Fatigue loading of tendon results in collagen kinking and denaturation but does not change local tissue mechanics

Fatigue loading is a primary cause of tendon degeneration, which is characterized by the disruption of collagen fibers and the appearance of abnormal (e.g., cartilaginous, fatty, calcified) tissue deposits. The formation of such abnormal deposits, which further weakens the tissue, suggests that resident tendon cells acquire an aberrant phenotype in response to fatigue damage and the resulting altered mechanical microenvironment. While fatigue loading produces clear changes in collagen organization and molecular denaturation, no data exist regarding the effect of fatigue on the local tissue mechanical properties. Therefore, the objective of this study was to identify changes in the local tissue stiffness of tendons after fatigue loading. We hypothesized that fatigue damage would reduce local tissue stiffness, particularly in areas with significant structural damage (e.g., collagen denaturation). We tested this hypothesis by identifying regions of local fatigue damage (i.e., collagen fiber kinking and molecular denaturation) via histologic imaging and by measuring the local tissue modulus within these regions via atomic force microscopy (AFM). Counter to our initial hypothesis, we found no change in the local tissue modulus as a consequence of fatigue loading, despite widespread fiber kinking and collagen denaturation. These data suggest that immediate changes in topography and tissue structure – but not local tissue mechanics – initiate the early changes in tendon cell phenotype as a consequence of fatigue loading that ultimately culminate in tendon degeneration.     

Fatigue of bovine trabecular bone

Fatigue loading of bone, from the activities of daily living in the elderly, or from prolonged exercise in the young, can lead to increased risk of fracture. Elderly patients with osteoporosis are particularly prone to fragility fractures of the vertebrae, where load is carried primarily by trabecular bone. In this study, specimens of bovine trabecular bone were loaded in compressive fatigue at four different normalized stresses to one of six maximum strains. The resulting change in modulus and residual strain accumulation were measured over the life of the fatigue test. The number of cycles to reach a given maximum compressive strain increased with decreasing normalized stress. Modulus reduction and specimen residual strain increased with increasing maximum compressive strain, but few differences were observed between specimens loaded to the same maximum strain at different normalized stresses.     

Fatigue microdamage in bovine trabecular bone

Microdamage, in the form of small cracks, may accumulate in trabecular bone loaded in fatigue. Specimens of bovine trabecular bone were loaded in compressive fatigue at one of four normalized stresses and loading was stopped after the specimens reached one of six maximum strains. Microdamage was identified using a fluorochrome staining technique, and microdamage parameters, including the number of damaged trabeculae and the damaged area fraction, were measured. No microdamage was observed during loading to strains below the yield strain; at higher strains, all microdamage parameters increased with increasing maximum compressive strain. Few significant differences were observed in the type or amount of microdamage accumulation between specimens loaded to the same maximum strain at different normalized stresses; however, more trabecular fractures were observed at high numbers of cycles, which corresponded to low normalized stresses.     

Near-terminal creep damage does not substantially influence fatigue life under physiological loading

Cortical bone specimens were damaged using repeated blocks of tensile creep loading until a near-terminal amount of creep damage was generated (corresponding to a reduction in elastic modulus of 15%). One group of cortical bone specimens was submitted to the near-terminal damage protocol and subsequently underwent fatigue loading in tension with a maximum strain of 2000 με (Damage Fatigue, n=5). A second group was submitted to cyclic fatigue loading but was not pre-damaged (Control Fatigue, n=5). All but one specimen (a damaged specimen) reached run-out (10 million cycles, 7.7 days). No significant differences in microscopic cracks or other tissue damage were observed between the two groups or between either group and additional, completely unloaded specimens. Our results suggest that damage in cortical bone allograft that is not obvious or associated with a stress riser may not substantially affect its fatigue life under physiologic loading.     

The relationships between cyclic fatigue loading, changes in initial mechanical properties, and the in vivo temporal mechanical response of the rat patellar tendon

Damage accumulation underlies tendinopathy. Animal models of overuse injuries do not typically control loads applied to the tendon. Our in vivo model in the rat patellar tendon allows direct control of the loading applied to the tendon. Despite this advantage, natural variation among tendons results in different amounts of damage induced by the same loading protocol. Our objectives were to (1) assess changes in the initial mechanical parameters (hysteresis, stiffness of the loading and unloading load-displacement curves, and elongation) after fatigue loading to identify parameters that are indicative of the induced damage, and (2) evaluate the relationships between these identified initial damage indices with the stiffness 7 day after loading. Left patellar tendons of adult, female retired breeder, Sprague-Dawley rats (n = 68) were fatigue loaded per our previously published in vivo fatigue loading protocol. To induce a range of damage, fatigue loading consisted of either 5, 100, 500 or 7200 cycles that ranged from 1 N to 40 N. Diagnostic tests were applied before and immediately after fatigue loading, and after 45 min of recovery to deduce recoverable and non-recoverable changes in initial damage indices. Relationships between these initial damage indices and the 7-day stiffness (at sacrifice) were determined. Day-0 hysteresis, loading and unloading stiffness exhibited cycle-dependent changes. Initial hysteresis loss correlated with the 7-day stiffness. k-means cluster analysis demonstrated a relationship between 7-day stiffness and day-0 hysteresis and unloading stiffness. This analysis also separated samples that exhibited low from high damage in response to both high or low number of cycles; a key delineation for interpretation of the biological response in future studies. Identifying initial parameters that reflect the induced damage is critical since the ability of the tendon to repair depends on the damage induced and the number of applied loading cycles.     

Compressive fatigue and endurance of juvenile bovine articular cartilage explants

Articular cartilage is an enduring tissue. For most individuals, articular cartilage facilitates a lifetime of pain-free ambulation, supporting millions of loading cycles from activities of daily living. Although early studies into osteoarthritis focused on the role of mechanical fatigue in articular cartilage degeneration, much is still unknown regarding its strength and endurance characteristics. The compressive strength of juvenile, bovine articular cartilage explants was determined to be loading rate-dependent, reaching a maximum strength of 29.5 ± 4.8 MPa at a strain rate of 0.10 %/sec. The fatigue and endurance properties of articular cartilage were characterized utilizing a material testing system, as well as a custom, validated instrument termed the two degrees-of-freedom endurance meter (endurometer). These instruments characterized fatigue in articular cartilage explants at loading levels ranging from 10 to 80 % strength (%S), up to 100,000 cycles. Cartilage explants displayed characteristics of fatigue – fatigue life increased as the loading magnitude decreased. All explants failed within 14,000 cycles at loading levels between 50 and 80 %S. At 10 and 20 %S, all explants endured 100,000 loading cycles. There was no significant difference in equilibrium compressive modulus between run-out explants and unloaded controls, although the pooled modulus increased in response to testing. Histological staining and biochemical assays revealed no material changes in collagen, sulfated glycosaminoglycan, or hydration content between unloaded controls and explants cyclically loaded to run-out. These results suggest articular cartilage may have a putative endurance limit of 20 %S (5.86 MPa), with implications for articular cartilage biomechanics and mechanobiology.     

Monitoring micrometer-scale collagen organization in rat-tail tendon upon mechanical strain using second harmonic microscopy

We continuously monitored the microstructure of a rat-tail tendon during stretch/relaxation cycles. To that purpose, we implemented a new biomechanical device that combined SHG imaging and mechanical testing modalities. This multi-scale experimental device enabled simultaneous visualization of the collagen crimp morphology at the micrometer scale and measurement of macroscopic strain-stress response. We gradually increased the ultimate strain of the cycles and showed that preconditioning mostly occurs in the first stretching. This is accompanied by an increase of the crimp period in the SHG image. Our results indicate that preconditioning is due to a sliding of microstructures at the scale of a few fibrils and smaller, that changes the resting length of the fascicle. This sliding can reverse on long time scales. These results provide a proof of concept that continuous SHG imaging performed simultaneously with mechanical assay allows analysis of the relationship between macroscopic response and microscopic structure of tissues.     

Early response to tendon fatigue damage accumulation in a novel in vivo model

This study describes the development and application of a novel rat patellar tendon model of mechanical fatigue for investigating the early in vivo response to tendon subfailure injury. Patellar tendons of adult female Sprague-Dawley rats were fatigue loaded between 1–35 N using a custom-designed loading apparatus. Patellar tendons were subjected to Low-, Moderate- or High-level fatigue damage, defined by grip-to-grip strain measurement. Molecular response was compared with that of a laceration-repair injury. Histological analyses showed that progression of tendon fatigue involves formation of localized kinked fiber deformations at Low damage, which increased in density with presence of fiber delaminations at Moderate damage, and fiber angulation and discontinuities at High damage levels. RT-PCR analysis performed at 1- and 3-day post-fatigue showed variable changes in type I, III and V collagen mRNA expression at Low and Moderate damage levels, consistent with clinical findings of tendon pathology and were modest compared with those observed at High damage levels, in which expression of all collagens evaluated were increased markedly. In contrast, only type I collagen expression was elevated at the same time points post-laceration. Findings suggest that cumulative fatigue in tendon invokes a different molecular response than laceration. Further, structural repair may not be initiated until reaching end-stage fatigue life, where the repair response may unable to restore the damaged tendon to its pre-fatigue architecture.     

Damage in trabecular bone at small strains

Evidence that damage decreases bone quality, increases fracture susceptibility, and serves as a remodeling stimulus motivates further study of what loading magnitudes induce damage in trabecular bone. In particular, whether damage occurs at the smaller strains characteristic of habitual, as opposed to traumatic, loading is not known. The overall goal of this study was to characterize damage accumulation in trabecular bone at small strains (0.20 - 0.45% strain). A continuum damage mechanics approach was taken whereby damage was quantified by changes in modulus and residual strain. Human vertebral specimens (n = 7) were tested in compression using a multi-cycle load - unload protocol in which the maximum applied strain for each cycle, epsilonmax, was increased incrementally from epsilonmax = 0.20% on the first loading cycle to epsilonmax = 0.45% on the last cycle. Modulus and residual strain were measured for each cycle. Both changes in modulus and residual strains commenced at small strains, beginning as early as 0.24 and 0.20% strain, respectively. Strong correlations between changes in modulus and residual strains were observed (r = 0.51 - 0.98). Fully nonlinear, high-resolution finite element analyses indicated that even at small apparent strains, tissue-level strains were sufficiently high to cause local yielding. These results demonstrate that damage in trabecular bone occurs at apparent strains less than half the apparent compressive yield strain reported previously for human vertebral trabecular bone. Further, these findings imply that, as a consequence of the highly porous trabecular structure, tissue yielding can initiate at very low apparent strains and that this local failure has detectable and negative consequences on the apparent mechanical properties of trabecular bone.     

In situ fibril stretch and sliding is location-dependent in mouse supraspinatus tendons

Tendons are able to transmit high loads efficiently due to their finely optimized hierarchical collagen structure. Two mechanisms by which tendons respond to load are collagen fibril sliding and deformation (stretch). Although many studies have demonstrated that regional variations in tendon structure, composition, and organization contribute to the full tendon׳s mechanical response, the location-dependent response to loading at the fibril level has not been investigated. In addition, the instantaneous response of fibrils to loading, which is clinically relevant for repetitive stretch or fatigue injuries, has also not been studied. Therefore, the purpose of this study was to quantify the instantaneous response of collagen fibrils throughout a mechanical loading protocol, both in the insertion site and in the midsubstance of the mouse supraspinatus tendon. Utilizing a novel atomic force microscopy-based imaging technique, tendons at various strain levels were directly visualized and analyzed for changes in fibril d-period with increasing tendon strain. At the insertion site, d-period significantly increased from 0% to 1% tendon strain, increased again from 3% to 5% strain, and decreased after 5% strain. At the midsubstance, d-period increased from 0% to 1% strain and then decreased after 7% strain. In addition, fibril d-period heterogeneity (fibril sliding) was present, primarily at 3% strain with a large majority occurring in the tendon midsubstance. This study builds upon previous work by adding information on the instantaneous and regional-dependent fibrillar response to mechanical loading and presents data proposing that collagen fibril sliding and stretch are directly related to tissue organization and function.     

Mechanical damage to the intervertebral disc annulus fibrosus subjected to tensile loading

Damage of the annulus fibrosus is implicated in common spinal pathologies. The objective of this study was to obtain a quantitative relationship between both the number of cycles and the magnitude of tensile strain resulting in damage to the annulus fibrosus. Four rectangular tensile specimens oriented in the circumferential direction were harvested from the outer annulus of 8 bovine caudal discs (n = 32) and subjected to one of four tensile testing protocols: (i) ultimate tensile strain (UTS) test; (ii) baseline cyclic test with 4 series of 400 cycles of baseline cyclic loading (peak strain = 20% UTS); (iii & iv) acute and fatigue damage cyclic tests consisting of 4 x 400 cycles of baseline cyclic loading with intermittent loading to 1 and 100 cycles, respectively, with peak tensile strain of 40%, 60%, and 80% UTS. Normalized peak stress for all mechanically loaded specimens was reduced from 0.89 to 0.11 of the baseline control levels, and depended on the magnitude of damaging strain and number of cycles at that damaging strain. Baseline, acute, and fatigue protocols resulted in permanent deformation of 3.5%, 6.7% and 9.6% elongation, respectively. Damage to the laminate structure of the annulus in the absence of biochemical activity in this study was assessed using histology, transmission electron microscopy, and biochemical measurements and was most likely a result of separation of annulus layers (i.e., delamination). Permanent elongation and stress reduction in the annulus may manifest in the motion segment as sub-catastrophic damage including increased neutral zone, disc bulging, and loss of nucleus pulposus pressure. The preparation of rectangular tensile strip specimens required cutting of collagen fibers and may influence absolute values of results, however, it is not expected to affect the comparisons between loading groups or dose-response reported.     

A phenomenological model for predicting fatigue life in bovine trabecular bone

Cyclic loading of bone during daily activities can lead to fatigue degradation and increased risk of fracture in both the young and elderly population. Damage processes under cyclic loading in trabecular bone result in the reduction of the elastic modulus and accumulation of residual strain. These effects increase with increasing stress levels, leading to a progressive reduction in fatigue life. The present work analyzes the effect of stress and strain variation on the above damage processes in bovine trabecular bone, and develops a phenomenological model relating fatigue life to the imposed stress level. The elastic modulus reduction of the bone specimens was observed to depend on the maximum compressive strain, while the rate of residual strain accumulation was a function of the stress level. A model was developed for the upper and lower bounds of bone elastic modulus reduction with increasing number of cycles, at each stress range. The experimental observations were described well by the model. The model predicted the bounds of the fatigue life with change in fatigue stress. The decrease in the fatigue life with increasing stress was related to corresponding increases in the residual strain accumulation rates at the elevated stress levels. The model shows the validity of fatigue predictions from relatively few cyclic experiments, by combining trends observed in the monotonic and the cyclic tests. The model also presents a relatively simple procedure for predicting the endurance limit for bovine trabecular bone specimens.     

Systematic error in mechanical measures of damage during four-point bending fatigue of cortical bone

Accumulation of fatigue microdamage in cortical bone specimens is commonly measured by a modulus or stiffness degradation after normalizing tissue heterogeneity by the initial modulus or stiffness of each specimen measured during a preloading step. In the first experiment, the initial specimen modulus defined using linear elastic beam theory (LEBT) was shown to be nonlinearly dependent on the preload level, which subsequently caused systematic error in the amount and rate of damage accumulation measured by the LEBT modulus degradation. Therefore, the secant modulus is recommended for measurements of the initial specimen modulus during preloading. In the second experiment, different measures of mechanical degradation were directly compared and shown to result in widely varying estimates of damage accumulation during fatigue. After loading to 400,000 cycles, the normalized LEBT modulus decreased by 26% and the creep strain ratio decreased by 58%, but the normalized secant modulus experienced no degradation and histology revealed no significant differences in microcrack density. The LEBT modulus was shown to include the combined effect of both elastic (recovered) and creep (accumulated) strain. Therefore, at minimum, both the secant modulus and creep should be measured throughout a test to most accurately indicate damage accumulation and account for different damage mechanisms. Histology revealed indentation of tissue adjacent to roller supports, with significant sub-surface damage beneath large indentations, accounting for 22% of the creep strain on average. The indentation of roller supports resulted in inflated measures of the LEBT modulus degradation and creep. The results of this study suggest that investigations of fatigue microdamage in cortical bone should avoid the use of four-point bending unless no other option is possible.     

Strain-related collagen gene expression in human osteoblast-like cells

The gene expression of cells in the musculoskeletal system, such as in bone, cartilage, ligament and tendon, is profoundly affected by mechanical loading. Previous studies have demonstrated that the expression of many genes, including collagen types I and III, are affected by mechanical strain in diverse cell types, such as human osteoblast-like SaOs-2 cells. However, whether the effect of mechanical loading on collagen gene expression is strain-related remains unclear. The goal of this study was to determine the relationship between mechanical strain and the gene expression of collagen types I and III in SaOs-2 cells. A Flexercell cellular mechanical loading system was used to subject SaOs-2 cells to equibiaxial cyclic tensile stress at a rate of 0.5 Hz with various strains of 5%, 7.5%, 10%, and 12.5% for 24 h. The relative amount of mRNA of both collagen I and collagen III increased at 5% strain compared with that of the control. As the strain increased, the relative amount of mRNA of collagen I remained stable at strain levels up to 12.5%. However, the mRNA for collagen III began to drop when the strain was greater than 5%, until a 10% strain was reached. From the application of a 10% strain through the maximum loading of a 12.5% strain, the relative amount of collagen III mRNA remained stable at amounts lower than that of the control. Thus, the gene expression of collagen types I and III responds differentially to mechanical strain at various magnitudes.     

Arterial Extracellular Matrix: A Mechanobiological Study of the Contributions and Interactions of Elastin and Collagen

The complex network structure of elastin and collagen extracellular matrix (ECM) forms the primary load bearing components in the arterial wall. The structural and mechanobiological interactions between elastin and collagen are important for properly functioning arteries. Here, we examined the elastin and collagen organization, realignment, and recruitment by coupling mechanical loading and multiphoton imaging. Two-photon excitation fluorescence and second harmonic generation methods were performed with a multiphoton video-rate microscope to capture real time changes to the elastin and collagen structure during biaxial deformation. Enzymatic removal of elastin was performed to assess the structural changes of the remaining collagen structure. Quantitative analysis of the structural changes to elastin and collagen was made using a combination of two-dimensional fast Fourier transform and fractal analysis, which allows for a more complete understanding of structural changes. Our study provides new quantitative evidence, to our knowledge on the sequential engagement of different arterial ECM components in response to mechanical loading. The adventitial collagen exists as large wavy bundles of fibers that exhibit fiber engagement after 20% strain. The medial collagen is engaged throughout the stretching process, and prominent elastic fiber engagement is observed up to 20% strain after which the engagement plateaus. The fiber orientation distribution functions show remarkably different changes in the ECM structure in response to mechanical loading. The medial collagen shows an evident preferred circumferential distribution, however the fiber families of adventitial collagen are obscured by their waviness at no or low mechanical strains. Collagen fibers in both layers exhibit significant realignment in response to unequal biaxial loading. The elastic fibers are much more uniformly distributed and remained relatively unchanged due to loading. Removal of elastin produces similar structural changes in collagen as mechanical loading. Our study suggests that the elastic fibers are under tension and impart an intrinsic compressive stress on the collagen.     

Arterial Extracellular Matrix: A Mechanobiological Study of the Contributions and Interactions of Elastin and Collagen

The complex network structure of elastin and collagen extracellular matrix (ECM) forms the primary load bearing components in the arterial wall. The structural and mechanobiological interactions between elastin and collagen are important for properly functioning arteries. Here, we examined the elastin and collagen organization, realignment, and recruitment by coupling mechanical loading and multiphoton imaging. Two-photon excitation fluorescence and second harmonic generation methods were performed with a multiphoton video-rate microscope to capture real time changes to the elastin and collagen structure during biaxial deformation. Enzymatic removal of elastin was performed to assess the structural changes of the remaining collagen structure. Quantitative analysis of the structural changes to elastin and collagen was made using a combination of two-dimensional fast Fourier transform and fractal analysis, which allows for a more complete understanding of structural changes. Our study provides new quantitative evidence, to our knowledge on the sequential engagement of different arterial ECM components in response to mechanical loading. The adventitial collagen exists as large wavy bundles of fibers that exhibit fiber engagement after 20% strain. The medial collagen is engaged throughout the stretching process, and prominent elastic fiber engagement is observed up to 20% strain after which the engagement plateaus. The fiber orientation distribution functions show remarkably different changes in the ECM structure in response to mechanical loading. The medial collagen shows an evident preferred circumferential distribution, however the fiber families of adventitial collagen are obscured by their waviness at no or low mechanical strains. Collagen fibers in both layers exhibit significant realignment in response to unequal biaxial loading. The elastic fibers are much more uniformly distributed and remained relatively unchanged due to loading. Removal of elastin produces similar structural changes in collagen as mechanical loading. Our study suggests that the elastic fibers are under tension and impart an intrinsic compressive stress on the collagen.     

Mechanical strain modulates extracellular matrix degradation and byproducts in an isoform-specific manner

Many studies have shown that mechanical forces can alter collagen degradation by proteases, and this mechanochemical effect may potentially serve an important role in determining extracellular matrix content and organization in load-bearing tissues. However, it is not yet known whether mechano-sensitive degradation depends on particular protease isoforms, nor is it yet known whether particular degradation byproducts can be altered by mechanical loading. In this study, we tested the hypothesis that different types of proteases exhibit different sensitivities to mechanical loading both in degradation rates and byproducts. Decellularized porcine pericardium samples were treated with human recombinant matrix metalloproteinases-1, ?8, ?9, cathepsin K, or a protease-free control while subjected to different levels of strain in a planar, biaxial mechanical tester. Tissue degradation was monitored by tracking the decay in mechanical stresses during displacement control tests, and byproducts were assessed by mass spectrometry analysis of the sample supernatant after degradation. Our key finding shows that cathepsin K-mediated degradation of collagenous tissue was enhanced with increasing strain, while MMP1-, MMP8-, and MMP9-mediated degradation were first decreased and then increased by strain. Degradation induced changes in tissue mechanical properties, and proteomic analysis revealed strain-sensitive degradome signatures with different ECM byproducts released at low vs. high strains. This evidence suggests a potentially new type of mechanobiology wherein mechanical forces alter the degradation products that can provide important signaling feedback functions during tissue remodeling.     

Rest-inserted loading rapidly amplifies the response of bone to small increases in strain and load cycles.

We hypothesized that a 10-s rest interval (at zero load) inserted between each load cycle would increase the osteogenic effects of mechanical loading near previously identified thresholds for strain magnitude and cycle numbers. We tested our hypothesis by subjecting the right tibiae of female C57BL/6J mice (16 wk, n = 70) to exogenous mechanical loading within a peri-threshold physiological range of strain magnitudes and load cycle numbers using a noninvasive murine tibia loading device. Bone responses to mechanical loading were determined via dynamic histomorphometry. More specifically, we contrasted bone formation induced by cyclic vs. rest-inserted loading (10-s rest at zero load inserted between each load cycle) by first varying peak strains (1,000, 1,250, or 1,600 micro epsilon) at fixed cycle numbers (50 cycles/day, 3 days/wk for 3 wk) and then varying cycle numbers (10, 50, or 250 cycles/day) at a fixed strain magnitude (1,250 micro epsilon). Within the range of strain magnitudes tested, the slope of periosteal bone formation rate (p.BFR/BS) with increasing strain magnitudes was significantly increased by rest-inserted compared with cyclical loading. Within the range of load cycles tested, the slope of p.BFR/BS with increasing load cycles of rest-inserted loading was also significantly increased by rest-inserted compared with cyclical loading. In sum, the data of this study indicate that inserting a 10-s rest interval between each load cycle amplifies bone's response to mechanical loading, even within a peri-threshold range of strain magnitudes and cycle numbers.     

Potential strain-dependent mechanisms defining matrix alignment in healing tendons

Tendon mechanical function after injury and healing is largely determined by its underlying collagen structure, which in turn is dependent on the degree of mechanical loading experienced during healing. Experimental studies have shown seemingly conflicting outcomes: although collagen content steadily increases with increasing loads, collagen alignment peaks at an intermediate load. Herein, we explored potential collagen remodeling mechanisms that could give rise to this structural divergence in response to strain. We adapted an established agent-based model of collagen remodeling in order to simulate various strain-dependent cell and collagen interactions that govern long-term collagen content and fiber alignment. Our simulation results show two collagen remodeling mechanisms that give rise to divergent collagen content and alignment in healing tendons: (1) strain-induced collagen fiber damage in concert with increased rates of deposition at higher strains, or (2) strain-dependent rates of enzymatic degradation. These model predictions identify critical future experiments needed to isolate each mechanism’s specific contribution to the structure of healing tendons.     

Fatigue injury risk in anterior cruciate ligament of target side knee during golf swing

A golf-related ACL injury can be linked with excessive golf play or practice because such over-use by repetitive golf swing motions can increase damage accumulation to the ACL bundles. In this study, joint angular rotations, forces, and moments, as well as the forces and strains on the ACL of the target-side knee joint, were investigated for ten professional golfers using the multi-body lower extremity model. The fatigue life of the ACL was also predicted by assuming the estimated ACL force as a cyclic load. The ACL force and strain reached their maximum values within a short time just after ball-impact in the follow-through phase. The smaller knee flexion, higher internal tibial rotation, increase of the joint compressive force and knee abduction moment in the follow-through phase were shown as to lead an increased ACL loading. The number of cycles to fatigue failure (fatigue life) in the ACL might be several thousands. It is suggested that the excessive training or practice of swing motion without enough rest may be one of factors to lead to damage or injury in the ACL by the fatigue failure. The present technology can provide fundamental information to understand and prevent the ACL injury for golf players.