Human Patellar Tendon Research Articles

THE EFFECT OF LIFE‐LONG ENDURANCE EXERCISE ON COLLAGEN CROSS‐LINKING OF THE HUMAN PATELLAR TENDON

IntroductionIt remains unknown if life‐long habitual endurance exercise influences the accumulation of Advanced Glycation Endproducts (AGE) cross‐links that are closely associated with aging and disease in connective tissue.PurposeTo examine the effect of aging and life‐long habitual endurance exercise on the collagen cross‐linking of the human patellar tendon.MethodsWe investigated 13 healthy injury free master athletes (old trained men, OT; age 59–75 years, running distance of 49±3 km/wk over 29±3 yrs (mean±SEM)), 12 old untrained controls (OC; matched to OT for BMI and age) and 10 young men matched for current running distance (young trained,YT; age 21–34, 48±4 km/week) and 12 young untrained controls (YC; matched to YT for BMI and age). Percutaneous tendon biopsies were obtained and analyzed for enzymatic (HP&LP) cross‐links, pentosidine, and collagen concent from the patellar tendon. A 2‐way ANOVA was used for statistical analysis.ResultsPentosidine increased with age (P<0.001)(Pentosidine: OT, 39±4; OC, 48±3; YT, 11±2 and YC, 9±1 mmol/mol collagen). There was an interaction between age and training (P=0.019), such that master athletes (OT) had a lower tendon pentosidine than OC (P = 0.006).ConclusionThese are the first data to demonstrate AGE cross‐links in tendon in male master athletes are lower compared to old untrained controls. The results suggest that life‐long habitual endurance exercise can counteract the aging process in tendon and thereby possibly reducing the risk of injury. Research support: Danish Association of Rheumatism.

Production of a sterilised decellularised tendon allograft for clinical use

Application of a high-level decontamination or sterilisation procedure and cell removal technique to tendon allograft can reduce the concerns of disease transmission, immune reaction, and may improve remodelling of the graft after implantation. The decellularised matrix can also be used as a matrix for tendon tissue engineering. One such sterilisation factor, Peracetic acid (PAA) has the advantage of not producing harmful reaction residues. The aim of this study was to evaluate the effects of PAA treatment and a cell removal procedure on the production of tendon matrix. Human patellar tendons, thawed from frozen were treated respectively as: Group 1, control with no treatment; Group 2, sterilised with PAA (0.1% (w/v) PAA for 3h) Group 3, decellularised (incubation successively in hypotonic buffer, 0.1% (w/v) sodium dodecyl sulphate, and a nuclease solution); Group 4, decellularised and PAA sterilised. Histological analysis showed that no cells were visible after the decellularisation treatment. The integrity of tendon structure was maintained after decellularisation and PAA sterilisation, however, the collagen waveform was slightly loosened. No contact cytotoxicity was found in any of the groups. Determination of de-natured collagen showed no significant increase when compared with the control. This suggested that the decellularisation and sterilisation processing procedures did not compromise the major properties of the tendon. The sterilised, decellularised tendon could be suitable for clinical use.

1E14 ヒト膝蓋腱の力学的性質に及ぼす高強度筋力トレーニングの影響(GS6-2:軟組織のバイオメカニクス(2))

1E14 ヒト膝蓋腱の力学的性質に及ぼす高強度筋力トレーニングの影響(GS6-2:軟組織のバイオメカニクス(2))

Human tenocytes are stimulated to proliferate by acetylcholine through an EGFR signalling pathway

Studies of human patellar and Achilles tendons have shown that primary tendon fibroblasts (tenocytes) not only have the capacity to produce acetylcholine (ACh) but also express muscarinic ACh receptors (mAChRs) through which ACh can exert its effects. In patients with tendinopathy (chronic tendon pain) with tendinosis, the tendon tissue is characterised by hypercellularity and angiogenesis, both of which might be influenced by ACh. In this study, we have tested the hypothesis that ACh increases the proliferation rate of tenocytes through mAChR stimulation and have examined whether this mechanism operates via the extracellular activation of the epidermal growth factor receptor (EGFR), as shown in other fibroblastic cells. By use of primary human tendon cell cultures, we identified cells expressing vimentin, tenomodulin and scleraxis and found that these cells also contained enzymes related to ACh synthesis and release (choline acetyltransferase and vesicular acetylcholine transporter). The cells furthermore expressed mAChRs of several subtypes. Exogenously administered ACh stimulated proliferation and increased the viability of tenocytes in vitro. When the cells were exposed to atropine (an mAChR antagonist) or the EGFR inhibitor AG1478, the proliferative effect of ACh decreased. Western blot revealed increased phosphorylation, after ACh stimulation, for both EGFR and the extracellular-signal-regulated kinases 1 and 2. Given that tenocytes have been shown to produce ACh and express mAChRs, this study provides evidence of a possible autocrine loop that might contribute to the hypercellularity seen in tendinosis tendon tissue.

Chlorhexidine Gluconate Cleansing Has No Effect on the Structural Properties of Human Patellar Tendon Allografts

If an anterior cruciate ligament graft somehow becomes contaminated intraoperatively, soaking it in 4% chlorhexidine gluconate has been shown to be the most popular and efficacious method for sterilization before implantation. The purpose of this study was to evaluate the effects of a chlorhexidine soak on the structural properties of human patellar tendon allografts. Sixteen human patellar tendon allografts were randomly split into 2 groups of 8. Grafts in 1 group were soaked in 4% chlorhexidine gluconate for 30 minutes, and the other grafts were kept moist in normal saline-soaked gauze. Data on preload width, preload thickness, elongation, ultimate tensile load, and stiffness were obtained through measurement and mechanical testing of the grafts. Graft donor ages ranged from 29 to 43 years. There was no difference in the mean values of graft dimensions of the chlorhexidine-exposed group versus the normal saline-exposed group before mechanical testing (width of 9.48 mm v 9.56 mm, P = .89; thickness of 4.01 mm v 4.57 mm, P = .34). Graft elongation was not statistically different between the groups (2.52 mm v 1.43 mm, P = .27). No statistically significant difference was noted between the ultimate tensile load (2,219 N v 1,878 N, P = .36) or stiffness (274.3 N/mm v 297.0 N/mm, P = .63) of the grafts in both groups. Structural properties of human patellar tendon allografts are not significantly affected by soaking in 4% chlorhexidine gluconate for 30 minutes. Surgeons wishing to treat an inadvertently contaminated graft intraoperatively with 4% chlorhexidine may do so without concern that such treatment will impact graft strength.

The effects of dexamethasone on human patellar tendon stem cells: Implications for dexamethasone treatment of tendon injury

Injection of Dexamethasone (Dex) is commonly used in clinics to treat tendon injury such as tendinopathy because of its anti-inflammatory capabilities. However, serious adverse effects have been reported as a result of Dex treatment, such as impaired tendon healing and tendon rupture. Using both in vitro and in vivo approaches, this study was to determine the effects of Dex treatment on the proliferation and differentiation of human tendon stem cells (hTSCs), which can directly impact tendon healing. We found that Dex treatment stimulated cell proliferation at lower concentrations (<1,000 nM), whereas a high concentration (1,000 nM) decreased cell proliferation. Moreover, at all concentrations used (5, 10, 100, and 1,000 nM), Dex treatment induced non-tenocyte differentiation of hTSCs, as evidenced by a change in cell shape, a nearly complete suppression of collagen type I expression, and an upregulation of non-tenocyte related genes (PPARγ and Sox-9), which was especially evident when higher concentrations (>10 nM) of Dex were used. Implantation of Dex-treated hTSCs for a short time (3 weeks) resulted in the extensive formation of fatty tissues, cartilage-like tissues, and bony tissues. These findings suggest that Dex treatment in clinics may cause a paradoxical effect on the injured tendons it is supposed to treat: by inducing non-tenocyte differentiation of hTSCs, Dex treatment depletes the stem cell pool and leads to the formation of non-tendinous tissues (e.g., fatty and cartilage-like tissues), which make tendon susceptible to rupture.

Repetitive mechanical stretching modulates transforming growth factor-β induced collagen synthesis and apoptosis in human patellar tendon fibroblasts

The cellular and molecular mechanisms underlying the development of tendinopathy are not clear, but inflammatory mediators produced by tendon fibroblasts in response to repetitive mechanical loading may be an important factor for this illness. In this study, we explored the effect of cyclic mechanical stretching on collagen synthesis and apoptosis of human patellar tendon fibroblasts (HPTFs). The role of a candidate inflammatory mediator, transforming growth factor-β1 (TGFβ1), which we identified in a cytokine antibody array, in collagen synthesis and apoptosis during repetitive mechanical stretching was also investigated. Our results showed that there was a significant increase in collagen type I synthesis at 4% and 8% stretch. Significantly, enhancement of apoptosis may account for the observed decrease in fibroblast numbers after 8% stretching. Furthermore, the exogenous addition of an anti-TGFβ1 antibody or gene silencing by si-TGFβ1 eliminated the increase in collagen type I production and activities of caspases during apoptosis under cyclic uniaxial stretching conditions. These results suggest that TGFβ1 may take part in the increase of cellular production of collagen type I and apoptosis during the development of tendinopathy. Furthermore, caspase 8 mediates activation of caspase 3 and poly ADP-ribose polymerase (PARP) cleavage during TGFβ1-induced apoptosis in stretching HPTFs.

The effects of immobilization on the mechanical properties of the patellar tendon in younger and older men

BackgroundIt remains unknown if inactivity changes the mechanical properties of the human patellar tendon in younger and older healthy persons. The purpose was to examine the effects of short-term unilateral immobilization on the structural and mechanical properties of the patellar tendon in older men and younger men, in vivo. MethodsEight older men and eight younger men underwent 14days of unilateral immobilization. All individuals were assessed on both sides before and after the intervention. MRI was used to assess whole patellar tendon dimensions. The mechanical properties of the patellar tendon were assessed using simultaneous force and ultrasonographic measurements during isometric ramp contractions. FindingsIn older men, tendon stiffness [Pre: mean 2949 (SD 799) vs. Post: mean 2366 (SD 774) Nmm−1, P<0.01] and Young's Modulus [Pre: mean 1.2 (SD 0.3) vs. Post: mean 1.0 (SD 0.3) GPa, P<0.05] declined with immobilization on the immobilized side. On the control side, tendon stiffness [Pre: mean 3340 (SD 1209) vs. Post: mean 2230 (SD 503), P<0.01] and Young's Modulus [Pre: mean 1.5 (SD 0.4) vs. Post: mean 0.9 (SD 0.3) GPa, P<0.05] also decreased with immobilization. In younger men, tendon stiffness [Pre: 3622 (SD 1760) vs. Post: mean 2910 (SD 1528) Nmm−1, P<0.01] and Young's Modulus [Pre: mean 1.7 (SD 1.1) vs. Post: mean 1.4 (SD 0.8) GPa, P<0.05] decreased only on the immobilized side. InterpretationShort-term immobilization led to impaired mechanical properties of the patellar tendon on the immobilized side in both younger men and older men, which can influence the function of the muscle–tendon complex.

Cryopreservation with glycerol improves the in vitro biomechanical characteristics of human patellar tendon allografts

To evaluate the in vitro biomechanical characteristics of patellar tendon ligaments (BTB) when stored as fresh frozen or as glycerol cryopreserved allografts. Seventy patellar tendons were harvested from 35 cadaveric human donors and randomly assigned into seven groups. Grafts in group FRESH were mechanically tested within 2 h of harvesting. FROZ-3, FROZ-6, and FROZ-9 were deep-frozen to -80 °C for 3, 6, and 9 months, respectively. Grafts in groups CRYO-3, CRYO-6, and CRYO-9 were initially incubated with 10% glycerol in a phosphate-buffered saline for 1 h and then stored in glycerol solution (10% glycerol in PBS) at -80 °C for 3, 6, and 9 months, respectively. Grafts were mechanically tested with two cycling modes (50-250 °N and 150-500 °N) and then loaded to failure. Cryopreserved grafts demonstrated more consistent results and expressed lower elongation rates after both cycling loading protocols compared to their frozen counterparts at all storage times. During load-to-failure analysis, ultimate stiffness levels were predominantly higher (23.9-61.5%) in cryopreserved grafts compared with frozen grafts, and ultimate stress levels were 26% (13.3-47.7%) higher, regardless of the storage time. Moreover, cryopreserved grafts revealed similar ultimate elongation and uniformly higher ultimate stiffness and ultimate stress levels compared to fresh grafts. The results of this in vitro study demonstrated superior mechanical properties of cryopreserved grafts compared to frozen grafts within a preservation period of 9 months. Cryopreservation with glycerol solution might be used to further improve the quality of preserved soft-tissue allografts.

Sterilization with electron beam irradiation influences the biomechanical properties and the early remodeling of tendon allografts for reconstruction of the anterior cruciate ligament (ACL)

Although allografts for anterior cruciate ligament (ACL) replacement have shown advantages compared to autografts, their use is limited due to the risk of disease transmission and the limitations of available sterilization methods. Gamma sterilization has shown detrimental effects on graft properties at the high doses required for sufficient pathogen inactivation. In our previous in vitro study on human patellar tendon allografts, Electron beam (Ebeam) irradiation showed less detrimental effects compared to gamma sterilization (Hoburg et al. in Am J Sports Med 38(6):1134-1140, 2010). To investigate the biological healing and restoration of the mechanical properties of a 34 kGy Ebeam treated tendon allograft twenty-four sheep underwent ACL replacement with either a 34 kGy Ebeam treated allograft or a non-sterilized fresh frozen allograft. Biomechanical testing of stiffness, ultimate failure load and AP-laxity as well as histological analysis to investigate cell, vessel and myofibroblast-density were performed after 6 and 12 weeks. Native sheep ACL and hamstring tendons (HAT, each n = 9) served as controls. The results of a previous study analyzing the remodeling of fresh frozen allografts (n = 12) and autografts (Auto, n = 18) with the same study design were also included in the analysis. Statistics were performed using Mann-Whitney U test followed by Bonferroni-Holm correction. Results showed significantly decreased biomechanical properties during the early remodeling period in Ebeam treated grafts and this was accompanied with an increased remodeling activity. There was no recovery of biomechanical function from 6 to 12 weeks in this group in contrast to the results observed in fresh frozen allografts and autografts. Therefore, high dose Ebeam irradiation investigated in this paper cannot be recommended for soft tissue allograft sterilization.

7A16 Magnetic Resonance Imagingを用いたヒト膝蓋腱長さ変化の生体内解析(OS1 筋骨格系のバイオメカニクス1)

7A16 Magnetic Resonance Imagingを用いたヒト膝蓋腱長さ変化の生体内解析(OS1 筋骨格系のバイオメカニクス1)

Mechanical properties of human patellar tendon at the hierarchical levels of tendon and fibril

Tendons are strong hierarchical structures, but how tensile forces are transmitted between different levels remains incompletely understood. Collagen fibrils are thought to be primary determinants of whole tendon properties, and therefore we hypothesized that the whole human patellar tendon and its distinct collagen fibrils would display similar mechanical properties. Human patellar tendons (n = 5) were mechanically tested in vivo by ultrasonography. Biopsies were obtained from each tendon, and individual collagen fibrils were dissected and tested mechanically by atomic force microscopy. The Young's modulus was 2.0 ± 0.5 GPa, and the toe region reached 3.3 ± 1.9% strain in whole patellar tendons. Based on dry cross-sectional area, the Young's modulus of isolated collagen fibrils was 2.8 ± 0.3 GPa, and the toe region reached 0.86 ± 0.08% strain. The measured fibril modulus was insufficient to account for the modulus of the tendon in vivo when fibril content in the tendon was accounted for. Thus, our original hypothesis was not supported, although the in vitro fibril modulus corresponded well with reported in vitro tendon values. This correspondence together with the fibril modulus not being greater than that of tendon supports that fibrillar rather than interfibrillar properties govern the subfailure tendon response, making the fibrillar level a meaningful target of intervention. The lower modulus found in vitro suggests a possible adverse effect of removing the tissue from its natural environment. In addition to the primary work comparing the two hierarchical levels, we also verified the existence of viscoelastic behavior in isolated human collagen fibrils.

Tensile Force Transmission in Human Patellar Tendon Fascicles Is Not Mediated by Glycosaminoglycans

Correct mechanical function of tendons is essential to human physiology and therefore the mechanical properties of tendon have been a subject of research for many decades now. However, one of the most fundamental questions remains unanswered: How is load transmitted through the tendon? It has been suggested that the proteoglycan-associated glycosaminoglycans (GAGs) found on the surface of the collagen fibrils may be an important transmitter of load, but existing results are ambiguous and have not investigated human tendons. We have used a small-scale mechanical testing system to measure the mechanical properties of fascicles from human patellar tendon at two different deformation rates before and after removal of GAGs by treatment with chondroitinase ABC. Efficiency of enzyme treatment was quantified using dimethylmethylene blue assay. Removal of at least 79% of the GAGs did not significantly change the tendon modulus, relative energy dissipation, peak stress, or peak strain. The effect of deformation rate was not modulated by the treatment either, indicating no effect on viscosity. These results suggest that GAGs cannot be considered mediators of tensile force transmission in the human patellar tendon, and as such, force transmission must either take place through other matrix components or the fibrils must be mechanically continuous at least to the tested length of 7 mm.

Tensile Mechanical Measurements on Individual Collagen Fibrils

Introduction: There is a hierarchical order in tendon ranging from the collagen molecule to the whole tendon. We have developed a method using the atomic force microscope (AFM) together with a novel data acquisition technique to quantify the sub-failure mechanical properties of individual collagen fibrils. Here we use this method to investigate how the saline environment affects tensile force transmission. Methods: Human patellar tendon tissue was obtained from a healthy young male. Six fascicles and six fibrils were mechanically tested sub-failure to determine modulus and relative energy dissipation in PBS solutions of 0.02, 0.15 and 1 M concentration as well as two HEPES buffers containing NaCl or NaCl+CaCl2. Mechanical testing was performed to 4% strain. Results: The tensile properties of collagen fibrils are highly resistant to changes in their saline environment. The only consistent response to environment composition was a minor (<5%) increase in relative energy dissipation in 1 M PBS. This is in sharp contrast to previous reports of more than 100% increase in radial compressive modulus of fibrils in 1 M salt solution, and more than 100% increase in energy dissipation of AFM pulling experiments in the presence of Ca2+.

Effects of cyclic loading on the tensile properties of human patellar tendon

Bone–patellar tendon-bone (BPTB) graft is a popular choice for ACL reconstruction. These grafts are subjected to cyclic loading during the activities of daily living. Significant knee laxity is observed in reconstructed knee shortly after reconstruction. The source of this laxity is not clear. The change in the tensile properties of the graft due to cyclic loading can be one of the reasons for the change in knee laxity. Twenty patellar tendons from fresh frozen cadaver knees were cyclically loaded at a stress amplitude equivalent to 33% of the failure strength of the contralateral patellar tendon for 5000 cycles at 1.4 Hz. They were then tested in tension to failure. Failure properties and the low load properties such as toe-region modulus were calculated. The results were compared with those of contralateral patellar tendons that were not subjected to cyclic loading before testing to failure. Fatigue loading did not alter the failure and low load properties with the exception of failure strain which decreased by about 10% (P < .05). Cyclically loaded patellar tendons with higher tissue mass density possess higher strength, modulus of elasticity, toughness, and transition stress (P < .05). The results indicate that there is no significant change in graft properties because of cyclic loading with the above load magnitude. The change in knee laxity observed after reconstruction, hence, is not because of change in graft properties due to moderate cyclic loading. Other factors, such as plastic deformation (yielding) of the graft, might play a role in increased knee laxity after reconstruction.

8G-10 ヒト膝蓋腱に作用する負荷の生体内計測(OS-3 軟組織及びその構成要素のバイオメカニクス(1))

8G-10 ヒト膝蓋腱に作用する負荷の生体内計測(OS-3 軟組織及びその構成要素のバイオメカニクス(1))

8C-16 MRIを用いたヒト膝蓋腱の生体内伸長測定(OS-2(2) 臨床応用を目指した骨・関節力学(3))

8C-16 MRIを用いたヒト膝蓋腱の生体内伸長測定(OS-2(2) 臨床応用を目指した骨・関節力学(3))

In vivoUltrasonographic Evaluation of Patellar Tendon Stiffness after Anterior Cruciate Ligament Reconstruction with Patellar Tendon Autograft

Background:Tendon mechanical properties have been used to evaluate the effects of therapies on the healing of animal tendons, but these measures have not been convenient to record in vivo in humans due to their invasive nature. The aims of this study were to assess the capability of an ultrasonography technique to track the change in stiffness of the healing human patellar tendon and to assess the correlation between stiffness parameters and clinical recovery measurements.Method:Ten subjects undergoing anterior cruciate ligament reconstruction with a patellar tendon autograft were recruited for the study as well as 10 healthy control subjects. The surgical tendons' mechanical properties were evaluated at 2 and 6 months after surgery with a two-scan ultrasonography technique. A paired sign-rank test was performed to compare the change in biomechanical parameters and clinical recovery measurements across time between surgical and control groups.Results:Tendon stiffness of the surgical group increased from ~71% to ~94% when compared to the stiffness of the contralateral tendons at the first visit; however, the difference was not significant (P= 0.18). Significant improvements were shown in all the clinical recovery outcomes. Poor correlation was found between the clinical recovery measurements and tendon stiffness.Conclusions:No significant change in patellar tendon stiffness with healing was detected over the 2–6 month period after surgery, suggesting the tendon stiffness recovery in humans proceeds more slowly than other clinical measures.

In vivo Ultrasonographic Evaluation of Patellar Tendon Stiffness after Anterior Cruciate Ligament Reconstruction with Patellar Tendon Autograft

Background: Tendon mechanical properties have been used to evaluate the effects of therapies on the healing of animal tendons, but these measures have not been convenient to record in vivo in humans due to their invasive nature. The aims of this study were to assess the capability of an ultrasonography technique to track the change in stiffness of the healing human patellar tendon and to assess the correlation between stiffness parameters and clinical recovery measurements. Method: Ten subjects undergoing anterior cruciate ligament reconstruction with a patellar tendon autograft were recruited for the study as well as 10 healthy control subjects. The surgical tendons' mechanical properties were evaluated at 2 and 6 months after surgery with a two-scan ultrasonography technique. A paired sign-rank test was performed to compare the change in biomechanical parameters and clinical recovery measurements across time between surgical and control groups. Results: Tendon stiffness of the surgical group increased from ~71% to ~94% when compared to the stiffness of the contralateral tendons at the first visit; however, the difference was not significant P = 0.18. Significant improvements were shown in all the clinical recovery outcomes. Poor correlation was found between the clinical recovery measurements and tendon stiffness. Conclusions: No significant change in patellar tendon stiffness with healing was detected over the 2--6 month period after surgery, suggesting the tendon stiffness recovery in humans proceeds more slowly than other clinical measures.

Tensile Properties of Human Collagen Fibrils and Fascicles Are Insensitive to Environmental Salts

To carry out realistic in vitro mechanical testing on anatomical tissue, a choice has to be made regarding the buffering environment. Therefore, it is important to understand how the environment may influence the measurement to ensure the highest level of accuracy. The most physiologically relevant loading direction of tendon is along its longitudinal axis. Thus, in this study, we focus on the tensile mechanical properties of two hierarchical levels from human patellar tendon, namely: individual collagen fibrils and fascicles. Investigations on collagen fibrils and fascicles were made at pH 7.4 in solutions of phosphate-buffered saline at three different concentrations as well as two HEPES buffered solutions containing NaCl or NaCl + CaCl 2. An atomic force microscope technique was used for tensile testing of individual collagen fibrils. Only a slight increase in relative energy dissipation was observed at the highest phosphate-buffered saline concentration for both the fibrils and fascicles, indicating a stabilizing effect of ionic screening, but changes were much less than reported for radial compression. Due to the small magnitude of the effects, the tensile mechanical properties of collagen fibrils and fascicles from the patellar tendon of mature humans are essentially insensitive to environmental salt concentration and composition at physiological pH.