May Chung

Static Compressive Loading Reduces the mRNA Expression of Type II and X Collagen in Rat Growth-Plate Chondrocytes During Postnatal Growth

The mechanisms by which chondrocytes modulate longitudinal bone growth are not well understood. This in vitro study investigated the effects of loading on the mRNA expression pattern of key molecular components of the growth-plate related to the extracellular matrix (type II and type X collagen) and the PTH-PTHrP feedback loop. Short-term static compressive loading was applied to rat proximal tibial growth-plate explants. Four age groups at specific developmental stages were investigated. The spatial variation in the mRNA expression was compared among loaded explants, their contralateral sham controls, and uncultured growth plates from normal animals. Basic cell metabolism (18S rRNA) was unaffected by load. Results indicated a narrower spatial distribution of mRNA expression of type II collagen throughout the growth plate; similarly, a narrowed distribution of expression of type X collagen was noted in the lower hypertrophic zone of the growth-plate. This suggests that mechanical compression influences chondrocytes of the hypertrophic zone to alter their expression of specific genes encoding proteins of the extracellular matrix, while PTH-PTHrP receptor mRNA, a regulatory protein, remained unaffected by loading. The effects of compression were similar at the different stages of growth, suggesting that additional factors may be involved in the clinical progression of skeletal deformities observed during growth spurts. Although this study was done in vitro and limited to static loading, it furthers our understanding of growth-plate mechanobiology as a first step toward providing a scientific rationale for treating progressive musculoskeletal deformities.

Metabolic profiling of synovial fluid in a unilateral ovine model of anterior cruciate ligament reconstruction of the knee suggests biomarkers for early osteoarthritis

Joint injuries and subsequent osteoarthritis (OA) are the leading causes of chronic joint disease. In this work, we explore the possibility of applying magnetic resonance spectroscopy‐based metabolomics to detect host responses to an anterior cruciate ligament (ACL) reconstruction injury in synovial fluid in an ovine model. Using multivariate statistical analysis, we were able to distinguish post‐injury joint samples (ACL and sham surgery) from the uninjured control samples, and as well the ACL surgical samples from sham surgery. In all samples there were 65 metabolites quantified, of which six could be suggested as biomarkers for early post‐injury degenerative changes in the knee joints: isobutyrate, glucose, hydroxyproline, asparagine, serine, and uridine. Our results raise a cautionary note indicating that surgical interventions into the knee can result in metabolic alterations that need to be distinguished from those caused by the early onset of OA. Our findings illustrate the potential application of metabolomics as a diagnostic and prognostic tool for detection of injuries to the knee joint. The ability to detect a unique pattern of metabolic changes in the synovial fluid of sheep offers the possibility of extending the approach to precision medicine protocols in patient populations in the future.

Single intra-articular dexamethasone injection immediately post-surgery in a rabbit model mitigates early inflammatory responses and post-traumatic osteoarthritis-like alterations.

Despite surgical reconstruction of the anterior cruciate ligament, a significant number of patients will still develop post‐traumatic osteoarthritis (PTOA). Our objective was to determine if mitigating aspects of the acute phase of inflammation following a defined knee surgery with a single administration of a glucocorticoid could prevent the development of PTOA‐like changes within an established rabbit model of surgically induced PTOA. An early and late post‐surgical time‐point was investigated in this study (48 h and 9 weeks post‐surgery) in which the following groups were repeated (each n = 6, for a total of 24 rabbits per time‐point, and 48 rabbits used in the study): control (age/sex matched), sham (arthrotomy), drill injury (arthrotomy + two drill holes to a non‐cartilaginous area of the femoral notch), and drill injury + single intra‐articular (IA) injection of dexamethasone (DEX). At 48 h post‐surgery, DEX treatment significantly lowered the mRNA levels for a subset of pro‐inflammatory mediators, and significantly lowered the histological grade. Nine weeks post surgery, DEX treatment significantly lowered the histological scores (presented as effect size) for synovium (3.8), lateral femoral condyle (3.9), and lateral tibial cartilage (5.1) samples. Thus, DEX likely acts to prevent injury induced inflammation that could contribute to subsequent joint damage.

Indirect Injury Stimulates Scar Formation-Adaptation or Pathology?

In several animal models of osteoarthritis induced by cruciate ligament transection, a dense, scar-like tissue mass forms rapidly on the medial side of the knee joint. This mass mimics clinical fibrosis that sometimes occurs after joint surgery. It is unknown exactly why this medial tissue mass forms and what cells are involved in its formation. This study characterizes this medial mass by histology, biochemistry, and the expression of types I and III collagen mRNA. The medial mass is compared with the medial collateral ligament (MCL) and the MCL epiligament in anterior cruciate-transected and unoperated joints, and to normal skin and skin scar. The morphology of the medial mass resembled the epiligament and skin scar more than the MCL. The concentration of DNA and RNA and the RNA-DNA ratio were elevated dramatically in the medial mass compared with all other tissues including skin scar. However, the mRNA copy number and ratio of collagen types I and III mRNAs did not differ significantly among the medial mass, MCL, epiligament, and skin in either the control or the operated joints. The response of the medial mass, MCL, and MCL epiligament to cruciate transaction involves both hyperplasia and hypertrophy, but without a dramatic shift in cell phenotype. The medial mass may be a useful mimic or model of intraarticular adhesions, hypertrophic scars, ligament sprains, and arthrofibrosis.

Normal sheep synovium has similar appearances and constitutive expression of inflammatory cytokines within and between knee joints: a baseline histological and molecular analysis

Abstract Clinical evidence suggests that synovium can add to adjacent articular cartilage damage, potentially contributing to the development of osteoarthritis (OA). Inflammation of the synovium (synovitis) is dependent on the type of injury sustained, the time after injury and concomitant changes in other joint tissues. To define the role of synovitis in OA development, there is a need for baseline measures that can reliably distinguish synovial inflammation from normal synovium both within and between joints. This study tested the hypothesis that normal synovium from distinct anatomical locations in young and adult sheep is homogeneous with respect to consistently low molecular expression of the inflammatory mediators – tumour necrosis factor alpha (TNF-α) and interleukins (IL) such as IL-1β, IL-1Ra, IL-6 and IL-8. Additionally, maturation will not influence the expression of these select inflammatory biomarkers. Samples of synovium from four anatomic locations (medial and lateral margins, suprapatellar pouch (patella region), posterior to the posterior cruciate ligament, from each joint of 5 adult and 4 immature animals were graded histologically or analyzed for mRNA expression of inflammatory cytokines. Histologically, no evidence of synovitis was noted although some variance in sub-intimal fibrosis was observed between sample locations in mature sheep. Molecular expression of all inflammatory mediators was low and homogeneously expressed at constitutive levels in all sample locations. These findings confirm the hypothesis that the normal sheep synovium is a homogeneous tissue throughout the joint and establishes the baseline expression levels for several pro-inflammatory mediators in both immature and mature sheep.

Post-natal Molecular Adaptations in Anteromedial and Posterolateral Bundles of the Ovine Anterior Cruciate Ligament: One Structure with Two Parts or Two Distinct Ligaments?

The human anterior cruciate ligament (ACL) is a composite structure of two anatomically distinct bundles: an anteromedial (AM) and posterolateral (PL) bundles. Tendons are often used as autografts for surgical reconstruction of ACL following severe injury. However, despite successful surgical reconstruction, some people experience re-rupture and later development of osteoarthritis. Understanding the structure and molecular makeup of normal ACL is essential for its optimal replacement. Reportedly the two bundles display different tensions throughout joint motion and may be fundamentally different. This study assessed the similarities and differences in ultrastructure and molecular composition of the AM and PL bundles to test the hypothesis that the two bundles of the ACL develop unique characteristics with maturation. ACLs from nine mature and six immature sheep were compared. The bundles were examined for mRNA and protein levels of collagen types I, III, V, and VI, and two proteoglycans. The fibril diameter composition of the two bundles was examined with transmission electron microscopy. Maturation does alter the molecular and structural composition of the two bundles of ACL. Although the PL band appears to mature slower than the AM band, no significant differences were detected between the bundles in the mature animals. We thus reject our hypothesis that the two ACL bundles are distinct. The two anatomically distinct bundles of the sheep ACL can be considered as two parts of one structure at maturity and material that would result in a structure of similar functionality can be used to replace each ACL bundle in the sheep.

Posttraumatic Osteoarthritis Development and Progression in an Ovine Model of Partial Anterior Cruciate Ligament Transection and Effect of Repeated Intra-articular Methylprednisolone Acetate Injections on Early Disease:

Background: Partial anterior cruciate ligament (p-ACL) ruptures are a common injury of athletes. However, few preclinical models have investigated the natural history and treatment of p-ACL injuries. Purpose: To (1) demonstrate whether a controlled p-ACL injury model (anteromedial band transection) develops progressive gross morphological and histological posttraumatic osteoarthritis (PTOA)–like changes at 20 and 40 weeks after the injury and (2) investigate the efficacy of repeated (0, 5, 10, and 15 weeks) intra-articular injections of methylprednisolone acetate (MPA; 80 mg/mL) in the mitigation of potential PTOA-like changes after p-ACL transection. Study Design: Controlled laboratory study. Methods: Twenty-one 3- to 5-year-old female Suffolk-cross sheep were allocated to 4 groups: (1) nonoperative controls (n = 5), (2) 20 weeks after p-ACL transection (n = 5), (3) 40 weeks after p-ACL transection (n = 6), and (4) 20 weeks after p-ACL transection + MPA (n = 5). Gross morphological grading and histological analyses were conducted. mRNA expression levels for inflammatory, degradative, and structural molecules were assessed. Results: p-ACL transection led to significantly more combined gross damage (P = .008) and significant aggregate histological damage (P = .009) at 40 weeks after p-ACL transection than the nonoperative controls, and damage was progressive over time. Macroscopically, MPA appeared to slightly mitigate gross damage at 20 weeks after p-ACL transection in some animals. However, microscopic analysis revealed that repeated MPA injections after p-ACL transection led to significant loss in proteoglycan content compared with the nonoperative controls and 20 weeks after p-ACL transection (P = .008 and P = .008, respectively). Conclusion: p-ACL transection led to significant gross and histological damage by 40 weeks, which was progressive over time. Multiple repeated MPA injections were not appropriate to mitigate injury-related damage in a p-ACL transection ovine model as significant proteoglycan loss was observed in MPA-treated knees. Clinical Relevance: A p-ACL injury leads to slow and progressive PTOA-like joint damage, and multiple repeated injections of glucocorticoids may be detrimental to the knee joint in the long term.

Mechanical Loading Uniquely Up-Regulates MMP-13 in Rat Supraspinatus Tendon But Not Achilles Tendon

Tendon injuries and chronic pain, known as tendinopathy, are extremely common in supraspinatus and Achilles tendons. Despite their prevalence, the pathogenesis of these conditions is poorly understood. One of most common cited factors for injuries to the rotator cuff tendons is “impingement”, namely, mechanical compression of the supraspinatus tendon by the overlying coracoacromial arch [1]. To date, few studies have investigated the molecular processes underlying this condition.Copyright