Deep Learning-Assisted Identification of Femoroacetabular Impingement (FAI) on Routine Pelvic Radiographs.

To use a novel deep learning system to localize the hip joints and detect findings of cam-type femoroacetabular impingement (FAI). A retrospective search of hip/pelvis radiographs obtained in patients to evaluate for FAI yielded 3050 total studies. Each hip was classified separately by the original interpreting radiologist in the following manner: 724 hips had severe cam-type FAI morphology, 962 moderate cam-type FAI morphology, 846 mild cam-type FAI morphology, and 518 hips were normal. The anteroposterior (AP) view from each study was anonymized and extracted. After localization of the hip joints by a novel convolutional neural network (CNN) based on the focal loss principle, a second CNN classified the images of the hip as cam positive, or no FAI. Accuracy was 74% for diagnosing normal vs. abnormal cam-type FAI morphology, with aggregate sensitivity and specificity of 0.821 and 0.669, respectively, at the chosen operating point. The aggregate AUC was 0.736. A deep learning system can be applied to detect FAI-related changes on single view pelvic radiographs. Deep learning is useful for quickly identifying and categorizing pathology on imaging, which may aid the interpreting radiologist.

RNA-Binding Protein LIN28a Regulates New Myocyte Formation in the Heart Through Long Noncoding RNA-H19.

BACKGROUND: Developmental cardiac tissue holds remarkable capacity to regenerate after injury and consists of regenerative mononuclear diploid cardiomyocytes. On maturation, mononuclear diploid cardiomyocytes become binucleated or polyploid and exit the cell cycle. Cardiomyocyte metabolism undergoes a profound shift that coincides with cessation of regeneration in the postnatal heart. However, whether reprogramming metabolism promotes persistence of regenerative mononuclear diploid cardiomyocytes enhancing cardiac function and repair after injury is unknown. Here, we identify a novel role for RNA-binding protein LIN28a, a master regulator of cellular metabolism in cardiac repair after injury. METHODS: LIN28a overexpression was tested using mouse transgenesis on postnatal cardiomyocyte numbers, cell cycle, and response to apical resection injury. With the use of neonatal and adult cell culture systems and adult and Mosaic Analysis with Double Markers myocardial injury models in mice, the effect of LIN28a overexpression on cardiomyocyte cell cycle and metabolism was tested. Last, isolated adult cardiomyocytes from LIN28a and wild-type mice 4 days after myocardial injury were used for RNA-immunoprecipitation sequencing. RESULTS: LIN28a was found to be active primarily during cardiac development and rapidly decreases after birth. LIN28a reintroduction at postnatal day (P) 1, P3, P5, and P7 decreased maturation-associated polyploidization, nucleation, and cell size, enhancing cardiomyocyte cell cycle activity in LIN28a transgenic pups compared with wild-type littermates. Moreover, LIN28a overexpression extended cardiomyocyte cell cycle activity beyond P7 concurrent with increased cardiac function 30 days after apical resection. In the adult heart, LIN28a overexpression attenuated cardiomyocyte apoptosis, enhanced cell cycle activity, cardiac function, and survival in mice 12 weeks after myocardial infarction compared with wild-type littermate controls. Instead, LIN28a small molecule inhibitor attenuated the proreparative effects of LIN28a on the heart. Neonatal rat ventricular myocytes overexpressing LIN28a mechanistically showed increased glycolysis, ATP production, and levels of metabolic enzymes compared with control. LIN28a immunoprecipitation followed by RNA-immunoprecipitation sequencing in cardiomyocytes isolated from LIN28a-overexpressing hearts after injury identified long noncoding RNA-H19 as its most significantly altered target. Ablation of long noncoding RNA-H19 blunted LIN28a-induced enhancement on cardiomyocyte metabolism and cell cycle activity. CONCLUSIONS: Collectively, LIN28a reprograms cardiomyocyte metabolism and promotes persistence of mononuclear diploid cardiomyocytes in the injured heart, enhancing proreparative processes, thereby linking cardiomyocyte metabolism to regulation of ploidy/nucleation and repair in the heart.

High fat diet causes inferior vertebral structure and function without disc degeneration in RAGE-KO mice.

Back pain and spinal pathologies are associated with obesity in juveniles and adults, yet studies identifying causal relationships are lacking and none investigate sex differences. This study determined if high fat (HF) diet causes structural and functional changes to vertebrae and intervertebral discs (IVDs); if these changes are modulated in mice with systematic ablation for the receptor for advanced glycation endproducts (RAGE-KO); and if these changes are sex-dependent. Wild-type (WT) and RAGE-KO mice were fed a low fat (LF) or HF diet for 12 weeks starting at 6 weeks, representing the juvenile population. HF diet led to weight/fat gain, glucose intolerance, and increased cytokine levels (IL-5, MIG, and RANTES); with less fat gain in RAGE-KO females. Most importantly, HF diet reduced vertebral trabecular bone volume fraction and compressive and shear moduli, without a modifying effect of RAGE-KO, but with a more pronounced effect in females. HF diet caused reduced cortical area fraction only in WT males. Neither HF diet nor RAGE-KO affected IVD degeneration grade. Biomechanical properties of coccygeal motion segments were affected by RAGE-KO but not diet, with some interactions identified. In conclusion, HF diet resulted in inferior vertebral structure and function with some sex differences, no IVD degeneration, and few modifying effects of RAGE-KO. These structural and functional deficiencies with HF diet provide further evidence that diet can affect spinal structures and may increase the risk for spinal injury and degeneration with aging and additional stressors. Back pain and spinal pathologies are associated with obesity in juveniles and adults, yet studies identifying causal relationships are lacking and none investigate sex differences.

Cortical Bone Derived Stem Cells Modulate Cardiac Fibroblast Response via miR-18a in the Heart After Injury.

The adult heart following injury such as a myocardial infarction forms a fibrotic scar associated with transformation of resident cardiac fibroblasts into myofibroblast, accelerating cardiac remodeling and dysfunction. Cell therapies provide a novel direction for the enhancement of cardiac structure and function but remain poorly described in terms of the effect on resident cardiac fibroblasts. We have shown cortical bone derived stem cells (CBSCs) exhibit an ability to repair the heart after myocardial injury together with reduced scar formation. Nevertheless, whether CBSCs possess ability to modulate resident fibroblast response after myocardial injury remains untested. OBJECTIVE: To determine the effect of secreted factors from CSBCs to attenuate myofibroblast formation in the heart after injury. METHODS AND RESULTS: CBSCs were injected in mice after myocardial infarction which demonstrated reduced fibrosis as determined by Masson's trichrome and Picro-Sirius red staining. In parallel, decreased expression of myofibroblast markers such as Acta2 was observed compared to PBS injected mice. To determine the effect of CBSCs on cardiac fibrosis, adult mouse cardiac fibroblasts were isolated from C57BL/6 mice, primed with CBSC pre-conditioned media for 12 h, and treated with 10ng TGF-ß for 48 h to mimic cardiac injury. Decreased expression of Acta2, periostin and CTGF was observed in adult cardiac fibroblasts cultured in CBSC medium compared to control cells. Additionally, analysis of myofibroblast markers such as vimentin and pSMAD/SMAD was also decreased compared to control cells. To determine the mechanism, we looked for enriched miRNA in CBSCs that can mediate anti fibrotic response after injury. Results showed significantly increased expression of miR-18a in CBSCs. The upregulation of miR-18a was also validated in adult fibroblasts treated with CBSCs compared to control cells. Adult fibroblasts treated with mimic for miR-18a followed by TGF-ß showed significant decrease in myofibroblast formation while miR-18a inhibitor completely inhibited the effect of CBSC medium. CONCLUSION: CBSCs reduce fibroblast to myofibroblast transition and differentiation in adult cardiac fibroblasts via miR-18a-5p. This finding reveals a new avenue for cell therapies to target myocardial scar modulation and provides a resolution for the cardiac repair response after injury in the adult myocardium.

Leptin signaling and the intervertebral disc: Sex dependent effects of leptin receptor deficiency and Western diet on the spine in a type 2 diabetes mouse model.

Type 2 diabetes and obesity are associated with back pain in juveniles and adults and are implicated in intervertebral disc (IVD) degeneration. Hypercaloric Western diets are associated with both obesity and type 2 diabetes. The objective of this study was to determine if obesity and type 2 diabetes result in spinal pathology in a sex-specific manner using in vivo diabetic and dietary mouse models. Leptin is an appetite-regulating hormone, and its deficiency leads to polyphagia, resulting in obesity and diabetes. Leptin is also associated with IVD degeneration, and increased expression of its receptor was identified in degenerated IVDs. We used young, leptin receptor deficient (Db/Db) mice to mimic the effect of diet and diabetes on adolescents. Db/Db and Control mice were fed either Western or Control diets, and were sacrificed at 3 months of age. Db/Db mice were obese, while only female mice developed diabetes. Female Db/Db mice displayed altered IVD morphology, with increased intradiscal notochordal band area, suggesting delayed IVD cell proliferation and differentiation, rather than IVD degeneration. Motion segments from Db/Db mice exhibited increased failure risk with decreased torsional failure strength. Db/Db mice also had inferior bone quality, which was most prominent in females. We conclude that obesity and diabetes due to impaired leptin signaling contribute to pathological changes in vertebrae, as well as an immature IVD phenotype, particularly of females, suggesting a sex-dependent role of leptin in the spine.

Advanced glycation end products cause RAGE-dependent annulus fibrosus collagen disruption and loss identified using in situ second harmonic generation imaging in mice intervertebral disk in vivo and in organ culture models.

Aging and diabetes are associated with increased low-back pain and intervertebral disk (IVD) degeneration yet causal mechanisms remain uncertain. Advanced glycation end products (AGEs), which accumulate in IVDs from aging and are implicated in diabetes-related disorders, alter collagen and induce proinflammatory conditions. A need exists for methods that assess IVD collagen quality and degradation in order to better characterize specific structural changes in IVDs due to AGE accumulation and to identify roles for the receptor for AGEs (RAGE). We used multiphoton microscopy with second harmonic generation (SHG), collagen-hybridizing peptide (CHP), and image analysis methods to characterize effects of AGEs and RAGE on collagen quality and quantity in IVD annulus fibrosus (AF). First, we used SHG imaging on thin sections with an in vivo dietary mouse model and determined that high-AGE (H-AGE) diets increased AF fibril disruption and collagen degradation resulting in decreased total collagen content, suggesting an early degenerative cascade. Next, we used in situ SHG imaging with an ex vivo IVD organ culture model of AGE challenge on wild type and RAGE-knockout (RAGE-KO) mice and determined that early degenerative changes to collagen quality and degradation were RAGE dependent. We conclude that AGE accumulation leads to RAGE-dependent collagen disruption in the AF and can initiate molecular and tissue level collagen disruption. Furthermore, SHG and CHP analyzes were sensitive to collagenous alterations at multiple hierarchical levels due to AGE and may be useful in identifying additional contributors to collagen damage in IVD degeneration processes.

Spatial mapping of collagen content and structure in human intervertebral disk degeneration.

Collagen plays a key structural role in both the annulus fibrosus (AF) and nucleus pulposus (NP) of intervertebral disks (IVDs). Changes in collagen content with degeneration suggest a shift from collagen type II to type I within the NP, and the activation of pro-inflammatory factors is indicative of fibrosis throughout. While IVD degeneration is considered a fibrotic process, an increase in collagen content with degeneration, reflective of fibrosis, has not been demonstrated. Additionally, changes in collagen content and structure in human IVDs with degeneration have not been characterized with high spatial resolution. The collagen content of 23 human lumbar L2/3 or L3/4 IVDs was quantified using second harmonic generation imaging (SHG) and multiple image processing algorithms, and these parameters were correlated with the Rutges histological degeneration grade. In the NP, SHG intensity increased with degeneration grade, suggesting fibrotic collagen deposition. In the AF, the entropy of SHG intensity was reduced with degeneration indicating increased collagen uniformity and suggesting less-organized lamellar structure. Collagen orientation entropy decreased throughout most IVD regions with increasing degeneration grade, further supporting a loss in collagen structural complexity. Overall, SHG imaging enabled visualization and quantification of IVD collagen content and organization with degeneration. There was an observed shift from an initially complex structure to more uniform structure with loss of microstructural elements and increased NP collagen polarity, suggesting fibrotic remodeling.

Phlpp1 is associated with human intervertebral disc degeneration and its deficiency promotes healing after needle puncture injury in mice.

Back pain is a leading cause of global disability and is strongly associated with intervertebral disc (IVD) degeneration (IDD). Hallmarks of IDD include progressive cell loss and matrix degradation. The Akt signaling pathway regulates cellularity and matrix production in IVDs and its inactivation is known to contribute to a catabolic shift and increased cell loss via apoptosis. The PH domain leucine-rich repeat protein phosphatase (Phlpp1) directly regulates Akt signaling and therefore may play a role in regulating IDD, yet this has not been investigated. The aim of this study was to investigate if Phlpp1 has a role in Akt dysregulation during IDD. In human IVDs, Phlpp1 expression was positively correlated with IDD and the apoptosis marker cleaved Caspase-3, suggesting a key role of Phlpp1 in the progression of IDD. In mice, 3 days after IVD needle puncture injury, Phlpp1 knockout (KO) promoted Akt phosphorylation and cell proliferation, with less apoptosis. At 2 and 8 months after injury, Phlpp1 deficiency also had protective effects on IVD cellularity, matrix production, and collagen structure as measured with histological and immunohistochemical analyses. Specifically, Phlpp1-deletion resulted in enhanced nucleus pulposus matrix production and more chondrocytic cells at 2 months, and increased IVD height, nucleus pulposus cellularity, and extracellular matrix deposition 8 months after injury. In conclusion, Phlpp1 has a role in limiting cell survival and matrix degradation in IDD and research targeting its suppression could identify a potential therapeutic target for IDD.

Sex Differences in Rat Intervertebral Disc Structure and Function Following Annular Puncture Injury.

STUDY DESIGN: A rat puncture injury intervertebral disc (IVD) degeneration model with structural, biomechanical, and histological analyses. OBJECTIVE: To determine if males and females have distinct responses in the IVD after injury. SUMMARY OF BACKGROUND DATA: Low back pain (LBP) and spinal impairments are more common in women than men. However, sex differences in IVD response to injury have been underexplored, particularly in animal models where sex differences can be measured without gender confounds. METHODS: Forty-eight male and female Sprague Dawley rats underwent sham, single annular puncture with tumor necrosis factor α (TNFα) injection (1×), or triple annular puncture with TNFα injection (3×) surgery. Six weeks after surgery, lumbar IVDs were assessed by radiologic IVD height, spinal motion segment biomechanical testing, histological degeneration grading, second harmonic generation (SHG) imaging, and immunofluorescence for fibronectin and α-smooth muscle actin. RESULTS: Annular puncture injuries significantly increased degenerative grade and IVD height loss for males and females, but females had increased degeneration grade particularly in the annulus fibrosus (AF). Despite IVD height loss, biomechanical properties were largely unaffected by injury at 6 weeks. However, biomechanical measures sensitive to outer AF differed by sex after 3× injury-male IVDs had greater torsional stiffness, torque range, and viscoelastic creep responses. SHG intensity of outer AF was reduced after injury only in female IVDs, suggesting sex differences in collagen remodeling. Both males and females exhibited decreased cellularity and increased fibronectin expression at injury sites. CONCLUSION: IVD injury results in distinct degeneration and functional healing responses between males and females. The subtle sex differences identified in this animal model suggest differences in response to IVD injury that might explain some of the variance observed in human LBP, and demonstrate the need to better understand differences in male and female IVD degeneration patterns and pain pathogenesis. LEVEL OF EVIDENCE: N/A.

Dietary advanced glycation end-product consumption leads to mechanical stiffening of murine intervertebral discs.

Back pain is a leading cause of disability and is strongly associated with intervertebral disc (IVD) degeneration. Reducing structural disruption and catabolism in IVD degeneration remains an important clinical challenge. Pro-oxidant and structure-modifying advanced glycation end-products (AGEs) contribute to obesity and diabetes, which are associated with increased back pain, and accumulate in tissues due to hyperglycemia or ingestion of foods processed at high heat. Collagen-rich IVDs are particularly susceptible to AGE accumulation due to their slow metabolic rates, yet it is unclear whether dietary AGEs can cross the endplates to accumulate in IVDs. A dietary mouse model was used to test the hypothesis that chronic consumption of high AGE diets results in sex-specific IVD structural disruption and functional changes. High AGE diet resulted in AGE accumulation in IVDs and increased IVD compressive stiffness, torque range and failure torque, particularly for females. These biomechanical changes were likely caused by significantly increased AGE crosslinking in the annulus fibrosus, measured by multiphoton imaging. Increased collagen damage measured with collagen hybridizing peptide did not appear to influence biomechanical properties and may be a risk factor as these animals age. The greater influence of high AGE diet on females is an important area of future investigation that may involve AGE receptors known to interact with estrogen. We conclude that high AGE diets can be a source for IVD crosslinking and collagen damage known to be important in IVD degeneration. Dietary modifications and interventions that reduce AGEs warrant further investigation and may be particularly important for diabetics, in whom AGEs accumulate more rapidly.