A biochemical study of the distribution of collagen and its crosslinks in knee ligaments and the patellar tendon.

PURPOSE: The purpose of this study was to investigate biochemical differences in collagen crosslinks from different locations within the ligaments and a tendon of the human knee. MATERIALS AND METHODS: The anterior cruciate ligament (ACL), posterior cruciate ligament (PCL), medial collateral ligament (MCL), lateral collateral ligament (LCL), and patellar tendon (PT) were obtained from 24 cadavers (13 men and 11 women) whose average age at the time of death was 84.8 years. Ligaments and PT samples were obtained from the femoral and tibial insertions and the midsubstance. Hydroxyproline (Hyp) and collagen crosslinks, including pyridinoline (Pyr) and pentosidine (Pen), were compared among the different sites. RESULTS: The midsubstance Hyp concentration was greater than at the femoral and tibial insertions in the ACL (p = 0.00124 and 0.000255, respectively) and PCL (p = 0.00036 and 0.042, respectively). The Pyr:collagen ratio did not differ among sites in any of the ligaments or PT. The Pen:collagen ratio at the midsubstance was greater than at the femoral and tibial insertions in the ACL (p = 0.00022 and 0.00025, respectively) and LCL (p = 0.000081 and 0.000021, respectively) and was greater at the femoral insertion in the MCL (p = 0.00010). CONCLUSIONS: The mature collagen crosslink Pyr was not different in distribution in knee ligaments and the PT. Pen increased at the midsubstance ligaments and the PT. As increased Pen may represent ligament degeneration, this may indicate that degeneration may progress more rapidly at the midsubstance than at the insertion sites of a ligament.

Greater glycosaminoglycan content in human patellar tendon biopsies is associated with more pain and a lower VISA score.

BACKGROUND: People with patellar tendinopathy experience chronic pain and activity limitation, but a pertinent biochemical marker correlated with these clinical features has not been identified. The Victoria Institute of Sport Assessment (VISA) questionnaire is a condition-specific patient-rated outcome measure. Since the quantity of glycosaminoglycans (GAGs) increases with advancing tendon pathology, we hypothesised that there would be a correlation between the quantity of GAGs in the patellar tendon and the VISA score. METHODS: Tissue biopsies from athletes with chronic patellar tendinopathy (confirmed by clinical examination and MRI) were recruited (n=7), as well as controls with no history of knee pain (n=4). The quantity of sulphated GAGs in the human patellar tendons was determined with a dimethyl methylene blue (DMMB) assay; this method was first validated with rat tendon tissue. The extent and distribution of GAG species and proteoglycans (decorin, versican and aggrecan) in the human tendon biopsies were examined using immunohistochemistry. RESULTS: Greater sulphated GAG content of the patellar tendon was correlated with the greater tendon dysfunction (R(2)=0.798). The quantity of aggrecan in the tendon, a chondroitin sulphate-rich proteoglycan, also increased with advancing tendon pathology. CONCLUSIONS: Increased GAGs in the pathological human patellar tendon are related to a worse clinical status. These findings indicate that the VISA score reflects the extent of tendon tissue pathology.

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.

Lower strength of the human posterior patellar tendon seems unrelated to mature collagen cross-linking and fibril morphology.

The human patellar tendon is frequently affected by tendinopathy, but the etiology of the condition is not established, although differential loading of the anterior and posterior tendon may be associated with the condition. We hypothesized that changes in fibril morphology and collagen cross-linking would parallel differences in material strength between the anterior and posterior tendon. Tendon fascicles were obtained from elective ACL surgery patients and tested micromechanically. Transmission electron microscopy was used to assess fibril morphology, and collagen cross-linking was determined by HPLC and calorimetry. Anterior fascicles were markedly stronger (peak stress: 54.3 +/- 21.2 vs. 39.7 +/- 21.3 MPa; P < 0.05) and stiffer (624 +/- 232 vs. 362 +/- 170 MPa; P < 0.01) than posterior fascicles. Notably, mature pyridinium type cross-links were less abundant in anterior fascicles (hydroxylysylpyridinoline: 0.859 +/- 0.197 vs. 1.416 +/- 0.250 mol/mol, P = 0.001; lysylpyridinoline: 0.023 +/- 0.006 vs. 0.035 +/- 0.006 mol/mol, P < 0.01), whereas pentosidine and pyrrole concentrations showed no regional differences. Fibril diameters tended to be larger in anterior fascicles (7.819 +/- 2.168 vs. 4.897 +/- 1.434 nm(2); P = 0.10). Material properties did not appear closely related to cross-linking or fibril morphology. These findings suggest region-specific differences in mechanical, structural, and biochemical properties of the human patellar tendon.

Mechanical properties and collagen cross-linking of the patellar tendon in old and young men.

Age-related loss in muscle mass and strength impairs daily life function in the elderly. However, it remains unknown whether tendon properties also deteriorate with age. Cross-linking of collagen molecules provides structural integrity to the tendon fibrils and has been shown to change with age in animals but has never been examined in humans in vivo. In this study, we examined the mechanical properties and pyridinoline and pentosidine cross-link and collagen concentrations of the patellar tendon in vivo in old (OM) and young men (YM). Seven OM (67 +/- 3 years, 86 +/- 10 kg) and 10 YM (27 +/- 2 years, 81 +/- 8 kg) with a similar physical activity level (OM 5 +/- 6 h/wk, YM 5 +/- 2 h/wk) were examined. MRI was used to assess whole tendon dimensions. Tendon mechanical properties were assessed with the use of simultaneous force and ultrasonographic measurements during ramped isometric contractions. Percutaneous tendon biopsies were taken and analyzed for hydroxylysyl pyridinoline (HP), lysyl pyridinoline (LP), pentosidine, and collagen concentrations. We found no significant differences in the dimensions or mechanical properties of the tendon between OM and YM. Collagen concentrations were lower in OM than in YM (0.49 +/- 0.27 vs. 0.73 +/- 0.14 mg/mg dry wt; P < 0.05). HP concentrations were higher in OM than in YM (898 +/- 172 vs. 645 +/- 183 mmol/mol; P < 0.05). LP concentrations were higher in OM than in YM (49 +/- 38 vs. 16 +/- 8 mmol/mol; P < 0.01), and pentosidine concentrations were higher in OM than in YM (73 +/- 13 vs. 11 +/- 2 mmol/mol; P < 0.01). These cross-sectional data raise the possibility that age may not appreciably influence the dimensions or mechanical properties of the human patellar tendon in vivo. Collagen concentration was reduced, whereas both enzymatic and nonenzymatic cross-linking of concentration was elevated in OM vs. in YM, which may be a mechanism to maintain the mechanical properties of tendon with aging.

Changes in the composition of the extracellular matrix in patellar tendinopathy.

OBJECTIVE: To compare the chemical levels and mRNA expression of proteoglycan and collagen in normal human patellar tendons and tendons exhibiting chronic overuse tendinopathy. METHODS: Sulfated glycosaminoglycan and hydroxyproline content were investigated by spectrophotometric measurement using papain-digested samples. Deglycosylated proteoglycan core proteins were analysed by Western blot using specific antibodies. Total mRNA isolated from samples of frozen tendons was assayed by relative quantitative RT-PCR for decorin, biglycan, fibromodulin, versican, aggrecan, and collagens Type I, II and III and normalised to glyceraldehyde-3-phosphate dehydrogenase. RESULTS: There was a significant increase in sulfated glycosaminoglycan content in pathologic tendons compared to normal. This was attributed to an increased deposition of the large aggregating proteoglycans versican and aggrecan and the small proteoglycans biglycan and fibromodulin, but not decorin. Aggrecan and versican were extensively degraded in both normal and pathologic tendons, biglycan was more fragmented in the pathologic tendons while predominantly intact fibromodulin and decorin were present in normal and pathologic tendons. There was a greater range in total collagen content but no change in the level of total collagen in pathologic tendons. There were no significant differences between the pathologic and normal tendon for all genes, however p values close to 0.05 indicated a trend in downregulation of Type I collagen and fibromodulin, and upregulation in versican and Type III genes in pathologic tissue. CONCLUSION: The changes in proteoglycan and collagen levels observed in patellar tendinopathy appear to be primarily due to changes in the metabolic turnover of these macromolecules. Changes in the expression of these macromolecules may not play a major role in this process.

Impact of sex and chronic resistance training on human patellar tendon dry mass, collagen content, and collagen cross-linking.

Collagen content and cross-linking are believed to be major determinants of tendon structural integrity and function. Sex and chronic resistance training have been shown to alter tendon function and may also alter the key structural features of tendon. Patellar tendon biopsies were taken from untrained men [n = 8, 1 repetition maximum (RM) = 53 +/- 3 kg], untrained women (n = 8, 1 RM = 29 +/- 2 kg), and resistance-trained (10 +/- 1 yr of training) men (n = 8, 1 RM = 71 +/- 6 kg). Biopsies were analyzed for dry mass, collagen content, and collagen cross-linking (hydroxylysylpyridinoline). We hypothesized that these elements of tendon structure would be lower in women than men, whereas chronic resistance training would increase these parameters in men. Tendon dry mass was significantly lower in women than men (343 +/- 5 vs. 376 +/- 8 microg dry mass/mg tendon wet wt, P < 0.01) and was not influenced by chronic resistance training (P > 0.05). The lower tendon dry mass in women tended to reduce (P = 0.08) collagen content per tendon wet weight. Collagen content of the tendon dry mass was not influenced by sex or resistance training (P > 0.05). Similarly, cross-linking of collagen was unaltered (P > 0.05) by sex or training. Although sex alters the water content of patellar tendon tissue, any changes in tendon function with sex or chronic resistance training in men do not appear to be explained by alterations in collagen content or cross-linking of collagen within the dry mass component of the tendon.

In vitro reversal of the load-bearing properties of lipid-depleted articular cartilage following exposure to phospholipid surfactant solutions.

BACKGROUND: A microscopic layer of surface active phospholipids overlays the articular cartilage of the knee. Its depletion in osteoarthritic joints results in loss of lubrication and load-bearing efficiency. We hypothesize that exposure of articular cartilage to the dominant unsaturated phospholipid component of knee surfactant can regenerate the load-bearing properties of the tissue. This was evaluated by studying the stress-strain and stiffness characteristics of normal intact and lipid-depleted cartilage exposed to lipid-based surfactants for known durations. METHODS: Normal intact, lipid-depleted and surfactant-treated bovine articular cartilage specimens were compressed at 0.5mm/min to a maximum strain of 40% and their stress-strain and stiffness data were compared. FINDINGS: The stiffness of lipid-depleted samples increased by 40% on average relative to the normal; after exposure of the same samples to saturated surfactant for one and 24h, the average stiffness decreased by 25% and 62%, respectively from this high value. On the other hand, exposure of delipidized specimens to a mixture of selected unsaturated surfactant species for one and 24h resulted in a reduction of 85% and 90% in the stiffness of the depilidized samples respectively, largely reversing the effect of lipid removal to a level much closer to that of the normal intact cartilage and therefore better than that obtained with incubation in the saturated surfactant. INTERPRETATION: Lipid loss in articular cartilage results in a consistent increase in stiffness relative to normal tissue stiffness. This consequence of lipid loss can be partially reversed by the reintroduction of surface active phospholipids. The results of this study show that the lipid components of cartilage play an important role in determining the compliance of the loaded tissue.

Biochemical study of collagen and its crosslinks in the anterior cruciate ligament and the tissues used as a graft for reconstruction of the anterior cruciate ligament.

Among tissue grafts used for reconstruction of the anterior cruciate ligament (ACL), the pateller tendon (PT) and semitendinosus tendon (ST) are commonly used. It was thought that there were differences in the biochemical composition and process of healing between PT and ST. The aim of this study was to investigate the biochemical difference between ACL and the graft tissues used for reconstruction of the ACL. Hydroxyproline and crosslinks of collagen and elastin were measured from samples of 29 knees from cadavers preserved in formalin solutions. The results of measurements were hydroxyproline: ACL 0.522, PT 0.577, ST 0.463 (micromol/mg dry weight); pyridinoline/collagen: ACL 0.381, PT 0.272, ST 0.244 (mol/mol); and pentosidine/collagen: ACL 0.0434, PT 0.0558, ST 0.0799 (mol/mol). The biochemical properties of PT was not so different from ST. Pentosidine also was measured in the present study to aid in the comparison of the ligament and tendons of the knee joint.

The tensile properties of collagen fascicles harvested from regenerated and residual tissues in the patellar tendon after removal of the central third.

The central one-third portion of the patellar tendon (PT) is commonly used for the reconstruction of the anterior cruciate ligament. For better understanding of the healing mechanisms of the PT, tensile properties of collagen fascicles harvested from the healing PT were studied. A rectangular defect was made at the central third portion in each right PT in the skeletally mature rabbit. At 6 and 12 weeks, tensile tests were performed on fascicles from the tissue regenerated in the defect and the non-resected, residual tissue. The elastic modulus and tensile strength of fascicles from the regenerated tissue gradually increased in a fashion similar to the bulk regenerated tissue. The properties of fascicles from the residual tissue were similar to those from normal tendons, which was very much different from those of the bulk residual tissue that were greatly deteriorated by the removal of the central portion.

Extracellular matrix modulates expression of cell-surface proteoglycan genes in fibroblasts.

Cell-surface proteoglycans are involved in many functions, including interactions with components of the extracellular microenvironment. They also act as coreceptors that bind and modify the actions of various growth factors, cytokines, and the extracellular matrix (ECM). This study investigated the regulation by the ECM of the expression of cell-surface proteoglycans (CD44, syndecan-1-4, betaglycan, glypican-1). We examined the changes in the expression levels of cell-surface proteoglycan genes in intact tendon, monolayer culture, and under various culture conditions. There was a significant increase in the expression of CD44 and syndecan-4 mRNAs during cell isolation from the tendon. With the switch to a 3D culture environment, there was a significant increase in the expression of CD44 at each passage point relative to its expression in 2D at those passage points. Syndecan-4 mRNA also increased steadily at each passage point in 3D culture environment. This influence on cell surface proteoglycans gene expression may indicate that collagen gel culture mimics in vivo tendon environment. This study provides further insight into the regulation of cell-surface proteoglycans in ligament and tendon fibroblasts by the ECM and 3D culture conditions.

The effect of gamma irradiation on anterior cruciate ligament allograft biomechanical and biochemical properties in the caprine model at time zero and at 6 months after surgery.

BACKGROUND: High levels of gamma irradiation are required to eliminate the risk of bacterial and viral transmission during implantation of musculoskeletal allografts. The effects of high levels of gamma irradiation on anterior cruciate ligament allograft biomechanics are still not known. HYPOTHESIS: High-dose gamma irradiation (4 Mrad) adversely affects anterior cruciate ligament allograft biomechanics at surgery and at 6 months after surgery and affects biochemistry at 6 months. STUDY DESIGN: Controlled laboratory study. METHODS: Bilateral anterior cruciate ligament reconstructions were performed in 18 adult goats, with one knee receiving an irradiated patellar tendon allograft (4 Mrad) and the other receiving a frozen control allograft (0 Mrad). In 6 recipients (time zero group), graft pairs were tested immediately after sacrifice, and load relaxation of the femur-allograft-tibia preparation was measured during cyclic anterior displacement. Twelve recipients received bilateral anterior cruciate ligament reconstructions, staged 2 months apart, and were sacrificed a mean of 6 months postoperatively. Load relaxation and tensile failure testing were performed, followed by allograft biochemistry assessment. RESULTS: At time zero, irradiated grafts showed less load relaxation than did contralateral controls, but by 6 months, the trend had reversed because of decreases in control graft relaxation, with no changes in irradiated graft relaxation. By 6 months, irradiated grafts showed lower stiffness and maximum force compared to controls but no differences in modulus, maximum stress, or biochemistry. CONCLUSION: High levels of gamma irradiation affect anterior cruciate ligament allograft subfailure viscoelastic and structural properties but not material or biochemical properties over time. CLINICAL RELEVANCE: Although high levels of gamma irradiation may inactivate infectious agents, this treatment is not a feasible clinical option because of altered allograft biomechanics.

The tensile and stress relaxation responses of human patellar tendon varies with specimen cross-sectional area.

In order to provide insight into the mechanical response of the collagen fascicle structures in tendon, a series of constant strain rate and constant displacement, stress relaxation mechanical tests were performed on sequentially sectioned human patellar tendon specimens (protocol 1) and specimens with both small (approximately 1 mm2) and large (approximately 20 mm2) cross-sectional areas (protocol 2). These data described the stress relaxation and constant strain rate tensile responses as a function of cross-sectional area and water content. The experimental data suggested that small portions of tendon exhibit a higher tensile modulus, a slower rate of relaxation and a lower amount of relaxation in comparison to larger specimens from the same location in the same tendon. The decrease in relaxation response and the increase in tensile modulus with decreasing cross-sectional area was nonlinear. These data suggest that there may be structures other than the subfascicle, such as the epitenon and other connective tissue components, which influence the tensile and stress relaxation responses in tendon.

Early biological effect of in vivo gene transfer of platelet-derived growth factor (PDGF)-B into healing patellar ligament.

To define the early biological effect of in vivo introduction of the PDGF-B gene on the healing of ligaments, a HVJ-liposome suspension containing platelet-derived growth factor (PDGF)-B cDNA was injected directly into the injured patellar ligament of 14-week-old male Wistar rats. Rats were killed at 1, 4 and 8 weeks for the morphological analysis of angiogenesis by laminin immunohistochemistry and of collagen deposition by Masson's Trichrome staining and collagen I immunohistochemistry. PDGF-B gene transfer caused the enhanced expression of PDGF in healing ligament up to 4 weeks after transfection, leading to an initial promotion of angiogenesis and subsequent enhanced collagen deposition in the wound. Enhanced and accelerated matrix synthesis in the PDGF-B gene introduced healing ligament suggests that this gene transfer technique may be a potentially useful tool for improving soft tissue repair.

Chondro-osseous metaplasia, bone density and patellar cartilage proteoglycan content in the osteoarthritis of STR/ORT mice.

The evolution of osteoarthritis (OA) was compared in male and female STR/ORT mice and in male CBA mice with particular emphasis on the changes in para-articular structures which occur before the classical degenerative phenomena in the articular cartilage of the knee. In male STR but not female STR mice or male CBA mice, chondro-osseous metaplasia was found in the tendinous structures which surround the joint and in the major ligamentous entheses such as the patellar ligament. This change was detectable from the age of three months. By contrast, changes in the articular surfaces of the knee were never detected before four months while the total patellar proteoglycan content in male STR animals increased from three months. Line density profile measurements on radiographs of the knees revealed that changes in the bone density of the sub-chondral zone of the articular surfaces only increased in affected animals after the development of severe OA changes in the joint. It is suggested that the model of OA which develops in male STR/ORT mice is secondary to soft tissue changes, particularly chondro-osseous metaplasia in para-articular structures.