Structural Consequences of a Partial Anterior Cruciate Ligament Injury on Remaining Joint Integrity: Evidence for Ligament and Bone Changes Over Time in an Ovine Model.

BACKGROUND: Severe injury to the knee joint often results in accelerated posttraumatic osteoarthritis (PTOA). In an ovine knee injury model, altered kinematics and degradation of the cartilage have been observed at 20 and 40 weeks after partial anterior cruciate ligament (ACL) transection (p-ACL Tx) surgery. However, changes to the integrity of the remaining intact intra-articular ligaments (posterolateral [PL] band and posterior cruciate ligament [PCL]) as well as the subchondral bone after anteromedial (AM) band Tx remain to be characterized. PURPOSE: (1) To investigate histological alterations to the remaining intact intra-articular ligaments, the synovium, and the infrapatellar fat pad (IPFP) and (2) to quantify subchondral bone changes at the contact surfaces of the proximal tibia at 20 and 40 weeks after AM band Tx. STUDY DESIGN: Descriptive laboratory study. METHODS: Mature female Suffolk cross sheep were allocated into 3 groups: nonoperative controls (n = 6), 20 weeks after partial ACL transection (p-ACL Tx; n = 5), and 40 weeks after p-ACL Tx (n = 6). Ligament, synovium, and IPFP sections were stained and graded. Tibial subchondral bone microarchitecture was assessed using high-resolution peripheral quantitative computed tomography. RESULTS: p-ACL Tx of the AM band led to significant change in histological scores of the PL band and the PCL at 20 weeks after p-ACL Tx (P = .031 and P = .033, respectively) and 40 weeks after p-ACL Tx (P = .011 and P = .029) as compared with nonoperative controls. Alterations in inflammatory cells and collagen fiber orientation contributed to the greatest extent of the combined histological score in the PL band and PCL. p-ACL Tx did not lead to chronic activation of the synovium or IPFP. Trabecular bone mineral density was strongly inversely correlated with combined gross morphological damage in the top and middle layers of the subchondral bone in the lateral tibial plateau for animals at 40 weeks after p-ACL Tx. CONCLUSION: p-ACL Tx influences the integrity (biology and structure) of remaining intact intra-articular ligaments and bone microarchitecture in a partial knee injury ovine model. CLINICAL RELEVANCE: p-ACL Tx leads to alterations in structural integrity of the remaining intact ligaments and degenerative changes in the trabecular bone mineral density, which may be detrimental to the injured athlete's knee joint in the long term.

Methylprednisolone acetate mitigates IL1ß induced changes in matrix metalloproteinase gene expression in skeletally immature ovine explant knee tissues.

OBJECTIVE AND DESIGN: This study aimed at evaluating the effect of methylprednisolone (MPA) on messenger ribonucleic acid (mRNA) expression levels in immature ovine knee joint tissue explants following interleukin (IL)1ß induction and to assess responsiveness of the explants. MATERIAL OR SUBJECTS: Explants were harvested from the articular cartilage, synovium, and infrapatellar fat pad (IPFP) from immature female sheep. TREATMENT: Methylprednisolone. METHODS: The samples were allocated into six groups: (1) control, (2) MPA (10-3 M), (3) MPA (10-4 M), (4) IL1ß, (5) IL1ß + 10-3 M MPA, or (6) IL1ß + 10-4 M MPA. mRNA expression levels for molecules relevant to inflammation, cartilage degradation/anabolism, activation of innate immunity, and adipose tissue/hormones were quantified. Fold changes with MPA treatment were compared via the comparative CT method. RESULTS: Methylprednisolone treatment significantly suppressed MMPs consistently across the cartilage (MMP1, MMP3, and MMP13), synovium (MMP1 and MMP3), and IPFP (MMP13) (all p < 0.05). Other genes that were less consistently suppressed include endogenous IL1ß (cartilage) and IL6 (IPFP) (all p < 0.05), and others not affected either by IL-1 exposure or subsequent MPA include TGFß1, TLR4, and adipose-related molecules. CONCLUSIONS: Methylprednisolone significantly mitigated IL1ß induced mRNA expression for MMPs in the immature cartilage, synovium, and IPFP, but the extent of the responsiveness was tissue-, location-, and gene-specific.

Stress Measurements on the Articular Cartilage Surface Using Fiber Optic Technology and In-Vivo Gait Kinematics.

It has been hypothesized a change in stress on the cartilage of a joint is a significant factor in the initiation and progression of post-traumatic osteoarthritis. Without a reliable method for measuring stress, this hypothesis has largely gone untested. In this study, a novel, repeatable, and reliable method for measuring stress on the surface of articular cartilage in articular joints is presented. Small Fiber Bragg Grating (FBG) sensors capable of measuring normal stress between contact surfaces in diarthrodial joints were developed and validated. The small size of these sensors (diameter of 125-300 µm and sensing length of 1 mm) allows them to be inserted into the joint space without the removal of biomechanically relevant structures. In-vitro stresses on the surface of the cartilage for both healthy and surgically damaged joints were measured after implantation of the FBG sensors using in vivo generated gait kinematic data and a 6-degrees of freedom parallel robot. Along with our capability to reproduce in vivo motions accurately and the improvements in fiber optic technology, this study describes the first direct measurement of stress in a joint using in vivo gait kinematics.

Three-dimensional in vivo kinematics and finite helical axis variables of the ovine stifle joint following partial anterior cruciate ligament transection.

Partial anterior cruciate ligament (p-ACL) rupture is a common injury, but the impact of a p-ACL injury on in vivo joint kinematics has yet to be determined in an animal model. The in vivo kinematics of the ovine stifle joint were assessed during 'normal' gait, and at 20 and 40 weeks after p-ACL transection (Tx). Gross morphological scoring of the knee was conducted. p-ACL Tx creates significant progressive post-traumatic osteoarthritis (PTOA)-like damage by 40 weeks. Statistically significant increases for flexion angles at hoof-strike (HS) and mid-stance (MST) were seen at 20 weeks post p-ACL Tx and the HS and hoof-off (HO) points at 40 weeks post p-ACL-Tx, therefore increased flexion angles occurred during stance phase. Statistically significant increases in posterior tibial shift at the mid-flexion (MF) and mid-extension (ME) points were seen during the swing phase of the gait cycle at 40 weeks post p-ACL Tx. Correlation analysis showed a strong and significant correlation between kinematic changes (instabilities) and gross morphological score in the inferior-superior direction at 40 weeks post p-ACL Tx at MST, HO, and MF. Further, there was a significant correlation between change in gross morphological combined score (ΔGCS) and the change in location of the helical axis in the anterior direction (ΔsAP) after p-ACL Tx for all points analyzed through the gait cycle. This study quantified in vivo joint kinematics before and after p-ACL Tx knee injury during gait, and demonstrated that a p-ACL knee injury leads to both PTOA-like damage and kinematic changes.

Alterations in Joint Angular Velocity Following Traumatic Knee Injury in Ovine Models.

Little effort has been directed towards the consequence of an injury on joint angular velocity. We hypothesized that the magnitude of knee joint angular velocity would be decreased after injury. Four injury groups were investigated in an ovine model: (1) anterior cruciate ligament (ACL) and medial collateral ligament (MCL) transection (ACL/MCL Tx) (n = 5), (2) lateral meniscectomy (Mx) (n = 5), (3) partial ACL transection (p-ACL Tx) (n = 5), and (4) partial-ACL and MCL transection (p-ACL/MCL Tx) (n = 5). The magnitude of the angular velocities decreased in the subjects of all groups at multiple points of the gait cycle. The maximum angular velocities during stance and the maximum extension angular velocities during swing were decreased in 15/20 and 17/20 subjects, respectively. There were strong correlations between morphological osteoarthritis scores and the reduction in the maximum extension angular velocities during swing 40 weeks post-p-ACL Tx and 20 weeks post-ACL/MCL Tx. There was no correlation between the decrease of the angular velocity and morphological osteoarthritis scores in the Mx group and the p-ACL/MCL Tx group. The reduction in angular velocity may be a helpful addition as a surrogate measure of OA risk after ACL injury, and could have clinical significance after further investigation in humans.

Are re-injured ligaments equivalent mechanically to injured ligaments: The role of re-injury severity?

The consequences of ligament re-injury have received limited attention. Although the mechanical properties of injured ligaments improve over time, these properties are never fully recaptured, rendering these injured ligaments susceptible to re-injury. Previous injury is a significant risk factor for recurrent injury, and this re-injury can result in longer absence from activity than the initial injury. A rabbit medial collateral ligament model was used to compare mechanically re-injured right medial collateral ligaments to injured left medial collateral ligaments. Two groups of different re-injury severity were investigated: 'minor' re-injury comparing transection re-injured right medial collateral ligaments to transection injured left medial collateral ligaments; 'major' re-injury comparing gap re-injured right medial collateral ligaments to transection injured left medial collateral ligaments. Initial injuries for both groups were right medial collateral ligament transections 1 week before re-injury. After 5-6 weeks of healing, mechanical testing was performed to determine (dimensionally) cross-sectional area; (structurally) medial collateral ligament laxity, failure load, and stiffness; and (materially) cyclic creep strain and failure stress. Because we wanted to evaluate whether the mechanical properties of re-injured ligaments were equivalent or, at least, no worse than injured ligaments, we used equivalence/noninferiority testing. This approach evaluates a research hypothesis of equivalence, rather than difference, and determines whether comparisons are 'statistically equivalent', 'noninferior', or 'potentially inferior'. Transection re-injured and gap re-injured ligaments were 'statistically equivalent' structurally to transection injured ligaments. Transection re-injured ligaments were 'noninferior' both materially and dimensionally to transection injured ligaments. Gap re-injured ligaments were 'potentially inferior' both materially and dimensionally to transection injured ligaments. Two differences between the re-injuries, which affect healing, may explain the mechanical outcomes: the presence or lack of healing products and the proximity of ligament ends at the time of re-injury. Our findings suggest that (in the short term) there is a severity of re-injury below which there is no additional disadvantage to the healing process, mechanical behaviour, and resulting potential for re-injury.

Correlation between translational and rotational kinematic abnormalities and osteoarthritis-like damage in two in vivo sheep injury models.

The relations between kinematic abnormalities and post traumatic osteoarthritis have not yet been clearly elucidated. This study was conducted to determine the finite helical axes parameters and the tibiofemoral translation vector in the knee joints of two surgically induced injury sheep models: anterior cruciate ligament and medial collateral ligament transection (ACL/MCL Tx) (n = 5) and lateral meniscectomy (n = 5). We hypothesized that morphological damage in the experimental joints would be correlated to alterations in these kinematic variables. There was no strong evidence that morphological damage to the joints 20 weeks post ACL/MCL transection or meniscectomy was correlated with alterations in the finite helical axes variables. Nevertheless, significant correlations were found between the morphological damage to the joints and the magnitude of the change in the translation vectors after ACL/MCL transection (significant correlations (p = 0.005) during stance and trends (p < 0.1) at all points analyzed during swing). It can be concluded that: (1) osteoarthritic-like morphological damage after ACL/MCL transection is more critically correlated to the absolute tibiofemoral translational change and (2) alterations in analyzed kinematic variables cannot solely define osteoarthritis risk after meniscal injuries. From a clinical perspective, our results suggest that the magnitude of the change in the translation vector, which is independent of the coordinate system and combines the effects of the three translational degrees of freedom, i.e. medial-lateral, anterior-posterior and inferior-superior, would be an osteoarthritis risk factor after ligament injury, and requires validation in humans.

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.

Enumeration and Localization of Mesenchymal Progenitor Cells and Macrophages in Synovium from Normal Individuals and Patients with Pre-Osteoarthritis or Clinically Diagnosed Osteoarthritis.

Osteoarthritis (OA) is a degenerative disorder characterized by chondrocyte apoptosis and degeneration of articular cartilage resulting in loss of mobility and pain. Inflammation plays a key role in the development and progression of OA both on the side of apoptosis and repair, while its exact role in pathogenesis has yet to be fully elucidated. Few studies have examined the cellular composition (inflammatory cells and/or progenitor cells) in the synovium of patients with pre-OA (asymptomatic with cartilage damage). Therefore, in the current study, mesenchymal progenitor cells (MPCs) and macrophages were enumerated within normal, pre-OA and OA synovium. No differences were observed between MPCs in normal vs. pre-OA, however, fewer macrophages were observed in pre-OA vs. normal synovium. Osteoarthritic synovium contained greater numbers of both MPCs and macrophages. Interestingly, the localization of MPCs and macrophages was affected by disease severity. In normal and pre-OA synovium, MPCs and macrophages co-localized, while in OA synovium, MPCs and macrophage populations were spatially distinct. Examining the cellular interactions between MPCs and macrophages in synovium may be essential for understanding the role of these cells in the onset and/or pathogenesis of the disease. This study has provided a first step by examining these cell types both spatially and temporally (e.g., disease severity). Further cellular and molecular studies will be needed to determine the functions of these cells in the context of disease and in relation to each other and the joint as a whole.

Use of pre-clinical surgically induced models to understand biomechanical and biological consequences of PTOA development.

Post-traumatic osteoarthritis (PTOA) development is often observed following traumatic knee injuries involving key stabilising structures such as the cruciate ligaments or the menisci. Both biomechanical and biological alterations that follow knee injuries have been implicated in PTOA development, although it has not been possible to differentiate clearly between the two causal factors. This review critically examines the outcomes from pre-clinical lapine and ovine injury models arising in the authors' laboratories and differing in severity of PTOA development and progression. Specifically, we focus on how varying severity of knee injuries influence the subsequent alterations in kinematics, kinetics, and biological outcomes. The immediate impact of injury on the lubrication capacity of the joint is examined in the context of its influence on biomechanical alterations, thus linking the biological changes to abnormal kinematics, leading to a focus on the potential areas for interventions to inhibit or prevent development of the disease. We believe that PTOA results from altered cartilage surface interactions where biological and biomechanical factors intersect, and mitigating acute joint inflammation may be critical to prolonging PTOA development. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:454-465, 2017.

Location and gene-specific effects of methylprednisolone acetate on mitigating IL1ß-induced inflammation in mature ovine explant knee tissue.

OBJECTIVE AND DESIGN: To determine the ability of methylprednisolone acetate (MPA) to influence interleukin 1ß (IL1ß)-induced gene expression in ovine knee joint tissues. MATERIAL OR SUBJECTS: Ovine articular cartilage, synovium, and infrapatellar fat pad (IPFP) explants. TREATMENT: Explants were treated with 10-3 M or 10-4 M MPA. METHODS: Explant treatment groups: (1) control (DMEM); (2) inflammation (IL1ß); (3) IL1ß + 10-3 M MPA; or (4) IL1ß + 10-4 M MPA. Cell viability was assessed pre- and post-treatment. Expression of mRNA levels for inflammatory, degradative, anabolic, innate immunity, and adipose-related molecules was quantified via qPCR, and analyzed via the comparative C T method. RESULTS: Except for IL8 in a subset of cartilage locations, matrix metalloproteinases (MMPs) were the only genes consistently affected by MPA. MPA mitigated IL1ß-induced MMP3 expression levels in all regions of the articular cartilage, and in the synovium and IPFP, while MMP1 mRNA expression levels were significantly decreased with MPA after IL1ß in the tibial plateau and synovium, but paradoxical increases in the IPFP. MMP13 mRNA expression levels exhibited significant decreases with MPA after IL1ß in the femoral condyles, tibial plateau, synovium, and IPFP. CONCLUSIONS: MPA treatment suppressed IL1ß-induced mRNA levels for MMPs in articular cartilage, synovium, and IPFP and was found to be tissue-, location-, and gene-specific.

The infrapatellar fat pad is affected by injury induced inflammation in the rabbit knee: use of dexamethasone to mitigate damage.

OBJECTIVE AND DESIGN: The health of the infrapatellar fat pad (IFP) has been linked to pain, joint inflammation, and the onset of post-traumatic osteoarthritis. Thus, early inflammation effects on the IFP could have long term sequelae on joint integrity. This study was designed to characterize the natural history of the IFP in a model of surgically induced knee injury and inflammation, and to test the efficacy of one intra-articular (IA) administration of dexamethasone (DEX) immediately following surgery. METHODS: An IA bone drill hole injury to the rabbit knee was conducted and immediately treated with DEX (n = 12). Early and late post-surgical time-points were investigated (48 h and 9 weeks) and the outcome measures were analysis of IFP histology, mRNA levels for relevant molecules, and protein levels for a subset of cytokines. Data were analyzed against a surgical control (injury without treatment; n = 12), a surgical sham (capsular incision only; n = 12), and normal control (n = 6). TREATMENT: Single IA injection of DEX (0.5 mg/kg), administered at the completion of surgery. RESULTS: IFPs from injured joints exhibited significantly increased cellularity and early fibrosis at 48 h post surgery. While the histological inflammation from a capsular incision alone resolved, knee injured animals progressed to a significantly more fibrotic IFP by 9 weeks. DEX significantly lowered histological scores at 48 h, but not at the 9 weeks. DEX did not influence mRNA levels for IL-1ß, 6, and 8, however, protein analysis indicated that IL-8 levels were lower in DEX treated joints. DEX resulted in significantly elevated expression of mRNA for MCP-1, leptin, and VEGF. CONCLUSION: One IA administration of a glucocorticoid appears to mitigate the initial inflammation within the joint, but is not sufficient to protect the joint to 9 weeks post-surgery.

Ligament and meniscus loading in the ovine stifle joint during normal gait.

BACKGROUND: The ovine stifle joint is an ideal preclinical model to study knee joint biomechanics. Knowledge of the ovine ligamentous and meniscal loading during normal gait is currently limited. METHODS: The in vivo kinematics of the ovine stifle joint (N=4) were measured during "normal" gait using a highly accurate instrumented spatial linkage (ISL, 0.3±0.2mm). These motions were reproduced in vitro using a unique robotic testing platform and the loads carried by the anterior/posterior cruciate ligaments (ACL/PCL), medial/lateral collateral ligaments (MCL/LCL), and medial/lateral menisci (MM/LM) during gait were determined. RESULTS: Considerable inter-subject variability in tissue loads was observed. The load in the ACL was near zero at hoof-strike (0% gait) and reached a peak (100 to 300N) during early-stance (~10% gait). The PCL reached a peak load (200 to 500N) just after hoof-strike (~5% gait) and was mostly unloaded throughout the remainder of stance. Load in the MCL was substantially lower than the cruciate ligaments, reaching a maximum of 50 to 100N near the beginning of stance. The LCL carried a negligible amount of load through the entire gait cycle. There was also a major contribution of the MM and LM to load transfer from the femur to the tibia during normal gait. The total meniscal load reached a maximum average between 350 and 550N during gait. CONCLUSION: Knowledge of joint function during normal motion is essential for understanding normal and pathologic joint states. The considerable variability in the magnitudes and patterns of tissue loads among animals simulates clinical variability in humans. LEVEL OF EVIDENCE: III.

An instrumented spatial linkage for measuring knee joint kinematics.

BACKGROUND: In this study, the design and development of a highly accurate instrumented spatial linkage (ISL) for kinematic analysis of the ovine stifle joint is described. The ovine knee is a promising biomechanical model of the human knee joint. METHODS: The ISL consists of six digital rotational encoders providing six degrees of freedom (6-DOF) to its motion. The ISL makes use of the complete and parametrically continuous (CPC) kinematic modeling method to describe the kinematic relationship between encoder readings and the relative positions and orientation of its two ends. The CPC method is useful when calibrating the ISL, because a small change in parameters corresponds to a small change in calculated positions and orientations and thus a smaller optimization error, compared to other kinematic models. The ISL is attached rigidly to the femur and the tibia for motion capture, and the CPC kinematic model is then employed to transform the angle sensor readings to relative motion of the two ends of the linkage, and thereby, the stifle joint motion. RESULTS: The positional accuracy for ISL after calibration and optimization was 0.3±0.2mm (mean +/- standard deviation). The ISL was also evaluated dynamically to ensure that accurate results were maintained, and achieved an accuracy of 0.1mm. CONCLUSIONS: Compared to the traditional motion capture methods, this system provides increased accuracy, reduced processing time, and ease of use. Future work will be on the application of the ISL to the ovine gait and determination of in vivo joint motions and tissue loads. CLINICAL RELEVANCE: Accurate measurement of knee joint kinematics is essential in understanding injury mechanisms and development of potential preventive or treatment strategies.

Relationship between increased in vivo meniscal loads and abnormal tibiofemoral surface alignment in ACL deficient sheep is varied.

The aim of this study was to quantify how abnormal dynamic tibiofemoral surface alignment affects the load bearing function of menisci in vivo. Using a sheep model of ACL deficiency, we tested the hypothesis that increased in vivo meniscal loads correlate with greater tibiofemoral surface alignment abnormality. Stifle kinematics were recorded using a bone-mounted instrumented spatial linkage in four sheep before, and at four and twenty weeks (w) after ACL transection. A parallel robotic manipulator was used to quantify stifle kinetics by reproducing each animal׳s in vivo kinematics and measuring tissue loads during gait. Meniscal resultant loads were estimated from the change in joint reaction force after sequentially removing load-bearing tissues. Tibiofemoral subchondral surfaces were then traced and modeled using thin plate splines. Proximity disturbance is a surface interaction measure used to quantify dynamic tibiofemoral surface alignment abnormality. ACL transection increased meniscal loads by 30-145% at 20w post-ACL transection, whereas the degree of dynamic tibiofemoral subchondral surface alignment varied between sheep. Positive and significant correlations between increased meniscal loads and proximity disturbance values >10mm were observed (R2=0.04-0.57; p≤0.05). Our results suggest that the proximity disturbance measure reflects abnormal meniscal loads following ACL injury; however given the range of R2 values, perturbations in dynamic tibiofemoral subchondral surface alignment do not explain abnormal joint kinetics entirely, and point to the presence of other dynamic compensatory mechanisms that may have a significant bearing on in vivo joint function and long-term joint health.

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.

Alterations in Hoffa's fat pad induced by an inflammatory response following idealized anterior cruciate ligament surgery.

OBJECTIVE: To determine whether inflammation following anterior cruciate ligament (ACL) reconstruction leads to long-term pathological changes in the infrapatellar fat pad (IPFP or Hoffa's fat pad) which could compromise the integrity of the knee joint. MATERIALS AND METHODS: Sixteen mature sheep underwent anatomic idealized ACL reconstruction surgery (ACL-R) and were sacrificed at 2 weeks (n = 9) and 20 weeks (n = 7) post-ACL-R. Five additional animals served as unoperated controls. A histological grading protocol was developed to quantify the changes in the IPFP post-injury. mRNA expression levels for key markers of inflammation, angiogenesis and tissue regeneration were assessed by qPCR. RESULTS: The IPFP exhibited altered cellularity and fibrosis at 2 and 20 weeks post-ACL-R. Immunohistochemistry detected macrophage-like cells in the IPFP which were increased at 20 weeks. Specific pro-inflammatory cytokines and IPFP specific adipokines exhibited changes indicating early inflammation mediated alterations. Elevations in CD105 mRNA levels at 2 weeks corroborated the increases in neovascularization observed in the IPFP following injury. CONCLUSIONS: Sustained long-term pathological changes stemming from inflammation are present in IPFP tissue after ACL-R surgery and may compromise the long-term integrity of the knee joint.

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.

Increased meniscal loading after anterior cruciate ligament transection in vivo: a longitudinal study in sheep.

INTRODUCTION: Meniscal injury has been well documented as a frequent consequence of both acute and chronic ACL deficiency. The purpose of this study was to evaluate the effect of ACL deficiency on meniscal loads in vivo and determine how these loads would change over time after ACL injury. METHODS: The in vivo kinematics of the stifle joint of five sheep were measured during normal gait, as well as 4 and 20 weeks after ACL transection. A unique robotic testing platform was then programmed to reproduce all the previously recorded kinematics and the loads carried by medial and lateral menisci during gait were estimated. RESULTS: The results demonstrated a significant increase in both medial and lateral meniscal loads 20 weeks following ACL transection, mainly during mid-stance phase of gait (p = 0.007 and p = 0.003, respectively), with interesting inter-subject variability. A moderate correlation (R(2) ≥ 0.5) between in situ meniscal loads and anterior tibial translations was also detected over time after injury, increased translations post injury generally corresponded to larger meniscal loads. CONCLUSION: The dramatic increase in meniscal loads long term post ACL transection probably explains the meniscal changes or injuries reported clinically in many chronic ACL-deficient knees.

A new measure of tibiofemoral subchondral bone interactions that correlates with early cartilage damage in injured sheep.

We have demonstrated previously that chondral damage is associated with increased knee surface velocities following ligament and meniscus injuries in sheep. We tested the hypothesis that cartilage damage scores would correlate with a new bone surface interaction measure that captures complex changes in tibiofemoral alignment, "proximity disturbance" (PD). Six sheep underwent combined anterior cruciate and medial collateral ligament transection (ACL/MCLx), five complete lateral meniscectomy (Mx), and four sham arthrotomy (Sham). Tibiofemoral subchondral bone surfaces were modeled, and the post-operative changes in relative separation of the surfaces (i.e., "proximity") were derived from subject-specific in vivo 3D stifle kinematics. Surface areas of regions of near contact were determined, and PD was calculated as the range of change in tibiofemoral proximity, divided by normalized overlapping proximity surface areas between baseline and post-operative time points. Cartilage morphology was graded at dissection. ACL/MCLx PD was significantly elevated relative to Mx and Shams, and correlated with cartilage damage (r(2) = 0.88-0.98). Although not statistically significant, Mx PD values tended to be higher than those of Shams, and correlated with cartilage damage. Results from both injury models suggest that increasing change in tibiofemoral surface alignment may be increasingly deleterious to long-term cartilage health in sheep.