Citations

Cells establish and sustain structural and functional integrity of the genome to support cellular identity and prevent malignant transformation. In this review, we present a strategic overview of epigenetic regulatory mechanisms including histone modifications and higher order chromatin organization (HCO) that are perturbed in breast cancer onset and progression. Implications for dysfunctions that occur in hormone regulation, cell cycle control, and mitotic bookmarking in breast cancer are considered, with an emphasis on epithelial‐to‐mesenchymal transition and cancer stem cell activities. The architectural organization of regulatory machinery is addressed within the contexts of translating cancer‐compromised genomic organization to advances in breast cancer risk assessment, diagnosis, prognosis, and identification of novel therapeutic targets with high specificity and minimal off target effects.

ObjectiveTo assess the biologic effects of lidocaine on the viability, proliferation, and function of human adipose tissue–derived mesenchymal stromal/stem cells (MSCs) in vitro.MethodsAdipose‐derived MSCs from three donors were exposed to lidocaine at various dilutions (2 mg/mL to 8 mg/mL) and exposure times (0.5 to 4 hours). Cell number and viability, mitochondrial activity, and real‐time reverse‐transcriptase quantitative polymerase chain reaction (RT‐qPCR) were analyzed at 0 (immediate effects) or 24 and 48 hours (recovery effects) after treatment with lidocaine.ResultsTrypan blue staining showed that increasing concentrations of lidocaine decreased the number of observable viable cells. 3‐[4,5,dimethylthiazol‐2‐yl]‐5‐[3‐carboxymethoxy‐phenyl]‐2‐[4‐sulfophenyl]‐2H‐tetrazolium (MTS) assays revealed a concentration‐ and time‐ dependent decline of mitochondrial activity and proliferative ability. Gene expression analysis by RT‐qPCR revealed that adipose‐derived MSCs exposed to lidocaine express robust levels of stress response/cytoprotective genes. However, higher concentrations of lidocaine caused a significant downregulation of these genes. No significant differences were observed in expression of extracellular matrix (ECM) markers COL1A1 and DCN except for COL3A1 (P < .05). Levels of messenger RNA (mRNA) for proliferation markers (CCNB2, HIST2H4A, P < .001) and MKI67 (P < .001) increased at 24 and 48 hours. Expression levels of several transcription factors— including SP1, PRRX1, and ATF1—were modulated in the same manner. MSC surface markers CD44 and CD105 demonstrated decreased expression immediately after treatment, but at 24 and 48 hours postexposure, the MSC markers showed no significant difference among groups.ConclusionLidocaine is toxic to MSCs in a dose‐ and time‐ dependent manner. MSC exposure to high (4‐8 mg/mL) concentrations of lidocaine for prolonged periods can affect their biologic functions. Although the exposure time in vivo is short, it is essential to choose safe concentrations when applying lidocaine along with MSCs to avoid compromising the viability and potency of the stem cell therapy.

Abstract Osteosarcomas are bone tumors that frequently metastasize to the lung. Aberrant expression of the transcription factor, runt‐related transcription factor 2 (RUNX2), is a key pathological feature in osteosarcoma and associated with loss of p53 and miR‐34 expression. Elevated RUNX2 may transcriptionally activate genes mediating tumor progression and metastasis, including the RUNX2 target gene osteopontin ( OPN/SPP1 ). This gene encodes a secreted matricellular protein produced by osteoblasts to regulate bone matrix remodeling and tissue calcification. Here we investigated whether and how the RUNX2/OPN axis regulates lung metastasis of osteosarcoma. Importantly, RUNX2 depletion attenuates lung metastasis of osteosarcoma cells in vivo. Using next‐generation RNA‐sequencing, protein‐based assays, as well as the loss‐ and gain‐of‐function approaches in selected osteosarcoma cell lines, we show that osteopontin messenger RNA levels closely correlate with RUNX2 expression and that RUNX2 controls the levels of secreted osteopontin. Elevated osteopontin levels promote heterotypic cell–cell adhesion of osteosarcoma cells to human pulmonary microvascular endothelial cells, but not in the presence of neutralizing antibodies. Collectively, these findings indicate that the RUNX2/OPN axis regulates the ability of osteosarcoma cells to attach to pulmonary endothelial cells as a key step in metastasis of osteosarcoma cells to the lung.

AbstractThe RUNX1 transcription factor has recently been shown to be obligatory for normal development. RUNX1 controls the expression of genes essential for proper development in many cell lineages and tissues including blood, bone, cartilage, hair follicles, and mammary glands. Compromised RUNX1 regulation is associated with many cancers. In this review, we highlight evidence for RUNX1 control in both invertebrate and mammalian development and recent novel findings of perturbed RUNX1 control in breast cancer that has implications for other solid tumors. As RUNX1 is essential for definitive hematopoiesis, RUNX1 mutations in hematopoietic lineage cells have been implicated in the etiology of several leukemias. Studies of solid tumors have revealed a context‐dependent function for RUNX1 either as an oncogene or a tumor suppressor. These RUNX1 functions have been reported for breast, prostate, lung, and skin cancers that are related to cancer subtypes and different stages of tumor development. Growing evidence suggests that RUNX1 suppresses aggressiveness in most breast cancer subtypes particularly in the early stage of tumorigenesis. Several studies have identified RUNX1 suppression of the breast cancer epithelial‐to‐mesenchymal transition. Most recently, RUNX1 repression of cancer stem cells and tumorsphere formation was reported for breast cancer. It is anticipated that these new discoveries of the context‐dependent diversity of RUNX1 functions will lead to innovative therapeutic strategies for the intervention of cancer and other abnormalities of normal tissues.

Abstract Background The use of mesenchymal stem cells (MSCs) in clinical applications for the treatment of musculoskeletal disease is steadily increasing in office-based practice. The so-called “first generation” of MSCs is defined as autologous stem cells that have undergone minimal manipulation and are used for a homologous purpose. Systematic reviews of the clinical trials completed to date of such MSCs enable practitioners to better understand what is currently known about the outcomes and side effects of such treatments. Study Design A systematic review of human clinical studies of office-based MSC therapy for the treatment of painful degenerative musculoskeletal conditions. Methods A search of the Ovid MEDLINE, EMBASE, and Scopus databases was conducted from 2006 through September 2016. Seven hundred sixty-one records were identified from database searching, and two records from reference review of included papers. Studies with human subjects that evaluated treatment of musculoskeletal disease with minimally manipulated MSCs were included. Results Eight studies were included in this review based on selection criteria. A total of 941 patients were included, 841 of whom received cellular products, and no significant adverse events were reported. Symptomatology generally improved, though no differences were seen over controls where present. Conclusion Support in the literature is strongest for the use of bone marrow aspirate concentrate (BMAC) injections for the treatment of knee pain, but applications of the use of BMAC and peripheral blood–derived MSCs for the treatment of hip pain, tendon pain, and disc pain have all been reported. Further research is required, with large randomized controlled trials.

ABSTRACT The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell‐released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV‐associated functional activities. Finally, a checklist is provided with summaries of key points.

ABSTRACT Developing novel approaches to treat skeletal disorders requires an understanding of how critical molecular factors regulate osteoblast differentiation and bone remodeling. We have reported that (1) retinoic acid receptor-related orphan receptor beta (Rorβ) is upregulated in bone samples isolated from aged mice and humans in vivo; (2) Rorβ expression is inhibited during osteoblastic differentiation in vitro; and (3) genetic deletion of Rorβ in mice results in preservation of bone mass during aging. These data establish that Rorβ inhibits osteogenesis and that strict control of Rorβ expression is essential for bone homeostasis. Because microRNAs (miRNAs) are known to play important roles in the regulation of gene expression in bone, we explored whether a predicted subset of nine miRNAs regulates Rorβ expression during both osteoblast differentiation and aging. Mouse osteoblastic cells were differentiated in vitro and assayed for Rorβ and miRNA expression. As Rorβ levels declined with differentiation, the expression of many of these miRNAs, including miR-219a-5p, was increased. We further demonstrated that miR-219a-5p was decreased in bone samples from old (24-month) mice, as compared with young (6-month) mice, concomitant with increased Rorβ expression. Importantly, we also found that miR-219a-5p expression was decreased in aged human bone biopsies compared with young controls, demonstrating that this phenomenon also occurs in aging bone in humans. Inhibition of miR-219a-5p in mouse calvarial osteoblasts led to increased Rorβ expression and decreased alkaline phosphatase expression and activity, whereas a miR-219a-5p mimic decreased Rorβ expression and increased osteogenic activity. Finally, we demonstrated that miR-219a-5p physically interacts with Rorβ mRNA in osteoblasts, defining Rorβ as a true molecular target of miR-219a-5p. Overall, our findings demonstrate that miR-219a-5p is involved in the regulation of Rorβ in both mouse and human bone. © 2018 American Society for Bone and Mineral Research.

Background: The preoperative assessment of cartilage lesions is critical to surgical planning and decision making. The accurate radiographic determination of acetabular cartilage damage has remained elusive for modern imaging modalities, including magnetic resonance imaging (MRI) and magnetic resonance arthrography (MRA). While risk factors have been individually described, no multivariable system exists for predicting high-grade cartilage damage. Purpose: To determine the preoperative predictors of grade 3 to 4 acetabular labrum articular disruption (ALAD) lesions. Study Design: Case-control study; Level of evidence, 3. Cohort study (diagnosis); Level of evidence, 1. Methods: Retrievable radiographs were reviewed from primary hip arthroscopic procedures performed at 2 high-volume institutions between December 2007 and April 2017. The predictive value of demographic and radiographic factors for the intraoperative documentation of ALAD grade 3 to 4 damage was analyzed and entered into a multivariable model, and a statistically guided scoring system for the damage risk was created using the Akaike information criterion. The scoring system was then prospectively validated on 167 patients who underwent primary hip arthroscopy between April 2017 and February 2018. Results: A total of 652 primary hip arthroscopic procedures in 614 patients (390 female, 224 male; mean age, 33.2 ± 12.5 years; mean body mass index, 26.9 ± 5.5 kg/m 2 ) from 2007 to 2017 were analyzed. Male sex (odds ratio [OR], 3.11; P < .01), age ≥35 years (OR, 1.96; P < .01), cam morphology (alpha angle >55°) (OR, 2.96; P < .01), and Tönnis grade 1 to 2 (grade 1: OR, 4.14; P < .01, and grade 2: OR, 9.29; P < .01) were univariate risk factors for intraoperatively documented high-grade damage. A multivariable scoring system, the Rapidly Assessed Predictor of Intraoperative Damage (RAPID) score (0-5 points), was generated based on sex, Tönnis grade, and cam morphology. Patients with increasing RAPID scores had an increasing risk of damage, with a 10.5% risk for those with 0 points and an 88.0% risk for those with 5 points ( P < .01). The area under the curve was 0.75 for the study group and 0.76 for the validation group ( P = .94). Conclusion: While preoperative MRI has diagnostic value for hip arthroscopic surgery, the RAPID score provides added benefit as a readily employable, in-clinic system for predicting high-grade cartilage damage. The discriminatory value of the RAPID score compares favorably with previous MRI and MRA studies. This information will help the clinician and patient plan for high-grade damage and identify potential targets for cartilage treatment.

Background Twelve paired fresh frozen cadaveric wrists were randomized to a 360-degree tenodesis repair group or the 360-degree tenodesis repair with an internal brace (suture tape) construct. Case Description The specimens were preloaded to 5 N and subsequently biomechanically loaded to failure, at a rate of 0.1 mm/s on a jig that allowed for axial load. The maximum load and mode of failure were recorded. Load to failure in the 360 tenodesis group with internal brace was 283.47 ± 100.25 N, compared with the 360 tenodesis group only, whose yield strength was 143.61 ± 90.54 N. The mode of failure within the internal brace construct was either through knot slippage, graft disruption, or bone separation from strength testing construct. The 360 tenodesis group tended to fail via graft slippage or graft rupture. Literature Review The management of scapholunate instability can be a difficult problem to treat. Traditionally, many of the surgical reconstructions have focused upon dorsal ligament reconstruction with Kirschner (K) wire fixation. This results in prolonged immobilization of the wrist with varied outcomes, in part due to the multiaxial instability that may persist due to concomitant volar ligament disruption. To address this instability, surgical techniques have been devised that address both the volar and dorsal ligament injuries. Clinical Relevance Scapholunate reconstruction with a 360-degree tenodesis and internal brace augmentation (SLITT procedure) provided superior biomechanical stability than tenodesis alone.

Abstract Expression of Runx2/p57 is a hallmark of the osteoblast‐lineage identity. Although several regulators that control the expression of Runx2/p57 during osteoblast‐lineage commitment have been identified, the epigenetic mechanisms that sustain this expression in differentiated osteoblasts remain to be completely determined. Here, we assess epigenetic mechanisms associated with Runx2/p57 gene transcription in differentiating MC3T3 mouse osteoblasts. Our results show that an enrichment of activating histone marks at the Runx2/p57 P1 promoter is accompanied by the simultaneous interaction of Wdr5 and Utx proteins, both are components of COMPASS complexes. Knockdown of Wdr5 and Utx expression confirms the activating role of both proteins at the Runx2‐P1 promoter. Other chromatin modifiers that were previously described to regulate Runx2/p57 transcription in mesenchymal precursor cells (Ezh2, Prmt5, and Jarid1b proteins) were not found to contribute to Runx2/p57 transcription in full‐committed osteoblasts. We also determined the presence of additional components of COMPASS complexes at the Runx2/p57 promoter, evidencing that the Mll2/COMPASS‐ and Mll3/COMPASS‐like complexes bind to the P1 promoter in osteoblastic cells expressing Runx2/p57 to modulate the H3K4me1 to H3K4me3 transition.