Markers of Pain in 3 Different Murine Models of Posttraumatic Osteoarthritis: A Comparison Using Gait Analysis, Imaging, Histology, and Gene Expression.

BACKGROUND: Animal models play an important role in studying posttraumatic osteoarthritis (PTOA) disease progression. Different models exist, such as destabilization of the medial meniscus (DMM), anterior cruciate ligament (ACL) surgical transection (ACLs), and noninvasive ACL rupture. PURPOSE: To study the effects of PTOA on nociception in 3 different murine models and to relate these findings to macroscopic and microscopic changes in joint tissues. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 42 male C57BL/6 mice, 12 weeks old, were randomly assigned to 4 groups: intact control (n = 10), DMM (n = 10), ACLs (n = 11), and closed ACL rupture (ACLc; n = 11) groups. Gait analysis was performed on 5 mice from the DMM group and 6 mice from ACLs and ACLc groups at 0, 1, 2, 4, 8, and 12 weeks after injury. At the 12-week time point, all mice underwent radiographs and then either micro-computed tomography imaging followed by histology and immunohistochemistry or gene expression analysis of the dorsal root ganglion and tibialis anterior muscle. RESULTS: The peripheral and central pain markers were expressed at significantly higher levels in the synovium of both ACL injury groups when compared with the DMM group. Muscle atrophy genes were significantly upregulated in the ACL injury groups. Pain-related gait behavior started at 4 weeks for the ACL rupture groups and at 12 weeks for the DMM group. High-resolution radiographic imaging and histology demonstrated divergent changes in bone microstructure between the ACLs and DMM groups, suggesting different mechanical loading environments in these models. CONCLUSION: The principal finding of this study is the presence of markers of nociception at both the gene and the protein levels, with earlier expression in the ACL injury groups when compared with the DMM group. The second finding of this study is that the noninvasive ACL rupture model demonstrated changes comparable with those of the commonly used surgical ACL transection model, supporting use of this clinically realistic model in future studies of PTOA. CLINICAL RELEVANCE: Quantitative clinical outcomes (imaging, pain scale, gait changes) related to osteoarthritis severity in an animal study, allowing for better understanding of clinical outcomes of osteoarthritis progression after ACL injuries in humans.

The effect of cryoprotectant vehicle solution on cartilage cell viability following vitrification.

Osteochondral allografts are often used to repair large articular cartilage defects to prevent or delay the onset of osteoarthritis. This approach is limited by the timely acquisition and use of allograft tissue since standard hypothermic protocols allow for a maximum storage of 4 weeks. Vitrification is a proven technique for the long-term preservation of cells and tissues, but requires careful determination of parameters to be successful, particularly for articular cartilage. One parameter that is infrequently considered is the choice of cryoprotectant vehicle solution. The aim of this study was to evaluate the impact of a subset of vehicle solutions on an established vitrification protocol for articular cartilage. These solutions were phosphate-buffered saline (PBS), Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12 (DMEM), X-VIVO, and Unisol-CV (UCV). Both the solution pH at various points throughout vitrification and the cell viability of porcine articular cartilage slices following vitrification were measured. Using randomized block ANOVA, it was found that the normalized cell viability of articular cartilage vitrified in UCV was significantly greater than that of PBS (p < 0.05) and may be greater than those of DMEM and X-VIVO (p < 0.1). There was no correlation between pH parameters and cell viability, although significant differences between calculated pH parameters were identified. These results provide information to guide the design of effective vitrification protocols for articular cartilage.

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.

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.

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.

Fibril deformation under load of the rabbit Achilles tendon and medial collateral ligament femoral entheses.

Microscopic visualization under load of the region connecting ligaments/tendons to bone, the enthesis, has been performed previously; however, specific investigation of individual fibril deformation may add insight to such studies. Detailed visualization of fibril deformation would inform on the mechanical strategies employed by this tissue in connecting two mechanically disparate materials. Clinically, an improved understanding of enthesis mechanics may help guide future restorative efforts for torn or injured ligaments/tendons, where the enthesis is often a point of weakness. In this study, a custom ligament/tendon enthesis loading device was designed and built, a unique method of sample preparation was devised, and second harmonic and two-photon fluorescence microscopy were used to capture the fibril-level load response of the rabbit Achilles tendon and medial collateral ligament femoral entheses. A focus was given to investigation of the mechanical problem of fibril embedment. Resultant images indicate a rapid (occurring over approximately 60 µm) change in fibril orientation at the interface of ligament/tendon and calcified fibrocartilage early in the loading regime, before becoming relatively constant. Such a change in fibril angle helps confirm the materially graded region demonstrated by others, while, in this case, providing additional insight into fibril bending. We speculate that the scale of the mechanical problem (i.e., fibril diameters being on the order of 250 nm) allows fibrils to bend over the small (relative to the imaging field of view, but large relative to fibril diameter) distances observed; thus, potentially lessening required embedment lengths. Nevertheless, this behavior merits further investigation to be confirmed. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2506-2515, 2018.

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.