies of vocal fold tissues

Vibratory function of the vocal folds is largely determined by the rheological properties or viscoelastic shear properties of the vocal fold lamina propria. To date, investigation of the sample size estimation and statistical experimental design for vocal fold rheological studies is nonexistent. The current work provides the closed-form sample size formulas for two major study designs (i.e. paired and two-group designs) in vocal fold research. Our results demonstrated that the paired design could greatly increase the statistical power compared to the two-group design. By comparing the variance of estimated treatment effect, this study also confirms that ignoring within-subject and within-vocal fold correlations during rheological data analysis will likely increase type I errors. Finally, viscoelastic shear properties of intact and scarred rabbit vocal fold lamina propria were measured and used to illustrate theoretical findings in a realistic scenario and project sample size requirement for future studies.     

Elastic energy storage in unmineralized and mineralized extracellular matrices (ECMs): a comparison between molecular modeling and experimental measurements

In order to facilitate locomotion and limb movement many animals store energy elastically in their tendons. In the turkey, much of the force generated by the gastrocnemius muscle is stored as elastic energy during tendon deformation and not within the muscle. As limbs move, the tendons are strained causing the collagen fibers in the extracellular matrices to be strained. During growth, avian tendons mineralize in the portions distal to the muscle and show increased tensile strength, modulus, and energy stored per unit strain as a result. In this study the energy stored in unmineralized and mineralized collagen fibers was measured and compared to the amount of energy stored in molecular models. Elastic energy storage values calculated using the molecular model were slightly higher than those obtained from collagen fibers, but display the same increases in slope as the fiber data. We hypothesize that these increases in slope are due to a change from the stretching of flexible regions of the collagen molecule to the stretching of less flexible regions. The elastic modulus obtained from the unmineralized molecular model correlates well with elastic moduli of unmineralized collagen from other studies. This study demonstrates the potential importance of molecular modeling in the design of new biomaterials.     

Collagen fibril diameter distribution does not reflect changes in the mechanical properties of in vitro stress-deprived tendons

The purpose of this study was to determine if an association exists between the tensile properties and the collagen fibril diameter distribution in in vitro stress-deprived rat tail tendons. Rat tail tendons were paired into two groups of 21 day stress-deprived and 0 time controls and compared using transmission electron microscopy (n = 6) to measure collagen fibril diameter distribution and density, and mechanical testing (n =6) to determine ultimate stress and tensile modulus. There was a statistically significant decrease in both ultimate tensile strength (control: 17.95+/-3.99 MPa, stress-deprived: 6.79+/-3.91 MPa) and tensile modulus (control: 312.8+/-89.5 MPa, stress-deprived: 176.0+/-52.7 MPa) in the in vitro stress-deprived tendons compared to controls. However, there was no significant difference between control and stress-deprived tendons in the number of fibrils per tendon counted, mean fibril diameter, mean fibril density, or fibril size distribution. The results of this study demonstrate that the decrease in mechanical properties observed in in vitro stress-deprived rat tail tendons is not correlated with the collagen fibril diameter distribution and, therefore, the collagen fibril diameter distribution does not, by itself, dictate the decrease in mechanical properties observed in in vitro stress-deprived rat tail tendons.     

Structure and function of tuna tail tendons

The caudal tendons in tunas and other scombrid fish link myotomal muscle directly to the caudal fin rays, and thus serve to transfer muscle power to the hydrofoil-like tail during swimming. These robust collagenous tendons have structural and mechanical similarity to tendons found in other vertebrates, notably the leg tendons of terrestrial mammals. Biochemical studies indicate that tuna tendon collagen is composed of the (alpha1)(2),alpha2 heterotrimer that is typical of vertebrate Type I collagen, while tuna skin collagen has the unusual alpha1,alpha2,alpha3 trimer previously described in the skin of some other teleost species. Tuna collagen, like that of other fish, has high solubility due to the presence of an acid-labile intermolecular cross-link. Unlike collagen in mammalian tendons, no differences related to cross-link maturation were detected among tendons in tuna ranging from 0.05 to 72 kg (approx. 0.25-6 years). Tendons excised post-mortem were subjected to load cycling to determine the modulus of elasticity and resilience (mean of 1.3 GPa and 90%, respectively). These material properties compare closely to those of leg tendons from adult mammals that can function as effective biological springs in terrestrial locomotion, but the breaking strength is substantially lower. Peak tendon forces recorded during steady swimming appear to impose strains of much less than 1% of tendon length, and no more than 1.5% during bursts. Thus, the caudal tendons in tunas do not appear to function as elastic storage elements, even at maximal swimming effort.     

Shear Elastic Modulus on Patellar Tendon Captured from Supersonic Shear Imaging: Correlation with Tangent Traction Modulus Computed from Material Testing System and Test–Retest Reliability

Characterization of the elastic properties of a tendon could enhance the diagnosis and treatment of tendon injuries. The purpose of this study was to examine the correlation between the shear elastic modulus on the patellar tendon captured from a Supersonic Shear Imaging (SSI) and the tangent traction modulus computed from a Material testing system (MTS) on 8 fresh patellar pig tendons (Experiment I). Test–retest reliability of the shear elastic modulus captured from the SSI was established in Experiment II on 22 patellar tendons of 11 healthy human subjects using the SSI. Spearman Correlation coefficients for the shear elastic modulus and tangent traction modulus ranged from 0.82 to 1.00 (all p<0.05) on the 8 tendons. The intra and inter-operator reliabilities were 0.98 (95% CI: 0.93–0.99) and 0.97 (95% CI: 0.93–0.98) respectively. The results from this study demonstrate that the shear elastic modulus of the patellar tendon measured by the SSI is related to the tangent traction modulus quantified by the MTS. The SSI shows good intra and inter-operator repeatability. Therefore, the present study shows that SSI can be used to assess elastic properties of a tendon.     

Cross-Linking in Collagen by Nonenzymatic Glycation Increases the Matrix Stiffness in Rabbit Achilles Tendon

Nonenzymatic glycation of connective tissue matrix proteins is a major contributor to the pathology of diabetes and aging. Previously the author and colleagues have shown that nonenzymatic glycation significantly enhances the matrix stability in the Achilles tendon (Reddy et al., 2002, Arch. Biochem. Biophys., 399, 174–180). The present study was designed to gain further insight into glycation-induced collagen cross-linking and its relationship to matrix stiffness in the rabbit Achilles tendon. The glycation process was initiated by incubating the Achilles tendons (n = 6) in phosphate-buffered saline containing ribose, whereas control tendons (n = 6) were incubated in phosphate-buffered saline without ribose. Eight weeks following glycation, the biomechanical attributes as well as the degree of collagen cross-linking were determined to examine the potential associations between matrix stiffness and molecular properties of collagen. Compared to nonglycated tendons, the glycated tendons showed increased maximum load, stress, strain, Young''s modulus of elasticity, and toughness indicating that glycation increases the matrix stiffness in the tendons. Glycation of tendons resulted in a considerable decrease in soluble collagen content and a significant increase in insoluble collagen and pentosidine. Analysis of potential associations between the matrix stiffness and degree of collagen cross-linking showed that both insoluble collagen and pentosidine exhibited a significant positive correlation with the maximum load, stress, and strain, Young''s modulus of elasticity, and toughness (r values ranging from .61 to .94) in the Achilles tendons. However, the soluble collagen content present in neutral salt buffer, acetate buffer, and acetate buffer containing pepsin showed an inverse relation with the various biomechanical attributes tested (r values ranging from .22 to .84) in the Achilles tendons. The results of the study demonstrate that glycation-induced collagen cross-linking is directly associated with the increased matrix stiffness and other mechanical attributes of the tendon.     

Possible role of decorin glycosaminoglycans in fibril to fibril force transfer in relative mature tendons--a computational study from molecular to microstructural level

Experimental studies on immature tendons have shown that the collagen fibril net is discontinuous. Manifold evidences, despite not being conclusive, indicate that mature tissue is discontinuous as well. According to composite theory, there is no requirement that the fibrils should extend from one end of the tissue to the other; indeed, an interfibrillar matrix with a low elastic modulus would be sufficient to guarantee the mechanical properties of the tendon. Possible mechanisms for the stress-transfer involve the interfibrillar proteoglycans and can be related to the matrix shear stress and to electrostatic non-covalent forces. Recent studies have shown that the glycosaminoglycans (GAGs) bound to decorin act like bridges between contiguous fibrils connecting adjacent fibril every 64-68 nm; this architecture would suggest their possible role in providing the mechanical integrity of the tendon structure. The present paper investigates the ability of decorin GAGs to transfer forces between adjacent fibrils. In order to test this hypothesis the stiffness of chondroitin-6-sulphate, a typical GAG associated to decorin, has been evaluated through the molecular mechanics approach. The obtained GAG stiffness is piecewise linear with an initial plateau at low strains (<800%) and a high stiffness region (3.1 x 10(-11)N/nm) afterwards. By introducing the calculated GAG stiffness in a multi-fibril model, miming the relative mature tendon architecture, the stress-strain behaviour of the collagen fibre was determined. The fibre incremental elastic modulus obtained ranges between 100 and 475 MPa for strains between 2% and 6%. The elastic modulus value depends directly on the fibril length, diameter and inversely on the interfibrillar distance. In particular, according to the obtained results, the length of the fibril is likely to play the major role in determining stiffness in mature tendons.     

Functionally distinct tendons have different biomechanical,biochemical and histological responses to in vitro unloading

Tendons with different in vivo functions are known to have different baseline biomechanics, biochemistry and ultrastructure, and these can be affected by changes in loading. However it is not know whether different tendon types respond in the same, or different ways, to changes in loading.This study performed in vitro un-loading (stress deprivation) in culture on ovine medial extensor tendons (MET, a positional tendon), and superficial and deep digital flexor tendons (SDFTs and DDFTs, with energy-storing and intermediate functions respectively), for 21 days (n = 14 each). Tensile strength and elastic modulus were then measured, followed by biochemical assays for sulphated glycosaminoglycan (sGAG) and hydroxyproline content. Histological inspection for cell morphology, cell density and collagen alignment was also performed.The positional tendon (MET) had a significant reduction (∼50%) in modulus and strength (P < 0.001) after in vitro stress-deprivation, however there were no significant effects on the energy-storing tendons (SDFT and DDFT). In contrast, sGAG was not affected in the MET, but was reduced in the SDFT and DDFT (P < 0.001). All tendons lost compactness and collagen organisation, and had reduced cell density, but these were more rapid in the MET than the SDFT and DDFT.These results suggest that different tendon types respond to identical stimuli in different ways, thus; (i) the results from an experiment in one tendon type may not be as applicable to other tendon types as previously thought, (ii) positional tendons may be particularly vulnerable to clinical stress-deprivation, and (iii) graft tendon source may affect the biological response to loading in ligament and tendon reconstruction.     

Influence of decorin on the mechanical, compositional, and structural properties of the mouse patellar tendon

The interactions of small leucine-rich proteoglycans (SLRPs) with collagen fibrils, their association with water, and their role in fibrillogenesis suggests that SLRPs may play an important role in tendon mechanics. Some studies have assessed the role of SLRPs in the mechanical response of the tendon, but the relationships between sophisticated mechanics, assembly of collagen, and SLRPs have not been well characterized. Decorin content was varied in a dose dependent manner using decorin null, decorin heterozygote, and wild type mice. Quantitative measures of mechanical (tension and compression), compositional, and structural changes of the mouse patellar tendon were evaluated. Viscoelastic, tensile dynamic modulus was increased in the decorin heterozygous tendons compared to wild type. These tendons also had a significant decrease in total collagen and no structural changes compared to wild type. Decorin null tendons did not have any mechanical changes; however, a significant decrease in the average fibril diameter was found. No differences were seen between genotypes in elastic or compressive properties, and all tendons demonstrated viscoelastic mechanical dependence on strain rate and frequency. These results suggest that decorin, a member of the SLRP family, plays a role in tendon viscoelasticity that cannot be completely explained by its role in collagen fibrillogenesis. In addition, reductions in decorin do not cause large changes in indentation compressive properties, suggesting that other factors contribute to these properties. Understanding these relationships may ultimately help guide development of tissue engineered constructs or treatment modalities.     

Blood variables in adult stumptail macaques (Macaca arctoides) living in a captive group; annual variability

Monthly blood samples were collected for more than two years from adult stumptail macaques living in a large captive group. The social organization, feeding pattern, and food composition were stable throughout the study period. No seasonal variability was observed for any of the 30 blood variables studied. It appeared that for each variable, within-animal variance was small relative to between-animal variance. A table of means and reference ranges is presented and discussed.     

Fracture Mechanics of Collagen Fibrils: Influence of Natural Cross-Links

Tendons are important load-bearing structures, which are frequently injured in both sports and work. Type I collagen fibrils are the primary components of tendons and carry most of the mechanical loads experienced by the tissue, however, knowledge of how load is transmitted between and within fibrils is limited. The presence of covalent enzymatic cross-links between collagen molecules is an important factor that has been shown to influence mechanical behavior of the tendons. To improve our understanding of how molecular bonds translate into tendon mechanics, we used an atomic force microscopy technique to measure the mechanical behavior of individual collagen fibrils loaded to failure. Fibrils from human patellar tendons, rat-tail tendons (RTTs), NaBH₄ reduced RTTs, and tail tendons of Zucker diabetic fat rats were tested. We found a characteristic three-phase stress-strain behavior in the human collagen fibrils. There was an initial rise in modulus followed by a plateau with reduced modulus, which was finally followed by an even greater increase in stress and modulus before failure. The RTTs also displayed the initial increase and plateau phase, but the third region was virtually absent and the plateau continued until failure. The importance of cross-link lability was investigated by NaBH₄ reduction of the rat-tail fibrils, which did not alter their behavior. These findings shed light on the function of cross-links at the fibril level, but further studies will be required to establish the underlying mechanisms.     

Axial speed of sound for the monitoring of injured equine tendons: a preliminary study

Equine superficial digital flexor tendons (SDFT) are often injured, and they represent an excellent model for human sport tendinopathies. While lesions can be precisely diagnosed by clinical evaluation and ultrasonography, a prognosis is often difficult to establish; the knowledge of the injured tendon's mechanical properties would help in anticipating the outcome. The objectives of the present study were to compare the axial speed of sound (SOS) measured in vivo in normal and injured tendons and to investigate their relationship with the tendons' mechanical parameters, in order to assess the potential of quantitative axial ultrasound to monitor the healing of the injured tendons. SOS was measured in vivo in the right fore SDFTs of 12 horses during walk, before and 3.5 months after the surgical induction of a bilateral core lesion. The 12 horses were then euthanized, their SDFTs isolated and tested in tension to measure their elastic modulus and maximal load (and corresponding stress). SOS significantly decreased from 2179.4 ± 31.4 m/s in normal tendons to 2065.8 ± 67.1 m/s 3.5 months after the surgical induction, and the tendons' elastic modulus (0.90 ± 0.17 GPa) was found lower than what has been reported in normal tendons. While SOS was not correlated to tendon maximal load and corresponding stress, the SOS normalized on its value in normal tendons was correlated to the tendons' elastic modulus. These preliminary results confirm the potential of axial SOS in helping the functional assessment of injured tendon.     

A model for the stability and creep of organic materials

A model is presented for the thermally assisted breaking of a number of bonds arranged in parallel and stressed by an individual soft spring each. Using a simplified potential for the bond it is shown that in equilibrium there are two definite regions of elastic behavior: one with all bonds intact, the other with a variable fraction of bonds broken, therefore with a tangent modulus steadily decreasing with applied stress. Criteria are given for the existence of these regions. Beyond these regions time-dependent creep to rupture is found, limited, in turn, by the theoretical fracture strength, the stress necessary for fracture without any thermal assistance, beyond which a bound state is impossible. The time-to-fracture for creep rupture is calculated and an example of the time evolution of the accelerating creep given. The results of the calculations are applied to experimental data on Wallaby tendons by Wang and Ker (J. Exp. Biol. 198 (1995) 831) and data estimated for the bond potential depth, the theoretical fracture strength and the number density of bonds involved as well as the elastic modulus of the ensemble. Values are derived under the assumption of one deformation mechanism being dominant--e.g., (sub-)fibril sliding or sliding of collagen molecules along one another--but the model cannot definitely distinguish between mechanisms.     

Muscle Function in vivo: A Comparison of Muscles used for Elastic Energy Savings versus Muscles Used to Generate Mechanical Power1

SYNOPSIS. The function of muscles used to generate force economicallyand facilitate elastic energy savings in their tendons is comparedwith muscles that function to produce mechanical power. Theunderlying architectural design of the muscle and its tendon(if present) dictate much of their functional capacity and rolein animal locomotion. Using methods that allow direct recordingsof muscle force and fiber length change, the functional designof muscle-tendon systems can now be investigated in vivo. Thesestudies reveal that, in the case of wallaby hindleg muscles,the fibers can maintain sufficient stiffness during tendon stretchand recoil to ensure useful elastic energy recovery and savingsof metabolic energy. In the case of the pectoralis muscle ofpigeons, although isometric or active lengthening of the muscle'sfibers may occur late in the upstroke of the wing beat cycleto enhance force development, the fibers shorten extensivelyduring the downstroke (up to 35% of their resting length) toproduce mechanical power for aerodynamic lift and thrust. Oscillatorylength change, with force enhancement during active lengtheningmay be a general feature of muscles that power aerial and aquaticlocomotion. Similarly, force enhancement by active lengtheningis likely to be important to the design and function of musclesthat primarily generate force to minimize energy expenditure/unitforce generated, as well as for elastic energy savings withina long tendon. Architectural features of muscle-tendon unitsfor effective elastic energy savings, however, are likely toconstrain locomotor performance when mechanical work is required,as when an animal accelerates, either limiting performance orrequiring the recruitment of functional agonists with greatermechanical power generating capability (i.e., longer fibers)     

Elastic energy storage in an unmineralized collagen type I molecular model with explicit solvation and water infiltration

Collagen type I is a structural protein that provides tensile strength to tendons and ligaments. Type I collagen molecules form collagen fibers, which are viscoelastic and can therefore store energy elastically via molecular elongation and dissipate viscous energy through molecular rearrangement and fibrillar slippage. The ability to store elastic energy is important for the resiliency of tendons and ligaments, which must be able to deform and revert to their initial lengths with changes in load.In an earlier paper by one of the present authors, molecular modeling was used to investigate the role of mineralization upon elastic energy storage in collagen type I. Their collagen model showed a similar trend to their experimental data but with an over-estimation of elastic energy storage. Their simulations were conducted in vacuum and employed a distance-dependent dielectric function. In this study, we performed a re-evaluation of Freeman and Silver's model data incorporating the effects of explicit solvation and water infiltration, in order to determine whether the model data could be improved with a more accurate representation of the solvent and osmotic effects. We observed an average decrease in the model's elastic energy storage of 45.1%±6.9% in closer proximity to Freeman and Silver's experimental data. This suggests that although the distance-dependent dielectric implicit solvation approach was favored for its increased speed and decreased computational requirements, an explicit representation of water may be necessary to more accurately model solvent interactions in this particular system. In this paper, we discuss the collagen model described by Freeman and Silver, the present model building approach, the application of the present model to that of Freeman and Silver, and additional assumptions and limitations.     

Tensile properties of calcified and uncalcified avian tendons

The mechanical properties of turkey and heron leg tendons have been investigated in dynamic tensile tests. Heron tendons have properties similar to those found for various mammalian tendons. The Young's modulus and the density of turkey tendons increase with increasing calcification. Ultimate tensile stresses are similar to those found for uncalcified tendon, but Young's modulus may reach about 16 GPa, a value normally associated with bone. Calcification lowers the amount of strain energy that can be stored temporarily in the tendons of the legs. The contribution made by elastic strain energy storage to lowering the cost of running is reduced.     

Effect of preconditioning and stress relaxation on local collagen fiber re-alignment: inhomogeneous properties of rat supraspinatus tendon

Repeatedly and consistently measuring the mechanical properties of tendon is important but presents a challenge. Preconditioning can provide tendons with a consistent loading history to make comparisons between groups from mechanical testing experiments. However, the specific mechanisms occurring during preconditioning are unknown. Previous studies have suggested that microstructural changes, such as collagen fiber re-alignment, may be a result of preconditioning. Local collagen fiber re-alignment is quantified throughout tensile mechanical testing using a testing system integrated with a polarized light setup, consisting of a backlight, 90 deg-offset rotating polarizer sheets on each side of the test sample, and a digital camera, in a rat supraspinatus tendon model, and corresponding mechanical properties are measured. Local circular variance values are compared throughout the mechanical test to determine if and where collagen fiber re-alignment occurred. The inhomogeneity of the tendon is examined by comparing local circular variance values, optical moduli and optical transition strain values. Although the largest amount of collagen fiber re-alignment was found during preconditioning, significant re-alignment was also demonstrated in the toe and linear regions of the mechanical test. No significant changes in re-alignment were seen during stress relaxation. The insertion site of the supraspinatus tendon demonstrated a lower linear modulus and a more disorganized collagen fiber distribution throughout all mechanical testing points compared to the tendon midsubstance. This study identified a correlation between collagen fiber re-alignment and preconditioning and suggests that collagen fiber re-alignment may be a potential mechanism of preconditioning and merits further investigation. In particular, the conditions necessary for collagen fibers to re-orient away from the direction of loading and the dependency of collagen reorganization on its initial distribution must be examined.     

Pulmonary artery relative area change is inversely related to ex vivo measured arterial elastic modulus in the canine model of acute pulmonary embolization

A low relative area change (RAC) of the proximal pulmonary artery (PA) over the cardiac cycle is a good predictor of mortality from right ventricular failure in patients with pulmonary hypertension (PH). The relationship between RAC and local mechanical properties of arteries, which are known to stiffen in acute and chronic PH, is not clear, however. In this study, we estimated elastic moduli of three PAs (MPA, LPA and RPA: main, left and right PAs) at the physiological state using mechanical testing data and correlated these estimated elastic moduli to RAC measured in vivo with both phase-contrast magnetic resonance imaging (PC-MRI) and M-mode echocardiography (on RPA only). We did so using data from a canine model of acute PH due to embolization to assess the sensitivity of RAC to changes in elastic modulus in the absence of chronic PH-induced arterial remodeling. We found that elastic modulus increased with embolization-induced PH, presumably a consequence of increased collagen engagement, which corresponds well to decreased RAC. Furthermore, RAC was inversely related to elastic modulus. Finally, we found MRI and echocardiography yielded comparable estimates of RAC. We conclude that RAC of proximal PAs can be obtained from either MRI or echocardiography and a change in RAC indicates a change in elastic modulus of proximal PAs detectable even in the absence of chronic PH-induced arterial remodeling. The correlation between RAC and elastic modulus of proximal PAs may be useful for prognoses and to monitor the effects of therapeutic interventions in patients with PH.     

Stress wave velocities in bovine patellar tendon.

The velocity of longitudinal stress waves in an elastic body is given by the square root of the ratio of its elastic modulus to its density. In tendinous and ligamentous tissue, the elastic modulus increases with strain and with strain rate. Therefore, it was postulated that stress wave velocity would also increase with increasing strain and strain rate. The purpose of this study was to determine the velocity of stress waves in tendinous tissue as a function of strain and to compare these values to those predicted using the elastic modulus derived from quasi-static testing. Five bovine patellar tendons were harvested and potted as bone-tendon-bone specimens. Quasi-static mechanical properties were determined in tension at a deformation rate of 100 mm/s. Impact loading was employed to determine wave velocity at various strain levels, achieved by preloading the tendon. Following impact, there was a measurable delay in force transmission across the specimen and this delay decreased with increasing tendon strain. The wave velocities at tendon strains of 0.0075, 0.015, and 0.0225 were determined to be 260 +/- 52 m/s, 360 +/- 71 m/s, and 461 +/- 94 m/s, respectively. These velocities were significantly (p < 0.01) faster than those predicted using elastic moduli derived from the quasi-static tests by 52, 45, and 41 percent, respectively. This study has documented that stress wave velocity in patellar tendon increases with increasing strain and is underestimated with a modulus estimated from quasi-static testing.     

Differential cross-linking and radio-protective effects of genipin on mature bovine and human patella tendons

Gamma irradiation is a proven sterilization method, but is not widely used on allografts for anterior cruciate ligament reconstruction (e.g., patella tendon) due to radiation-induced decreases in mechanical strength. Addressing this drawback would improve the safety and supply of allografts to meet current and future demand. It was hypothesized that genipin-induced collagen cross-linking would increase the tensile modulus of patella tendon tissue such that 5 MRad gamma irradiation would not reduce the tissue mechanical strength below the original untreated values. Optimized genipin treatment increased the tensile modulus of bovine tendons by ~2.4-fold. After irradiation, genipin treated tissue did not significantly differ from native tissue, proving the hypothesis. Optimized genipin treatment of human tendons increased the tensile modulus by ~1.3-fold. After irradiation, both control and genipin-treated tissues possessed ~50–60% of their native tendon modulus, disproving the hypothesis. These results highlight possible age- and species- dependent effects of genipin cross-linking on tendon tissue. Cross-linking of human allografts may be beneficial only in younger donor tissues. Future research is warranted to better understand the mechanisms and applications of collagen cross-linking for clinical use.