Publications by authors named "Amel Dudakovic"

78 Publications

Mechanical strain-mediated reduction in RANKL expression is associated with RUNX2 and BRD2.

Gene 2020 Dec 16;763S:100027. Epub 2020 Jan 16.

Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN, USA; Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA. Electronic address:

Mechanical loading-related strains trigger bone formation by osteoblasts while suppressing resorption by osteoclasts, uncoupling the processes of formation and resorption. Osteocytes may orchestrate this process in part by secreting sclerostin (SOST), which inhibits osteoblasts, and expressing receptor activator of nuclear factor-κB ligand (RANKL/TNFSF11) which recruits osteoclasts. Both SOST and RANKL are targets of the master osteoblastic transcription factor RUNX2. Subjecting human osteoblastic Saos-2 cells to strain by four point bending down-regulates their expression of SOST and RANKL without altering RUNX2 expression. RUNX2 knockdown increases basal SOST expression, but does not alter SOST down-regulation following strain. Conversely, RUNX2 knockdown does not alter basal RANKL expression, but prevents its down-regulation by strain. Chromatin immunoprecipitation revealed RUNX2 occupies a region of the RANKL promoter containing a consensus RUNX2 binding site and its occupancy of this site decreases following strain. The expression of epigenetic acetyl and methyl writers and readers was quantified by RT-qPCR to investigate potential epigenetic bases for this change. Strain and RUNX2 knockdown both down-regulate expression of the bromodomain acetyl reader BRD2. BRD2 and RUNX2 co-immunoprecipitate, suggesting interaction within regulatory complexes, and BRD2 was confirmed to interact with the RUNX2 promoter. BRD2 also occupies the RANKL promoter and its occupancy was reduced following exposure to strain. Thus, RUNX2 may contribute to bone remodeling by suppressing basal SOST expression, while facilitating the acute strain-induced down-regulation of RANKL through a mechanosensitive epigenetic loop involving BRD2.
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http://dx.doi.org/10.1016/j.gene.2020.100027DOI Listing
December 2020

Constitutive activation of NF-κB inducing kinase (NIK) in the mesenchymal lineage using Osterix (Sp7)- or Fibroblast-specific protein 1 (S100a4)-Cre drives spontaneous soft tissue sarcoma.

PLoS One 2021 22;16(7):e0254426. Epub 2021 Jul 22.

Musculoskeletal Research Center, Washington University School of Medicine, St. Louis, MO, United States of America.

Aberrant NF-κB signaling fuels tumor growth in multiple human cancer types including both hematologic and solid malignancies. Chronic elevated alternative NF-κB signaling can be modeled in transgenic mice upon activation of a conditional NF-κB-inducing kinase (NIK) allele lacking the regulatory TRAF3 binding domain (NT3). Here, we report that expression of NT3 in the mesenchymal lineage with Osterix (Osx/Sp7)-Cre or Fibroblast-Specific Protein 1 (FSP1)-Cre caused subcutaneous, soft tissue tumors. These tumors displayed significantly shorter latency and a greater multiple incidence rate in Fsp1-Cre;NT3 compared to Osx-Cre;NT3 mice, regardless of sex. Histological assessment revealed poorly differentiated solid tumors with some spindled patterns, as well as robust RelB immunostaining, confirming activation of alternative NF-κB. Even though NT3 expression also occurs in the osteolineage in Osx-Cre;NT3 mice, we observed no bony lesions. The staining profiles and pattern of Cre expression in the two lines pointed to a mesenchymal tumor origin. Immunohistochemistry revealed that these tumors stain strongly for alpha-smooth muscle actin (αSMA), although vimentin staining was uniform only in Osx-Cre;NT3 tumors. Negative CD45 and S100 immunostains precluded hematopoietic and melanocytic origins, respectively, while positive staining for cytokeratin 19 (CK19), typically associated with epithelia, was found in subpopulations of both tumors. Principal component, differential expression, and gene ontology analyses revealed that NT3 tumors are distinct from normal mesenchymal tissues and are enriched for NF-κB related biological processes. We conclude that constitutive activation of the alternative NF-κB pathway in the mesenchymal lineage drives spontaneous sarcoma and provides a novel mouse model for NF-κB related sarcomas.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0254426PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8297882PMC
July 2021

Lamin A/C Is Dispensable to Mechanical Repression of Adipogenesis.

Int J Mol Sci 2021 Jun 19;22(12). Epub 2021 Jun 19.

Department of Mechanical & Biomedical Engineering, Boise State University, 1910 University Drive, MS-2085, Boise, ID 83725, USA.

Mesenchymal stem cells (MSCs) maintain the musculoskeletal system by differentiating into multiple lineages, including osteoblasts and adipocytes. Mechanical signals, including strain and low-intensity vibration (LIV), are important regulators of MSC differentiation via control exerted through the cell structure. Lamin A/C is a protein vital to the nuclear architecture that supports chromatin organization and differentiation and contributes to the mechanical integrity of the nucleus. We investigated whether lamin A/C and mechanoresponsiveness are functionally coupled during adipogenesis in MSCs. siRNA depletion of lamin A/C increased the nuclear area, height, and volume and decreased the circularity and stiffness. Lamin A/C depletion significantly decreased markers of adipogenesis (adiponectin, cellular lipid content) as did LIV treatment despite depletion of lamin A/C. Phosphorylation of focal adhesions in response to mechanical challenge was also preserved during loss of lamin A/C. RNA-seq showed no major adipogenic transcriptome changes resulting from LIV treatment, suggesting that LIV regulation of adipogenesis may not occur at the transcriptional level. We observed that during both lamin A/C depletion and LIV, interferon signaling was downregulated, suggesting potentially shared regulatory mechanism elements that could regulate protein translation. We conclude that the mechanoregulation of adipogenesis and the mechanical activation of focal adhesions function independently from those of lamin A/C.
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http://dx.doi.org/10.3390/ijms22126580DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8234021PMC
June 2021

Ezh2 knockout in mesenchymal cells causes enamel hyper-mineralization.

Biochem Biophys Res Commun 2021 Aug 16;567:72-78. Epub 2021 Jun 16.

Department of Oral Biology, Rutgers School of Dental Medicine, NJ, USA. Electronic address:

Enhancer of zeste homolog 2 (EZH2) is the catalytic core of polycomb repressive complex 2 (PRC2), which primarily methylates lysine 27 on histone H3 (H2K27me3), generating transcriptionally suppressed heterochromatin. Since EZH2 suppresses expression of genes involved in dentin formation, we examined the role of EZH2 in tooth development. Intriguingly, microCT analysis of teeth from mice with conditional Ezh2 knockout in uncommitted mesenchymal cells showed hyper-mineralization of enamel, which is produced by the epithelial-lineage cells, ameloblasts. Scanning electron microscopy analysis and nano-indentation of the incisor enamel from knockout mice revealed smaller inter-rod spaces and higher hardness compared to wild type enamel, respectively. Interestingly, expression of the calcium channel subunit gene, Orai2, was decreased compared to its competitor, Orai1, both in knockout mouse incisors and the ex vivo culture of ameloblasts with the surrounding tissues under EZH2 inhibition. Moreover, histological analysis of incisor from knockout mice showed decreased ameloblastin and expedited KLK4 expression in the ameloblasts. These observations suggest that EZH2 depletion in dental mesenchymal cells reduces enamel matrix formation and increases enamel protease activity from ameloblasts, resulting in enamel hyper-mineralization. This study demonstrates the significant role of the suppressive H3K27me3 mark for heterochromatin on enamel formation.
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http://dx.doi.org/10.1016/j.bbrc.2021.06.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8290883PMC
August 2021

Surface Roughness of Titanium Orthopedic Implants Alters the Biological Phenotype of Human Mesenchymal Stromal Cells.

Tissue Eng Part A 2021 Aug 16. Epub 2021 Aug 16.

Department of Orthopedic Surgery, Mayo Clinic, Rochester, Minnesota, USA.

Metal orthopedic implants are largely biocompatible and generally achieve long-term structural fixation. However, some orthopedic implants may loosen over time even in the absence of infection. fixation failure is multifactorial, but the fundamental biological defect is cellular dysfunction at the host-implant interface. Strategies to reduce the risk of short- and long-term loosening include surface modifications, implant metal alloy type, and adjuvant substances such as polymethylmethacrylate cement. Surface modifications (e.g., increased surface rugosity) can increase osseointegration and biological ingrowth of orthopedic implants. However, the localized responses of cells to implant surface modifications need to be better characterized. As an model for investigating cellular responses to metallic orthopedic implants, we cultured mesenchymal stromal/stem cells on clinical-grade titanium disks (Ti6Al4V) that differed in surface roughness as high (porous structured), medium (grit blasted), and low (bead blasted). Topological characterization of clinically relevant titanium (Ti) materials combined with differential mRNA expression analyses (RNA-seq and real-time quantitative polymerase chain reaction) revealed alterations to the biological phenotype of cells cultured on titanium structures that favor early extracellular matrix production and observable responses to oxidative stress and heavy metal stress. These results provide a descriptive model for the interpretation of cellular responses at the interface between native host tissues and three-dimensionally printed modular orthopedic implants, and will guide future studies aimed at increasing the long-term retention of such materials after total joint arthroplasty.
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http://dx.doi.org/10.1089/ten.TEA.2020.0369DOI Listing
August 2021

Multiple pharmacological inhibitors targeting the epigenetic suppressor enhancer of zeste homolog 2 (Ezh2) accelerate osteoblast differentiation.

Bone 2021 09 30;150:115993. Epub 2021 Apr 30.

Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN, USA; Center for Regenerative Medicine, Mayo Clinic, Rochester, MN, USA; Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA. Electronic address:

Skeletal development and bone formation are regulated by epigenetic mechanisms that either repress or enhance osteogenic commitment of mesenchymal stromal/stem cells and osteoblasts. The transcriptional suppressive trimethylation of histone 3 lysine 27 (H3K27me3) hinders differentiation of pre-committed osteoblasts. Osteoblast maturation can be stimulated by genetic loss of the H3K27 methyltransferase Ezh2 which can also be mimicked pharmacologically using the classical Ezh2 inhibitor GSK126. Identification of other Ezh2 inhibitors (iEzh2) that enhance osteogenic potential would increase chemical options for developing new bone stimulatory compounds. In this study, we examined a panel of iEzh2s and show that all eight inhibitors we tested are capable of accelerating osteoblast differentiation to different degrees at concentrations that are well below cytotoxic concentrations. Inhibition of Ezh2 is commensurate with loss of cellular H3K27me3 levels while forced expression of Ezh2 reverses the effect of Ezh2 suppression. Reduced Ezh2 function by siRNA depletion of Ezh2 mRNA and protein levels also stimulates osteoblastogenesis, consistent with the specificity of iEzh2 to target the active site of Ezh2. Diminished Ezh2 levels preempt the effects of iEzh2s on H3K27me3. GSK126, EPZ-6438 and siRNA depletion of Ezh2 each are effective in reducing H3K27me3 levels. However, EPZ-6438 is more potent than GSK126 in stimulating osteoblastogenesis, as reflected by increased extracellular matrix mineralization. Collectively, our data indicate that Ezh2 inhibitors properly target Ezh2 consistent with their biochemical affinities. The range of compounds capable of promoting osteogenesis presented in this study offers the opportunity to develop diverse bone anabolic strategies for distinct clinical scenarios, including spine fusion, non-union of bone and dental implant enhancement.
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http://dx.doi.org/10.1016/j.bone.2021.115993DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8217219PMC
September 2021

Fibroblastic differentiation of mesenchymal stem/stromal cells (MSCs) is enhanced by hypoxia in 3D cultures treated with bone morphogenetic protein 6 (BMP6) and growth and differentiation factor 5 (GDF5).

Gene 2021 Jul 20;788:145662. Epub 2021 Apr 20.

Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN, United States; Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, United States. Electronic address:

Introduction: Culture conditions and differentiation cocktails may facilitate cell maturation and extracellular matrix (ECM) secretion and support the production of engineered fibroblastic tissues with applications in ligament regeneration. The objective of this study is to investigate the potential of two connective tissue-related ligands (i.e., BMP6 and GDF5) to mediate collagenous ECM synthesis and tissue maturation in vitro under normoxic and hypoxic conditions based on the hypothesis that BMP6 and GDF5 are components of normal paracrine signalling events that support connective tissue homeostasis.

Methods: Human adipose-derived MSCs were seeded on 3D-printed medical-grade polycaprolactone (PCL) scaffolds using a bioreactor and incubated in media containing GDF5 and/or BMP6 for 21 days in either normoxic (5% oxygen) or hypoxic (2% oxygen) conditions. Constructs were harvested on Day 3 and 21 for cell viability analysis by live/dead staining, structural analysis by scanning electron microscopy, mRNA levels by RTqPCR analysis, and in situ deposition of proteins by immunofluorescence microscopy.

Results: Pro-fibroblastic gene expression is enhanced by hypoxic culture conditions compared to normoxic conditions. Hypoxia renders cells more responsive to treatment with BMP6 as reflected by increased expression of ECM mRNA levels on Day 3 with sustained expression until Day 21. GDF5 was not particularly effective either in the absence or presence of BMP6.

Conclusions: Fibroblastic differentiation of MSCs is selectively enhanced by BMP6 and not GDF5. Environmental factors (i.e., hypoxia) also influenced the responsiveness of cells to this morphogen.
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http://dx.doi.org/10.1016/j.gene.2021.145662DOI Listing
July 2021

Elevated Expression of Plasminogen Activator Inhibitor (PAI-1/SERPINE1) is Independent from rs1799889 Genotypes in Arthrofibrosis.

Meta Gene 2021 Jun 5;28. Epub 2021 Mar 5.

Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN, United States.

Arthrofibrosis is characterized by excessive extracellular matrix deposition in patients with total knee arthroplasties (TKAs) and causes undesirable joint stiffness. The pathogenesis of arthrofibrosis remains elusive and currently there are no diagnostic biomarkers for the pathological formation of this connective tissue. Fibrotic soft tissues are known to have elevated levels of plasminogen activator inhibitor-1 (PAI-1) (encoded by ), a secreted serine protease inhibitor that moderates extracellular matrix remodeling and tissue homeostasis. The 4G/5G insertion/deletion (rs1799889) is a well-known polymorphism that directly modulates PAI-1 levels. Homozygous 4G/4G allele carriers typically have higher PAI-1 levels and may predispose patients to soft tissue fibrosis (e.g., liver, lung, and kidney). Here, we examined the genetic contribution of the rs1799889 polymorphism to musculoskeletal fibrosis in arthrofibrotic (n = 100) and non-arthrofibrotic (n = 100) patients using Sanger Sequencing. Statistical analyses revealed that the allele frequencies of the rs1799889 polymorphism are similar in arthrofibrotic and non-arthrofibrotic patient cohorts. Because the fibrosis related rs1799889 polymorphism is independent of arthrofibrosis susceptibility in TKA patients, the possibility arises that fibrosis of joint connective tissues may involve unique genetic determinants distinct from those linked to classical soft tissue fibrosis.
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http://dx.doi.org/10.1016/j.mgene.2021.100877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8011541PMC
June 2021

Low-Dose Tamoxifen Induces Significant Bone Formation in Mice.

JBMR Plus 2021 Mar 20;5(3):e10450. Epub 2021 Jan 20.

Department of Medicine University of North Carolina Chapel Hill NC USA.

Use of the selective estrogen receptor modulator Tamoxifen (TAM) is a mainstay to induce conditional expression of Cre recombinase in transgenic laboratory mice. To excise β-catenin in 28-day-old male and female Prrx1-CreER/β-catenin mice (C57BL/6), we utilized TAM at 150 mg/kg; despite β-catenin knockout in MSC, we found a significant increase in trabecular and cortical bone volume in all genders. Because TAM was similarly anabolic in KO and control mice, we investigated a dose effect on bone formation by treating wild-type mice (WT C57BL/6, 4 weeks) with TAM (total dose 0, 20, 40, 200 mg/kg via four injections). TAM increased bone in a dose-dependent manner analyzed by micro-computed tomography (μCT), which showed that, compared to control, 20 mg/kg TAM increased femoral bone volume fraction (bone volume/total volume [BV/TV]) (21.6% ± 1.5% to 33% ± 2.5%; 153%,  < 0.005). With TAM 40 mg/kg and 200 mg/kg, BV/TV increased to 48.1% ± 4.4% (223%,  < 0.0005) and 58% ± 3.8% (269%,  < 0.0001) respectively, compared to control. Osteoblast markers increased with 200 mg/kg TAM: (224%,  < 0.0001), (166%,  < 0.0001), (223%,  < 0.0001), and (228%,  < 0.0001). Osteoclasts per bone surface (Oc#/BS) nearly doubled at the lowest TAM dose (20 mg/kg), but decreased to <20% control with 200 mg/kg TAM. Our data establish that use of TAM at even very low doses to excise a floxed target in postnatal mice has profound effects on trabecular and cortical bone formation. As such, TAM treatment is a major confounder in the interpretation of bone phenotypes in conditional gene knockout mouse models. © 2020 The Authors. published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbm4.10450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7990151PMC
March 2021

Is Essential for Patterning of Multiple Musculoskeletal Tissues but Dispensable for Tendon Differentiation.

Stem Cells Dev 2021 Jun 27;30(11):601-609. Epub 2021 Apr 27.

Research Division, Shriners Hospital for Children, Portland, Oregon, USA.

An efficient musculoskeletal system depends on the precise assembly and coordinated growth and function of muscles, skeleton, and tendons. However, the mechanisms that drive integrated musculoskeletal development and coordinated growth and differentiation of each of these tissues are still being uncovered. Epigenetic modifiers have emerged as critical regulators of cell fate differentiation, but so far almost nothing is known about their roles in tendon biology. Previous studies have shown that epigenetic modifications driven by Enhancer of zeste homolog 2 (EZH2), a major histone methyltransferase, have significant roles in vertebrate development including skeletal patterning and bone formation. We now find that targeting through the limb mesenchyme also has significant effects on tendon and muscle patterning, likely reflecting the essential roles of early mesenchymal cues mediated by for coordinated patterning and development of all tissues of the musculoskeletal system. Conversely, loss of in the tendon cells did not disrupt overall tendon structure or collagen organization suggesting that tendon differentiation and maturation are independent of signaling.
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http://dx.doi.org/10.1089/scd.2020.0209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8165461PMC
June 2021

Alterations of mesenchymal stromal cells in cerebrospinal fluid: insights from transcriptomics and an ALS clinical trial.

Stem Cell Res Ther 2021 03 18;12(1):187. Epub 2021 Mar 18.

Department of Neurology, Mayo Clinic, 200 First St. SW, Rochester, MN, 55905, USA.

Background: Mesenchymal stromal cells (MSCs) have been studied with increasing intensity as clinicians and researchers strive to understand the ability of MSCs to modulate disease progression and promote tissue regeneration. As MSCs are used for diverse applications, it is important to appreciate how specific physiological environments may stimulate changes that alter the phenotype of the cells. One need for neuroregenerative applications is to characterize the spectrum of MSC responses to the cerebrospinal fluid (CSF) environment after their injection into the intrathecal space. Mechanistic understanding of cellular biology in response to the CSF environment may predict the ability of MSCs to promote injury repair or provide neuroprotection in neurodegenerative diseases.

Methods: In this study, we characterized changes in morphology, metabolism, and gene expression occurring in human adipose-derived MSCs cultured in human (hCSF) or artificial CSF (aCSF) as well as examined relevant protein levels in the CSF of subjects treated with MSCs for amyotrophic lateral sclerosis (ALS).

Results: Our results demonstrated that, under intrathecal-like conditions, MSCs retained their morphology, though they became quiescent. Large-scale transcriptomic analysis of MSCs revealed a distinct gene expression profile for cells cultured in aCSF. The aCSF culture environment induced expression of genes related to angiogenesis and immunomodulation. In addition, MSCs in aCSF expressed genes encoding nutritional growth factors to expression levels at or above those of control cells. Furthermore, we observed a dose-dependent increase in growth factors and immunomodulatory cytokines in CSF from subjects with ALS treated intrathecally with autologous MSCs.

Conclusions: Overall, our results suggest that MSCs injected into the intrathecal space in ongoing clinical trials remain viable and may provide a therapeutic benefit to patients.
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http://dx.doi.org/10.1186/s13287-021-02241-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7977179PMC
March 2021

Cell Surface Glycoprotein CD24 Marks Bone Marrow-Derived Human Mesenchymal Stem/Stromal Cells with Reduced Proliferative and Differentiation Capacity In Vitro.

Stem Cells Dev 2021 03;30(6):325-336

Department of Internal Medicine, Erasmus MC, University Medical Center Rotterdam, Rotterdam, the Netherlands.

Bone marrow-derived mesenchymal stem/stromal cells (BMSCs) are fundamental to bone regenerative therapies, tissue engineering, and postmenopausal osteoporosis. Donor variation among patients, cell heterogeneity, and unpredictable capacity for differentiation reduce effectiveness of BMSCs for regenerative cell therapies. The cell surface glycoprotein CD24 exhibits the most prominent differential expression during osteogenic versus adipogenic differentiation of human BMSCs. Therefore, CD24 may represent a selective biomarker for subpopulations of BMSCs with increased osteoblastic potential. In undifferentiated human BMSCs, CD24 cell surface expression is variable among donors (range: 2%-10%) and increased by two to fourfold upon osteogenic differentiation. Strikingly, FACS sorted CD24 cells exhibit delayed mineralization and reduced capacity for adipocyte differentiation. RNAseq analysis of CD24 and CD24 BMSCs identified a limited number of genes with increased expression in CD24 cells that are associated with cell adhesion, motility, and extracellular matrix. Downregulated genes are associated with cell cycle regulation, and biological assays revealed that CD24 cells have reduced proliferation. Hence, expression of the cell surface glycoprotein CD24 identifies a subpopulation of human BMSCs with reduced capacity for proliferation and extracellular matrix mineralization. Functional specialization among BMSCs populations may support their regenerative potential and therapeutic success by accommodating cell activities that promote skeletal tissue formation, homeostasis, and repair.
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http://dx.doi.org/10.1089/scd.2021.0027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7984936PMC
March 2021

Combination of BMP2 and EZH2 Inhibition to Stimulate Osteogenesis in a 3D Bone Reconstruction Model.

Tissue Eng Part A 2021 Aug 12;27(15-16):1084-1098. Epub 2021 Jan 12.

Department of Orthopedic Surgery, Mayo Clinic, Rochester, Minnesota, USA.

High concentrations of bone morphogenetic protein 2 (BMP2) in bone regeneration cause adverse events (e.g, heterotopic bone formation and acute inflammation). This study examines novel epigenetic strategies (i.e., EZH2 inhibition) for augmenting osteogenesis, thereby aiming to reduce the required BMP2 dose for bone regeneration and minimize these adverse effects. Human bone marrow-derived mesenchymal stem cells (BMSCs) were grown on three-dimensional (3D)-printed medical-grade polycaprolactone scaffolds and incubated in osteogenic media containing 50 ng/mL BMP2 and/or 5 μM GSK126 (EZH2 inhibitor) for 6 days ( = 3 per group and timepoint). Constructs were harvested for realtime quantitative polymerase chain reaction analysis at Day 10 and immunofluorescence (IF) microscopy at Day 21. After pretreating for 6 days and maintaining in osteogenic media for 4 days, BMSC-seeded scaffolds were also implanted in an immunocompromised subcutaneous murine model ( = 39; 3/group/donor and 3 control scaffolds) for histological analysis at 8 weeks. Pretreatment of BMSCs with BMP2 and BMP2/GSK126 costimulated expression of osteoblast-related genes (e.g., , , , and ), as well as protein accumulation (e.g., collagen type 1/ and osteocalcin/) based on IF staining. While implantation for 8 weeks did not result in bone formation, increased angiogenesis was observed in BMP2 and BMP2/GSK126 groups. This study finds that BMP2 and GSK126 costimulate osteogenic differentiation of MSCs on 3D scaffolds and may contribute to enhanced vascularization when implanted to support bone formation. Thus, epigenetic priming with EZH2 inhibitors may have translational potential in bone healing by permitting a reduction of BMP2 dosing to mitigate its side effects. Impact statement While autografts are still the gold standard for bone reconstruction, tissue availability and donor morbidity are significant limitations. Previous attempts to use high concentrations of bone morphogenetic protein 2 (BMP2) have been shown to cause adverse events such as excessive bone formation and acute inflammation. Overall, the utilization of EZH2 inhibitors to modulate gene expression in favor of bone healing has been demonstrated in a tissue engineering strategy. Our study will pave the way to developing tissue engineering strategies involving GSK126 as an adjuvant to increase the effects of BMP2 for stimulating cells of interest on a three-dimensional scaffold for bone regeneration.
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http://dx.doi.org/10.1089/ten.TEA.2020.0218DOI Listing
August 2021

Myeloma-Modified Adipocytes Exhibit Metabolic Dysfunction and a Senescence-Associated Secretory Phenotype.

Cancer Res 2021 02 20;81(3):634-647. Epub 2020 Nov 20.

Maine Medical Center Research Institute, Scarborough, Maine.

Bone marrow adipocytes (BMAd) have recently been implicated in accelerating bone metastatic cancers, such as acute myelogenous leukemia and breast cancer. Importantly, bone marrow adipose tissue (BMAT) expands with aging and obesity, two key risk factors in multiple myeloma disease prevalence, suggesting that BMAds may influence and be influenced by myeloma cells in the marrow. Here, we provide evidence that reciprocal interactions and cross-regulation of myeloma cells and BMAds play a role in multiple myeloma pathogenesis and treatment response. Bone marrow biopsies from patients with multiple myeloma revealed significant loss of BMAT with myeloma cell infiltration of the marrow, whereas BMAT was restored after treatment for multiple myeloma. Myeloma cells reduced BMAT in different preclinical murine models of multiple myeloma and using myeloma cell-adipocyte cocultures. In addition, multiple myeloma cells altered adipocyte gene expression and cytokine secretory profiles, which were also associated with bioenergetic changes and induction of a senescent-like phenotype. , senescence markers were also increased in the bone marrow of tumor-burdened mice. BMAds, in turn, provided resistance to dexamethasone-induced cell-cycle arrest and apoptosis, illuminating a new possible driver of myeloma cell evolution in a drug-resistant clone. Our findings reveal that bidirectional interactions between BMAds and myeloma cells have significant implications for the pathogenesis and treatment of multiple myeloma. Targeting senescence in the BMAd or other bone marrow cells may represent a novel therapeutic approach for treatment of multiple myeloma. SIGNIFICANCE: This study changes the foundational understanding of how cancer cells hijack the bone marrow microenvironment and demonstrates that tumor cells induce senescence and metabolic changes in adipocytes, potentially driving new therapeutic directions.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-1088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854508PMC
February 2021

A Potential Theragnostic Regulatory Axis for Arthrofibrosis Involving Adiponectin (ADIPOQ) Receptor 1 and 2 (ADIPOR1 and ADIPOR2), TGFβ1, and Smooth Muscle α-Actin (ACTA2).

J Clin Med 2020 Nov 17;9(11). Epub 2020 Nov 17.

Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN 55905, USA.

(1) Background: Arthrofibrosis is a common cause of patient debility and dissatisfaction after total knee arthroplasty (TKA). The diversity of molecular pathways involved in arthrofibrosis disease progression suggest that effective treatments for arthrofibrosis may require a multimodal approach to counter the complex cellular mechanisms that direct disease pathogenesis. In this study, we leveraged RNA-seq data to define genes that are suppressed in arthrofibrosis patients and identified adiponectin () as a potential candidate. We hypothesized that signaling pathways activated by ADIPOQ and the cognate receptors ADIPOR1 and ADIPOR2 may prevent fibrosis-related events that contribute to arthrofibrosis. (2) Methods: Therefore, ADIPOR1 and ADIPOR2 were analyzed in a TGFβ1 inducible cell model for human myofibroblastogenesis by both loss- and gain-of-function experiments. (3) Results: Treatment with AdipoRon, which is a small molecule agonist of ADIPOR1 and ADIPOR2, decreased expression of collagens (, , and ) and the myofibroblast marker smooth muscle α-actin (ACTA2) at both mRNA and protein levels in basal and TGFβ1-induced cells. (4) Conclusions: Thus, ADIPOR1 and ADIPOR2 represent potential drug targets that may attenuate the pathogenesis of arthrofibrosis by suppressing TGFβ-dependent induction of myofibroblasts. These findings also suggest that AdipoRon therapy may reduce the development of arthrofibrosis by mediating anti-fibrotic effects in joint capsular tissues.
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http://dx.doi.org/10.3390/jcm9113690DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7698546PMC
November 2020

Biological functions of chromobox (CBX) proteins in stem cell self-renewal, lineage-commitment, cancer and development.

Bone 2021 02 24;143:115659. Epub 2020 Sep 24.

Department of Orthopedic Surgery, Mayo Clinic College of Medicine, Mayo Clinic, Rochester, MN, United States of America.

Epigenetic regulatory proteins support mammalian development, cancer, aging and tissue repair by controlling many cellular processes including stem cell self-renewal, lineage-commitment and senescence in both skeletal and non-skeletal tissues. We review here our knowledge of epigenetic regulatory protein complexes that support the formation of inaccessible heterochromatin and suppress expression of cell and tissue-type specific biomarkers during development. Maintenance and formation of heterochromatin critically depends on epigenetic regulators that recognize histone 3 lysine trimethylation at residues K9 and K27 (respectively, H3K9me3 and H3K27me3), which represent transcriptionally suppressive epigenetic marks. Three chromobox proteins (i.e., CBX1, CBX3 or CBX5) associated with the heterochromatin protein 1 (HP1) complex are methyl readers that interpret H3K9me3 marks which are mediated by H3K9 methyltransferases (i.e., SUV39H1 or SUV39H2). Other chromobox proteins (i.e., CBX2, CBX4, CBX6, CBX7 and CBX8) recognize H3K27me3, which is deposited by Polycomb Repressive Complex 2 (PRC2; a complex containing SUZ12, EED, RBAP46/48 and the methyl transferases EZH1 or EZH2). This second set of CBX proteins resides in PRC1, which has many subunits including other polycomb group factors (PCGF1, PCGF2, PCGF3, PCGF4, PCGF5, PCGF6), human polyhomeotic homologs (HPH1, HPH2, HPH3) and E3-ubiquitin ligases (RING1 or RING2). The latter enzymes catalyze the subsequent mono-ubiquitination of lysine 119 in H2A (H2AK119ub). We discuss biological, cellular and molecular functions of CBX proteins and their physiological and pathological activities in non-skeletal cells and tissues in anticipation of new discoveries on novel roles for CBX proteins in bone formation and skeletal development.
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http://dx.doi.org/10.1016/j.bone.2020.115659DOI Listing
February 2021

Engineering Cartilage Tissue by Co-culturing of Chondrocytes and Mesenchymal Stromal Cells.

Methods Mol Biol 2021 ;2221:53-70

Department of Developmental BioEngineering, TechMed Centre, University of Twente, Enschede, The Netherlands.

Co-culture of chondrocytes and mesenchymal stromal cells (MSCs) has been shown to be beneficial in engineering cartilage tissue in vitro. In these co-cultures, MSCs increase the proliferation and matrix deposition of chondrocytes. The MSCs accomplish this beneficial effect by so-called trophic actions. Thus, large cartilage constructs can be made with a relatively small number of chondrocytes. In this chapter, we describe different methods for making co-cultures of MSCs and chondrocytes. We also provide detailed protocols for analyzing MSC-chondrocyte co-cultures with cell tracking, proliferation assays, species-specific polymerase chain reactions (PCR), rheological analysis, compression analysis, RNA-sequencing analysis, short tandem repeats analysis, and biochemical examination.
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http://dx.doi.org/10.1007/978-1-0716-0989-7_5DOI Listing
March 2021

Genetic background dependent modifiers of craniosynostosis severity.

J Struct Biol 2020 12 23;212(3):107629. Epub 2020 Sep 23.

Department of Orthodontics and Pediatric Dentistry, School of Dentistry, University of Michigan, Ann Arbor, MI, United States. Electronic address:

Craniosynostosis severity varies in patients with identical genetic mutations. To understand causes of this phenotypic variation, we backcrossed the FGFR2 mouse model of Crouzon syndrome onto congenic C57BL/6 and BALB/c backgrounds. Coronal suture fusion was observed in C57BL/6 (88% incidence, p < .001 between genotypes) but not in BALB/c FGFR2 mutant mice at 3 weeks after birth, establishing that that the two models differ in phenotype severity. To begin identifying pre-existing modifiers of craniosynostosis severity, we compared transcriptome signatures of cranial tissues from C57BL/6 vs. BALB/c FGFR2 mice. We separately analyzed frontal bone with coronal suture tissue from parietal bone with sagittal suture tissues because the coronal suture but not the sagittal suture fuses in FGFR2 mice. The craniosynostosis associated Twist and En1 transcription factors were down-regulated, while Runx2 was up-regulated, in C57BL/6 compared to BALB/c tissues, which could predispose to craniosynostosis. Transcriptome analyses under the GO term MAPK cascade revealed that genes associated with calcium ion channels, angiogenesis, protein quality control and cell stress response were central to transcriptome differences associated with genetic background. FGFR2 and HSPA2 protein levels plus ERK1/2 activity were higher in cells isolated from C57BL/6 than BALB/c cranial tissues. Notably, the HSPA2 protein chaperone is central to craniofacial genetic epistasis, and we find that FGFR2 protein is abnormally processed in primary cells from FGFR2 but not FGFR2 mice. Therefore, we propose that differences in protein quality control responses may contribute to genetic background influences on craniosynostosis phenotype severity.
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http://dx.doi.org/10.1016/j.jsb.2020.107629DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7885185PMC
December 2020

Autophagy Is Involved in Mesenchymal Stem Cell Death in Coculture with Chondrocytes.

Cartilage 2020 Jul 22:1947603520941227. Epub 2020 Jul 22.

Department of Developmental BioEngineering, University of Twente, Enschede, Netherlands.

Objective: Cartilage formation is stimulated in mixtures of chondrocytes and human adipose-derived mesenchymal stromal cells (MSCs) both and . During coculture, human MSCs perish. The goal of this study is to elucidate the mechanism by which adipose tissue-derived MSC cell death occurs in the presence of chondrocytes.

Methods: Human primary chondrocytes were cocultured with human MSCs derived from 3 donors. The cells were cultured in monoculture or coculture (20% chondrocytes and 80% MSCs) in pellets (200,000 cells/pellet) for 7 days in chondrocyte proliferation media in hypoxia (2% O). RNA sequencing was performed to assess for differences in gene expression between monocultures or coculture. Immune fluorescence assays were performed to determine the presence of caspase-3, LC3B, and P62.

Results: RNA sequencing revealed significant upregulation of >90 genes in the 3 cocultures when compared with monocultures. STRING analysis showed interconnections between >50 of these genes. Remarkably, 75% of these genes play a role in cell death pathways such as apoptosis and autophagy. Immunofluorescence shows a clear upregulation of the autophagic machinery with no substantial activation of the apoptotic pathway.

Conclusion: In cocultures of human MSCs with primary chondrocytes, autophagy is involved in the disappearance of MSCs. We propose that this sacrificial cell death may contribute to the trophic effects of MSCs on cartilage formation.
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http://dx.doi.org/10.1177/1947603520941227DOI Listing
July 2020

Inhibition of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) stimulates osteoblastogenesis by potentiating bone morphogenetic protein 2 (BMP2) responses.

J Cell Physiol 2021 02 19;236(2):1195-1213. Epub 2020 Jul 19.

Research Centre, Shriners Hospital for Children - Canada, Montreal, Quebec, Canada.

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is a pleiotropic enzyme involved in DNA repair, cell cycle control, and transcription regulation. A potential role for DNA-PKcs in the regulation of osteoblastogenesis remains to be established. We show that pharmacological inhibition of DNA-PKcs kinase activity or gene silencing of Prkdc (encoding DNA-PKcs) in murine osteoblastic MC3T3-E1 cells and human adipose-derived mesenchymal stromal cells markedly enhanced osteogenesis and the expression of osteoblast differentiation marker genes. Inhibition of DNA-PKcs inhibited cell cycle progression and increased osteogenesis by significantly enhancing the bone morphogenetic protein 2 response in osteoblasts and other mesenchymal cell types. Importantly, in vivo pharmacological inhibition of the kinase enhanced bone biomechanical properties. Bones from osteoblast-specific conditional Prkdc-knockout mice exhibited a similar phenotype of increased stiffness. In conclusion, DNA-PKcs negatively regulates osteoblast differentiation, and therefore DNA-PKcs inhibitors may have therapeutic potential for bone regeneration and metabolic bone diseases.
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http://dx.doi.org/10.1002/jcp.29927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7680411PMC
February 2021

Mechanical strain-mediated reduction in RANKL expression is associated with RUNX2 and BRD2.

Gene X 2020 Dec 16;5:100027. Epub 2020 Jan 16.

Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN, USA.

Mechanical loading-related strains trigger bone formation by osteoblasts while suppressing resorption by osteoclasts, uncoupling the processes of formation and resorption. Osteocytes may orchestrate this process in part by secreting sclerostin (SOST), which inhibits osteoblasts, and expressing receptor activator of nuclear factor-κB ligand (RANKL/TNFSF11) which recruits osteoclasts. Both SOST and RANKL are targets of the master osteoblastic transcription factor RUNX2. Subjecting human osteoblastic Saos-2 cells to strain by four point bending down-regulates their expression of SOST and RANKL without altering RUNX2 expression. RUNX2 knockdown increases basal SOST expression, but does not alter SOST down-regulation following strain. Conversely, RUNX2 knockdown does not alter basal RANKL expression, but prevents its down-regulation by strain. Chromatin immunoprecipitation revealed RUNX2 occupies a region of the RANKL promoter containing a consensus RUNX2 binding site and its occupancy of this site decreases following strain. The expression of epigenetic acetyl and methyl writers and readers was quantified by RT-qPCR to investigate potential epigenetic bases for this change. Strain and RUNX2 knockdown both down-regulate expression of the bromodomain acetyl reader BRD2. BRD2 and RUNX2 co-immunoprecipitate, suggesting interaction within regulatory complexes, and BRD2 was confirmed to interact with the RUNX2 promoter. BRD2 also occupies the RANKL promoter and its occupancy was reduced following exposure to strain. Thus, RUNX2 may contribute to bone remodeling by suppressing basal SOST expression, while facilitating the acute strain-induced down-regulation of RANKL through a mechanosensitive epigenetic loop involving BRD2.
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http://dx.doi.org/10.1016/j.gene.2020.100027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7285908PMC
December 2020

Inhibition of the epigenetic suppressor EZH2 primes osteogenic differentiation mediated by BMP2.

J Biol Chem 2020 06 24;295(23):7877-7893. Epub 2020 Apr 24.

Department of Orthopedic Surgery, Mayo Clinic, Rochester, Minnesota, USA

Bone-stimulatory therapeutics include bone morphogenetic proteins ( BMP2), parathyroid hormone, and antibody-based suppression of WNT antagonists. Inhibition of the epigenetic enzyme enhancer of zeste homolog 2 (EZH2) is both bone anabolic and osteoprotective. EZH2 inhibition stimulates key components of bone-stimulatory signaling pathways, including the BMP2 signaling cascade. Because of high costs and adverse effects associated with BMP2 use, here we investigated whether BMP2 dosing can be reduced by co-treatment with EZH2 inhibitors. Co-administration of BMP2 with the EZH2 inhibitor GSK126 enhanced differentiation of murine (MC3T3) osteoblasts, reflected by increased alkaline phosphatase activity, Alizarin Red staining, and expression of bone-related marker genes ( Bglap and Phospho1). Strikingly, co-treatment with BMP2 (10 ng/ml) and GSK126 (5 μm) was synergistic and was as effective as 50 ng/ml BMP2 at inducing MC3T3 osteoblastogenesis. Similarly, the BMP2-GSK126 co-treatment stimulated osteogenic differentiation of human bone marrow-derived mesenchymal stem/stromal cells, reflected by induction of key osteogenic markers ( Osterix/SP7 and IBSP). A combination of BMP2 (300 ng local) and GSK126 (5 μg local and 5 days of 50 mg/kg systemic) yielded more consistent bone healing than single treatments with either compound in a mouse calvarial critical-sized defect model according to results from μCT, histomorphometry, and surgical grading of qualitative X-rays. We conclude that EZH2 inhibition facilitates BMP2-mediated induction of osteogenic differentiation of progenitor cells and maturation of committed osteoblasts. We propose that epigenetic priming, coupled with bone anabolic agents, enhances osteogenesis and could be leveraged in therapeutic strategies to improve bone mass.
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http://dx.doi.org/10.1074/jbc.RA119.011685DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7278362PMC
June 2020

Lumbar intervertebral disc mRNA sequencing identifies the regulatory pathway in patients with disc herniation and spondylolisthesis.

Gene 2020 Aug 30;750:144634. Epub 2020 Mar 30.

Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN, United States; Department of Biochemistry & Molecular Biology, Mayo Clinic, Rochester, MN, United States. Electronic address:

Lumbar degenerative disc disease (DDD) is a multifaceted progressive condition and often accompanied by disc herniation (DH) and/or degenerative spondylolisthesis (DS). Given the high prevalence of the disease (up to 20% according to some estimates) and the high costs associated with its care, there is a need to explore novel therapies such as regenerative medicine. Exploring these novel therapies first warrants investigation of molecular pathways underlying these disorders. Here, we show results from next generation RNA sequencing (RNA-seq) on mRNA isolated from 10 human nucleus pulposus (NP) samples of lumbar degenerated discs (DH and DS; n = 5 for each tissue) and other musculoskeletal tissues (Bone, cartilage, growth plate, and muscle; n = 7 for each tissue). Pathway and network analyses based on gene ontology (GO) terms were used to identify the biological functions of differentially expressed mRNAs. A total of 701 genes were found to be significantly upregulated in lumbar NP tissue compared to other musculoskeletal tissues. These differentially expressed mRNAs were primarily involved in DNA damage, immunity and G1/S transition of mitotic cell cycle. Interestingly, DH-specific signaling genes showed major network in chemotactic (e.g., CXCL10, CXCL11, IL1RL2 and IL6) and matrix-degrading pathway (e.g., MMP16, ADAMTSL1, 5, 8, 12, and 15), while DS-specific signaling genes were found to be those involved in cell adhesion (e.g., CDH1, EPHA1 and EFNA2) and inflammatory cytokines (e.g., CD19, CXCL5, CCL24, 25 and XCL2). Our findings provide new leads for therapeutic drug discovery that would permit optimization of medical or pharmacological intervention for cases of lumbar DDD.
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http://dx.doi.org/10.1016/j.gene.2020.144634DOI Listing
August 2020

Functional expression of ZNF467 and PCBP2 supports adipogenic lineage commitment in adipose-derived mesenchymal stem cells.

Gene 2020 May 4;737:144437. Epub 2020 Feb 4.

Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN, United States; Department of Medical Genetics, Mayo Clinic, Rochester, MN, United States; Department of Molecular Medicine, Mayo Clinic, Rochester, MN, United States. Electronic address:

Bone marrow-derived mesenchymal stromal/stem cells (BMSCs) have the potential to be employed in many different skeletal therapies. A major limitation to utilizing BMSCs as a therapeutic strategy in human disease and tissue regeneration is the low cell numbers obtained from initial isolation necessitating multiple cell passages that can lead to decreased cell quality. Adipose-derived mesenchymal stromal/stem cells (AMSCs) have been proposed as an alternative cell source for regenerative therapies; however the differentiation capacity of these cells differs from BMSCs. To understand the differences between BMSCs and AMSCs, we compared the global gene expression profiles of BMSCs and AMSCs and identified two genes, PCBP2 and ZNF467 that were differentially expressed between AMSCs and BMSCs. We demonstrate that PCBP2 and ZNF467 impact adipogenic but not osteogenic differentiation, further supporting evidence that AMSCs and BMSCs appear to be adapted to their microenvironment.
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http://dx.doi.org/10.1016/j.gene.2020.144437DOI Listing
May 2020

β-Catenin Preserves the Stem State of Murine Bone Marrow Stromal Cells Through Activation of EZH2.

J Bone Miner Res 2020 06 24;35(6):1149-1162. Epub 2020 Feb 24.

Department of Medicine, University of North Carolina Chapel Hill, Raleigh, NC, USA.

During bone marrow stromal cell (BMSC) differentiation, both Wnt signaling and the development of a rigid cytoskeleton promote commitment to the osteoblastic over adipogenic lineage. β-catenin plays a critical role in the Wnt signaling pathway to facilitate downstream effects on gene expression. We show that β-catenin was additive with cytoskeletal signals to prevent adipogenesis, and β-catenin knockdown promoted adipogenesis even when the actin cytoskeleton was depolymerized. β-catenin also prevented osteoblast commitment in a cytoskeletal-independent manner, with β-catenin knockdown enhancing lineage commitment. Chromatin immunoprecipitation (ChIP)-sequencing demonstrated binding of β-catenin to the promoter of enhancer of zeste homolog 2 (EZH2), a key component of the polycomb repressive complex 2 (PRC2) complex that catalyzes histone methylation. Knockdown of β-catenin reduced EZH2 protein levels and decreased methylated histone 3 (H3K27me3) at osteogenic loci. Further, when EZH2 was inhibited, β-catenin's anti-differentiation effects were lost. These results indicate that regulating EZH2 activity is key to β-catenin's effects on BMSCs to preserve multipotentiality. © 2020 American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbmr.3975DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7295671PMC
June 2020

The epigenetic reader Brd4 is required for osteoblast differentiation.

J Cell Physiol 2020 06 23;235(6):5293-5304. Epub 2019 Dec 23.

Department of Orthopedic Surgery, Mayo Clinic, Rochester, Minnesota.

Transcription networks and epigenetic mechanisms including DNA methylation, histone modifications, and noncoding RNAs control lineage commitment of multipotent mesenchymal progenitor cells. Proteins that read, write, and erase histone tail modifications curate and interpret the highly intricate histone code. Epigenetic reader proteins that recognize and bind histone marks provide a crucial link between histone modifications and their downstream biological effects. Here, we investigate the role of bromodomain-containing (BRD) proteins, which recognize acetylated histones, during osteogenic differentiation. Using RNA-sequencing (RNA-seq) analysis, we screened for BRD proteins (n = 40) that are robustly expressed in MC3T3 osteoblasts. We focused functional follow-up studies on Brd2 and Brd4 which are highly expressed in MC3T3 preosteoblasts and represent "bromodomain and extra terminal domain" (BET) proteins that are sensitive to pharmacological agents (BET inhibitors). We show that small interfering RNA depletion of Brd4 has stronger inhibitory effects on osteoblast differentiation than Brd2 loss as measured by osteoblast-related gene expression, extracellular matrix deposition, and alkaline phosphatase activity. Similar effects on osteoblast differentiation are seen with the BET inhibitor +JQ1, and this effect is reversible upon its removal indicating that this small molecule has no lasting effects on the differentiation capacity of MC3T3 cells. Mechanistically, we find that Brd4 binds at known Runx2 binding sites in promoters of bone-related genes. Collectively, these findings suggest that Brd4 is recruited to osteoblast-specific genes and may cooperate with bone-related transcription factors to promote osteoblast lineage commitment and maturation.
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http://dx.doi.org/10.1002/jcp.29415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8128078PMC
June 2020

Challenges in the Measurement and Interpretation of Serum Titanium Concentrations.

Biol Trace Elem Res 2020 Jul 6;196(1):20-26. Epub 2019 Nov 6.

Department of Orthopedic Surgery, Mayo Clinic, 200 1st Street, SW, Rochester, MN, 55905, USA.

The measurement of circulating metal ion levels in total hip arthroplasty patients continues to be an area of clinical interest. National regulatory agencies have recommended measurement of circulating cobalt and chromium concentrations in metal-on-metal bearing symptomatic total hip arthroplasty patients. However, the clinical utility of serum titanium (Ti) measurements is less understood due to wide variations in reported values and methodology. Fine-scale instrumentation for detecting in situ Ti levels continues to improve and has transitioned from graphite furnace atomic absorption spectroscopy to inductively coupled plasma optical emission spectrometry or inductively coupled plasma mass spectrometry. Additionally, analytical interferences, variable sample types, and non-standardized sample collection methods complicate Ti measurement and underlie the wide variation in reported levels. Normal reference ranges and pathologic ranges for Ti levels remain to be established quantitatively. However, before these ranges can be recognized and implemented, methodological standardization is necessary. This paper aims to provide background and recommendations regarding the complexities of measurement and interpretation of circulating Ti levels in total hip arthroplasty patients.
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http://dx.doi.org/10.1007/s12011-019-01891-4DOI Listing
July 2020

Knockdown of formin mDia2 alters lamin B1 levels and increases osteogenesis in stem cells.

Stem Cells 2020 01 6;38(1):102-117. Epub 2019 Nov 6.

Department of Medicine, University of North Carolina Chapel Hill, Chapel Hill, North Carolina.

Nuclear actin plays a critical role in mediating mesenchymal stem cell (MSC) fate commitment. In marrow-derived MSCs, the principal diaphanous-related formin Diaph3 (mDia2) is present in the nucleus and regulates intranuclear actin polymerization, whereas Diaph1 (mDia1) is localized to the cytoplasm and controls cytoplasmic actin polymerization. We here show that mDia2 can be used as a tool to query actin-lamin nucleoskeletal structure. Silencing mDia2 affected the nucleoskeletal lamin scaffold, altering nuclear morphology without affecting cytoplasmic actin cytoskeleton, and promoted MSC differentiation. Attempting to target intranuclear actin polymerization by silencing mDia2 led to a profound loss in lamin B1 nuclear envelope structure and integrity, increased nuclear height, and reduced nuclear stiffness without compensatory changes in other actin nucleation factors. Loss of mDia2 with the associated loss in lamin B1 promoted Runx2 transcription and robust osteogenic differentiation and suppressed adipogenic differentiation. Hence, mDia2 is a potent tool to query intranuclear actin-lamin nucleoskeletal structure, and its presence serves to retain multipotent stromal cells in an undifferentiated state.
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http://dx.doi.org/10.1002/stem.3098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6993926PMC
January 2020

Hypothermia and nutrient deprivation alter viability of human adipose-derived mesenchymal stem cells.

Gene 2020 Jan 5;722:144058. Epub 2019 Sep 5.

Department of Orthopedic Surgery, Mayo Clinic College of Medicine, Mayo Clinic, Rochester, MN, United States of America; Department of Biochemistry & Molecular Biology, Mayo Clinic College of Medicine, Mayo Clinic, Rochester, MN, United States of America. Electronic address:

Purpose: Adipose-derived mesenchymal stem cells (MSCs) are attractive biological agents in regenerative medicine. To optimize cell therapies, it is necessary to determine the most effective delivery method for MSCs. Therefore, we evaluated the biological properties of MSCs after exposure to various temperatures to define optimal storage conditions prior to therapeutic delivery of MSCs.

Design: Prospective observational study.

Methods And Materials: Adherent and non-adherent MSCs were incubated at multiple temperatures (i.e., 4, 23 and 37 °C) in Lactated Ringers (LR) solution lacking essential cell growth ingredients, or in culture media which is optimized for cell growth. Cells were assessed either after the temperature changes (4 h) or after recovery (24 h). Metabolic activity of MSCs, cell number and expression of representative mRNA biomarkers were evaluated to assess the biological effects of temperature. We monitored changes in mRNAs expression related to cytoprotective- or stress-related responses (e.g., FOS, JUN, ATF1, ATF4, EGR1, EGR2, MYC), proliferation (e.g., HIST2H4, CCNB2), and extracellular matrix production (ECM; e.g., COL3A1, COL1A1) by quantitative real time reverse-transcriptase polymerase chain reaction (RT-qPCR) analysis.

Results: Our study demonstrates that storing MSCs in Lactated Ringers (LR) solution for 4 h decreases cell number and metabolic activity. The number of viable MSCs decreased significantly when cultured at physiological temperature (37 °C) and severe hypothermia (4 °C), while cells grown at ambient temperature (23 °C) exhibited the least detrimental effects. There were no appreciable biological differences in mRNA markers for proliferation or ECM deposition at any of the temperatures. However, biomarkers related to cytoprotective- or stress-responses were selectively elevated depending on temperature or media type (i.e., LR versus standard media).

Conclusion: The biological impact of nutrient-free media and temperature changes after 4 h exposure persists after a 24 h recovery period. Hence, storage temperature and media conditions should be optimized to improve effective dosing of MSCs.
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http://dx.doi.org/10.1016/j.gene.2019.144058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7309368PMC
January 2020

A Versatile Protocol for Studying Anterior Cruciate Ligament Reconstruction in a Rabbit Model.

Tissue Eng Part C Methods 2019 04;25(4):191-196

1 Department of Orthopedic Surgery, Mayo Clinic, Rochester, Minnesota.

Anterior cruciate ligament (ACL) injuries are frequent, as >200,000 injuries occur in the United States alone each year. Owing to the risks for associated meniscus and cartilage damage, ACL injuries are a significant source of both orthopedic care and research. Given the extended recovery course after ACL injury, which often lasts 1-2 years, and is associated with limited participation in sports and activities of daily living for patients, there is a critical need for the evolution of new and improved methods for ACL repair. Subsequently, animal models of ACL reconstruction (ACLR) play a key role in the development and initial trialing of novel ACL interventions. This article provides a clear operative description and associated illustrations for a validated, institutional animal care and use committee, and veterinarian approved and facile model of ACLR to serve researchers investigating ACLR.
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http://dx.doi.org/10.1089/ten.TEC.2018.0357DOI Listing
April 2019
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