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.

Impaired Annulus Fibrosus Development and Vertebral Fusion Cause Severe Scoliosis in Mice with Deficiency of c-Jun NH2-Terminal Kinases 1 and 2.

Mitogen-activated protein kinases, including c-Jun NH2-terminal kinase (JNK), play an important role in the development and function of a large variety of tissues. The skeletal phenotype of JNK1 and JNK2 double-knockout (dKO) mice (JNK1fl/flCol2-Cre/JNK2-/-) and control genotypes were analyzed at different embryonic and postnatal stages. JNK1/2 dKO mice displayed a severe scoliotic phenotype beginning during development that was grossly apparent around weaning age. Alcian blue staining at embryonic day 17.5 showed abnormal fusion of the posterior spinal elements. In adult mice, fusion of vertebral bodies and of spinous and transverse processes was noted by micro-computed tomography, Alcian blue/Alizarin red staining, and histology. The long bones developed normally, and histologic sections of growth plate and articular cartilage revealed no significant abnormalities. Histologic sections of the vertebral column at embryonic days 15.5 and 17.5 revealed an abnormal organization of the annulus fibrosus in the dKOs, with chondrocyte-like cells and fusion of dorsal processes. Spinal sections in 10-week-old dKO mice showed replacement of intervertebral disk structures (annulus fibrosus and nucleus pulposus) by cartilage and bone tissues, with cells staining for markers of hypertrophic chondrocytes, including collagen X and runt-related transcription factor 2. These findings demonstrate a requirement for both JNK1 and JNK2 in the normal development of the axial skeleton. Loss of JNK signaling results in abnormal endochondral bone formation and subsequent severe scoliosis.

Tiludronate and clodronate do not affect bone structure or remodeling kinetics over a 60 day randomized trial.

BACKGROUND: Tiludronate and clodronate are FDA-approved bisphosphonate drug therapies for navicular disease in horses. Although clinical studies have determined their ability to reduce lameness associated with skeletal disorders in horses, data regarding the effect on bone structure and remodeling is lacking. Additionally, due to off-label use of these drugs in young performance horses, effects on bone in young horses need to be investigated. Therefore, the purpose of this randomized, experimental pilot study was to determine the effect of tiludronate and clodronate on normal bone cells, structure and remodeling after 60 days in clinically normal, young horses. Additionally, the effect of clodronate on bone healing 60 days after an induced defect was investigated. RESULTS: All horses tolerated surgery well, with no post-surgery lameness and all acquired biopsies being adequate for analyses. Overall, tiludronate and clodronate did not significantly alter any bone structure or remodeling parameters, as evaluated by microCT and dynamic histomorphometry. Tiludronate did not extensively impact bone formation or resorption parameters as evaluated by static histomorphometry. Similarly, clodronate did not affect bone formation or resorption after 60 days. Sixty days post-defect, healing was minimally affected by clodronate. CONCLUSIONS: Tiludronate and clodronate do not appear to significantly impact bone tissue on a structural or cellular level using standard dose and administration schedules.

An Anatomy Pre-Course Predicts Student Performance in a Professional Veterinary Anatomy Curriculum.

Little to no correlation has been identified between previous related undergraduate coursework or outcomes on standardized tests and performance in a veterinary curriculum, including anatomy coursework. Therefore, a relatively simplistic method to predict student performance before entrance would be advantageous to many. The purpose of this study was to evaluate whether there is a correlation between performance in a veterinary anatomy pre-course and subsequent performance within a professional anatomy curriculum. Incoming first-year veterinary students at the Louisiana State University School of Veterinary Medicine were asked to participate in a free weeklong pre-course, before the start of the semester. The pre-course covered the musculoskeletal anatomy of the canine thoracic limb using dissection-based methods. Student performance, as evaluated by test grades in the pre-course, did indeed correlate with test grades in professional veterinary anatomy courses. A significant and positive correlation was identified between pre-course final exam performance and performance on examinations in each of 3 professional anatomy courses. Qualitative analyses of student comments pertaining to their experience within the pre-course indicated differences in the perceived benefits of the pre-course between high-, middle-, and low-performing students. These varied perceptions may provide predictive feedback as well as guidance for supporting lower performing students. Together, these results indicate that performance in a weeklong pre-course covering only a small portion of canine anatomy is a strong predictor of performance within a professional anatomy curriculum. In addition, the pre-course differentially affected student perceptions of their learning experience.

Loss of Oncostatin M Signaling in Adipocytes Induces Insulin Resistance and Adipose Tissue Inflammation in Vivo.

Oncostatin M (OSM) is a multifunctional gp130 cytokine. Although OSM is produced in adipose tissue, it is not produced by adipocytes. OSM expression is significantly induced in adipose tissue from obese mice and humans. The OSM-specific receptor, OSM receptor ß (OSMR), is expressed in adipocytes, but its function remains largely unknown. To better understand the effects of OSM in adipose tissue, we knocked down Osmr expression in adipocytes in vitro using siRNA. In vivo, we generated a mouse line lacking Osmr in adiponectin-expressing cells (OSMR(FKO) mice). The effects of OSM on gene expression were also assessed in vitro and in vivo OSM exerts proinflammatory effects on cultured adipocytes that are partially rescued by Osmr knockdown. Osm expression is significantly increased in adipose tissue T cells of high fat-fed mice. In addition, adipocyte Osmr expression is increased following high fat feeding. OSMR(FKO) mice exhibit increased insulin resistance and adipose tissue inflammation and have increased lean mass, femoral length, and bone volume. Also, OSMR(FKO) mice exhibit increased expression of Osm, the T cell markers Cd4 and Cd8, and the macrophage markers F4/80 and Cd11c Interestingly, the same proinflammatory genes induced by OSM in adipocytes are induced in the adipose tissue of the OSMR(FKO) mouse, suggesting that increased expression of proinflammatory genes in adipose tissue arises both from adipocytes and other cell types. These findings suggest that adipocyte OSMR signaling is involved in the regulation of adipose tissue homeostasis and that, in obesity, OSMR ablation may exacerbate insulin resistance by promoting adipose tissue inflammation.

Assessment of tuber coxae bone biopsy in the standing horse.

OBJECTIVE: To describe a biopsy technique in standing horses with minimal morbidity that consistently provides a substantial bone biopsy with intact, undamaged architecture. STUDY DESIGN: Experimental, prospective study. ANIMALS: Ten Thoroughbred horses. METHODS: Biopsies were obtained from the tuber coxae of 10 sedated, standing horses using an oscillating saw. Bilateral biopsies, separated by 60 days, were evaluated with micro-computed tomography (microCT). The first biopsy was prepared for decalcified histology; the second for undecalcified histology. Both biopsies were evaluated qualitatively for histologic quality. RESULTS: The biopsy technique did not result in any significant complications, was well tolerated and all biopsies were of good histologic quality. CONCLUSION: Cortical and trabecular bone biopsies can be successfully collected from the tuber coxa using a simple technique that creates minimal morbidity and allows sequential samples to be collected. The biopsies were larger than those described previously, provided adequate bone for multiple histologic sections, and had intact, undamaged architecture on examination with microCT and light microscopy.

Obesity inhibits the osteogenic differentiation of human adipose-derived stem cells.

BACKGROUND: Craniomaxillofacial defects secondary to trauma, tumor resection, or congenital malformations are frequent unmet challenges, due to suboptimal alloplastic options and limited autologous tissues such as bone. Significant advances have been made in the application of adipose-derived stem/stromal cells (ASCs) in the pre-clinical and clinical settings as a cell source for tissue engineering approaches. To fully realize the translational potential of ASCs, the identification of optimal donors for ASCs will ensure the successful implementation of these cells for tissue engineering approaches. In the current study, the impact of obesity on the osteogenic differentiation of ASCs was investigated. METHODS: ASCs isolated from lean donors (body mass index <25; lnASCs) and obese donors (body mass index >30; obASCs) were induced with osteogenic differentiation medium as monolayers in an estrogen-depleted culture system and on three-dimensional scaffolds. Critical size calvarial defects were generated in male nude mice and treated with scaffolds implanted with lnASCs or obASCs. RESULTS: lnASCs demonstrated enhanced osteogenic differentiation in monolayer culture system, on three-dimensional scaffolds, and for the treatment of calvarial defects, whereas obASCs were unable to induce similar levels of osteogenic differentiation in vitro and in vivo. Gene expression analysis of lnASCs and obASCs during osteogenic differentiation demonstrated higher levels of osteogenic genes in lnASCs compared to obASCs. CONCLUSION: Collectively, these results indicate that obesity reduces the osteogenic differentiation capacity of ASCs such that they may have a limited suitability as a cell source for tissue engineering.

Pre- and post-examination reflections of first-year medical students in an integrated medical anatomy course.

Due to the rigor and pace of undergraduate medical anatomy courses, it is not uncommon for students to struggle and fail initially. However, repetition of coursework places an additional burden on the student, instructor, and institution. The purpose of this study was to compare the exam preparation strategies of repeating and non-repeating students to identify areas where struggling students can be supported prior to course failure. As part of their integrated anatomy course, first-year medical students at Indiana University completed a metacognitive Practice-Based Learning and Improvement (PBLI) assignment prior to and after their first exam. In the PBLIs, students were asked to reflect on their exam preparation strategies, confidence, and satisfaction, as well as their predicted and actual exam performance. PBLI responses from non-repeating and repeating students were then analyzed quantitatively and qualitatively. A total of 1802 medical students were included in this study, including 1751 non-repeating and 51 repeating students. Based on their PBLI responses, non-repeating students were appropriately confident, somewhat satisfied, and more accurate when predicting their exam performance. Repeating students were overconfident, dissatisfied, and inaccurate when predicting their first exam performance on their initial, unsuccessful attempt but were more successful on their second, repeat attempt. Qualitative analysis revealed that repeating students aimed to improve their studying by modifying their existing study strategies and managing their time more effectively. In conjunction with other known risk factors, these insights into repeater and non-repeater exam preparation practices can help anatomy educators better identify and support potential struggling students.

Integrated Case-Based Learning Session for Breast and Upper Limb Anatomy.

Introduction: Medical students are frequently introduced to medical school curricula through anatomy coursework, which often includes histology and embryology content. As medical education has increasingly emphasized integration of content areas, use of activities such as case-based learning (CBL) sessions has grown. Little published work has demonstrated the effectiveness of CBL sessions in integrating anatomy, embryology, and histology on first-year medical students' ability to improve content mastery and adapt their study techniques. Methods: We developed a CBL session that included anatomy, embryology, and histology content covering the upper extremity and breast pathology that was taught to incoming first-year medical students (N = 51) during a prematriculation program in the summers of 2022 and 2023. The session involved completion of an individual pre- and postsession quiz; group completion of clinical cases involving image interpretation, matching exercises, and construction of diagrams, flowcharts, or tables; and a postsession survey with Likert-style and free-response questions about preparation and session effectiveness. Results: Postsession quiz scores significantly improved (p < .001). On the postsession survey (response rate: 59%), students commented that they enjoyed the real-life application and integration of the cases and that the sessions improved their understanding of the connections between content areas. Other comments demonstrated that students were evaluating and adapting their study approach in preparation for the sessions, often using techniques introduced and practiced in the sessions. Discussion: CBL sessions can provide opportunities to incoming first-year medical students to practice, adapt, and evaluate study techniques while delivering integrated content.

Clinically relevant doses of tiludronate do not affect bone remodelling in pasture-exercised horses.

BACKGROUND: Bisphosphonates are widely used in equine athletes to reduce lameness associated with skeletal disorders. Widespread off-label use has led to concern regarding potential negative effects on bone healing, but little evidence exists to support or refute this. OBJECTIVES: To investigate the influence of clinically relevant doses of tiludronate on bone remodelling and bone healing. STUDY DESIGN: Randomised, controlled in vivo experiments. METHODS: Each horse had a single tuber coxae biopsied (Day 0), then were divided into a treatment (IV tiludronate) or control (IV saline) group. Treatments were administered 30 and 90 days following initial biopsy. Biopsy of the tuber coxae was repeated on Day 60 to evaluate bone healing following a single treatment. Oxytetracycline was administered on Days 137 and 147 to label bone formation. The contralateral tuber coxae was biopsied on Day 150 to evaluate effects of repeated treatment. Bone biopsies were evaluated with micro-computed tomography and/or dynamic histomorphometry using standard techniques. RESULTS: Nineteen horses completed the study, with no complications following the biopsies and treatments. No significant differences in the trabecular bone parameters or bone formation rate were observed between treatment groups. MAIN LIMITATIONS: The use of a first-generation bisphosphonate may mean some effects of these drugs are underrepresented using this model. The results pertain to the tuber coxae and may not reflect injury or the healing response that occurs in long bones in training or racing. CONCLUSIONS: In this model, tiludronate did not affect normal bone remodelling in the horse, despite repeat dosages.

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.

Determining baseline anatomy knowledge among professional allied health students.

Health professional students often struggle with anatomy coursework despite undergraduate coursework in anatomy. Educators must identify early on whether students may struggle in order to target remediation. The purpose of the study was to elucidate whether an assessment tool administered before the start of a professional anatomy course correlated with allied health students' course performance. Students over four years were given a quiz covering anatomy knowledge they were expected to know upon matriculation to their professional program. A supplemental data form was administered at the course's conclusion to identify prior anatomy experience and topics in which students felt deficient. Pre-quiz scores significantly correlated with examination performance throughout the course. Students reported feeling most deficient in neurobiology (54.9%) and anatomy terminology (39.1%). Videos were created to target these deficient knowledge areas; students who watched the videos did better in course assessments than those who did not. Most respondents (98.0%) recommended students take an undergraduate anatomy course prior to starting a health professional program. These results indicate that a quiz assessing anatomy knowledge among matriculating students may identify students with the potential to struggle in a professional anatomy course early on. Responses outlined areas in which students felt deficient, which allows educators to target topics early with intervention tools such as the review videos in this study. Finally, most respondents strongly recommended undergraduate coursework in anatomy prior to starting a professional health program, which outlines students' recognition that a solid foundation in anatomical knowledge is important to success in professional programs.

Promoting metacognition in an allied health anatomy course.

Metacognition, the ability to self-regulate one's learning and performance, has been shown to improve student outcomes. Anatomy is recognized as one of the toughest courses in allied health curricula, and students could benefit from metacognitive activities. The purpose of this study was to explore the changes in metacognition of allied health students in an anatomy course and identify which groups need support with this skill. First-year physician assistant (MPAS), physical therapy (DPT), and occupational therapy (OTD) students (n = 129) were invited to participate. At the beginning and end of the course, students completed a questionnaire including the metacognitive awareness inventory (MAI) that assesses metacognition. Students were also asked to reflect on their examination performances using a modified Likert scale and participated in reflective discussion boards to encourage development of metacognitive skills, which were thematically analyzed. Paired metacognition scores had increased significantly by the end of the course. However, middle-performers anticipated high grades and were less satisfied with their grade, indicating a disconnect in their metacognition compared to high- and low-performers. Students' receptiveness to modifying study strategies to improve performance declined throughout the course; by mid-way through, they relied more on existing strategies. Increasing time constraints were frequently cited as a major factor when considering study strategies and modification of such strategies. To maximize the effectiveness of metacognitive activities, they should be positioned early in the course when students are most receptive. In addition, middle performers may benefit from additional support to improve metacognition.

Adult stem cell mobilization enhances intramembranous bone regeneration: a pilot study.

BACKGROUND: Stem cell mobilization, which is defined as the forced egress of stem cells from the bone marrow to the peripheral blood (PB) using chemokine receptor agonists, is an emerging concept for enhancing tissue regeneration. However, the effect of stem cell mobilization by a single injection of the C-X-C chemokine receptor type 4 (CXCR4) antagonist AMD3100 on intramembranous bone regeneration is unclear. QUESTIONS/PURPOSES: We therefore asked: Does AMD3100 mobilize adult stem cells in C57BL/6 mice? Are stem cells mobilized to the PB after marrow ablation? And does AMD3100 enhance bone regeneration? METHODS: Female C57BL/6 mice underwent femoral marrow ablation surgery alone (n = 25), systemic injection of AMD3100 alone (n = 15), or surgery plus AMD3100 (n = 57). We used colony-forming unit assays, flow cytometry, and micro-CT to investigate mobilization of mesenchymal stem cells, endothelial progenitor cells, and hematopoietic stem cells to the PB and bone regeneration. RESULTS: AMD3100 induced mobilization of stem cells to the PB, resulting in a 40-fold increase in mesenchymal stem cells. The marrow ablation injury mobilized all three cell types to the PB over time. Administration of AMD3100 led to a 60% increase in bone regeneration at Day 21. CONCLUSIONS: A single injection of a CXCR4 antagonist lead to stem cell mobilization and enhanced bone volume in the mouse marrow ablation model of intramembranous bone regeneration.

The glass ceiling thickens: the impact of COVID-19 on academic medicine faculty in the United States.

The inequities faced by women in academic Medicine before the COVID-19 pandemic are well established. However, there is little formal data regarding exactly how the pandemic has affected faculty. This cross-sectional study investigated the impact of the pandemic on responsibilities at home, work, and mental health according to gender identification, faculty rank, and faculty appointment. In February 2021, an online questionnaire was broadly distributed to academic medicine faculty. Respondents were asked to provide demographic data, answer questions about their responsibilities at home and work, mental health, and how the pandemic has influenced these. Respondents were also asked to document what their institution(s) can do to help faculty through the pandemic. Responses were analyzed via Pearson's chi-square tests and thematic analysis. Women faculty were more likely to be responsible for the care of others (70%, p = 0.014), and the impact was negative, especially for early career faculty (p = 0.019). Productivity in research, teaching, and clinical practice were negatively impacted, with women feeling this in clinical practice (p = 0.005), increased teaching load (p = 0.042), and inadequate work environment (p = 0.013). In the areas of self-care and mental health, women (p < 0.001), early career-faculty (p < 0.001), and clinical faculty (p = 0.029) were more negatively impacted. Early-career women were more likely to fear retribution. Five themes emerged, including Flexible Expectations, Support, Mental Health, Compensation, and Communication. Pre-pandemic stress and burnout were rampant, and this study demonstrates that academic medicine faculty are still suffering. It is the authors' hope that administrations can utilize these data to make informed decisions regarding policies enacted to assist populations who are most vulnerable to the effects of the pandemic.

Breaking barriers: The landscape of human and veterinary medical anatomy education and the potential for collaboration.

Despite human (HUM) and veterinary (VET) medical institutions sharing the goal of educating future clinicians, there is little collaboration between them regarding curricular and pedagogical practices during the preclinical/basic science training years. This may be, at least in part, due to a lack of understanding of each type of curriculum. This study presents data about curricula, student populations, pedagogical methodologies applied, and anatomy educators' training at both HUM and VET institutions. Preclinical curricula, admissions criteria, and student demographics were analyzed for 21 institutions in the United States having both HUM and VET schools. This dataset was augmented by a questionnaire sent to anatomists internationally, detailing anatomy curricula, pedagogies applied, and anatomy educators' training. Many curricular similarities between both training programs were identified, including anatomy education experiences. However, VET programs were found to include more preclinical coursework than HUM programs. Students who matriculate to VET or HUM schools have similar academic records, including prerequisite coursework and grade point average. Median HUM class size was significantly larger, and the percentage of women enrolled in VET institutions was significantly higher. Training of anatomy educators was identical with one exception: VET educators are far more likely to hold a clinical degree. This study elucidates the substantial similarities between VET and HUM programs, particularly in anatomy education, underscoring the potential for collaboration between both types of programs in areas such as interprofessional education, bioethics, zoonotic disease management, and postgraduate training.

Virtual learning impacts communication and teamwork.

BACKGROUND: Essential interpersonal skills, such as teamwork and communication, are increasingly emphasised in the curricula of various health professions' programmes. A push towards virtual learning has gained traction following COVID-19 online learning; however, the implications of this modality on the aforementioned skills remain unclear. APPROACH: Medical, physician assistant, physical therapy and occupational therapy students engaged in a four-part educational intervention aimed at promoting interprofessional teamwork, communication and role knowledge during an anatomy course taught in-person in 2019 and virtually in 2020. Students' perceptions of the intervention were explored through focus groups following each of these educational interventions using inductive coding. EVALUATION: A comparison of focus group and survey data collected in both years demonstrated less effective communication and teamwork. With respect to communication and teamwork in a virtual learning setting, the following subthemes were identified: Challenges ascribed to the virtual learning format, Feeling of missing out on in-person experiences, Less engagement and accountability, Lack of bonding and teamwork, Feeling uncomfortable, as well as Added difficulty regarding Conversing, Ensuring inclusivity, Hesitation and Inadvertently domineering discussions. Communication challenges stemming from the virtual learning format were identified as the primary hindrance. IMPLICATIONS: Therefore, virtual learning may be less effective than in-person with respect to cultivating communication and teamwork skills. In our context, and based on the evaluation, we have decided to assess the utility of virtual interactive sessions in the context of other activities in the curriculum to ensure in-person opportunities are available for students to cultivate the necessary communication and teamwork skills.

NOMENs land: The place of eponyms in the anatomy classroom.

The law of Non-Original Malappropriate Eponymous Nomenclature (NOMEN) states that no phenomenon is named after its discoverer. However, eponymous terms are rife in the anatomical and medical literature. In this viewpoint commentary, the authors discuss the history of anatomical eponyms, explain the additional cognitive load imposed by eponyms that can negatively impact student learning and explore the view that eponyms are "pale, male and stale" in the socially conscious 21st century. The authors probe two of the most common arguments used to keep eponyms in anatomy education: (1) clinicians use them because they are easy, and (2) eponyms remind us of anatomy's history. Educators, clinicians and students need to work together to progress this movement and bring a modern lens to this discussion. Based on the arguments presented in this commentary, the authors propose that eponyms should be removed from anatomy curricula, textbooks and have no place in the anatomy classroom.

Role of the Hypoxia-Inducible Factor Pathway in Normal and Osteoarthritic Meniscus and in Mice after Destabilization of the Medial Meniscus.

OBJECTIVE: Meniscus injury and the hypoxia-inducible factor (HIF) pathway are independently linked to osteoarthritis pathogenesis, but the role of the meniscus HIF pathway remains unclear. We sought to identify and evaluate HIF pathway response in normal and osteoarthritic meniscus and to examine the effects of Epas1 (HIF-2α) insufficiency in mice on early osteoarthritis development. METHODS: Normal and osteoarthritic human meniscus specimens were obtained and used for immunohistochemical evaluation and cell culture studies for the HIF pathway. Meniscus cells were treated with pro-inflammatory stimuli, including interleukins (IL)-1ß, IL-6, transforming growth factor (TGF)-α, and fibronectin fragments (FnF). Target genes were also evaluated with HIF-1α and HIF-2α (Epas1) overexpression and knockdown. Wild-type (n = 36) and Epas1+/- (n = 30) heterozygous mice underwent destabilization of the medial meniscus (DMM) surgery and were evaluated at 2 and 4 weeks postoperatively for osteoarthritis development using histology. RESULTS: HIF-1α and HIF-2α immunostaining and gene expression did not differ between normal and osteoarthritic meniscus. While pro-inflammatory stimulation significantly increased both catabolic and anabolic gene expression in the meniscus, HIF-1α and Epas1 expression levels were not significantly altered. Epas1 overexpression significantly increased Col2a1 expression. Both wild-type and Epas1+/- mice developed osteoarthritis following DMM surgery. There were no significant differences between genotypes at either time point. CONCLUSION: The HIF pathway is likely not responsible for osteoarthritic changes in the human meniscus. Additionally, Epas1 insufficiency does not protect against osteoarthritis development in the mouse at early time points after DMM surgery. The HIF pathway may be more important for protection against catabolic stress.

FGF21, not GCN2, influences bone morphology due to dietary protein restrictions.

BACKGROUND: Dietary protein restriction is emerging as an alternative approach to treat obesity and glucose intolerance because it markedly increases plasma fibroblast growth factor 21 (FGF21) concentrations. Similarly, dietary restriction of methionine is known to mimic metabolic effects of energy and protein restriction with FGF21 as a required mechanism. However, dietary protein has been shown to be required for normal bone growth, though there is conflicting evidence as to the influence of dietary protein restriction on bone remodeling. The purpose of the current study was to evaluate the effect of dietary protein and methionine restriction on bone in lean and obese mice, and clarify whether FGF21 and general control nonderepressible 2 (GCN2) kinase, that are part of a novel endocrine pathway implicated in the detection of protein restriction, influence the effect of dietary protein restriction on bone. METHODS: Adult wild-type (WT) or Fgf21 KO mice were fed a normal protein (18 kcal%; CON) or low protein (4 kcal%; LP) diet for 2 or 27 weeks. In addition, adult WT or Gcn2 KO mice were fed a CON or LP diet for 27 weeks. Young New Zealand obese (NZO) mice were placed on high-fat diets that provided protein at control (16 kcal%; CON), low levels (4 kcal%) in a high-carbohydrate (LP/HC) or high-fat (LP/HF) regimen, or on high-fat diets (protein, 16 kcal%) that provided methionine at control (0.86%; CON-MR) or low levels (0.17%; MR) for up to 9 weeks. Long bones from the hind limbs of these mice were collected and evaluated with micro-computed tomography (µCT) for changes in trabecular and cortical architecture and mass. RESULTS: In WT mice the 27-week LP diet significantly reduced cortical bone, and this effect was enhanced by deletion of Fgf21 but not Gcn2. This decrease in bone did not appear after 2 weeks on the LP diet. In addition, Fgf21 KO mice had significantly less bone than their WT counterparts. In obese NZO mice dietary protein and methionine restriction altered bone architecture. The changes were mediated by FGF21 due to methionine restriction in the presence of cystine, which did not increase plasma FGF21 levels and did not affect bone architecture. CONCLUSIONS: This study provides direct evidence of a reduction in bone following long-term dietary protein restriction in a mouse model, effects that appear to be mediated by FGF21.