Surgical menopause initiates molecular changes that do not result in mechanical changes in normal and healing ligaments.

OBJECTIVES: Ligaments which heal spontaneously have a healing process that is similar to skin wound healing. Menopause impairs skin wound healing and may likewise impair ligament healing. Our purpose in this study was to investigate the effect of surgical menopause on ligament healing in a rabbit medial collateral ligament model. METHODS: Surgical menopause was induced with ovariohysterectomy surgery in adult female rabbits. Ligament injury was created by making a surgical gap in the midsubstance of the medial collateral ligament. Ligaments were allowed to heal for six or 14 weeks in the presence or absence of oestrogen before being compared with uninjured ligaments. Molecular assessment examined the messenger ribonucleic acid levels for collagens, proteoglycans, proteinases, hormone receptors, growth factors and inflammatory mediators. Mechanical assessments examined ligament laxity, total creep strain and failure stress. RESULTS: Surgical menopause in normal medial collateral ligaments initiated molecular changes in all the categories evaluated. In early healing medial collateral ligaments, surgical menopause resulted in downregulation of specific collagens, proteinases and inflammatory mediators at 6 weeks of healing, and proteoglycans, growth factors and hormone receptors at 14 weeks of healing. Surgical menopause did not produce mechanical changes in normal or early healing medial collateral ligaments. With or without surgical menopause, healing ligaments exhibited increased total creep strain and decreased failure stress compared with uninjured ligaments. CONCLUSIONS: Surgical menopause did not affect the mechanical properties of normal or early healing medial collateral ligaments in a rabbit model. The results in this preclinical model suggest that menopause may result in no further impairment to the ligament healing process. Cite this article: Bone Joint Res 2015;4:38-44.

Changes of early post-traumatic osteoarthritis in an ovine model of simulated ACL reconstruction are associated with transient acute post-injury synovial inflammation and tissue catabolism.

The study described here tested the hypothesis that early intra-articular inflammation is associated with the development of post-traumatic osteoarthritis (PTOA) in a sheep model. We extended previously published work in which we investigated joint gross morphology and synovial mRNA expression of inflammatory and catabolic molecules 2 weeks after anatomic Anterior cruciate ligament (ACL) autograft reconstructive surgery (ACL-R). The same variables have been analyzed at 20 weeks post surgery together with new experimental variables at both time points. Animals were sacrificed at 20 weeks post ACL-R surgery and their joints graded for signs of PTOA. Synovial samples were harvested for histological grading plus mRNA and protein analysis for a panel of inflammatory and catabolic molecules. The mRNA expression levels for this panel plus connective tissue matrix turnover molecules were also investigated in cartilage samples. Results of gross morphological assessments at 20 weeks post surgery showed some changes consistent with early OA, but indicated little progression of damage from the 2 week time point. While significant alterations in mRNA levels for synovial inflammatory and catabolic molecules were detected at 2 weeks, values had normalized by 20 weeks. Similarly, all mRNA expression levels for inflammatory and catabolic molecules in articular cartilage had returned to normal levels by 20 weeks post ACL-R surgery. We conclude that synovial inflammatory processes are initiated very early after ACL-R surgery and may instigate events that lead to the gross cartilage and joint abnormalities observed as early as 2 weeks. However, the absence of sustained inflammation and joint instability may prevent OA progression.

Technical issues in using robots to reproduce joint specific gait.

Reproduction of the in vivo motions of joints has become possible with improvements in robot technology and in vivo measuring techniques. A motion analysis system has been used to measure the motions of the tibia and femur of the ovine stifle joint during normal gait. These in vivo motions are then reproduced with a parallel robot. To ensure that the motion of the joint is accurately reproduced and that the resulting data are reliable, the testing frame, the data acquisition system, and the effects of limitations of the testing platform need to be considered. Of the latter, the stiffness of the robot and the ability of the control system to process sequential points on the path of motion in a timely fashion for repeatable path accuracy are of particular importance. Use of the system developed will lead to a better understanding of the mechanical environment of joints and ligaments in vivo.

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.

Alterations to cell metabolism in connective tissues of the knee after ovariohysterectomy in a rabbit model: are there implications for the postmenopausal athlete?

BACKGROUND: Participation in regular exercise and athletic activities across the lifespan is encouraged to maintain the cardiovascular and musculoskeletal systems and general wellbeing. Before the menopause there is an increased risk of anterior cruciate ligament (ACL) injuries in female athletes, whereas there is an increased risk of joint diseases such as knee osteoarthritis after the menopause. Although there are few data regarding alterations in individual connective tissues of the knee in humans either before, during or after the menopause, it is possible to assess changes in experimental models following surgical menopause. OBJECTIVE: To assess changes in cell metabolism in the medial collateral ligament, ACL, patellar tendon, lateral and medial menisci, tibial plateau and femoral condyle articular cartilage and the synovium after surgical menopause in an experimental model system. METHODS: Panels of rabbits were subjected to ovariohysterectomy or sham operations, and RNA from each tissue was assessed for collagen, proteoglycan, proteinase, growth factor, sex hormone receptor and inflammatory mediator messenger RNA levels by reverse transcribed PCR. RESULTS: Unique alterations in cell metabolism were detected 2 months after surgical menopause and the pattern of significant changes was tissue specific (number of mRNA species altered, extent of changes, elevation/depression of changes). CONCLUSIONS: Changes in cell metabolism may alter the set point for the tissues of the knee and subsequently the functioning of the knee after the menopause. Such changes may contribute to an increased risk of injury and/or degenerative conditions. Further studies in pre and postmenopausal women athletes may also shed light on whether the present findings can be extrapolated to human populations.

Minimal promoter for the NAD+-specific glutamate dehydrogenase gene of Neurospora crassa.

The expression of the NAD+-specific glutamate dehydrogenase (NAD-GDH) gene of Neurospora crassa is subject to catabolite repression. To identify the minimal sequence necessary for promoter function, the 5'-flanking region of the NAD-GDH gene was screened for potential protein-binding sites. Fragments of DNA, containing sequences upstream from the ATG initiation codon, were employed as probes of Southwestern blots of total cellular protein from cells grown in media promoting repression and induction of NAD-GDH. Two polypeptides interacted differentially with a promoter probe; one was present in greater abundance in repressed cells and a higher relative level of the second was witnessed in induced cells. Electrophoretic mobility shift assays with labeled promoter fragments exhibited preferential interaction with proteins in the induced cultures. The upstream sequence containing the putative protein-binding sites was fused with the coding sequence of the green fluorescent protein (GFP). The resulting plasmid was introduced into the microconidia of an albino mutant of N. crassa by electroporation. Stable integration of the plasmid and_expression of GFP in the hyphae and conidia of the transformants were demonstrated by Southern and Western blot analysis and fluorescence microscopy.