Acute mechanical damage and the resulting joint contact abnormalities are central to the initiation and progression of post-traumatic osteoarthritis (PTOA). Study of PTOA is typically performed in vivo with replicate animals using artificially induced injury features. The goal of this work was to measure changes in a joint contact stress in the knee of a large quadruped after creation of a clinically realistic overload injury and a focal cartilage defect. Whole-joint overload was achieved by excising a 5-mm wedge of the anterior medial meniscus. Focal cartilage defects were created using a custom pneumatic impact gun specifically developed and mechanically characterized for this work. To evaluate the effect of these injuries on joint contact mechanics, Tekscan (Tekscan, Inc., South Boston, MA) measurements were obtained pre-operatively, postmeniscectomy, and postimpact (1.2-J) in a nonrandomized group of axially loaded cadaveric sheep knees. Postmeniscectomy, peak contact stress in the medial compartment is increased by 71% (p = 0.03) and contact area is decreased by 35% (p = 0.001); the center of pressure (CoP) shifted toward the cruciate ligaments in both the medial (p = 0.004) and lateral (p = 0.03) compartments. The creation of a cartilage defect did not significantly change any aspect of contact mechanics measured in the meniscectomized knee. This work characterizes the mechanical environment present in a quadrupedal animal knee joint after two methods to reproducibly induce joint injury features that lead to PTOA.
and enables coordinated endosomal escape and release of siRNA into cytoplasm to rapidly engage the RISC complex and simultaneously suppress both canonical (p65) and noncanonical (p100) NF-kB activities. To determine the articular residence time of NP, three 8-week old male C57BL/6J mice were injected i.a. with Cy5.5-labeled NP (0.1 mg) and particle fluorescence in the knee joint was followed non-invasively for 48 h by whole body IVIS imaging. To examine NF-kB activity after injury, right knees of 12 mice (3 per group) underwent compressive loading (6N for 60 cycles). Left knees served as controls. Mice were sacrificed at 12h, 24h, 48h and 14 days after loading and knee joints examined for phospho (P)-p65 and (P)-p100 expression. To study the effects of peptide-siRNA NP, 12 additional mice (4 per group) were treated i.a. with 15 uL of peptide-p65/p100 siRNA NP (0.1 ug total, group A), peptide-scrambled siRNA NP (0.1 ug, group B) or PBS control (group C) immediately after mechanical loading and on day 2 after loading. All mice were sacrificed at day 5 post-injury and processed for histology. TUNEL assay was used to detect cell apoptosis and immunohistochemistry was used to detect P-p65, P-p100. In addition, defective autophagy has been shown to contribute to chondrocyte death and cartilage degeneration in OA. We monitored autophagic activity with microtubule-associated protein light chain 3 (LC3). Results: We found that after a single i.a. dose of peptide-siRNA NP, IVIS signal persisted for at least 48h in the knee joint, indicating the persistence of the nanoparticles. In the injured knees loss of Safranin-O staining, an indication of cartilage degeneration, was not detected at the site of injury until day 5 post-injury. However, chondrocyte apoptosis at injury site occurred within the first 12h. P-p65 and P-p100 activation (upregulation) was observed only in the injured knees in and around the impact site at early time points (12e48h) and persists outside the cartilage impact site and in the synovium for at least two weeks. No such upregulation was found in the non-injured knees. Compared with the peptide-scrambled siRNA NP and PBS control groups, i.a. administration of peptide-NF-kB siRNA NP significantly lowered P-p65 and P-p100 fluorescent density on day 5 by~50% and 30%, respectively, and suppressed chondrocyte apoptosis, as evidenced by~50% reduction in the number of TUNEL-positive chondrocytes. Regarding autophagic activity peptide-NF-kB siRNA NP treatment protected against injuryinduced LC3 downregulation while there was no significant difference between the two control groups. Conclusions: These results provide proof-of-concept that early intervention with peptide-NF-kB siRNA NP can protect against chondrocyte apoptosis and preserve chondrocyte integrity by suppressing the inflammatory reaction induced by impact injury and modulating chondrocyte autophagic activity. This protective effect may translate to long-term suppression of PTOA.
Osteoarthritis is a chronic, deleterious disease of the joints. It currently affects nearly 25 million Americans. Clinically, osteoarthritis presents as joint pain and verified by radiographic evidence of joint space narrowing. Unfortunately, symptomatic osteoarthritis describes the later stages of disease, at which point irreversible cartilage and bone damage has occurred. Crosssectional imaging modalities offer the promise of visualizing early features of disease, enabling the development and evaluation of interventions to forestall or prevent degenerative change. Modalities of clinical interest include magnetic resonance imaging (MRI) and multi-detector computed tomography (MDCT). The following work describes the efficacy of MRI-derived measures for the identification and accurate quantification of local and whole joint changes in articular cartilage thickness changes in vivo. This was performed as part of a study investigating the diagnostic potential of clinical morphometric and compositional MRI to identify early features of osteoarthritis in a large animal model of traumatic knee joint injury. Surgically induced trauma consisted of a partial medial meniscectomy and blunt impact of either 0 J, 0.6 J, or 1.2 J to the weight-bearing cartilage of the medial femur. The study was six months in duration. To evaluate the accuracy of MRI-derived measures of cartilage thickness, imaging acquired at time of euthanasia was compared to high-resolution contrast-enhanced micro-computed tomography (micro CT). 3dimensional multimodal analysis demonstrated that morphometric MRI imaging is sensitive to sub-voxel changes in cartilage thickness. Therefore, MRI is a clinically relevant modality to quantify subtle cartilage damage, thereby presenting an opportunity to identify patients earlier in the disease process. vi
ms. 5mm width of subchondral trabecular bone of the medial joint was extracted (figure), and divided into 4 subregions: anterior, center, posterior, and medial. Trabecular bone microstructure at each region was measured by the dedicated software (TRI/3D-BON, Ratoc System Engineering Co., Ltd., Tokyo). Results: BV/TV (bone volume fraction) at the anterior, center, posterior, and medial regions were 29.5, 29.6, 15.5, 34.2 % respectively. Tb.Th (trabecular thickness) were 285, 294, 245, 338 mm, Tb.N (trabecular number) were 0.64, 0.58, 0.65, 0.61 /mm, and Conn.D (connectivity density) were 2.33, 2.38, 1.15, 2.48. Conclusions: Subchondral bones were increased particularly at the medial region of the medial proximal tibia, composed of thickened and well-connected trabecular bones. Subchondral bone microstructure measured by HR-pQCT may become a new imaging marker for OA knees.
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