Versican contributes to ligament formation of knee joints.

Versican is a large proteoglycan in the extracellular matrix. During embryonic stages, it plays a crucial role in the development of cartilage, heart, and dermis. Previously, we reported that Prx1-Vcan conditional knockout mice, lacking Vcan expression in mesenchymal condensation areas of the limb bud, show the impaired joint formation and delayed cartilage development. Here, we investigated their phenotype in adults and found that they develop swelling of the knee joint. Histologically, their newborn joint exhibited impaired formation of both anterior and posterior cruciate ligaments. Immunostaining revealed a decrease in scleraxis-positive cells in both articular cartilage and ligament of Prx1-Vcan knee joint, spotty patterns of type I collagen, and the presence of type II collagen concomitant with the absence of versican expression. These results suggest that versican expression during the perinatal period is required for cruciate ligaments' formation and that its depletion affects joint function in later ages.

Distribution, quantity and gene expression of mechanoreceptors in ligaments and tendons of knee joint in rabbits.

Here we investigated the morphology, quantity, distribution and gene expression of mechanoreceptors in the anterior cruciate ligament (ACL), posterior cruciate ligament (PCL), medial collateral ligament (MCL), lateral collateral ligament (LCL), patellar tendon (PaT) and popliteal tendon (PoT) of the knee joint. Twelve 6-month-old rabbits were divided into two groups. In one group, the ACL, PCL, MCL, LCL, PaT and PoT were collected to observe the morphology, distribution and quantity of mechanoreceptors. In another group, the ACL, PCL, MCL, LCL, PaT and PoT of bilateral knee joints were used to determine S100B, CGRP and NEFM gene levels. Five types of mechanoreceptors were observed including Ruffini corpuscles, Pacinian corpuscles, Golgi-tendon bodies, atypical mechanoreceptors and free nerve endings. The total amount of mechanoreceptors was significantly lower in MCL, LCL, PaT and PoT as compared with ACL and PCL (P < 0.001). All examined mechanoreceptors were present in ACL, PCL and LCL. However, no Pacinian corpuscles and Golgi-tendon bodies were found in MCL and PoT as well as Pacinian corpuscles were not observed in PaT. The present study indicated that the levels of NEFM was significantly lower in PCL, MCL, LCL, PaT and PoT as compared with ACL (P < 0.05), but there was no significant difference in CGRP level between ACL and other ligaments except LCL (P > 0.05). Thus, the quantity, type and gene expression of mechanoreceptors are different in various ligaments. Thus, the quantity and distribution of mechanoreceptors may be related to ligament's function.

Cruciate ligament, patellar tendon, and patella formation involves differential cellular sources and dynamics as joint cavitation proceeds.

BACKGROUND: Cruciate ligament (CL) and patellar tendon (PT) are important elements of the knee joint, uniting femur, patella, and tibia into a single functional unit. So far, knowledge on the developmental mechanism of CL, PT, and patella falls far behind other skeletal tissues. RESULTS: Here, employing various lineage tracing strategies we investigate the cellular sources and dynamics that drive CL, PT, and patella formation during mouse embryonic development. We show that Gdf5 and Gli1 are generally expressed in the same cell population that only contributes to CL, but not PT or patella development. In addition, Col2 is expressed in two independent cell populations before and after joint cavitation, where the former contributes to the CL and the dorsal part of the PT and the latter contributes to the patella. Moreover, Prrx1 is always expressed in CL and PT progenitors, but not patella progenitors where it is switched off after joint cavitation. Finally, we reveal that patella development employs different cellular dynamics before and after joint cavitation. CONCLUSIONS: Our findings delineate the expression changes of several skeletogenesis-related genes before and after joint cavitation, and provide an indication on the cellular dynamics underlying ligament, tendon, and sesamoid bone formation during embryogenesis.

The morphology and histology study on rabbit degenerated medial meniscus after posterior cruciate ligament rupture.

The morphology and histology changes in the medial meniscus after posterior cruciate ligament (PCL) rupture are poorly understood. Forty-eight rabbits were divided into matched mode pairs; each rabbit had an experimental side, in which the PCL was transacted, and a control side. At the 4, 8, 16 and 24 weeks after the PCL transection, each of the 12 rabbits was killed. Histology was performed to detect the expression of the tissue inhibitors of metalloproteinases-1 (TIMP-1), matrix metalloproteinase (MMP)-1 and MMP-13 in the medial meniscus. We found that medial meniscus displayed significant degenerative characteristics in morphology. The histological evaluation of the degeneration found that the expression levels of TIMP-1, MMP-1 and MMP-13 in the medial meniscus were higher in the experiment side than those in the control side (P<0.05). The expression of both TIMP-1 and MMP-13 was initially elevated and then decreased. The MMP-1 expression reached its peak swiftly and then maintained a relatively high level. There were clear time-dependent degenerative changes in the histology of the medial meniscus after PCL rupture. The high expression of TIMP-1, MMP-1 and MMP-13 in the cartilage may be responsible for the degeneration, and PCL rupture may trigger meniscus degradation and ultimately osteoarthritis.

Differential expressions of the lysyl oxidase family and matrix metalloproteinases-1, 2, 3 in posterior cruciate ligament fibroblasts after being co-cultured with synovial cells.

PURPOSE: The adult human posterior cruciate ligament (PCL) has poor functional healing response. The synovial tissue, which surrounds the PCL ligament, might be the major regulator of the microenvironment in the joint cavity after PCL injury, thus affecting the healing process. Here we establish a novel co-culture system for PCL fibroblasts and synovial cells (SC) in vitro to explore the direct influence of paracrine on PCL cells by characterizing the different expressions of the lysyl oxidase family (LOXs) and matrix metalloproteinases (MMP-1, 2, 3), which respectively facilitate extracellular matrix (ECM) repair and degradation. METHODS: Total RNA was harvested, reverse transcribed and assessed by semi-quantitative PCR and real-time PCR for the expression of LOXs and MMP-1, 2, 3 messenger RNAs. MMP-2 activity was assayed from the collected culture media samples by using zymography. RESULTS: We found co-culture could promote gene expressions of the LOXs and MMP-1, 2, 3 in normal PCL fibroblasts. But in injured PCL, we found that matrix crosstalk induced an increase of the MMP-1, 2, 3 expressions and a down-regulation of the LOXs. CONCLUSION: Based on these results, the crosstalk between PCL and SC strongly modified homeostatic balance of ECM and appeared to have a significant impact on PCL wound healing; decreased expression of cross-linking enzymes (LOXs) and increased expression of ECM-degrading proteinases (MMP-1, 2, 3) might be of great contribution to poor healing ability of PCL ligament.

Usefulness of specific OA biomarkers, thrombin-cleaved osteopontin, in the posterior cruciate ligament OA rabbit model.

OBJECTIVE: We undertook this study to determine whether thrombin-cleaved osteopontin (OPN) in synovial fluid (SF) represents a useful marker of osteoarthritis (OA) progression in the posterior cruciate ligament transection (PCLT) OA rabbit model. METHOD: PCLT was performed on the right knee joints of 48 rabbits. The rabbits were then sacrificed separately at 4, 8, 16, and 24 weeks post-surgery, when the joint was harvested and macroscopic and histological assessments of articular cartilage were performed. Thrombin-cleaved OPN product in SF was determined using Western blotting and the levels were measured using an enzyme-linked immunoassay. RESULTS: The macroscopic and histological scores for PCLT knees were already elevated 4 weeks after surgery and increased with time. Western blotting showed the presence of thrombin-cleaved OPN in SF from PCLT knees. Thrombin-cleaved OPN levels in SF were elevated at 4 weeks (P < 0.001) and were elevated peaking at 24 weeks (P < 0.00001) after PCLT compared to baseline. A positive significant correlation was found between thrombin-cleaved OPN levels and the macroscopic scores (8 weeks: ρ = 0.695, P = 0.012; 16 weeks: ρ = 0.751, P = 0.005; 24 weeks: ρ = 0.660, P = 0.020). Furthermore, the same correlation was noted between thrombin-cleaved OPN levels and the histological scores (4 weeks: ρ = 0.609, P = 0.036; 8 weeks: ρ = 0.662, P = 0.019; 16 weeks: ρ = 0.827, P = 0.001; 24 weeks: ρ = 0.813, P = 0.001). CONCLUSION: In this rabbit model of PCLT, thrombin-cleaved OPN levels in SF appear to provide a useful marker of OA disease severity and progression.

The extent of degeneration of cruciate ligament is associated with chondrogenic differentiation in patients with osteoarthritis of the knee.

OBJECTIVE: Degeneration in cruciate ligaments results from abnormal biomechanical stress and the aging process. Such degeneration is a common outcome in patients with osteoarthritis (OA) of the knee and contributes to the progression of OA. However, to date, there are no specific markers that can predict the extent of ligament degeneration. We hypothesized that the extent of degeneration has correlations to increased chondrogenic potential. METHODS: Twenty anterior cruciate ligaments (ACLs) and 30 posterior cruciate ligaments (PCLs) from 30 knees of 28 adult patients with OA at the time of total knee arthroplasty were used for the study. Degeneration was histologically assessed using a grading system. Expressions of Scleraxis (as a ligament cell marker) and Sry-type HMG box 9 (SOX9) (as a chondrogenic marker) were immunohistochemically assessed in each grade. RESULTS: We found the opposite expression pattern between Scleraxis and SOX9 according to the grade. The percentage of Scleraxis-positive cells decreased significantly by grade (60.9±23.7 in grade 1, 39.7±30.5 in grade 2, and 13.9±27.1 in grade 3, P<0.0001). In contrast, the percentage of SOX9-positive cells increased significantly by grade (2.5±4.9 in grade 1, 17.5±13.4 in grade 2, and 50.9±27.1 in grade 3, P<0.0001). Furthermore, co-localized expression of both Scleraxis and SOX9 was demonstrated in chondrocyte-like cells. CONCLUSIONS: This study indicates that chondrogenic differentiation is associated with the progression of degeneration in human ligaments. Our results suggest that the expression of SOX9 as a chondrogenic marker could be an indicator for the extent of degeneration in human ligaments. It remains to be elucidated whether suppression of chondrogenic differentiation can prevent progression of the degenerative process of cruciate ligaments in patients with OA.

Posterior cruciate mechanoreceptors in osteoarthritic and cruciate-retaining TKA retrievals: a pilot study.

BACKGROUND: Although normal cruciate ligaments and those in patients with osteoarthritic (OA) knees contain mechanoreceptors, it is unclear whether they are present after functioning in a cruciate-retaining total knee arthroplasty (TKA). QUESTIONS/PURPOSES: We therefore determined if the areas occupied by mechanoreceptors in the human posterior cruciate ligament (PCL) are similar in patients with osteoarthritis and in patients who have had TKA with retention of the PCL. METHODS: We identified five cruciate-retaining TKA specimens from a retrieval program and obtained five PCLs during cruciate-sacrificing TKA from patients with OA; the retrieved specimens had been in place 5 to 12 years. The whole en bloc PCL specimens were harvested for the study. These specimens were then sectioned to a thickness of 8 µm and mounted on microscope slides. Two transverse cross-sections from the distal third from each specimen 100 µm apart were then subjected to immunohistochemistry with neurofilament protein (NFP) and S-100 protein. RESULTS: All five PCL specimens in each group revealed multiple areas of positive stained elements with both S-100 protein and NFP immunohistochemical staining. Morphologically, these elements appear to correspond to Pacinilike, Golgilike, and fusiform types of mechanoreceptors. We observed no difference in positive staining mechanoreceptor elements as a percentage of area in the osteoarthritis and TKA groups. CONCLUSION: Mechanoreceptors appear to occupy similar areas before and after implantation of a TKA. CLINICAL RELEVANCE: If mechanoreceptors continue to function after cruciate-retaining TKA, then it may continue to participate in proprioception of the knee after TKA.

Mechanoreceptors found in a posterior cruciate ligament from a well-functioning total knee arthroplasty retrieval.

Histologic analysis of the posterior cruciate ligament has been reported in the normal and osteoarthritic knee but not after cruciate-retaining (CR) total knee arthroplasty (TKA). Retention of the posterior cruciate ligament during TKA has been debated as to whether it is beneficial in stability and function. If the presence of mechanoreceptors is shown to be maintained in CR TKA, then there may be an argument for retention. This case report used a retrieval of a well-functioning TKA specimen that had a CR TKA. To prove the presence of mechanoreceptors within the ligament, immunohistochemistry techniques using S100 protein and neurofilament protein were used. This specimen had pacini and lamellar type of mechanoreceptors present on immunohistochemistry analysis. The presence or retention of mechanoreceptors and innervations of the ligament may indicate an advantage when retained during TKA.

Sox9-expressing precursors are the cellular origin of the cruciate ligament of the knee joint and the limb tendons.

Sox9 expression defines cell progenitors in a variety of tissues during mouse embryogenesis. To establish a genetic tool for cell-lineage tracing and gene-function analysis, we generated mice in which the CreERT2 gene was targeted to the endogenous mouse Sox9 locus. In Sox9(CreERT2/+) ;R26R embryos, tamoxifen activated Cre recombinase exclusively in Sox9-expressing tissues. To determine the suitability of this mouse line for developmental stage-specific gene recombination, we investigated the cellular origins of the cruciate ligaments of the knee joint and the limb tendons, in which precursor cells have not been defined. The cells in these tissues were labeled after tamoxifen treatment before or at the stage of chondrogenic mesenchymal condensation, indicating that ligament and tendon cells originated from Sox9-expressing cells and that cell fate determination occurred at mesenchymal condensation. This mouse line is a valuable tool for the temporal genetic tracing of the progeny of, and inducible gene modification in Sox9-expressing cells.

Mechanoreceptors of the posterior cruciate ligament.

The mechanical role of the anterior and posterior cruciate ligaments in the passive and functional stability of the knee joint has been well documented. Both these knee joint ligaments contain Ruffini, Pacinian, Golgi and free nerve endings with different capabilities of providing the central nervous system with information regarding movement and position as well as chemical events. The posterior cruciate ligament provides 95% of the restraining force to a posterior tibial displacement, is significantly stronger than the other knee ligaments, and sensory nerve endings are located in the tibia and femoral bone insertions. This report aims to review the anatomy and physiology of the various mechanoreceptors of the posterior cruciate ligament, placing special emphasis on their role in knee joint stability. It concludes that the posterior crude ligament may not only serve as a 'mechanical stabilizer' of the knee joint, but also probably has an important 'sensory function' that should be taken into account when dealing with injuries to it.

Tendons and ligaments are anatomically distinct but overlap in molecular and morphological features--a comparative study in an ovine model.

Tendons and ligaments are similar in composition but differ in function. Simple anatomical definitions do not reflect the fact individual tendons and ligaments have unique properties due to their adaptation to a specific role. The patellar tendon is a structure of particular clinical interest. A null hypothesis was declared stating that the patellar tendon is not significantly different in terms of matrix composition and collagen fibril diameter to other tendons. The lateral and medial collateral ligaments (LCL, MCL), anterior and posterior cruciate ligaments (ACL, PCL), together with the long digital extensor, superficial digital extensor, and patellar tendons (LDET, SDFT, PT) were harvested from three cadaveric ovine hindlimbs. The extracellular matrix was assessed in terms of water, collagen, and total sulphated glycosaminoglycan (GAG) content. The organization of the collagen component was determined by an ultrastructural analysis of collagen fibril diameter distributions, together with values for the collagen fibril index (CFI) and mass-average diameter (MAD). There were significant differences between ligaments and tendons. The PT had a bimodal collagen fibril diameter distribution with CFI 72.9%, MAD 202 nm, water content 53.1%, GAG content 2.3 microg/mg, and collagen content 73.7%, which was not significantly different from the other tendons. The results of this study support the null hypothesis suggesting that the patellar tendon is similar to other tendons and demonstrate that tendons have different characteristics to ligaments.

Type II collagen deposition in cruciate ligament precedes osteoarthritis in the guinea pig knee.

OBJECTIVE: To examine the collagens in cruciate ligaments of young Dunkin-Hartley guinea pigs, to determine whether a change in specific collagen types is an early feature of the spontaneous osteoarthritis (OA), which consistently develops in the medial compartment of the knee in this strain. DESIGN: Collagen types I, II, III, IX, and XI were detected by immunofluorescence microscopy in the anterior and posterior cruciate ligaments of animals at 3, 4-5 and 12 weeks of age. Type II collagen in PCL was further analysed by confocal microscopy or biochemical assay after cyanogen bromide digestion, SDS-PAGE and immunoblotting. Interfibrillar proteoglycans were visualized by transmission electron microscopy. RESULTS: Collagen types I and III formed the bulk of fibrous mid-ligament tissue in all animals. Typical cartilage collagens, types II, IX and XI, were identified by immunolabeling where ligaments attached to tibial bone. Type II collagen, normally restricted to the fibrocartilage attachment sites, was also found at separate foci in anterior fiber bundles of the posterior cruciate ligament in 12-week-old animals. Biochemical data confirmed these observations which, together with electron microscopy showing large atypical proteoglycan structures, suggested the deposition of fibrocartilage within the fibrous mid-ligament. CONCLUSIONS: Cruciate ligaments, especially posterior cruciate ligament in Dunkin-Hartley guinea pigs synthesize cartilage-like matrix in mid-ligament prior to the appearance of classical signs of OA.

Does nitric oxide help explain the differential healing capacity of the anterior cruciate, posterior cruciate, and medial collateral ligaments?

This study compared the ability of rabbit medial collateral ligament, posterior cruciate ligament, and anterior cruciate ligament tissue to synthesize nitric oxide, and determined its effects on matrix synthesis, an important component of ligament repair. It is not known whether ligament cells can produce nitric oxide and, if so, whether it influences healing of ligament injuries. The anterior cruciate and posterior cruciate ligament tissue produced large amounts of nitric oxide in response to the inflammatory cytokine interleukin-1. Medial collateral ligament, in contrast, produced only modest amounts of nitric oxide. Furthermore, anterior cruciate ligament and, to some degree, posterior cruciate ligament synthesized nitric oxide spontaneously in culture, whereas medial collateral ligament never did so. When nitric oxide was supplied to these tissues, it strongly inhibited collagen synthesis by the two cruciate ligaments, but had little effect on collagen synthesis by the medial collateral ligament. Endogenously synthesized nitric oxide was also able to inhibit collagen synthesis as well as proteoglycan synthesis by the two cruciate ligaments, but had little effect on matrix synthesis by the medial collateral ligament. We propose a novel hypothesis, based on nitric oxide production and matrix synthesis, that may help explain why the two cruciate ligaments have such limited healing capacity compared with the medial collateral ligament.

Differential expression of integrin subunits in canine knee ligament fibroblasts.

A method for measuring the expression of integrin subunits on the cell surface of knee ligament fibroblasts was developed with use of flow cytometry and immunofluorescence. The ligament cells exhibited uniform size and density, as shown by forward and side-scatter properties, and showed minimal nonspecific binding of isotype control antibodies compared with unstained cells. All cells expressed the alpha5 integrin subunit; lateral collateral ligament cells stained with antibody to alpha5 showed a mean fluorescence intensity 2-fold higher than that of medial collateral ligament cells, 1.5-fold higher than that of posterior cruciate ligament cells, and 3-fold higher than that of anterior cruciate ligament cells, indicating a greater expression of the alpha5 subunit by lateral collateral ligament cells than by medial collateral, posterior cruciate, and anterior cruciate ligament cells. All cells expressed the beta1 integrin subunit; the expression by posterior cruciate ligament cells was 3-fold higher than that by medial collateral ligament or lateral collateral ligament cells and 5-fold higher than that by anterior cruciate ligament cells. All cells expressed the beta3 integrin subunit; the expression by posterior cruciate ligament cells was 1.5, 3, and 4.5-fold greater than that by lateral collateral, anterior cruciate, and medial collateral ligament cells, respectively. Our data suggest there is a differential expression of integrin subunits in knee ligament fibroblasts, and this in part may explain differences in their attachment and adherence to extracellular matrix molecules.

Quantitative evaluation of nutritional pathways for the posterior cruciate ligament and the lateral collateral ligament in rabbits.

The cruciate ligament of the knee receives its nutrition from a direct vascular supply and by permeation of nutrients from the synovial fluid. The contributions of these two routes as nutritional pathways are not known in detail. In this study, we injected [3H]methyl glucose as a tracer intravenously or directly into the knee of rabbits. Tracer concentrations in plasma, synovial fluid, the posterior cruciate ligament (PCL), and the lateral collateral ligament (LCL) were analysed by a pharmacokinetic compartment model. The contribution of [3H]methyl glucose permeation from the synovial fluid during steady state was calculated at 44.3% in the PCL and at 39.0% in the LCL. Although these results indicated that more than half the nutrition for both ligaments is provided by its vascular supply, synovial fluid permeation is also an important transport route for small molecules for the PCL and the LCL, which is an extra-articular structure.

Postnatal development of the cruciate ligament insertions in the rat knee. morphological evaluation and immunohistochemical study of collagens types I and II.

The postnatal structural remodelling and calcification patterns in the insertions (entheses) of both cruciate ligaments were studied in a rat model with histology and immunohistochemical analysis of collagens types I and II. In the neonate, both ligaments which labelled only for type I collagen attached to epiphyseal cartilage which solely labelled for collagen type II. The entheses calcified between days 20 and 35, and a subchondral bone plate formed under the entheses between days 30 and 55. Thus, within a period of 35 days the tissue to which the ligaments attached increased multifold in stiffness. Interestingly, the process of enthesial calcification and formation of compact bone did not happen simultaneously in both ligaments, not even synchronous at both ends of the same ligament or within a single insertion. This asynchronous calcification of the different knee ligament insertions may make the sudden change in mechanical environment at the entheses less dramatic for the ligaments and knee joint surfaces as anticipated from mechanical models. In addition, a fibrocartilaginous tissue, rich in collagen type II, formed in the ligament at a time when the epiphyseal cartilage was replaced by bone, and grew wider with time. The interposition of a fibrocartilaginous zone in the insertion may diminish the sudden change in stiffness between ligament soft tissue and hard bone.

Changes in the distribution of fibrillar collagens in the collateral and cruciate ligaments of the rabbit knee joint during fetal and postnatal development.

The collateral ligaments can be clearly distinguished in the 25-day fetal rabbit knee joint. Type I and V collagens are present in the extracellular matrix between the cells of the lateral and medial collateral ligaments and this distribution persists until the rabbit is skeletally mature. From 8 months onwards type III collagen is also present, particularly around the cells. Type I collagen mRNA is expressed by the cells from the 25-day fetal to 8-month-old adult ligament. The ligament sheath is composed of types III and V collagens. The cruciate ligaments are present between the femur and tibia in the 20-day fetus. The matrix is composed of types I and V collagens from the 25-day fetus until at 12- to 14-weeks postnatal, type III collagen appears in the pericellular regions together with type V. At 8 months and 2 years the amount of type III collagen has increased. All the cells express the mRNA for type I collagen at 12- to 14-weeks, but only isolated cells express this mRNA at 8 months. Thus, both the collateral and cruciate ligaments undergo changes in their complement of collagens during postnatal development and ageing. The implications of these complex interactions of different types of collagen are discussed in relation to healing and the surgical replacement of torn ligaments by tendons.

Expression of tenascin in joint-associated tissues during development and postnatal growth.

The extracellular matrix protein, tenascin, is selectively expressed in a variety of connective tissues during development. In this study, the distribution of tenascin in tissues contributing to the knee joint during embryonic development and postnatal growth in the rat has been investigated by immunohistochemistry. In recently formed embryonic knee joints, tenascin expression was abundant in the territorial matrix of superficial articular cartilage. Site of attachment of cruciate and patellar ligaments to cartilage were strongly stained; staining of ligaments weakened with distance from the attachment site. In rapidly growing 4-wk-old rats, tenascin was present in a fine line on the surface of articular cartilage, but at 10 wk of age tenascin staining was absent from most of the articular surface. In postnatal rats, there was strong tenascin staining of the synovial lining, but not of subintimal tissue. Cruciate ligaments were histologically fibrocartilaginous in 4 and 10-wk-old rats; within these ligaments strong pericellular tenascin staining was seen in association with rounded chondrocyte-like cells. Tenascin was absent from the cartilaginous growth plates of 4 and 10-wk-old rats, but intense tenascin staining was seen at the junction between epiphyseal bone and growth plate. Within the metaphysis, tenascin staining on bone surfaces increased with distance from the hypertrophic chondrocytes. Osteocytes within epiphyseal trabecular bone were strongly stained for tenascin, whereas those in the metaphysis were mostly unstained. The results presented here demonstrate that tenascin expression in joint-associated tissues changes markedly with cell type and stage of differentiation.