Changes in tissue sodium concentration and sodium relaxation times during the maturation of human knee cartilage: Ex vivo 23 Na MRI study at 10.5 T.

PURPOSE: To evaluate the influence of skeletal maturation on sodium (23 Na) MRI relaxation parameters and the accuracy of tissue sodium concentration (TSC) quantification in human knee cartilage. METHODS: Twelve pediatric knee specimens were imaged with whole-body 10.5 T MRI using a density-adapted 3D radial projection sequence to evaluate 23 Na parameters: B1 + , T1 , biexponential T 2 * $$ {\mathrm{T}}_2^{\ast } $$ , and TSC. Water, collagen, and sulfated glycosaminoglycan (sGAG) content were calculated from osteochondral biopsies. The TSC was corrected for B1 + , relaxation, and water content. The literature-based TSC (TSCLB ) used previously published values for corrections, whereas the specimen-specific TSC (TSCSP ) used measurements from individual specimens. 23 Na parameters were evaluated in eight cartilage compartments segmented on proton images. Associations between 23 Na parameters, TSCLB - TSCSP difference, biochemical content, and age were determined. RESULTS: From birth to 12 years, cartilage water content decreased by 18%; collagen increased by 59%; and sGAG decreased by 36% (all R2 ≥ 0.557). The short T 2 * $$ {\mathrm{T}}_2^{\ast } $$ ( T 2 * S $$ {{\mathrm{T}}_2^{\ast}}_{\mathrm{S}} $$ ) decreased by 72%, and the signal fraction relaxing with T 2 * S $$ {{\mathrm{T}}_2^{\ast}}_{\mathrm{S}} $$ ( fT 2 * S $$ {{\mathrm{fT}}_2^{\ast}}_{\mathrm{S}} $$ ) increased by 55% during the first 5 years but remained relatively stable after that. TSCSP was significantly correlated with sGAG content from biopsies (R2 = 0.739). Depending on age, TSCLB showed higher or lower values than TSCSP . The TSCLB - TSCSP difference was significantly correlated with T 2 * S $$ {{\mathrm{T}}_2^{\ast}}_{\mathrm{S}} $$ (R2 = 0.850), fT 2 * S $$ {{\mathrm{fT}}_2^{\ast}}_{\mathrm{S}} $$ (R2 = 0.651), and water content (R2 = 0.738). CONCLUSION: TSC and relaxation parameters measured with 23 Na MRI provide noninvasive information about changes in sGAG content and collagen matrix during cartilage maturation. Cartilage TSC quantification assuming fixed relaxation may be feasible in children older than 5 years.

Nuclear protein-1 is the common link for pathways activated by aging and obesity in chondrocytes: A potential therapeutic target for osteoarthritis.

Pathways leading to osteoarthritis (OA) are diverse depending on the risk factors involved; thus, developing OA therapeutics has been challenging. Here we report that nuclear protein-1 (Nupr1), a stress-inducible protein/transcription factor, is activated by pathways associated with obesity and aging in chondrocytes. Treatment of human chondrocytes with free fatty acids (palmitate and oleate; a model for high-fat diet/obesity) induced PERK signaling and increased expression of caspase-3, TRB3, and Nupr1. On the other hand, treatment of chondrocytes with menadione (oxidative stress inducer) induced oxidation of IRE1, activated antioxidant response (higher Nrf2 expression), and increased expression of Nupr1 and matrix metalloproteinases. Experimental OA was induced by destabilization of the medial meniscus (DMM) in the knee joints of Nupr1+/+ and Nupr1-/- mice. Loss of Nupr1 expression reduced the severity of cartilage lesions in this model. Together, our findings suggest that Nupr1 is a common factor activated by signaling pathways activated by obesity (ER stress) and age (oxidative stress) and a potential drug target for OA resulting from various risk factors.

High-fat diet induces endoplasmic reticulum stress to promote chondrocyte apoptosis in mouse knee joints.

Mice fed a high-fat diet (HFD) become obese and develop osteoarthritis (OA)-like lesions, including chondrocyte apoptosis, in the knee joints. However, the mechanism by which HFD/obesity induces chondrocyte apoptosis is not clearly understood. In the present study, male mice were fed a low-fat diet (LFD, 10% kcal), HFD (45% kcal), or a HFD administered with 0.5 g/kg bodyweight of 4-phenyl butyric acid (PBA, a small chaperone known to ease endoplasmic reticulum [ER] stress), via the drinking water. At the end of the 18-week study, stifle (knee) joints from all animals were collected, fixed, paraffin embedded, and sectioned. Immunostaining of joints from the HFD group showed increased expression of ER stress and apoptotic markers and increased expression of nuclear protein 1 and tribbles related protein-3 compared to the LFD group. Mice on HFD also showed higher percentage of chondrocyte death, lower chondrocyte numbers per cartilage area, and thickening of subchondral bone. Administration of PBA alleviated all of the HFD-induced symptoms. Our study demonstrated that HFD induces ER stress to promote chondrocyte death and subchondral bone thickening, which could be relieved by alleviating ER stress via PBA administration, suggesting that ER stress could play an important role in obesity-linked OA and could be targeted for OA therapeutics.

Naturally occurring osteoarthritis in male mice with an extended lifespan.

Aim: The purpose of this study was to evaluate whether pharmacologic treatments or genotypes shown to prolong murine lifespan ameliorate the severity of age-associated osteoarthritis.Materials and Methods: Male UM-HET3 mice were fed diets containing 17-α-estradiol, acarbose, nordihydroguaiaretic acid, or control diet per the National Institute on Aging Interventions Testing Program (ITP) protocol. Findings were compared to genetically long-lived male Ames dwarf mice. Stifles were analyzed histologically with articular cartilage structure (ACS) and safranin O scoring as well as with quantitative histomorphometry.Results: Depending on the experimental group, ITP mice were between 450 and 1150 days old at the time of necropsy and 12-15 animals were studied per group. Two age groups (450 and 750 days) with 16-20 animals per group were used for Ames dwarf studies. No differences were found in the ACS or safranin O scores between treatment and control groups in the ITP study. There was high variability in most of the histologic outcome measures. For example, the older UM-HET3 controls had ACS scores of 6.1 ± 5.8 (mean±SD) and Saf O scores of 6.8 ± 5.6. Nevertheless, 17-α-estradiol mice had larger areas and widths of subchondral bone compared to controls, and dwarf mice had less subchondral bone area and width and less articular cartilage necrosis than non-dwarf controls.Conclusions: UM-HET3 mice developed age-related OA but with a high degree of variability and without a significant effect of the tested ITP treatments. High variability was also seen in the Ames dwarf mice but differences in several measures suggested some protection from OA.

Quantitative susceptibility mapping detects neovascularization of the epiphyseal cartilage after ischemic injury in a piglet model of legg-calvé-perthes disease.

BACKGROUND: Legg-Calvé-Perthes disease (LCPD) is a childhood hip disorder thought to be caused by disruption of blood supply to the developing femoral head. There is potential for imaging to help assess revascularization of the femoral head. PURPOSE: To investigate whether quantitative susceptibility mapping (QSM) can detect neovascularization in the epiphyseal cartilage following ischemic injury to the developing femoral head. STUDY TYPE: Prospective. ANIMAL MODEL: Right femoral head ischemia was surgically induced in 6-week-old male piglets. The animals were sacrificed 48 hours (n = 3) or 4 weeks (n = 7) following surgery, and the operated and contralateral control femoral heads were harvested for ex vivo MRI. FIELD STRENGTH/SEQUENCE: Preclinical 9.4T MRI to acquire susceptibility-weighted 3D gradient echo (GRE) images with 0.1 mm isotropic spatial resolution. ASSESSMENT: The 3D GRE images were used to manually segment the cartilage overlying the femoral head and were subsequently postprocessed using QSM. Vessel volume, cartilage volume, and vessel density were measured and compared between operated and control femoral heads at each timepoint. Maximum intensity projections of the QSM images were subjectively assessed to identity differences in cartilage canal appearance, location, and density. STATISTICAL TESTS: Paired t-tests with Bonferroni correction were used (P < 0.008 considered significant). RESULTS: Increased vascularity of the epiphyseal cartilage following ischemic injury was clearly identified using QSM. No changes were detected 48 hours after surgery. Vessel volume, cartilage volume, and vessel density were all increased in the operated vs. control femoral heads 4 weeks after surgery (P = 0.001, 0.002, and 0.001, respectively). Qualitatively, the increase in vessel density at 4 weeks was due to the formation of new vessels that were organized in a brush-like orientation in the epiphyseal cartilage, consistent with the histological appearance of neovascularization. DATA CONCLUSION: QSM can detect neovascularization in the epiphyseal cartilage following ischemic injury to the femoral head. LEVEL OF EVIDENCE: 1 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2019;50:106-113.

Quantitative MRI Helps to Detect Hip Ischemia: Preclinical Model of Legg-Calvé-Perthes Disease.

Purpose To determine whether quantitative MRI relaxation time mapping techniques can help to detect ischemic injury to the developing femoral head. Materials and Methods For this prospective animal study conducted from November 2015 to February 2018, 10 male 6-week-old piglets underwent an operation to induce complete right femoral head ischemia. Animals were humanely killed at 48 hours (n = 2) or 4 weeks (n = 8) after the operation, and the operated and contralateral-control femoral heads were harvested and frozen. Thawed specimens were imaged at 9.4-T MRI by using T1, T2, T1 in the rotating frame (T1ρ), adiabatic T1ρ, relaxation along a fictitious field (RAFF), and T2* mapping and evaluated with histologic analysis. Paired relaxation time differences between the operated and control femoral heads were measured in the secondary ossification center (SOC), epiphyseal cartilage, articular cartilage, and metaphysis and were analyzed by using a paired t test. Results In the SOC, T1ρ and RAFF had the greatest percent increases in the operated versus control femoral heads at both 48 hours (112% and 72%, respectively) and 4 weeks (74% and 70%, respectively). In the epiphyseal and articular cartilage, T2, T1ρ, and RAFF were similarly increased at both points (range, 24%-49%). At 4 weeks, T2, T1ρ, adiabatic T1ρ, and RAFF were increased in the SOC (P = .004, .018, < .001, and .001, respectively), epiphyseal cartilage (P = .009, .008, .011, and .007, respectively), and articular cartilage (P = .005, .016, .033, and .018, respectively). Histologic assessment identified necrosis in SOC and deep layer of the epiphyseal cartilage at both points. Conclusion T2, T1 in the rotating frame, adiabatic T1 in the rotating frame, and relaxation along a fictitious field maps are sensitive in helping to detect ischemic injury to the developing femoral head. © RSNA, 2018 Online supplemental material is available for this article.

Effects of Monoclonal Antibodies against Nerve Growth Factor on Healthy Bone and Joint Tissues in Mice, Rats, and Monkeys: Histopathologic, Biomarker, and Microcomputed Tomographic Assessments.

Tanezumab, an anti-nerve growth factor (NGF) antibody, is in development for management of chronic pain. During clinical trials of anti-NGF antibodies, some patients reported unexpected adverse events requiring total joint replacements, resulting in a partial clinical hold on all NGF inhibitors. Three nonclinical toxicology studies were conducted to evaluate the effects of tanezumab or the murine precursor muMab911 on selected bone and joint endpoints and biomarkers in cynomolgus monkeys, Sprague-Dawley rats, and C57BL/6 mice. Joint and bone endpoints included histology, immunohistochemistry, microcomputed tomography (mCT) imaging, and serum biomarkers of bone physiology. Responses of bone endpoints to tanezumab were evaluated in monkeys at 4 to 30 mg/kg/week for 26 weeks and in rats at 0.2 to 10 mg/kg twice weekly for 28 days. The effects of muMab911 at 10 mg/kg/week for 12 weeks on selected bone endpoints were determined in mice. Tanezumab and muMab911 had no adverse effects on any bone or joint parameter. There were no test article-related effects on bone or joint histology, immunohistochemistry, or structure. Reversible, higher osteocalcin concentrations occurred only in the rat study. No deleterious effects were observed in joints or bones in monkeys, rats, or mice administered high doses of tanezumab or muMab911.

Oxidative Stress Promotes Peroxiredoxin Hyperoxidation and Attenuates Pro-survival Signaling in Aging Chondrocytes.

Oxidative stress-mediated post-translational modifications of redox-sensitive proteins are postulated as a key mechanism underlying age-related cellular dysfunction and disease progression. Peroxiredoxins (PRX) are critical intracellular antioxidants that also regulate redox signaling events. Age-related osteoarthritis is a common form of arthritis that has been associated with mitochondrial dysfunction and oxidative stress. The objective of this study was to determine the effect of aging and oxidative stress on chondrocyte intracellular signaling, with a specific focus on oxidation of cytosolic PRX2 and mitochondrial PRX3. Menadione was used as a model to induce cellular oxidative stress. Compared with chondrocytes isolated from young adult humans, chondrocytes from older adults exhibited higher levels of PRX1-3 hyperoxidation basally and under conditions of oxidative stress. Peroxiredoxin hyperoxidation was associated with inhibition of pro-survival Akt signaling and stimulation of pro-death p38 signaling. These changes were prevented in cultured human chondrocytes by adenoviral expression of catalase targeted to the mitochondria (MCAT) and in cartilage explants from MCAT transgenic mice. Peroxiredoxin hyperoxidation was observedin situin human cartilage sections from older adults and in osteoarthritic cartilage. MCAT transgenic mice exhibited less age-related osteoarthritis. These findings demonstrate that age-related oxidative stress can disrupt normal physiological signaling and contribute to osteoarthritis and suggest peroxiredoxin hyperoxidation as a potential mechanism.

Quantitative susceptibility mapping detects abnormalities in cartilage canals in a goat model of preclinical osteochondritis dissecans.

PURPOSE: To use quantitative susceptibility mapping (QSM) to investigate changes in cartilage canals in the distal femur of juvenile goats after their surgical transection. METHODS: Chondronecrosis was surgically induced in the right medial femoral condyles of four 4-day-old goats. Both the operated and control knees were harvested at 2, 3, 5, and 10 weeks after the surgeries. Ex vivo MRI scans were conducted at 9.4 Tesla using TRAFF (relaxation time along a fictitious field)-weighted fast spin echo imaging and QSM to detect areas of chondronecrosis and investigate cartilage canal abnormalities. Histological sections from these same areas stained with hematoxylin and eosin and safranin O were evaluated to assess the affected tissues. RESULTS: Both the histological sections and the TRAFF -weighted images of the femoral condyles demonstrated focal areas of chondronecrosis, evidenced by pyknotic chondrocyte nuclei, loss of matrix staining, and altered MR image contrast. At increasing time points after surgery, progressive changes and eventual disappearance of abnormal cartilage canals were observed in areas of chondronecrosis by using QSM. CONCLUSION: Abnormal cartilage canals were directly visualized in areas of surgically induced chondronecrosis. Quantitative susceptibility mapping enabled investigation of the vascular changes accompanying chondronecrosis in juvenile goats. Magn Reson Med 77:1276-1283, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Quantification and biodistribution of iron oxide nanoparticles in the primary clearance organs of mice using T1 contrast for heating.

PURPOSE: To use contrast based on longitudinal relaxation times (T1 ) or rates (R1 ) to quantify the biodistribution of iron oxide nanoparticles (IONPs), which are of interest for hyperthermia therapy, cell targeting, and drug delivery, within primary clearance organs. METHODS: Mesoporous silica-coated IONPs (msIONPs) were intravenously injected into 15 naïve mice. Imaging and mapping of the longitudinal relaxation rate constant at 24 h or 1 week postinjection were performed with an echoless pulse sequence (SWIFT). Alternating magnetic field heating measurements were also performed on ex vivo tissues. RESULTS: Signal enhancement from positive T1 contrast caused by IONPs was observed and quantified in vivo in liver, spleen, and kidney at concentrations up to 3.2 mg Fe/(g tissue wt.) (61 mM Fe). In most cases, each organ had a linear correlation between the R1 and the tissue iron concentration despite variations in intra-organ distribution, degradation, and IONP surface charge. Linear correlation between R1 and volumetric SAR in hyperthermia therapy was observed. CONCLUSION: The linear dependence between R1 and tissue iron concentration in major organs allows quantitative monitoring of IONP biodistribution in a dosage range relevant to magnetic hyperthermia applications, which falls into the concentration gap between CT and conventional MRI techniques. Magn Reson Med 78:702-712, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Wnt10b and Dkk-1 gene therapy differentially influenced trabecular bone architecture, soft tissue integrity, and osteophytosis in a skeletally mature rat model of osteoarthritis.

AIMS: Our goals in the current experiments were to determine if (a) upregulation of Wnt signaling would induce osteoarthritis changes in stable stifle joints and (b) if downregulation of Wnt signaling in destabilized joints would influence the progression of OA. METHODS: At 37 weeks of age, rats were injected in the stifle joint with a recombinant adeno-associated viral vector containing the Wnt-inhibitor Dkk-1 or a Wnt10b transgene. At 40 weeks of age, rats underwent surgical destabilization of the joint. At 50 weeks of age, stifle joints were submitted for micro-computed tomography and histopathological analysis. RESULTS: Injection of either Wnt10b or Dkk-1 transgenes in stable joints improved bone architectural parameters, but worsened soft tissue integrity. Osteophytosis was decreased by Dkk-1, but unchanged by Wnt10b. Destabilization negatively influenced bone architecture, increased osteophytosis, and decreased soft tissue integrity. Dkk-1 exacerbated the negative effects of destabilization, whereas Wnt10b had little effect on these parameters. Osteophytosis was improved, whereas soft tissue integrity was worsened by both transgenes in destabilized joints. CONCLUSIONS: The Wnt-inhibitor Dkk-1 does not appear to completely inhibit the effects of Wnt signaling on bone remodeling. In vivo upregulation of Wnt10b and its inhibitor, Dkk-1, can produce both parallel or contrasting phenotypic responses depending on the specific parameter measured and the fidelity of the examined joint. These observations elucidate different roles for Wnt signaling in stable versus destabilized joints and may help to explain the conflicting results previously reported for the role of Dkk-1 in joint disease.

Dystrophic Mineralization of Costal Cartilage in Hartley Guinea Pigs.

Hartley guinea pigs are widely used animal models of disease, particularly in studies of osteoarthritis. The purpose of this study was to investigate lesions in the costal cartilage from 16 male, 5- to 6-month-old Hartley guinea pigs. Routine histological sections from the costal cartilage and costochondral junction (longitudinal and cross sections) and sternum (for evaluation of bone marrow) were examined. All 16 (100%) animals had histological lesions involving the costal cartilage that included matrix degeneration and mineralization, reduced cellularity, and evidence of chondrocyte necrosis. Of the 16, 4 (25%) of the lesions contained blood vessels and 3 (19%) contained central osseous metaplasia. The cartilage lesions were accompanied by degeneration (sometimes with regeneration and/or fibrosis) in adjacent skeletal muscle in 15 of the 16 (94%) animals. The lesions in the costal cartilage were interpreted as dystrophic mineralization of unknown cause and appear to be incidental findings, although they bear some resemblance to lesions occurring in Tietze's disease in humans. The significance of the lesions in skeletal muscle is unclear. Histological lesions of cartilage matrix degeneration and mineralization in these sites have not, to our knowledge, been reported previously.

Proliferative Cartilaginous Lesions in the Calcaneal Tendons of huNOG Mice.

NOD/Shi-scid/IL-2Rγnull (NOG) mice are humanized with CD34+ hematopoietic cells (huNOG mice) and are commonly utilized for biological or medical research on human therapeutics. In the present study, nine 26-week-old, female huNOG mice were utilized for testing proprietary immune-modulating drugs over a 3-week period at the University of Washington. Two of the 9 mice developed unilateral swelling of a tibiotarsal joint with associated paresis of the affected limb. Full necropsies were performed after euthanasia at experimental end point, and routine tissues and affected tibiotarsal joints were evaluated. An expansile, multilobular mass composed primarily of chondroid cells associated with the calcaneal tendon was present within 1 tibiotarsal joint of both mice. A small focus of well-differentiated bone was present within the mass of 1 mouse. In addition, the calcaneal periosteum was expanded by a chondroid mass in 1 mouse. The cartilaginous masses associated with the calcaneal tendon were interpreted as a hyperplastic or low-grade neoplastic process accompanied by endochondral ossification, and the mass associated with the calcaneal periosteum was interpreted as chondroid metaplasia. Although the etiology of these lesions is unclear, their prevalence in 2/9 (22%) huNOG mice is interesting and may have biological significance for future studies involving the huNOG mouse model.

The Effect of Surgical Technique and Spacer Texture on Bone Regeneration: A Caprine Study Using the Masquelet Technique.

BACKGROUND: The Masquelet-induced-membrane technique is a commonly used method for treating segmental bone defects. However, there are no established clinical standards for management of the induced membrane before grafting. QUESTIONS/PURPOSES: Two clinically based theories were tested in a chronic caprine tibial defect model: (1) a textured spacer that increases the induced-membrane surface area will increase bone regeneration; and (2) surgical scraping to remove a thin tissue layer of the inner induced-membrane surface will enhance bone formation. METHODS: Thirty-two skeletally mature female goats were assigned to four groups: smooth spacer with or without membrane scraping and textured spacer with or without membrane scraping. During an initial surgical procedure (unilateral, left tibia), a defect was created excising bone (5 cm), periosteum (9 cm), and muscle (10 g). Segments initially were stabilized with an intramedullary rod and an antibiotic-impregnated polymethylmethacrylate spacer with a smooth or textured surface. Four weeks later, the spacer was removed and the induced-membrane was either scraped or left intact before bone grafting. Bone formation was assessed using micro-CT (total bone volume in 2.5-cm central defect region) as the primary outcome; radiographs and histologic analysis as secondary outcomes, with the reviewer blinded to the treatment groups of the samples being assessed 12 weeks after grafting. All statistical tests were performed using a linear mixed effects model approach. RESULTS: Micro-CT analysis showed greater bone formation in defects with scraped induced membrane (mean, 3034.5 mm3; median, 1928.0 mm3; quartile [Q]1-Q3, 273.3-2921.1 mm3) compared with defects with intact induced membrane (mean, 1709.5 mm3; median, 473.8 mm3; Q1-Q3, 132.2-1272.3 mm3; p = 0.034). There was no difference in bone formation between textured spacers (mean, 2405.5 mm3; median, 772.7 mm3; Q1-Q3, 195.9-2743.8 mm3) and smooth spacers (mean, 2473.2 mm3; median, 1143.6 mm3; Q1-Q3, 230.2-451.1 mm3; p = 0.917). CONCLUSIONS: Scraping the induced-membrane surface to remove the innermost layer of the induced-membrane increased bone regeneration. A textured spacer that increased the induced-membrane surface area had no effect on bone regeneration. CLINICAL RELEVANCE: Scraping the induced membrane during the second stage of the Masquelet technique may be a rapid and simple means of improving healing of segmental bone defects, which needs to be confirmed clinically.

Susceptibility weighted imaging of cartilage canals in porcine epiphyseal growth cartilage ex vivo and in vivo.

PURPOSE: High-resolution visualization of cartilage canals has been restricted to histological methods and contrast-enhanced imaging. In this study, the feasibility of non-contrast-enhanced susceptibility weighted imaging (SWI) for visualization of the cartilage canals was investigated ex vivo at 9.4 T, further explored at 7 and 3 T and demonstrated in vivo at 7 T, using a porcine animal model. METHODS: SWI scans of specimens of distal femur and humerus from 1 to 8 week-old piglets were conducted at 9.4 T using 3D-GRE sequence and SWI post-processing. The stifle joints of a 2-week old piglet were scanned ex vivo at 7 and 3 T. Finally, the same sites of a 3-week-old piglet were scanned, in vivo, at 7 T under general anesthesia using the vendor-provided sequences. RESULTS: High-contrast visualization of the cartilage canals was obtained ex vivo, especially at higher field strengths; the results were confirmed histologically. In vivo feasibility was demonstrated at 7 T and comparison of ex vivo scans at 3 and 7 T indicated feasibility of using SWI at 3 T. CONCLUSIONS: High-resolution 3D visualization of cartilage canals was demonstrated using SWI. This demonstration of fully noninvasive visualization opens new avenues to explore skeletal maturation and the role of vascular supply for diseases such as osteochondrosis.

Microarray analysis reveals age-related differences in gene expression during the development of osteoarthritis in mice.

OBJECTIVE: To better understand the contribution of age to the development of osteoarthritis (OA). METHODS: Surgical destabilization of the medial meniscus (DMM) was used to model OA in 12-week-old and 12-month-old male C57BL/6 mice. OA severity was evaluated histologically. RNA used for microarray and real-time polymerase chain reaction analysis was isolated from joint tissue collected from the medial side of the joint, including cartilage, meniscus, subchondral bone, and the joint capsule with synovium. Computational analysis was used to identify patterns of gene expression, and immunohistochemistry was used to evaluate tissue distribution of selected proteins. RESULTS: OA was more severe in older mice than in young mice. Only 55 genes showed a similar expression with DMM-induced OA in the 2 age groups, while 493 genes showed differential expression, the majority having increased expression in older mice. Functional categories for similarly expressed genes included extracellular matrix- and cell adhesion-related genes; differentially expressed genes included those related to muscle structure and development and immune response genes. Comparison of expression in sham-operated control joints revealed an age-related decrease in matrix gene expression and an increase in immune and defense response gene expression. Interleukin-33 was present in multiple joint tissue cells, while CCL21 was more localized to chondrocytes and meniscal cells. Periostin was found in the extracellular matrix of cartilage and meniscus. CONCLUSION: Age affects both the basal pattern of gene expression in joint tissues and the response to surgically induced OA. Examining tissue from the joint beyond only cartilage revealed novel genes and proteins that would be important to consider in OA.

Inflammation, apoptosis, and necrosis induced by neoadjuvant fas ligand gene therapy improves survival of dogs with spontaneous bone cancer.

Fas ligand (FasL) gene therapy for cancer has shown promise in rodents; however, its efficacy in higher mammals remains unknown. Here, we used intratumoral FasL gene therapy delivered in an adenovirus vector (Ad-FasL) as neoadjuvant to standard of care in 56 dogs with osteosarcoma. Tumors from treated dogs had greater inflammation, necrosis, apoptosis, and fibrosis at day 10 (amputation) compared to pretreatment biopsies or to tumors from dogs that did not receive Ad-FasL. Survival improvement was apparent in dogs with inflammation or lymphocyte-infiltration scores >1 (in a 3-point scale), as well as in dogs that had apoptosis scores in the top 50th percentile (determined by cleaved caspase-3). Survival was no different than that expected from standard of care alone in dogs with inflammation scores ≤1 or apoptosis scores in the bottom 50th percentile. Reduced Fas expression by tumor cells was associated with prognostically advantageous inflammation, and this was seen only in dogs that received Ad-FasL. Together, the data suggest that Ad-FasL gene therapy improves survival in a subset of large animals with naturally occurring tumors, and that at least in some tumor types like osteosarcoma, it is most effective when tumor cells fail to express Fas.

GEP constitutes a negative feedback loop with MyoD and acts as a novel mediator in controlling skeletal muscle differentiation.

Granulin-epithelin precursor (GEP) is an autocrine growth factor that has been implicated in embryonic development, tissue repair, tumorigenesis, and inflammation. Here we report that GEP was expressed in skeletal muscle tissue and its level was differentially altered in the course of C2C12 myoblast fusion. The GEP expression during myoblast fusion was a consequence of MyoD transcription factor binding to several E-box (CANNTG) sequences in the 5'-flanking regulatory region of GEP gene, followed by transcription. Recombinant GEP potently inhibited myotube formation from C2C12 myoblasts whereas the knockdown of endogenous of GEP via a siRNA approach accelerated the fusion of myoblasts to myotubes. Interestingly, the muscle fibers of GEP knockdown mice were larger in number but noticeably smaller in size when compared to the wild-type. Mechanistic studies revealed that during myoblast fusion, the addition of GEP led to remarkable reductions in the expressions of muscle-specific transcription factors, including MyoD. In addition, the regulation of myotube formation by GEP is mediated by the anti-myogenic factor JunB, which is upregulated following GEP stimulation. Thus, GEP growth factor, JunB, and MyoD transcription factor form a regulatory loop and act in concert in the course of myogenesis.

Naturally occurring osteochondrosis latens lesions identified by quantitative and morphological 10.5 T MRI in pigs.

Juvenile osteochondritis dissecans (JOCD) is a pediatric orthopedic disorder that involves the articular-epiphyseal cartilage complex and underlying bone. Clinical disease is often characterized by the presence of radiographically apparent osteochondral flaps and fragments. The existence of early JOCD lesions (osteochondrosis latens [OCL] and osteochondrosis manifesta [OCM]) that precede the development of osteochondral flaps and fragments is also well recognized. However, identification of naturally occurring OCL lesions (confined to cartilage) using noninvasive imaging techniques has not yet been accomplished. We hypothesized that 10.5 T magnetic resonance imaging (MRI) can identify naturally occurring OCL lesions at predilection sites in intact joints of juvenile pigs. Unilateral elbows and knees (stifles) were harvested from three pigs aged 4, 8, and 12 weeks, and scanned in a 10.5 T MRI to obtain morphological 3D DESS images, and quantitative T2 and T1ρ relaxation time maps. Areas with increased T2 and T1ρ relaxation times in the articular-epiphyseal cartilage complex were identified in 1/3 distal femora and 3/3 distal humeri and were considered suspicious for OCL or OCM lesions. Histological assessment confirmed the presence of OCL or OCM lesions at each of these sites and failed to identify additional lesions. Histological findings included necrotic vascular profiles associated with areas of chondronecrosis either confined to the epiphyseal cartilage (OCL, 4- and 8-week-old specimens) or resulting in a delay in endochondral ossification (OCM, 12-week-old specimen). Future studies with clinical MR systems (≤7 T) are needed to determine whether these MRI methods are suitable for the in vivo diagnosis of early JOCD lesions in humans.

Evaluation of lesion and overlying articular cartilage in patients with juvenile osteochondritis dissecans of the knee using quantitative diffusion MRI.

Current clinical MRI of patients with juvenile osteochondritis dissecans (JOCD) is limited by the low reproducibility of lesion instability evaluation and inability to predict which lesions will heal after nonoperative treatment and which will later require surgery. The aim of this study is to verify the ability of apparent diffusion coefficient (ADC) to detect differences in lesion microstructure between different JOCD stages, treatment groups, and healthy, unaffected contralateral knees. Pediatric patients with JOCD received quantitative diffusion MRI between January 2016 and September 2020 in this prospective research study. A disease stage (I-IV) and stability of each JOCD lesion was evaluated. ADCs were calculated in progeny lesion, interface, parent bone, cartilage overlying lesion, control bone, and control cartilage regions. ADC differences were evaluated using linear mixed models with Bonferroni correction. Evaluated were 30 patients (mean age, 13 years; 21 males), with 40 JOCD-affected and 12 healthy knees. Nine patients received surgical treatment after MRI. Negative Spearman rank correlations were found between ADCs and JOCD stage in the progeny lesion (ρ = -0.572; p < 0.001), interface (ρ = -0.324; p = 0.041), and parent bone (ρ = -0.610; p < 0.001), demonstrating the sensitivity of ADC to microstructural differences in lesions at different JOCD stages. We observed a significant increase in the interface ADCs (p = 0.007) between operative (mean [95% CI] = 1.79 [1.56-2.01] × 10-3 mm2 /s) and nonoperative group (1.27 [0.98-1.57] × 10-3 mm2 /s). Quantitative diffusion MRI detects microstructural differences in lesions at different stages of JOCD progression towards healing and reveals differences between patients assigned for operative versus nonoperative treatment.