Characterisation of key proteoglycans in the cranial cruciate ligaments (CCLs) from two dog breeds with different predispositions to CCL disease and rupture.

Cranial cruciate ligament disease and rupture (CCLD/R) is one of the most common orthopaedic conditions in dogs, eventually leading to osteoarthritis of the stifle joint. Certain dog breeds such as the Staffordshire bull terrier have an increased risk of developing CCLD/R. Previous studies into CCLD/R have found that glycosaminoglycan levels were elevated in cranial cruciate ligament (CCL) tissue from high-risk breeds when compared to the CCL from a low-risk breed to CCLD/R. Our objective was to determine specific proteoglycans/glycosaminoglycans in the CCL and to see whether their content was altered in dog breeds with differing predispositions to CCLD/R. Disease-free CCLs from Staffordshire bull terriers (moderate/high-risk to CCLD/R) and Greyhounds (low-risk to CCLD/R) were collected and key proteoglycan/glycosaminoglycans were determined by semi-quantitative Western blotting, quantitative biochemistry, quantitative reverse transcription polymerase chain reaction, and immunohistochemistry. Gene expression of fibromodulin (P = 0.03), aggrecan (P = 0.0003), and chondroitin-6-sulphate stubs (P = 0.01) were significantly increased, and for fibromodulin this correlated with an increase in protein content in Staffordshire bull terriers compared to Greyhound CCLs (P = 0.02). Decorin (P = 0.03) and ADAMTS-4 (P = 0.04) gene expression were significantly increased in Greyhounds compared to Staffordshire bull terrier CCLs. The increase of specific proteoglycans and glycosaminoglycans within the Staffordshire bull terrier CCLs may indicate a response to higher compressive loads, potentially altering their risk to traumatic injury. The higher decorin content in the Greyhound CCLs is essential for maintaining collagen fibril strength, while the increase of ADAMTS-4 indicates a higher rate of turnover helping to regulate normal CCL homeostasis in Greyhounds.

Investigation of fibrillin microfibrils in the canine cruciate ligament in dogs with different predispositions to ligament rupture.

Cranial cruciate ligament disease (CCLD) is the most common cause of pelvic limb lameness in dogs but its precise aetiopathogenesis is uncertain. Fibrillin microfibrils (FM) are complex macro-molecular assemblies found in many tissues including ligaments, where they are thought to play an important mechanical role. We hypothesised that FM ultrastructural variation correlates with the differing predisposition of canine breeds to CCLD. Non-diseased cranial and caudal cruciate ligaments (CCLs and CaCLs) were obtained from Greyhound (GH) and Staffordshire Bull Terrier (SBT) cadavers. Fibrillin microfibrils were extracted from the ligaments by bacterial collagenase digestion, purified by size-exclusion chromatography and subsequently visualized by atomic force microscopy (AFM). With AFM, FMs have a characteristic beads-on-a-string appearance. For each FM, periodicity (bead-bead distance) and length (number of beads/FM) was measured. Fibrillin microfibril length was found to be similar for GH and SBT, with non-significant inter-breed and inter-ligament differences. Fibrillin microfibril periodicity varied when comparing GH and SBT for CCL (GH 60.2 ± 1.4 nm; SBT 56.2 ± 0.8 nm) and CaCL (GH 55.5 ± 1.6 nm; SBT 61.2 ± 1.2 nm). A significant difference was found in the periodicity distribution when comparing CCL for both breeds (P < 0.00001), further, intra-breed differences in CCL vs CaCL were statistically significant within both breeds (P < 0.00001). The breed at low risk of CCLD exhibited a periodicity profile which may be suggestive of a repair and remodelling within the CCL.

Impact of Varied Factors on Iron, Nickel, Molybdenum and Vanadium Concentrations in the Knee Joint.

The aim of this study was to determine the concentrations of iron, nickel, molybdenum, and vanadium in the knee joint. We also examined the relationships between the concentrations of these metals in the knee joint and the influence of varied factors on the concentration of Fe, Ni, Mo, and V. The study of these trace elements is important, because these elements are used alone and in combination in diet supplements, and they are components of biomaterials implanted in medicine. The study materials, consisting of the spongy bone, cartilage, meniscus, anterior cruciate ligament (ACL), and infrapatellar fat pad, were obtained from 34 women and 12 men from northwestern Poland. The concentrations of Ni, Fe, Mo, and V were determined using spectrophotometric atomic absorption in inductively coupled argon plasma (ICP-AES). We found significantly higher Mo concentrations in the ACL of women than men. There was a significant difference in the Mo concentration in the spongy bone between patients from cities with fewer than 100,000 inhabitants and patients from cities with more than 100,000 residents. Iron concentrations in the spongy bone were higher in non-smoking patients and those who did not consume alcohol. Vanadium concentrations were higher in the infrapatellar fat pads in abstainers. In patients who had not undergone arthroscopy surgery, V concentration was lower in cartilage. The concentrations of V in the cartilage and infrapatellar fat pad were higher in osteoporotic patients than in non-osteoporotic patients. There were significant differences in Fe concentrations in the meniscus, with the lowest in osteoporotic patients. We noted lower Mo concentrations in the spongy bone of patients with rheumatoid arthritis. Furthermore, we noted some new interactions among metals in the studied structures of the knee joint. The results reported in this study show the influence of gender, place of residence, smoking, consumption of alcohol, arthroscopy surgery, osteoporosis, and rheumatoid arthritis on the Fe, Ni, Mo, and V concentrations in the studied structures of the knee joint.

A comparative study of the epiligament of the medial collateral and the anterior cruciate ligament in the human knee. Immunohistochemical analysis of collagen type I and V and procollagen type III.

BACKGROUND: Recent reports in rat models have shown that fibroblasts in the epiligament, an enveloping tissue of the ligament, are not static cells and play an important role during the early ligament healing of isolated grade III injury of the collateral ligaments of the knee. Fibroblasts produce collagen types I, III and V and infiltrate within the ligament body via the endoligament. In addition, similarities have been reported between the structure of the epiligament of the medial collateral ligament and anterior cruciate ligament of the knee in rat and in human. In line with the ascribed role of the epiligament tissue and the synthesis of these collagens and their role in ligament healing, the aim of this study was to determine their presence in the normal epiligament of the aforementioned ligaments in humans, to compare their differential expression and to present a novel hypothesis about the failure of healing of the anterior cruciate ligament in contrast to the medial collateral ligament. MATERIALS AND METHODS: We used samples from the mid-substance of the medial collateral and the anterior cruciate ligament of the knee joint, acquired from 12 fresh knee joints. Routine histological analysis was performed through hematoxylin and eosin stain, Mallory's trichrome stain and Van Gieson's stain. The immunohistochemical analysis was conducted using monoclonal antibodies against collagen type I and V and procollagen type III. The number of cells in the epiligament, endoligament and the ligament tissue was assessed quantitatively through a computerized system for image analysis NIS-Elements Advanced Research and Statistica software. RESULTS: Our observations revealed certain differences in the morphology of the epiligament, as well as variations in the expression of the investigated molecules. Expression of collagen type I was mostly low-positive (1+) in the epiligament and positive (2+) in the ligament tissue of both ligaments. Expression of procollagen type III was mostly positive (2+) in the epiligament and ligament tissue of the medial collateral ligament, low-positive (1+) in the epiligament and negative (0) in ligament tissue of the anterior cruciate ligament. Expression of collagen type V was predominantly low-positive (1+) in the epiligament and negative (0) in the ligament tissue of both ligaments. The immunoreactivity for all three molecules was always higher in the epiligament of the medial collateral ligament than that of the anterior cruciate ligament. CONCLUSIONS: The results of our study illustrate for the first time that fibroblasts in the human epiligament are indeed responsible for the synthesis of the main types of collagen participating in the early ligament healing, thus corresponding to previous data of the medial collateral ligament healing in animal models. The differences between the epiligament of the investigated ligaments could add a novel explanation for the failed anterior cruciate ligament healing.

Compositional mapping of the mature anterior cruciate ligament-to-bone insertion.

The anterior cruciate ligament (ACL)-to-bone interface constitutes a complex, multi-tissue structure comprised of contiguous ligament, non-mineralized fibrocartilage, mineralized fibrocartilage, and bone regions. This composite structure enables load transfer between structurally and functionally dissimilar tissues and is critical for ligament homeostasis and joint stability. Presently, there is a lack of quantitative understanding of the matrix composition and organization across this junction, especially after the onset of skeletal maturity. The objective of this study is to characterize the adult bovine ACL-to-bone interface using Fourier transform infrared spectroscopic imaging (FTIRI), testing the hypothesis that regional changes in collagen, proteoglycan, and mineral distribution, as well as matrix organization, persist at the mature insertion. It was observed that while collagen content increases continuously across the adult interface, collagen alignment decreases between ligament and bone. Proteoglycans were primarily localized to the fibrocartilage region and an exponential increase in mineral content was observed between the non-mineralized and mineralized regions. These observations reveal significant changes in collagen distribution and alignment with maturity, and these trends underscore the role of physiologic loading in postnatal matrix remodeling. Findings from this study provide new insights into interface organization and serve as benchmark design criteria for interface regeneration and integrative soft tissue repair. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2513-2523, 2017.

Rolled knitted scaffolds based on PLA-pluronic copolymers for anterior cruciate ligament reinforcement: A step by step conception.

The aim of this study was to prepare a new knitted scaffold from PLA-Pluronic block copolymers for anterior cruciate ligament reconstruction. The impact of sterilization methods (beta-ray and gamma-ray sterilization) on copolymers was first evaluated in order to take into account the possible damages due to the sterilization process. Beta-ray radiation did not significantly change mechanical properties in contrast to gamma-ray sterilization. It was shown that ACL cells proliferate onto these copolymers, demonstrating their cytocompatibility. Thirdly, in order to study the influence of shaping on mechanical properties, several shapes were created with copolymers yarns: braids, ropes and linear or rolled knitted scaffolds. The rolled knitted scaffold presented interesting mechanical characteristics, similar to native anterior cruciate ligament (ACL) with a 67 MPa Young's Modulus and a stress at failure of 22.5 MPa. These findings suggest that this three dimensional rolled knitted scaffold meet the mechanical properties of ligament tissues and could be suitable as a scaffold for ligament reconstruction. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 735-743, 2017.

Gold and Hydroxyapatite Nano-Composite Scaffolds for Anterior Cruciate Ligament Reconstruction: In Vitro Characterization.

Current anterior cruciate ligament (ACL) graft replacement materials often fail due to the lack of biological integration. While many newly developed extracellular matrix based scaffolds show good biocompatibility they often do not entice cellular remodeling and the rebuilding of a functional ligament. We have proposed the conjugation of gold nanoparticles (AuNP) and hydroxyapatite nanoparticles (nano-HAp) to acellular tissue to enhance cell attachment and proliferation while maintaining an improved degradation resistance and open microstructure. We are the first to investigate the double conjugation of AuNP and nano-HAp onto decellularized tissue to improve the tissue remodeling response. Decellularized porcine diaphragm was crosslinked with two types of nano-HAp and amine-functionalized AuNP with 1-ethyl-3-(3-dimethlaminopropyl) carbodiimide (EDC) crosslinker. Scaffolds were characterized using electron microscopy, differential scanning calorimetry, and fibroblast assays. Results demonstrated that scaffolds with nano-HAp have increased thermal stability at low levels of crosslinking. The open microstructure of the scaffold was not compromised allowing for cell migration while still providing increased degradation resistance. The addition of < 200 nm nano-HAp decreased cell viability compared to scaffolds without nanoparticles, but the addition of AuNP to scaffolds showed enhanced cell viability in the presence of < 200 nm nano-HAp. The addition of < 40 nm nano-HAp showed an increase in cell viability compared to scaffolds crosslinked without nanoparticles. It is concluded that attaching AuNP and < 40nm nano-HAp to extracellular matrices may improve overall properties.

PLA-poloxamer/poloxamine copolymers for ligament tissue engineering: sound macromolecular design for degradable scaffolds and MSC differentiation.

The treatment of anterior cruciate ligament (ACL) failures remains a current clinical challenge. The present study aims at providing suitable degradable scaffolds for ligament tissue engineering. First, we focus on the design and the evaluation of poly(lactide)/poloxamer or poly(lactide)/poloxamine multiblock copolymers selected and developed to have suitable degradation and mechanical properties to match ACL repair. In the second part, it is shown that the copolymers can be processed in the form of microfibers and scaffolds consisting of a combination of twisted/braided fibers to further modulate the mechanical properties and prepare scaffold prototypes suitable for ligament application. Finally, after assessment of their cytocompatibility, the polymer scaffolds are associated with mesenchymal stem cells (MSCs). MSC differentiation toward a ligament fibroblast phenotype is promoted by a dual stimulation including an inductive culture medium and cyclic mechanical loads. RT-qPCR analyses confirm the potential of our scaffolds and MSCs for ACL regeneration with upregulation of some differentiation markers including Scleraxis, Tenascin-C and Tenomodulin.

Degenerative changes of the cranial cruciate ligament harvested from dogs with cranial cruciate ligament rupture.

Degenerative cranial cruciate ligament (CCL) rupture is characterized histologically by degenerating extracellular matrix (ECM) and chondroid metaplasia. Here, we describe the progression of chondroid metaplasia and the changes in the expression of ECM components in canine CCL rupture (CCLR). CCLs from 26 stifle joints with CCLR (CCLR group) and normal CCLs from 12 young beagles (control group) were examined histologically and immunohistochemically for expression of type I (COLI), type II (COLII), type III collagen (COLIII) and Sry-type HMG box 9 (SOX9). Cell density and morphology of CCLs were quantified using hematoxylin-eosin staining. The percentage of round cells was higher in the CCLR group than in controls. COLI-positive areas were seen extensively in the connecting fibers, but weakly represented in the cytoplasm of normal CCLs. In the CCLR group, there were fewer COLI-positive areas, but many COLI-positive cells. The percentages of COLII-, COLIII- and SOX9-positive cells were higher in the CCLR group than in controls. The number of spindle cells with perinuclear halo was high in the CCLR group, and most of these cells were SOX9-positive. Deposition of COLI, the main ECM component of ligaments, decreased with increased COLIII expression in degenerated CCL tissue, which shows that the deposition of the ECM is changed in CCLR. On the contrary, expression of SOX9 increased, which may contribute to the synthesis of cartilage matrix. The expression of COLII and SOX9 in ligamentocytes showed that these cells tend to differentiate into chondrocytes.

Elastin content is high in the canine cruciate ligament and is associated with degeneration.

Cruciate ligaments (CLs) are primary stabilisers of the knee joint and canine cranial cruciate ligament disease (CCLD) and rupture is a common injury. Elastin fibres, composed of an elastin core and fibrillin containing microfibrils, are traditionally considered minor components of the ligament extracellular matrix (ECM). However, their content and distribution in CLs is unknown. The purposes of this study were to determine the elastin content of canine CLs and to ascertain its relationship to other biochemical components and histological architecture. Macroscopically normal CLs were harvested from Greyhounds (n=11), a breed with a low risk of CCLD. Elastin, collagen and sulfated glycosaminoglycan content were measured and histological scoring systems were developed to quantify ECM changes using a modified Vasseur score (mVS) and oxytalan fibre (bundles of microfibrils) staining. Elastin contents were 9.86 ± 3.97% dry weight in the cranial CL and 10.79 ± 4.37% in the caudal CL, respectively, and did not alter with advancing histological degeneration. All CLs demonstrated mild degenerative changes, with an average mVS score of 11.9 ± 3.3 (maximum 24). Increasing degeneration of the ligament ECM showed a positive correlation (r=0.690, P<0.001) with increased oxytalan fibre staining within the ECM. Elastin is an abundant protein in CLs forming a greater proportion of the ligament ECM than previously reported. The appearance of oxytalan fibres in degenerative CL ECM may reflect an adaptive or reparative response to normal or increased loads. This finding is important for future therapeutic or ligament replacement strategies associated with cranial CL injury.

Crimp frequency is strongly correlated to myofibroblast density in the human anterior cruciate ligament and its autologous tendon grafts.

PURPOSE: Collagen crimp is essential for maintaining viscoelastic properties of normal ligament and tendon tissue. The actin isoform α-smooth muscle actin (ASMA) has been identified in fibroblastic cells of these tissues. These highly differentiated cells, so-called myofibroblasts may transmit tensile forces to the extracellular matrix, thus it has been suggested that they are responsible for the wrinkling of the extracellular matrix and the formation of crimp. During anterior cruciate ligament (ACL) graft remodeling, crimp formation plays an integral role. Thus, it was our purpose to determine the relationship between myofibroblast density and crimp frequency in human tendon graft tissue and the ACL. METHODS: Different tendon grafts and ACLs were harvested from young human multi-organ donors immediately after death. Myofibroblasts were immunostained with a monoclonal antibody, and histomorphometry was performed using a digital imaging system. Crimp length was measured, and data were correlated. RESULTS: All tendons and ACLs showed a significant correlation of myofibroblast density and crimp frequency (R(2) 0.81-0.43). The strongest correlation was found for the patellar tendon, the poorest for the gracilis tendon. There is also evidence that the phenotype respectively the shape of myofibroblasts might be responsible for different stages of crimp formation. CONCLUSION: With the present investigation, we found that myofibroblasts might be involved in crimp formation and should be viewed as an integral part of normal tendon and ligament tissue. Furthermore, the shape of myofibroblasts may further indicate the contractile potency of the extracellular matrix, thus presenting a dynamic and variable crimp rather than a static situation. This study is an experimental study. In terms of clinical relevance all the mentioned tendons can be used as auto- or allografts for ACL reconstruction, nevertheless their microscopic structure and cellular population have yet not been adequately investigated and compared.

Effects of vibration on the proteome expression of anterior cruciate ligament cells.

Recent reports have suggested that vibration has beneficial effects on knee healing response; however, the biomechanism of these beneficial effects still need to be determined on the anterior cruciate ligament (ACL) cell level. In this study, we applied a 20 Hz vibration to ACL cells, which produced a 20% increase (P < 0.001) in cell activity and 17% increase (P < 0.001) in intracellular sulfated glycosaminoglycan levels. In the 20 Hz vibration-stimulated ACL cell group, eight up-regulated (100 ∼ 300%) protein spots were identified compared with the control group by proteomics analysis. Among these proteins, Annexin A2 and Prolyl 4 hydroxylase (PH4B) were shown to have a 71% and 16% higher expression, respectively, in the 20 Hz vibration-stimulated ACL cell group by Western blotting (P < 0.001). These results indicate that vibration produces a positive cellular environment, and Annexin A2 and prolyl 4 hydroxylase are expected to help ligament repair and ACL cell proliferation by controlling cell membrane and extracellular matrix formation.

Characterization of the anchoring morphology and mineral content of the anterior cruciate and medial collateral ligaments of the knee.

The manner in which ligament connects to bone remains an area of interest for researchers, bioengineers, and clinicians. Stable fixation of an anterior cruciate ligament (ACL) graft has been shown to be paramount to preventing excess anterior tibial translation and to restoring the normal kinematics of the knee joint. In this study, the surface area of attachment and the mineral characteristics of the ACL and medial collateral ligament (MCL) attachment sites were characterized to determine the factors that contributed to ligament attachment strength. Findings from this study indicated that the area of attachment of the ACL's insertion was significantly greater than the ligament's origin (95.8 mm(2) ± 21.5 vs. 73.2 mm(2) ± 16.2, P = 0.009). Additionally, the ACL was measured to have a greater surface area of attachment when compared with the MCL (84.5 mm(2) ± 18.8 vs. 58.2 mm(2) ± 23.8, P = 0.005); although, the MCL was observed to have a greater region of calcified fibrocartilage (CFC) than the ACL (533.0 µm ± 116.9 vs. 195.5 µm ± 36.6, P = 0.0003). No significant correlation was observed between the ligament's area of attachment and the thickness of the CFC region. Measurements of ash percent suggested that the boundary region, between the CFC and host bone, possessed the least mineral content for the three regions of interest. These data suggest that ligament attachment strength can be attributed to several factors, including the ligament's area of attachment, regional thickness, and mineral content of the CFC.

The effect of uniaxial cyclic tensile load on gene expression in canine cranial cruciate ligamentocytes.

OBJECTIVE: To determine the effects of uniaxial cyclic tensile load amplitude and duration on gene expression in cranial cruciate ligamentocytes cultured in monolayer. STUDY DESIGN: In vitro experimental study. ANIMALS: Adult dogs (n=9) weighing 20-35 kg. METHODS: Cranial cruciate ligaments (CCL, n=18) were aseptically collected, diced, digested using clostridial collagenase, and primary monolayer cultures were established. CCL cells were seeded at a concentration of 3 x 10(5) cells/mL onto a specialized collagen membrane. After 24 hours to allow attachment, ligamentocytes were subjected to 0%, 4%, or 8% uniaxial strain for 24 or 48 hours using a sinusoidal strain profile at 0.5 Hz. At the end of each time point, the ligamentocytes were harvested and analyzed for collagen 1 (COL1), collagen 3 (COL3), and matrix metalloproteinase-3 (MMP-3) gene expression using reverse transcriptase real-time polymerase chain reaction. RESULTS: Approximately 33% of CCL processed for this study yielded viable cell cultures compared with 100% of the medial collateral ligaments processed. For CCL cells under uniaxial strain, gene expression for COL1 was variable, but higher strains and longer time in culture resulted in increased COL1 expression. There were no significant differences found for COL3 at any time point or between strain regimens. In general, MMP-3 gene expression was increased early in tissue culture and at higher strains. CONCLUSIONS: COL1 and MMP-3 gene expression can be influenced by amplitude and duration of strain on CCL cells in monolayer culture. CLINICAL RELEVANCE: These data have implications for modeling and understanding canine cruciate ligament pathophysiology. In particular, MMP-3 could serve as a potential preventative or therapeutic target in cruciate disease.

Immunohistological evaluation of proprioceptive potential of the residual stump of injured anterior cruciate ligaments (ACL).

To evaluate proprioceptive potential in residual remnants, tissue harvested from ruptured ACLs in 63 consecutive patients was examined for evidence of residual proprioceptive fibres using H&E, and monoclonal antibodies to S-100 and NFP (neurofilament protein). Histological examination showed good subsynovial and intra-fascicular vascularity with free nerve endings in the majority. Morphologically normal mechanoreceptors (H&E) and proprioceptive fibres (positivity with monoclonal antibody for NFP) were found in 46% and 52.4% of stumps, respectively. A statistically significant correlation between injury duration and persistence of mechanoreceptors and proprioceptive fibres was noted. More fibres were seen where ACL remnant was adherent to PCL. Our study has shown that persistent residual proprioceptive fibres in injured ACLs (especially early cases with PCL adherence) are significant; not shaving ACL remnants may be of potential benefit during ACL reconstruction, as some re-innervation and recovery of proprioceptive potential may be possible, thus improving clinical outcomes.

Mucoid degeneration of the anterior cruciate ligament.

Mucoid degeneration of the anterior cruciate ligament (ACL) is a rare cause of knee pain. We report a case of a patient with mucoid degeneration of the ACL, presenting with posterior knee pain and no history of a major knee trauma. On clinical examination, the active range of motion showed a flexion deficit. The posterior knee pain was induced by passive hyperflexion of the knee. There was no evidence of ligamentary instability. MRI showed a diffuse thickening of the ACL with a nodular mass on the femoral insertion occupying the intercondylar notch, with increased signal intensity on both T1- and T2-weighted images. Arthroscopic evaluation showed a diffuse hypertrophy of the ACL, throughout the entire length of the posterolateral bundle (PLB). A yellowish homogenous mass on the femoral insertion of the ACL impinged on the posterior cruciate ligament (PCL) in flexion and occupied the entire intercondylar notch. We performed an arthroscopic debridement of the hypertrophied tissues as precisely as possible. This resulted in a nearly complete removal of the PLB and immediate relief of symptoms. Examination of knee stability after debridement showed a stable ACL. Arthroscopic debridement of the mucoid degeneration of the ACL proved to be a safe and effective method, without causing ligamentary instability in daily activities.

Increase in excitatory amino acid concentration and transporters expression in osteoarthritic knees of anterior cruciate ligament transected rabbits.

OBJECTIVE: The present study aimed to determine the role of excitatory amino acids (EAAs) and EAA transporters (EAATs) in an osteoarthritis (OA) model of rabbit knees. METHODS: OA was induced in New Zealand white male rabbits by anterior cruciate ligament transection (ACLT) in one knee of one hind limb; the other knee left unoperated. Rabbits that received ACLT of knee were assigned to the ACLT group (n=6), while a sham-operated group (n=6) underwent arthrotomy with no ACLT. Six naïve rabbits that received no surgery were used as normal control. The width of the knee joint was measured to determine the severity of joint inflammation. Before operation and at 10, 20, and 30 weeks after operation, knee joint dialysates were collected by microdialysis and assayed for EAAs by high-performance liquid chromatography. Gross morphology and histopathology and EAATs glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) expression in the articular cartilage of the knees were evaluated by immunohistochemistry and western blot analysis. RESULTS: In the ACLT knees, a significant increase in the joint width was observed (5.3+/-0.9 mm, P<0.05) at 30 weeks after operation, while the sham-operated and naïve knees showed no difference as compared with the basal values. The concentrations (microM) of aspartate and glutamate in knee dialysates at 30 weeks after ACLT in naïve, sham, and ACLT were 0.36+/-0.07 and 4.5+/-1.10; 0.38+/-0.09 and 4.61+/-1.11; 0.67+/-0.18 and 9.71+/-2.89, respectively. Levels of glutamate and aspartate in the dialysates obtained from the ACLT knees increased by 213.3+/-29.6% and 187.5+/-33.8% (P<0.05) when compared to those in the sham-operated knees. Both naïve and ACLT chondrocytes were positively stained by antibodies against GLAST and GLT-1. GLAST and GLT-1 protein expressions were significantly increased in the ACLT knees (P<0.05). CONCLUSION: Our findings indicate an involvement of EAAs and EAATs in the pathogenesis of OA in ACLT rabbits.

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.

Quantitation of estrogen receptors and relaxin binding in human anterior cruciate ligament fibroblasts.

The significantly higher incidence of anterior cruciate ligament (ACL) injuries in collegiate women compared with men may result from relative ligament laxity. Differences in estrogen and relaxin activity, similar to that seen in pregnancy, may account for this. We quantified estrogen receptors by flow cytometry and relaxin receptors by radioligand binding assay in human ACL cells and compared the presence of these receptors in males and females. ACL stumps were harvested from seven males and eight females with acute ACL injuries. The tissue was placed in M199 cell culture medium. Outgrowth cultures were obtained, and passage 2 cells were used for all studies. Estrogen receptor determination was performed using flow cytometry. Relaxin binding was performed in ACL cells derived from five female and male patients using I(125)-labeled relaxin. Estrogen receptors were identified by flow cytometry in 4 to 10% of ACL cells. Mean fluorescence of cells expressing estrogen receptors was approximately twice that of controls, with no significant differences between males and females. Relaxin studies showed low-level binding of I(125)-relaxin-labeled ACL cells. Relaxin binding was present in four out of five female ACL cells versus one out of five male ACL cells.

Morphological, biochemical and mechanical features of the cranial cruciate and lateral collateral ligaments in dogs.

The cruciate ligament and the collateral ligament play key roles in stabilization of the knee joint. Cases of serious knee joint problems presented at the, the Veterinary Teaching Hospital of Rakuno Gakuen University, Japan mostly involved rupture of the cranial cruciate ligament (CCL). Disorders in structural and biochemical components of the CCL were thought to be the causes of the knee problems. Morphological, biochemical and biomechanical features of the CCL and the lateral collateral ligament (LCL) were therefore analyzed. In the CCL, fibroblasts with ovoid and enlarged nuclei were observed mainly at the periphery of collagen bundles. The array of collagen fibrils in the LCL was slightly disoriented, but that of the CCL was tight and regular. In the LCL, the major groups of collagen fibrils were those with diameters of 70-80 and 120-130 nm. Most collagen fibrils in the CCL had diameters of 70-80 nm. The mean collagen diameters were 90 nm in the CCL and 105 nm in the LCL. The ratios of the noncollagen area to the area occupied by collagen fibrils were 43% in the CCL and 55% in the LCL. There was no difference between the amounts of HA or between the amounts of DS in two ligaments. However, the amount of CS in the CCL was about 17-times greater than that in the LCL. The expansion of and the resistance to tension exerted onto the CCL were less than those of the LCL. A high concentration of CS and low tensile strength due to small-sized collagen fibrils cause the CCL to rupture easily, especially when overextension of the knee joint occurs.