Paul Sciore

POSTMORTEM STABILITY OF TOTAL RNA ISOLATED FROM RABBIT LIGAMENT, TENDON AND CARTILAGE

The stability of RNA, particularly mRNA, in tissues is under complex regulation. Most studies to date have focused on very cellular tissues and not connective tissues such as ligaments, tendons and cartilage. As the availability of such tissues for transplantation or research purposes is frequently delayed following death, it is important to determine whether RNA stability in such tissues is influenced by time postmortem. To approach this question, skeletally mature NZW rabbits were used to investigate RNA integrity over time in dense, hypocellular connective tissues and in several hypercellular organ tissues such as brain, kidney, liver and lung. Samples were analyzed at varying intervals postmortem with respect to rRNA integrity by agarose gel electrophoresis and ethidium bromide staining and mRNA integrity by Northern blot analysis and RT-PCR. No degradation of rRNA or loss in integrity of mRNA for genes of low and high copy number was observed up to 96 h postmortem. These findings confirm that it is likely appropriate to use properly stored postmortem dense connective tissues for molecular biological investigations.

Altered levels of extracellular matrix molecule mRNA in healing rabbit ligaments

RT-PCR methods were used to amplify, semi-quantify, clone and sequence cDNA fragments specific for rabbit extracellular matrix molecules biglycan, collagen I, collagen III, decorin, lumican, versican, fibromodulin, and also glyceraldehyde-3-phosphate dehydrogenase (G3PDH), using RNA isolated from rabbit ligaments. Sequence analysis of two independent clones of PCR products was used to verify the identity of the cDNA. Semi-quantitative RT-PCR was used to study mRNA levels for these matrix molecules in normal and healing rabbit ligament at three, six, and fourteen weeks post-injury. The yield of RNA from the ligament scar was increased at three and six weeks post-injury, but it had returned to near normal levels by fourteen weeks. On a microgram RNA basis, it was demonstrated that biglycan, collagen I, collagen III and lumican mRNA levels are significantly elevated, versican mRNA levels significantly depressed, and decorin and fibromodulin mRNA levels showed no significant change in response to tissue injury in the ligament during the course of healing. These findings suggest that differential regulation of mRNA levels for these extracellular matrix molecules occurs during ligament healing.

Local Anesthetics Induce Chondrocyte Death in Bovine Articular Cartilage Disks in a Dose- and Duration-Dependent Manner

PURPOSE The purpose of this study was to evaluate the effect of various local anesthetics on chondrocyte viability in articular cartilage by use of a bovine disk model. METHODS Full-thickness bovine cartilage disks were isolated from the condylar surfaces of the radial-carpal joint by use of a 4-mm biopsy punch and were incubated in various concentrations of local anesthetics (e.g., bupivacaine) for varying amounts of time and stained for membrane integrity by use of ethidium bromide and SYTO 13 stain (Molecular Probes, Carlsbad, CA). Cell and nuclear morphology was assessed by transmission electron microscopy. RESULTS The addition of local anesthetics (i.e., 0.25% bupivacaine, 1% lidocaine, and 0.5% ropivacaine) to bovine articular cartilage disks had a negative effect on chondrocyte viability. Culturing bovine articular cartilage disks for increasing periods of time decreased chondrocyte viability for each of the local anesthetics, with significant negative correlations being shown between time of exposure to the drug and chondrocyte viability. These effects were also affected by the presence or absence of epinephrine in local anesthetic preparations. CONCLUSIONS Our results suggest that local anesthetics (i.e., bupivacaine, lidocaine, or ropivacaine) can have a detrimental effect on chondrocyte viability in bovine articular cartilage disks in a dose- and duration-dependent manner. CLINICAL RELEVANCE After arthroscopic surgery, it has been common practice to inject various local anesthetics into the joint for pain relief. Because adult chondrocytes have little or no capacity to regenerate, these results suggest that high-dose, long-term intra-articular administration of local anesthetics should be performed with caution.

Matrix metalloproteinase-13 expression in rabbit knee joint connective tissues: influence of maturation and response to injury.

The hypothesis of the present work was that expression of matrix metalloproteinase-13 (MMP-13, collagenase-3) would be induced during conditions involving important matrix remodeling such as ligament maturation, scar healing and joint instability. Therefore, MMP-13 expression in the medial collateral ligament (MCL) during the variable situations of tissue maturation and healing was assessed. MMP-13 expression in three intra-articular connective tissues of the knee (i.e. articular cartilage, menisci and synovium) following the transection of the anterior cruciate ligament of the knee was evaluated at 3 and 8 weeks post-injury. MMP-13 mRNA (semi-quantitative RT-PCR) and protein (immunohistochemistry and Western blotting) were detected in all of the tissues studied. Significantly higher MCL mRNA levels for MMP-13 were detected during the early phases of tissue maturation (i.e. 29 days in utero and 2-month-old rabbits) compared to later phases (5- and 12-month-old rabbits). This pattern of expression was recapitulated following MCL injury, with very high levels of expression in scar tissue at 3 weeks post-injury and then a decline to levels not significantly different from control values by 14 weeks. Elevated mRNA levels correlated with increased protein levels for MMP-13 in both menisci and synovium following the transection of the anterior cruciate ligament and during medial collateral ligament healing. These results indicate that MMP-13 expression is regulated by a number of variables and that high levels of expression occur in situations when connective tissue remodeling is very active.

Changes in mechanical loading lead to tendonspecific alterations in MMP and TIMP expression: influence of stress deprivation and intermittent cyclic hydrostatic compression on rat supraspinatus and Achilles tendons.

Background Tendinopathy commonly occurs in tendons with large in vivo loading demands like the Achilles tendon (AT) and supraspinatus tendon (SST). In addition to differences in their local anatomic environment, these tendons are designed for different loading requirements because of the muscles to which they attach, with the AT experiencing higher loads than the SST. One possible factor in the progression of tendinopathy is the interplay between mechanical loading and the regulation of enzymes that degrade the extracellular matrix (matrix metalloproteinases (MMPs)) and their inhibitors (tissue inhibitor of metalloprotienases (TIMPs)). Thus, overuse injuries may have different biological consequences in tendons designed for different in vivo loading demands. Aim In this study, the tendon-specific regulation of MMP-13, MMP-3 and TIMP-2 expression in rat AT and SST exposed to two different mechanical environments was investigated. Methods Rat AT and SST were exposed to stress deprivation (ie, detached from attachments) and intermittent cyclic hydrostatic compression (with attachments intact). Levels of MMP-13, MMP-3 and TIMP-2 mRNA were evaluated in time-zero control, attached, stressdeprived and “compressed” tendons. Results Stress deprivation led to elevated expression of MMP-13, MMP-3 and TIMP-2 in both tendons, although the magnitude of the increase was greater for the SST than the AT. Intermittent cyclic hydrostatic compression of attached tendons increased expression of MMP-13 in the SST, but not the AT. Conclusions The results of this study suggest that stress deprivation may be one contributor to the progression of tendinopathy in AT and SST, where the tendon designed for the lower in vivo loading demand (SST) was the most affected by a change in mechanical loading. The unique upregulation of MMP-13 with hydrostatic compression supports the impingement injury theory for rotator cuff tears.

PREGNANCY INDUCES COMPLEX CHANGES IN THE PATTERN OF MRNA EXPRESSION IN KNEE LIGAMENTS OF THE ADOLESCENT RABBIT

Knee laxity has been shown to increase during human pregnancy, and the laxity of the rabbit medial collateral ligament also increases during pregnancy. To determine whether the changes in tissue function could be related to alterations in the regulation of gene expression for a subset of relevant molecules in ligaments, RNA was isolated from the medial collateral(MCL) and anterior cruciate(ACL) ligaments of first time pregnant adolescent rabbits. Levels of mRNA for matrix molecules (collagen types I and III and the proteoglycans biglycan, decorin, versican and lumican), proteinases and inhibitors (collagenase, urokinase, PAI-1 and TIMP-1, -2 and -3), growth factors (bFGF, IGF-I, TGF-beta1 and ET-1), cytokines (IL-1beta and TNF) and enzymes responsible for important tissue mediators (COX-2 and iNOS) were assessed by semi-quantitative RT-PCR. In the MCL, levels of transcripts for all of the matrix molecules, growth factors and TIMPs 1 and 2 were significantly depressed at 29 days of pregnancy compared to age-matched non-pregnant controls. In contrast, transcripts for PAI-1 were elevated during pregnancy, while those for collagenase (MMP-1), urokinase, TIMP-3, IL-1beta, TNF, COX-2 and iNOS were not statistically altered. mRNA transcript levels rebounded by 7 days post-partum for most genes studied, indicating that the changes were rapidly reversible. For some molecules, transcript levels were again depressed at 18 days post-partum, indicating that regulatory mechanisms were still not stabilized. Analysis of mRNA from the ACL also revealed changes in the pattern of gene expression, with some similarities and differences from the MCL noted. These results indicate that pregnancy induces reversible changes in mRNA for matrix molecules in ligaments, but differences in responsiveness exist between different ligaments. The complexity of the changes observed indicates that there is probably no simple cause and effect relationship between laxity changes and the molecular alterations during pregnancy.

New understanding of the complex structure of knee menisci: Implications for injury risk and repair potential for athletes

Menisci help maintain the structural integrity of the knee. However, the poor healing potential of the meniscus following a knee injury can not only end a career in sports but lead to osteoarthritis later in life. Complete understanding of meniscal structure is essential for evaluating its risk for injury and subsequent successful repair. This study used novel approaches to elucidate meniscal architecture. The radial and circumferential collagen fibrils in the meniscus were investigated using novel tissue‐preparative techniques for light and electron microscopic studies. The results demonstrate a unique architecture based on differences in the packaging of the fundamental collagen fibrils. For radial arrays, the collagen fibrils are arranged in parallel into ∼10 μm bundles, which associate laterally to form flat sheets of varying dimensions that bifurcate and come together to form a honeycomb network within the body of the meniscus. In contrast, the circumferential arrays display a complex network of collagen fibrils arranged into ∼5 μm bundles. Interestingly, both types of architectural organization of collagen fibrils in meniscus are conserved across mammalian species and are age and sex independent. These findings imply that disruptions in meniscal architecture following an injury contribute to poor prognosis for functional repair.

Complex alterations in gene expression occur in the knee ligaments of the skeletally mature multiparous rabbit during pregnancy

Pregnancy is known to influence the function of ligaments of the knee in both humans and rabbits. During pregnancy, ligament laxity increases. The mechanism by which these changes in ligament function occur is unknown. The present study was undertaken to assess changes in the pattern of gene expression for a number of molecules which could be involved in the alterations in tissue function. Using RNA isolated from pregnant and age-matched nonpregnant rabbits, levels of mRNA for matrix molecules, proteinases and inhibitors, cytokines and growth factors and inducible nitric oxide synthase were assessed by semi-quantitative RT-PCR. The results indicate that the pregnancy-induced changes in the pattern of gene expression is very complex and differences in the changes observed for the medial collateral ligament and the anterior cruciate ligament may also indicate that ligament-specific effects of pregnancy occur. mRNA levels of some molecules were significantly elevated or suppressed, while others were unchanged, indicating that the changes observed were specific rather than general. While the extent of the molecules assessed was designed to be representative rather than exhaustive, the results indicate that there is likely no simple cause and effect relationship between the observed mechanical alterations during pregnancy and alterations in gene expression in the affected ligaments.

Gap junctions of the medial collateral ligament: structure, distribution, associations and function

Ligaments are composed of two major components: cells and extracellular matrix. The cells express gap junction proteins and are arranged into a series of rows that traverse the tissue, suggesting that all the cells of the tissue are functionally interconnected. The results of our study demonstrate that medial collateral ligament (MCL) cells do not have a uniform fusiform morphology or placement along a row of cells as previously suggested, but rather display a complex placement and form that weaves within the collagen matrix in a manner that is far more extensive and complex than previously appreciated. Within this morphological context, we find that MCL cells in vivo contain functional gap junctions (verified using fluorescence recovery after photobleaching) that are localized to sites of close cell–cell contact, and this pattern imparts or reflects a bipolarity inherent to each cell. When we studied ligament cells in conventional tissue culture we found that this bipolarity is lost, and the placement of gap junctions and their related proteins, as well as general cell morphology, is also altered. Finally, our study demonstrates, for the first time, that in addition to gap junctions, adherens junctions and desmosomes are also expressed by MCL cells both in vivo and in vitro and map to sites of cell–cell contact.

The use of an intra-articular depth guide in the measurement of partial thickness rotator cuff tears.

Purpose. The purpose of this study was to compare the accuracy of the conventional method for determining the percentage of partial thickness rotator cuff tears to a method using an intra-articular depth guide. The clinical utility of the intra-articular depth guide was also examined. Methods. Partial rotator cuff tears were created in cadaveric shoulders. Exposed footprint, total tendon thickness, and percentage of tendon thickness torn were determined using both techniques. The results from the conventional and intra-articular depth guide methods were correlated with the true anatomic measurements. Thirty-two patients were evaluated in the clinical study. Results. Estimates of total tendon thickness (r = 0.41, P = 0.31) or percentage of thickness tears (r = 0.67, P = 0.07) using the conventional method did not correlate well with true tendon thickness. Using the intra-articular depth guide, estimates of exposed footprint (r = 0.92, P = 0.001), total tendon thickness (r = 0.96, P = 0.0001), and percentage of tendon thickness torn (r = 0.88, P = 0.004) correlated with true anatomic measurements. Seven of 32 patients had their treatment plan altered based on the measurements made by the intra-articular depth guide. Conclusions. The intra-articular depth guide appeared to better correlate with true anatomic measurements. It may be useful during the evaluation and development of treatment plans for partial thickness articular surface rotator cuff tears.