van Wijnen, Andre J

AbstractThe Melatonin Osteoporosis Prevention Study (MOPS) demonstrated that nightly melatonin resulted in a time‐dependent decrease in equilibrium ratios of serum osteoclasts and osteoblasts in perimenopausal women. This study examines mechanisms related to the ratios of osteoblasts and osteoclasts using coculture models (transwell or layered) of human mesenchymal stem cell (MSC) and human peripheral blood monocytes (PBMCs). Human MSC/PBMC cocultures exposed to melatonin in osteogenic (OS+) medium for 21 days induced osteoblast differentiation and mineralization; however, only in layered cocultures did melatonin inhibit osteoclastogenesis. Melatonin effects were mediated through MT2 melatonin receptors, MEK1/2, and MEK5. In layered but not transwell cocultures, melatonin increased OPG:RANKL ratios by inhibiting RANKL, suggesting that contact with osteoclasts during osteoblastogenesis inhibits RANKL secretion. Melatonin modulated expression of ERK1/2, ERK5, β1 integrin, GLUT4, and IRβ that was dependent upon the type of coculture; however, in both cultures, melatonin increased RUNX2 and decreased PPARγ expression, indicating a role for metabolic processes that control osteogenic vs adipogenic cell fates of MSCs. Furthermore, melatonin also has osteoblast‐inducing effects on human adipose‐derived MSCs. In vivo, one‐year nightly melatonin (15 mg/L) given to neu female mice in their drinking water increased pErk1/2, pErk5, Runx2, and Opg and Rankl levels in bone consistent with melatonin's already reported bone‐enhancing effects. Finally, analysis of daily logs from the MOPS demonstrated a significant improvement in mood and perhaps sleep quality in women receiving melatonin vs placebo. The osteoblast‐inducing, bone‐enhancing effects of melatonin and improvement in quality of life suggest that melatonin is a safe and effective bone loss therapy.

Abstract Strategies for musculoskeletal tissue regeneration apply adult mesenchymal stem/stromal cells (MSCs) that can be sourced from bone marrow- and lipo-aspirates. Adipose tissue-derived MSCs are more easily harvested in the large quantities required for skeletal tissue-engineering approaches, but are generally considered to be less osteogenic than bone marrow MSCs. Therefore, we tested a new molecular strategy to improve their osteogenic lineage-differentiation potential using the fungal metabolite cytochalasin D (CytoD). We show that CytoD, which may function by redistributing the intracellular location of β-actin (ACTB), is a potent osteogenic stimulant as reflected by significant increases in alkaline phosphatase activity, extracellular matrix mineralization, and osteoblast-related gene expression (e.g., RUNX2, ALPL, SPARC, and TGFB3). RNA sequencing analyses of MSCs revealed that acute CytoD treatment (24 hours) stimulates a broad program of osteogenic biomarkers and epigenetic regulators. CytoD decreases mRNA and protein levels of the Polycomb chromatin regulator Enhancer of Zeste Homolog 2 (EZH2), which controls heterochromatin formation by mediating trimethylation of histone 3 lysine 27 (H3K27me3). Reduced EZH2 expression decreases cellular H3K27me3 marks indicating a global reduction in heterochromatin. We conclude that CytoD is an effective osteogenic stimulant that mechanistically functions by blocking both cytoplasmic actin polymerization and gene-suppressive epigenetic mechanisms required for the acquisition of the osteogenic phenotype in adipose tissue-derived MSCs. This finding supports the use of CytoD in advancing the osteogenic potential of MSCs in skeletal regenerative strategies.

Transplantation of autologous mesenchymal stem cells (MSCs) may be a viable option for treatment of several diseases. MSCs efficacy depends on adequate function of their mitochondria, which might be impaired in a noxious milieu. We hypothesized that obesity compromises MSCs mitochondrial structure and function, possibly via micro‐RNA (miRNA)‐based mechanisms. MSCs were collected from swine abdominal adipose tissue after 16 weeks of Lean or Obese diet (n = 7 each). Mitochondrial structure was assessed by electron microscopy and function by membrane potential and cytochrome‐c oxidase (COX)‐IV activity. Oxidative stress was assessed by Mito‐SOX and dihydroethidium staining. Next‐generation sequencing (RNA‐seq) was performed to identify miRNAs expression in MSCs, and predicted mitochondrial target genes were then identified (MitoCarta). Compared to Lean‐MSCs, mitochondria from Obese‐MSCs were smaller and showed cristae remodeling and loss. Mitochondrial membrane potential and COX‐IV activity decreased in Obese‐MSCs, associated with increased mitochondrial oxidative stress. RNA‐seq generated reads for 413 miRNAs, of which 5 miRNAs were upregulated in Obese‐MSCs (fold change >2, p < 0.05) and found to target 43 specific mitochondrial genes. Obesity impairs MSC mitochondrial structure and function, possibly mediated partly through miRNA‐induced mitochondrial gene regulation, leading to increased oxidative stress. Importantly, these alterations may limit the therapeutic use of autologous MSCs in subjects with obesity.

ABSTRACTDegenerative disk disease of the spine is a major cause of back pain and disability. Optimization of regenerative medical therapies for degenerative disk disease requires a deep mechanistic understanding of the factors controlling the structural integrity of spinal tissues. In this investigation, we sought to identify candidate regulatory genes controlling extracellular matrix synthesis in spinal tissues. To achieve this goal we performed high throughput next generation RNA sequencing on 39 annulus fibrosus and 21 nucleus pulposus human tissue samples. Specimens were collected from patients undergoing surgical discectomy for the treatment of degenerative disk disease. Our studies identified associations between extracellular matrix genes, growth factors, and other important regulatory molecules. The fibrous matrix characteristic of annulus fibrosus was associated with expression of the growth factors platelet derived growth factor beta (PDGFB), vascular endothelial growth factor C (VEGFC), and fibroblast growth factor 9 (FGF9). Additionally we observed high expression of multiple signaling proteins involved in the NOTCH and WNT signaling cascades. Nucleus pulposus extracellular matrix related genes were associated with the expression of numerous diffusible growth factors largely associated with the transforming growth signaling cascade, including transforming factor alpha (TGFA), inhibin alpha (INHA), inhibin beta A (INHBA), bone morphogenetic proteins (BMP2, BMP6), and others. Clinical significance: this investigation provides important data on extracellular matrix gene regulatory networks in disk tissues. This information can be used to optimize pharmacologic, stem cell, and tissue engineering strategies for regeneration of the intervertebral disk and the treatment of back pain. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1356–1369, 2018.

Discogenic low back pain (DLBP) is extremely common and costly. Effective treatments are lacking due to DLBP's unknown pathogenesis. Currently, there are no in vivo mouse models of DLBP, which restricts research in this field. The aim of this study was to establish a reliable DLBP model in mouse that captures the pathological changes in the disc and allows longitudinal pain testing. The model was generated by puncturing the mouse lumbar discs (L4/5, L5/6, and L6/S1) and removing the nucleus pulposus using a microscalpel under the microscope. Histology, molecular pathways, and pain‐related behaviors were examined. Over 12 weeks post‐surgery, animals displayed the mechanical, heat, and cold hyperalgesia along with decreased burrowing and rearing. Histology showed progressive disc degeneration with loss of disc height, nucleus pulposus reduction, proteoglycan depletion, and annular fibrotic disorganization. Immunohistochemistry revealed a substantial increase in inflammatory mediators at 2 and 4 weeks. Nerve growth factor was upregulated from 2 weeks to the end of the experiment. Nerve fiber ingrowth was induced in the injured discs after 4 weeks. Disc‐puncture also produced an upregulation of neuropeptides in dorsal root ganglia neurons and an activation of glial cells in the spinal cord dorsal horn. These findings indicate that the cellular and structural changes in discs, as well as peripheral and central nervous system plasticity, paralleled persistent, and robust behavioral pain responses. Therefore, this mouse DLBP model could be used to investigate mechanisms underlying discogenic pain, thereby facilitating effective drug screening and development of treatments for DLBP.

Abstract Mesenchymal stem cells (MSC) hold great potential for regenerative medicine because of their ability for self-renewal and differentiation into tissue-specific cells such as osteoblasts, chondrocytes, and adipocytes. MSCs orchestrate tissue development, maintenance and repair, and are useful for musculoskeletal regenerative therapies to treat age-related orthopedic degenerative diseases and other clinical conditions. Importantly, MSCs produce secretory factors that play critical roles in tissue repair that support both engraftment and trophic functions (autocrine and paracrine). The development of uniform protocols for both preparation and characterization of MSCs, including standardized functional assays for evaluation of their biological potential, are critical factors contributing to their clinical utility. Quality control and release criteria for MSCs should include cell surface markers, differentiation potential, and other essential cell parameters. For example, cell surface marker profiles (surfactome), bone-forming capacities in ectopic and orthotopic models, as well as cell size and granularity, telomere length, senescence status, trophic factor secretion (secretome), and immunomodulation, should be thoroughly assessed to predict MSC utility for regenerative medicine. We propose that these and other functionalities of MSCs should be characterized prior to use in clinical applications as part of comprehensive and uniform guidelines and release criteria for their clinical-grade production to achieve predictably favorable treatment outcomes for stem cell therapy.

AbstractFunctionalizing medical devices with polypeptides to enhance their performance has become important for improved clinical success. The extracellular matrix (ECM) adhesion protein vitronectin (VN) is an effective coating, although the chemistry used to attach VN often reduces its bioactivity. In vivo, VN binds the ECM in a sequence‐dependent manner with heparan sulfate (HS) glycosaminoglycans. We reasoned therefore that sequence‐based affinity chromatography could be used to isolate a VN‐binding HS fraction (HS9) for use as a coating material to capture VN onto implant surfaces. Binding avidity and specificity of HS9 were confirmed by enzyme‐linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR)‐based assays. Plasma polymerization of allylamine (AA) to tissue culture‐treated polystyrene (TCPS) was then used to capture and present HS9 as determined by radiolabeling and ELISA. HS9‐coated TCPS avidly bound VN, and this layered surface supported the robust attachment, expansion, and maintenance of human pluripotent stem cells. Compositional analysis demonstrated that 6‐O‐ and N‐sulfation, as well as lengths greater than three disaccharide units (dp6) are critical for VN binding to HS‐coated surfaces. Importantly, HS9 coating reduced the threshold concentration of VN required to create an optimally bioactive surface for pluripotent stem cells. We conclude that affinity‐purified heparan sugars are able to coat materials to efficiently bind adhesive factors for biomedical applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1887–1896, 2018.

ABSTRACTChronic low back pain is a major cause of disability and health care costs. Effective treatments are inadequate for many patients. Animal models are essential to further understanding of the pain mechanism and testing potential therapies. Currently, a number of preclinical models have been developed attempting to mimic aspects of clinical conditions that contribute to low back pain (LBP). This review focused on describing these animal models and the main behavioral tests for assessing pain in each model. Animal models of LBP can be divided into the following five categories: Discogenic LBP, radicular back pain, facet joint osteoarthritis back pain, muscle‐induced LBP, and spontaneous occurring LBP models. These models are important not only for enhancing our knowledge of how LBP is generated, but also for the development of novel therapeutic regimens to treat LBP in patients. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1305–1312, 2018.

Abstract The hematological malignancy acute myeloid leukemia (AML) interacts closely with osteoblasts within the protective bone marrow microenvironment. The bone marrow microenvironment protects tumor cells from chemotherapies, which can prevent sufficient eradication of tumor cells. To study the role of osteoblasts in the bone marrow microenvironment, our lab utilized a co-culture model of osteoblasts (MC3T3 osteoblast cell line) and acute myelogenous leukemia cells (KG1a AML cell line or AML patient samples from bone marrow aspirates). Osteoblasts were cultured with AML cells, or AML cells were cultured alone; AML cells were challenged with the standard chemotherapeutic agent cytarabine (Ara-C) at doses of 0µM, 0.1µM, 0.5µM, 1µM, 5µM, or 10µM in the presence or absence of osteoblasts; and AML cells were assayed by flow cytometry to assess cell death via annexin-V staining. Our lab has previously found that differentiating osteoblasts protect AML cells from an apoptosis inducing agent naturally present in the bone marrow. We now show that osteoblasts are also capable of protecting AML cells from the standard chemotherapeutic cytarabine. In addition, we have found that treatment of osteoblasts with the histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA) prevents these treated osteoblasts from protecting AML cells from cytarabine treatment, which is consistent with our lab’s previous findings that HDACi treatment inhibits protection of AML cells from an apoptosis inducing agent naturally present in the bone marrow. We have preliminary data that indicates that TAZS89A over-expression, a constitutively active form of TAZ, which is a transcriptional modulator that regulates osteoblast differentiation, may be sufficient to inhibit osteoblast mediated protection of AML cells from cytarabine. This finding would be consistent with the HDACi manipulated Nherf1-protein phosphatase 1α-TAZ signaling pathway that we have previously found to be sufficient to inhibit protection of AML cells from an apoptosis inducing agent naturally present in the bone marrow. Overall, these studies have delivered insights into the role of osteoblasts in protecting AML cells from chemotherapy in the bone marrow microenvironment and begun the characterization of mechanisms and targets responsible for the protective effects of osteoblasts. Manipulating differentiating osteoblasts within the bone marrow microenvironment therapeutically could aid in more complete destruction of the tumor cell burden and improve patient survival. Citation Format: Rosalie M. Sterner, Kimberly N. Kremer, Amel Dudakovic, Jennifer J. Westendorf, Andre J. van Wijnen, Karen E. Hedin. Osteoblasts protect AML cells from cytarabine-induced death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5930. doi:10.1158/1538-7445.AM2017-5930