Patellar Tendon Samples Research Articles

Upregulation of FABP4 induced inflammation in the pathogenesis of chronic tendinopathy

ObjectivesExcessive inflammation contributes to the pathogenesis of tendinopathy. Fatty acid binding protein 4 (FABP4) is a pro-inflammatory adipokine mediating various metabolic and inflammatory diseases. This study aimed to examine the expression of FABP4 and its association with the expressions of inflammatory cytokines in tendinopathy. The effects of a single injection of FABP4 on tendon pathology and inflammation were examined. The effect of FABP4 on the expressions of inflammatory cytokines and the effect of IL-1β on the expression of FABP4 in tendon-derived stem/progenitor cells (TDSCs) were also investigated. Methods1) Clinical patellar tendinopathy samples, healthy hamstring tendon samples, and healthy patellar tendon samples, 2) rotator cuff tendinopathy samples and healthy hamstring tendon samples; and 3) Achilles tendons of mice after saline or collagenase injection (CI) were stained for FABP4, IL-1β, IL-6, TNF-α and IL-10 by immunohistochemistry (IHC). For the rotator cuff tendinopathy samples, co-localization of FABP4 with IL-1β and TNF-α was done by immunofluorescent staining (IF). Mouse Achilles tendons injected with FABP4 or saline were collected for histology and IHC as well as microCT imaging post-injection. TDSCs were isolated from human and mouse tendons. The mRNA expressions of inflammatory cytokines in human and mouse TDSCs after the addition of FABP4 was quantified by qRT-PCR. The expression of FABP4 in TDSCs isolated from rotator cuff tendinopathy samples and healthy hamstring tendon samples was examined by IF. Mouse Achilles TDSCs were treated with IL-1β. The mRNA and protein expressions of FABP4 were examined by qRT-PCR and IF, respectively. ResultsThere was significant upregulation of FABP4 in the patellar tendinopathy samples and rotator cuff tendinopathy samples compared to their corresponding controls. FABP4 was mainly expressed in the pathological areas including blood vessels, hypercellular and calcified regions. The expressions of IL-1β and TNF-α increased in human rotator cuff tendinopathy samples and co-localized with the expression of FABP4. Collagenase induced tendinopathic-like histopathological changes and ectopic calcification in the mouse Achilles tendinopathy model. The expressions of inflammatory cytokines (IL-1β, IL-6, TNF-α, IL-10) and FABP4 increased in hypercellular region, round cells chondrocyte-like cells and calcified regions in the mouse Achilles tendons post-collagenase injection. A single injection of FABP4 in mouse Achilles tendons induced histopathological changes resembling tendinopathy, with increased cell rounding, loss of collagen fiber alignment, and additionally presence of chondrocyte-like cells and calcification post-injection. The expressions of IL1-β, IL-6, TNF-α and IL-10 increased in mouse Achilles tendons post-FABP4 injection. FABP4 increased the expressions of IL10, IL6, and TNFa in human TDSCs as well as the expressions of Il1b, Il6, and Il10 in mouse TDSCs. Human tendinopathy TDSCs expressed higher level of FABP4 compared to healthy hamstring TDSCs. Besides, IL-1β increased the expression of FABP4 in mouse TDSCs. ConclusionIn conclusion, an upregulation of FABP4 is involved in excessive inflammation and pathogenesis of tendinopathy. TDSCs is a potential source of FABP4 during tendon inflammation. Translation potential of this articleFABP4 can be a potential treatment target of tendinopathy.

Bi-Exponential 3D UTE-T1ρ Relaxation Mapping of Ex Vivo Human Knee Patellar Tendon at 3T.

Introduction: The objective of this study was to assess the bi-exponential relaxation times and fractions of the short and long components of the human patellar tendon ex vivo using three-dimensional ultrashort echo time T1ρ (3D UTE-T1ρ) imaging. Materials and Methods: Five cadaveric human knee specimens were scanned using a 3D UTE-T1ρ imaging sequence on a 3T MR scanner. A series of 3D UTE-T1ρ images were acquired and fitted using single-component and bi-component models. Single-component exponential fitting was performed to measure the UTE-T1ρ value of the patellar tendon. Bi-component analysis was performed to measure the short and long UTE-T1ρ values and fractions. Results: The single-component analysis showed a mean single-component UTE-T1ρ value of 8.4 ± 1.7 ms for the five knee patellar tendon samples. Improved fitting was achieved with bi-component analysis, which showed a mean short UTE-T1ρ value of 5.5 ± 0.8 ms with a fraction of 77.6 ± 4.8%, and a mean long UTE-T1ρ value of 27.4 ± 3.8 ms with a fraction of 22.4 ± 4.8%. Conclusion: The 3D UTE-T1ρ sequence can detect the single- and bi-exponential decay in the patellar tendon. Bi-component fitting was superior to single-component fitting.

Spatial gene expression in the adult rat patellar tendon

Tendons are dense connective tissues with relatively few cells which makes studying the molecular profile of the tissue challenging. There is not a consensus on the spatial location of various cell types within a tendon, nor the accompanying transcriptional profile. In the present study, we used two male rat patellar tendon samples for sequencing-based spatial transcriptomics to determine the gene expression profile. We integrated our data with a mouse Achilles single cell dataset to predict the cell type composition of the patellar tendon as a function of location within the tissue. The spatial location of the predicated cell types suggested that there were two populations of tendon fibroblasts, one located in the tendon midsubstance, while the other localized with red blood cells, pericytes, and immune cells to the tendon peripheral connective tissue. Of the highest expressed spatially variable genes, there were multiple genes with known function in tendon: Col1a1, Col1a2, Dcn, Fmod, Sparc, and Comp. Further, a novel spatially regulated gene (AABR07000398.1) with no known function was identified. The spatial gene expression of tendon associated genes (Scx, Thbs4, Tnmd, Can, Bgn, Lum, Adamts2, Lox, Ppib, Col2a1, Col3a1, Col6a2) was also visualized. Both patellar tendon samples had similar expression patterns for all these genes. This dataset provides new spatial insights into gene expression in a healthy tendon.

Tendon-Derived Progenitor Cells With Multilineage Potential Are Present Within Human Patellar Tendon.

Background:Progenitor cells serve as a promising source of regenerative potential in a variety of tissue types yet remain underutilized in tendinopathy. Tendon-derived progenitor cells (TDPCs) have previously been isolated from hamstring tendon but only as part of a concomitant medical procedure. Determining the presence of TDPCs in patellar tendon may facilitate clinical utilization of these cells because of the relative accessibility of this location for tissue harvest.Purpose:To characterize TDPCs in human patellar tendon samples.Study Design:Descriptive laboratory study.Methods:Human patellar tendon samples were obtained during elective knee surgery. TDPCs were isolated and seeded at an optimal low cell density and subcultured to confluence for up to 2 passages. Flow cytometry was used to analyze for the expression of CD90+, CD105+, CD44+, and CD31–, CD34–, and CD45– markers. The multilineage differentiation potential of TDPCs was tested in vitro via adipogenic, osteogenic, and chondrogenic culture with subsequent cytochemical staining for Oil Red O, Alizarin Red, and Alcian Blue, respectively. Enzyme-linked immunosorbent assay was used to quantify the amount of adiponectin, alkaline phosphatase, and SRY-box transcription factor 9 secreted into cell culture supernatant for further confirmation of lineage differentiation. Results were analyzed statistically using the 2-tailed Student t test.Results:TDPCs demonstrated near-uniform expression of CD90, CD105, and CD44 with minimal expression of CD34, CD31, and CD45. Adipogenic, osteogenic, and chondrogenic differentiation of TDPCs was confirmed using qualitative analysis. The expression of adiponectin, alkaline phosphatase, and SRY-box transcription factor 9 were significantly increased in differentiated cells versus undifferentiated TDPCs (P < .05).Conclusion:TDPCs can be successfully isolated from human patellar tendon samples, and they exhibit characteristics of multipotent progenitor cells.Clinical Relevance:These data demonstrate the promise of patellar tendon tissue as a source of progenitor cells for use in biologic therapies for the treatment of tendinopathy.

Effects of cyclic loading on the mechanical properties and failure of human patellar tendon

Patellar tendinopathy is a common overuse injury in sports such as volleyball, basketball, and long-distance running. Microdamage accumulation, in response to repetitive loading of the tendon, plays an important role in the pathophysiology of patellar tendinopathy. This damage presents mechanically as a reduction in Young’s modulus and an increase in residual strain. In this study, 19 human patellar tendon samples underwent cyclic testing in load control until failure, segmented by four ramped tests where digital image correlation (DIC) was used to assess anterior surface strain distributions. Ramped tests were performed prior to cyclic testing and at timepoints corresponding to 10%, 20%, and 30% of cyclic stiffness reduction. Young’s modulus significantly decreased and cyclic energy dissipation significantly increased over the course of cyclic testing. The DIC analysis illustrated a heterogeneous strain distribution, with strain concentrations increasing in magnitude and size over the course of cyclic testing. Peak stress and initial peak strain magnitudes significantly correlated with the number of cycles to failure (r2 = 0.65 and r2 = 0.57, respectively, p < 0.001); however, the rates of peak cyclic strain and modulus loss displayed the highest correlations with the number of cycles to failure (r2 = 96% and r2 = 86%, respectively, p < 0.001). The high correlation between the rates of peak cyclic strain and modulus loss suggest that non-invasive methods to continuously monitor tendon strain may provide meaningful predictions of overuse injury in the patellar tendon.

Comparison of water, hydroxyproline, uronic acid and elastin contents of bovine knee ligaments and patellar tendon and their relationships with biomechanical properties

Mechanical material properties of ligaments originate from their biochemical composition and structural organization. However, it is not yet fully elucidated how biochemical contents vary between knee ligaments and patellar tendon (PT) and how they relate with mechanical properties. The purpose of this study was to compare water, collagen, proteoglycan and elastin contents between bovine knee ligaments and PT and correlate them with tensile material properties.Hydroxyproline (collagen), uronic acid (proteoglycan) and elastin contents per wet and dry weights were measured using colorimetric biochemical methods for bovine knee ligament and PT samples (n = 10 knees). Direct comparison and correlation with multiple linear regression were performed against biomechanical properties measured in our earlier study.Anterior cruciate ligament (ACL) and PT exhibited lower hydroxyproline content per wet weight compared with other ligaments (p < 0.05). Cruciate ligaments had higher uronic acid content per dry weight compared with collateral ligaments (p < 0.05). Posterior cruciate ligament had higher elastin content than ACL (p < 0.05). Higher hydroxyproline content per wet weight implied higher Young's modulus, strength and toughness. Quantitatively, higher elastin content per wet weight predicted higher toe region nonlinearity and Young's modulus whereas higher uronic acid content per dry weight predicted lower Young's modulus, yield stress and toughness.Differences between ligaments in biochemical composition highlight differences in their physiological function and loading regimes. As expected, collagen content showed similar trend with stiffness and strength. The predictive role of proteoglycan and elastin contents on the mechanical properties might indicate their important functional role in ligaments.

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.

Expression of Wnt pathway mediators in metaplasic tissue in animal model and clinical samples of tendinopathy

Tissue metaplasia is observed in both ossified failed healing animal model and clinical samples of tendinopathy. The Wnt signalling pathway plays a vital role in pathological calcification. We hypothesized that the Wnt signalling pathway might contribute to tissue metaplasia and failed healing in tendinopathy. This study aimed to examine the spatial-temporal expression of Wnt pathway mediators in an ossified failed tendon healing animal model and clinical samples of tendinopathy. The effect of Wnt3a on the osteogenic differentiation of tendon-derived stem cells (TDSCs) was also examined. Ossified failed tendon healing was induced by the injection of collagenase into the patellar tendon of rats. At various times the tendons were harvested for immunohistochemical staining of Wnt3a, β-catenin, Lrp5 and Tcf1. Patellar tendon samples were obtained from 13 patients with patellar tendinopathy (11 unossified and 2 ossified) and 10 controls. Immunohistochemical staining of Wnt3a, β-catenin, Lrp5 and Tcf1 was similarly performed. Rat patellar TDSCs were treated with Wnt3a. The osteogenic differentiation of TDSCs was examined by ALP activity, alizarin red S staining and mRNA expression of osteogenic markers. There was increased expression of Wnt3a, β-catenin, Lrp5 and Tcf1 in the healing fibroblast-like cells, chondrocyte-like cells and ossified deposits in the animal model and in some clinical samples of tendinopathy. Wnt3a increased ALP activity, calcium nodule formation and expression of osteogenic markers in TDSCs. Activation of the Wnt signalling pathway and its effect on TDSCs might contribute to tissue metaplasia and failed healing in some cases of tendinopathy.

Expression of chondro-osteogenic BMPs in clinical samples of patellar tendinopathy

The pathogenesis of patellar tendinopathy remains unclear. Expression of BMP-2/-4/-7 was reported in an ossified failed tendon healing animal model of patellar tendinopathy. This study aimed to investigate the expression of these chondro-osteogenic BMPs in clinical samples of patellar tendinopathy. Patellar tendon samples were collected from 16 consecutive patients with patellar tendinopathy and 16 consecutive controls undergoing anterior cruciate ligament reconstruction with bone-patellar tendon-bone autograft in the authors' hospital after getting their consent. The expression of BMP-2/-4/-7 was examined in all samples using immunohistochemistry. Ossification observed in two tendinopathy samples was characterized by histology, alizarin red S staining, alcian blue staining, TRAP staining and immunohistochemical staining of Sox9, osteopontin (OPN) and osteocalcin (OCN). Regions of hypo- and hyper-cellularity and vascularity, with loss of crimp structure of collagen matrix, were observed in patellar tendinopathy samples. Round cells and in some cases, cells with typical chondrocyte phenotype were observed. For the ossified tendinopathy samples with positive alizarin red S staining, OPN-positive and Sox9-positive chondrocyte-like cells in alcian blue-stained extracellular matrix, OCN-positive osteoblast-like cells and TRAP-positive multi-nucleated cells were observed around the ossified deposits. No expression of BMP-2/-4/-7 was observed in healthy patellar tendons. However, the expression of BMP-2/-4/-7 was observed in all patellar tendinopathy samples with or without ossification. Clinical samples of patellar tendinopathy showed ectopic expression of BMP-2/-4/-7. This was not evident in control samples from healthy patellar tendons. Prognostic studies, Level III.

Lubricin Distribution in the Goat Infraspinatus Tendon: A Basis for Interfascicular Lubrication

This study was motivated by the need to better understand the tribology of the rotator cuff, as it could provide insights into degenerative processes and suggest therapeutic approaches to cuff disorders. The objective was to evaluate the distribution of a known lubricating protein in the infraspinatus tendon of the rotator cuff in adult goats. The hypothesis was that lubricin, also known as superficial zone protein or proteoglycan 4, serves as an interfascicular lubricant. Eight infraspinatus tendons were resected from eight Spanish goats, and five patellar tendons and articular cartilage samples were also resected from five of the goats. Samples were processed for immunolocalization of lubricin with use of a purified monoclonal antibody and for histological analysis with Masson trichrome staining for collagen. The locations of lubricin within the tendon were documented, and measurements were made of the distance to which lubricin was detected in the tendon, relative to the humeral insertion site, and the depth of lubricin staining into the fibrocartilage of the fascicle bordering the humeral joint space. Images from polarized light microscopy were used to measure the fascicle diameter and the crimp length. Lubricin was prominent in layers separating the fascicles in the infraspinatus tendon, with occasional intrafascicular staining. The fibrocartilaginous portion of the fascicle bordering the humeral joint space displayed diffuse lubricin staining. The Masson trichrome staining of the collagen in this lubricin-containing fibrocartilage indicated that it was not being tensioned at the time of fixation, whereas the collagen contained in the body of the tendon was under tension. The crimp of the fascicles near the humeral joint side of the tendon displayed a shorter peak-to-peak length than the crimp of the fascicles in the superior region of the tendon. Only the surface of two of the five patellar tendon samples stained for lubricin; there was no staining within these ligaments. The sheaths of the fascicles of the infraspinatus tendon near the bone insertion site contain lubricin, indicating that this lubricating protein may be facilitating interfascicular movement. The fact that the crimp pattern of fascicles changes with location in the tendon provides support for the supposition that fascicles move relative to one another as the tendon is loaded, underscoring the importance of a lubricating protein in the layer separating the fascicles.

VOCCs and TREK-1 ion channel expression in human tenocytes

Mechanosensitive and voltage-gated ion channels are known to perform important roles in mechanotransduction in a number of connective tissues, including bone and muscle. It is hypothesized that voltage-gated and mechanosensitive ion channels also may play a key role in some or all initial responses of human tenocytes to mechanical stimulation. However, to date there has been no direct investigation of ion channel expression by human tenocytes. Human tenocytes were cultured from patellar tendon samples harvested from five patients undergoing routine total knee replacement surgery (mean age: 66 yr; range: 63-73 yr). RT-PCR, Western blotting, and whole cell electrophysiological studies were performed to investigate the expression of different classes of ion channels within tenocytes. Human tenocytes expressed mRNA and protein encoding voltage-operated calcium channel (VOCC) subunits (Ca alpha(1A), Ca alpha(1C), Ca alpha(1D), Ca alpha(2)delta(1)) and the mechanosensitive tandem pore domain potassium channel (2PK(+)) TREK-1. They exhibit whole cell currents consistent with the functional expression of these channels. In addition, other ionic currents were detected within tenocytes consistent with the expression of a diverse array of other ion channels. VOCCs and TREK channels have been implicated in mechanotransduction signaling pathways in numerous connective tissue cell types. These mechanisms may be present in human tenocytes. In addition, human tenocytes may express other channel currents. Ion channels may represent potential targets for the pharmacological management of chronic tendinopathies.

Bone morphogenetic protein 13 stimulates cell proliferation and production of collagen in human patellar tendon fibroblasts

Background Recombinant human (rh) bone morphogenetic protein 13 (BMP13) has been shown to induce the formation of tendon and ligament tissues in animal experiments. The role of BMP13 in tissue regeneration in human tendons remains unexplored, however.Material and methods We collected healthy human patellar tendon samples for histological examination and tendon fibroblast culture. The cultured cells were incubated in the presence and absence of rhBMP13 and the effect of the protein on cell proliferation was measured using 5-bromo-2’-deoxyuridine uptake.Results BMP13 was detectable by immunohisto-chemical staining in healthy patellar tendon samples, and was located exclusively in active tenoblasts and perivascular mesenchymal cells but not in interstitial tenocytes. The expression of proliferating cell nuclear antigen (PCNA) and pro-collagen type I showed a similar distribution. In vitro studies showed that rhBMP13 can increase proliferation of tendon fibroblasts and increase the gene expression of pro-collagen type I in tendon fibroblast culture.Interpretation Our findings indicate that BMP13 may be involved in the matrix remodeling process in adult tendon, and that it may play a role in tissue regeneration in tendons.

Immunohistochemical Characterization of Cells in Adult Human Patellar Tendons

Cells in tendons are conventionally identified as elongated tenocytes and ovoid tenoblasts, but specific markers for these cells are not available. The roles and interplay of these cells in tendon growth, remodeling, and healing are not well established. Therefore, we proposed to characterize these cells with respect to cell turnover, extracellular matrix metabolism, and expression of growth factors. Here we examined 14 healthy human patellar tendon samples for the expression of various proteins in tenocytes and tenoblasts, which were identified as elongated tendon cells and ovoid tendon cells, respectively. Matrix metalloproteinase 1 (MMP1), procollagen type I (procol I), heat shock protein 47 (hsp47), bone morphogenetic protein 12 (BMP12), 13 (BMP13), and transforming growth factor beta1 (TGFbeta1) were detected by immunohistochemistry (IHC). An image analysis of the IHC staining for proliferation cell nuclear antigen (PCNA) and apoptotic cells was performed to determine the proliferation index and the apoptosis index in elongated and ovoid tendon cells. The ovoid tendon cells expressed higher levels of procol I, hsp47, MMP1, BMP12, BMP13, and TGFbeta1 than the elongated tendon cells. Both the proliferation index and the apoptosis index of ovoid tendon cells were higher than those of the elongated tendon cells. The results suggested that ovoid tendon cells, conventionally recognized as tenoblasts, were more active in matrix remodeling. The expression of BMP 12, BMP13 and TGFbeta1 might be associated with the different cellular activities of tenoblasts and tenocytes.

Paper #156 The effects of bone morphogenetic proteins 13 on human patellar tendon fibroblasts

Bone morphogenetic proteins (BMPs) are a family of proteins that can induce bone and cartilage formation during embryogenesis and tissue repair. Human recombinant (hr) BMP13 has been recently cloned and proved to promote the formation of tendon and ligament tissues in in vivo experiments. But the roles of BMP13 on tissue regeneration in tendons remain unexamined. In the present study, human patellar tendon samples were collected for histological examination and preparation of tendon fibroblast culture. Immunohistochemical staining showed that BMP12 was detected on healthy patellar tendon samples, chiefly located on active tenoblasts and perivascular mesenchymal cells, with a similar distribution as proliferation cell nuclear antigens (PCNA), pro-collagen I and decorin. In vitro studies on tendon fibroblast culture showed that hrBMP13 could increase cell proliferation and the gene expression of pro-collagen I and biglycan, but the gene expression of decorin was not affected. Our findings suggest that BMP13 may play a role in tendon healing. Bone morphogenetic proteins (BMPs) are a family of proteins that can induce bone and cartilage formation during embryogenesis and tissue repair. Human recombinant (hr) BMP13 has been recently cloned and proved to promote the formation of tendon and ligament tissues in in vivo experiments. But the roles of BMP13 on tissue regeneration in tendons remain unexamined. In the present study, human patellar tendon samples were collected for histological examination and preparation of tendon fibroblast culture. Immunohistochemical staining showed that BMP12 was detected on healthy patellar tendon samples, chiefly located on active tenoblasts and perivascular mesenchymal cells, with a similar distribution as proliferation cell nuclear antigens (PCNA), pro-collagen I and decorin. In vitro studies on tendon fibroblast culture showed that hrBMP13 could increase cell proliferation and the gene expression of pro-collagen I and biglycan, but the gene expression of decorin was not affected. Our findings suggest that BMP13 may play a role in tendon healing.

The roles of bone morphogenetic protein (BMP) 12 in stimulating the proliferation and matrix production of human patellar tendon fibroblasts

Recombinant human (rh) bone morphogenetic protein 12 (BMP12) is proved to induce the formation of tendon and ligament tissues in animal experiments. But the roles of BMP12 on tissue regeneration in human tendons remain unexplored. In the present study, healthy human patellar tendon samples were collected for histological examination and preparation of tendon fibroblast culture. Immunohistochemical staining showed that BMP12 was detected on healthy patellar tendon samples, only located on active tenoblasts and perivascular mesenchymal cells but not in interstitial tenocytes. The expression of PCNA and procollagen type I also exhibited a similar distribution. It indicates that BMP12 may be involved in matrix remodeling process in adult tissues. In vitro studies showed that rhBMP12 could increase proliferation of tendon fibroblasts and increase the gene expression of procollagen type I and type III, but decrease the gene expression of decorin in tendon fibroblasts culture. Our findings suggest that BMP12 may play a role in early phases of tissue regeneration in tendons.

Collagenases from Periarticular Ligaments and Tendon: Enzyme Levels during the Development of Joint Contracture

Our laboratories have previously demonstrated that normal rabbit periarticular ligaments, anterior cruciate ligaments (ACL), medial collateral ligaments (MCL) and patellar tendon (PT) secrete collagenase. In this current study we examined these connective tissues following an immobilization period of 4 weeks. In the ligaments producing collagenase, activity was expressed only in the control, not in the immobilized joint. Control and experimental patellar tendon samples produce collagenolytic activity, suggesting that the expression of enyzme is less affected in tendons as compared to ligaments. Characterization of these collagenases was carried out using an antiserum directed against rabbit synovial collagenase. We demonstrated that ligament (ACL) and tendon (PT) collagenases cross react with this antibody in a double immunodiffusion assay. Protein blots of PT, ACL and MCL collagenases identified one major species (Mr = 45,000) and a minor species (Mr = 50,000) of immunoreactive proteins in all three connective tissues. Differences between control and experimental enzyme levels appear to be due to less collagenase protein being produced by immobilized ligaments.