Using advanced racial and ethnic identity demographics to improve surveillance of work-related conditions in an occupational clinic setting.

BACKGROUND: Although racial and ethnic identities are associated with a multitude of disparate medical outcomes, surveillance of these subpopulations in the occupational clinic setting could benefit enormously from a more detailed and nuanced recognition of racial and ethnic identity. METHODS: The research group designed a brief questionnaire to capture several dimensions of this identity and collected data from patients seen for work-related conditions in four occupational medicine clinics from May 2019 through March 2020. Responses were used to calculate the sensitivity and specificity of extant racial/ethnic identity data within our electronic health records system, and were compared to participants' self-reported industry and occupation, coded according to North American Industry Classification System and Standard Occupational Classification System listings. RESULTS: Our questionnaire permitted collection of data that defined our patients' specific racial/ethnic identity with far greater detail, identified patients with multiple ethnic identities, and elicited their preferred language. Response rate was excellent (94.2%, n = 773). Non-White participants frequently selected a racial/ethnic subcategory (78.1%-92.2%). Using our race/ethnicity data as a referent, the electronic health record (EHR) had a high specificity (>87.1%), widely variable sensitivity (11.8%-82.2%), and poorer response rates (75.1% for race, 82.5% for ethnicity, as compared to 93.8% with our questionnaire). Additional analyses revealed some industries and occupations disproportionately populated by patients of particular racial/ethnic identities. CONCLUSIONS: Our project demonstrates the usefulness of a questionnaire which more effectively identifies racial/ethnic subpopulations in an occupational medicine clinic, permitting far more detailed characterization of their occupations, industries, and diagnoses.

Ezh2 Is Essential for Patterning of Multiple Musculoskeletal Tissues but Dispensable for Tendon Differentiation.

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 Ezh2 through the limb mesenchyme also has significant effects on tendon and muscle patterning, likely reflecting the essential roles of early mesenchymal cues mediated by Ezh2 for coordinated patterning and development of all tissues of the musculoskeletal system. Conversely, loss of Ezh2 in the tendon cells did not disrupt overall tendon structure or collagen organization suggesting that tendon differentiation and maturation are independent of Ezh2 signaling.

Occupational medicine clinical practice data reveal increased injury rates among Hispanic workers.

BACKGROUND: Minnesota has an ethnically diverse labor force, with the largest number of refugees per capita in the United States. In recent years, Minnesota has been and continues to be a major site for immigrant and refugee resettlement in the United States, with a large population of both immigrant and native born Hmong, Hispanic, and East Africans. This study seeks to evaluate the injury risk among the evolving minority workforce in the Minnesota Twin Cities region. METHODS: A retrospective cohort study identifying work-related injuries following pre-employment examinations was performed using electronic health records from a large multi-clinic occupational medicine practice. Preplacement examinations and subsequent work-related injuries were pulled from the electronic health record using representative ICD-10 codes for surveillance examinations and injuries. This study included patient records collected over a 2-year period from January 1, 2015, through December, 2016. The patients in this cohort worked in a wide-array of occupations including production, assembly, construction, law enforcement, among others. RESULTS: Hispanic minority workers were twice as likely to be injured at work compared with White workers. Hispanics were 2.89 times more likely to develop back injuries compared with non-Hispanic workers, and 1.86 times more likely to develop upper extremity injuries involving the hand, wrist, or elbow. CONCLUSION: Clinical practice data shows that Hispanic workers are at increased risk for work-related injuries in Minnesota. They were especially susceptible to back and upper extremity injuries. Lower injury rates in non-Hispanic minority workers, may be the result of injury underreporting and require further investigation.

Loss of histone methyltransferase Ezh2 stimulates an osteogenic transcriptional program in chondrocytes but does not affect cartilage development.

Ezh2 is a histone methyltransferase that suppresses osteoblast maturation and skeletal development. We evaluated the role of Ezh2 in chondrocyte lineage differentiation and endochondral ossification. Ezh2 was genetically inactivated in the mesenchymal, osteoblastic, and chondrocytic lineages in mice using the Prrx1-Cre, Osx1-Cre, and Col2a1-Cre drivers, respectively. WT and conditional knockout mice were phenotypically assessed by gross morphology, histology, and micro-CT imaging. Ezh2-deficient chondrocytes in micromass culture models were evaluated using RNA-Seq, histologic evaluation, and Western blotting. Aged mice with Ezh2 deficiency were also evaluated for premature development of osteoarthritis using radiographic analysis. Ezh2 deficiency in murine chondrocytes reduced bone density at 4 weeks of age but caused no other gross developmental effects. Knockdown of Ezh2 in chondrocyte micromass cultures resulted in a global reduction in trimethylation of histone 3 lysine 27 (H3K27me3) and altered differentiation in vitro RNA-Seq analysis revealed enrichment of an osteogenic gene expression profile in Ezh2-deficient chondrocytes. Joint development proceeded normally in the absence of Ezh2 in chondrocytes without inducing excessive hypertrophy or premature osteoarthritis in vivo In summary, loss of Ezh2 reduced H3K27me3 levels, increased the expression of osteogenic genes in chondrocytes, and resulted in a transient post-natal bone phenotype. Remarkably, Ezh2 activity is dispensable for normal chondrocyte maturation and endochondral ossification in vivo, even though it appears to have a critical role during early stages of mesenchymal lineage commitment.

Mapping molecular landmarks of human skeletal ontogeny and pluripotent stem cell-derived articular chondrocytes.

Tissue-specific gene expression defines cellular identity and function, but knowledge of early human development is limited, hampering application of cell-based therapies. Here we profiled 5 distinct cell types at a single fetal stage, as well as chondrocytes at 4 stages in vivo and 2 stages during in vitro differentiation. Network analysis delineated five tissue-specific gene modules; these modules and chromatin state analysis defined broad similarities in gene expression during cartilage specification and maturation in vitro and in vivo, including early expression and progressive silencing of muscle- and bone-specific genes. Finally, ontogenetic analysis of freshly isolated and pluripotent stem cell-derived articular chondrocytes identified that integrin alpha 4 defines 2 subsets of functionally and molecularly distinct chondrocytes characterized by their gene expression, osteochondral potential in vitro and proliferative signature in vivo. These analyses provide new insight into human musculoskeletal development and provide an essential comparative resource for disease modeling and regenerative medicine.

Extracellular matrix protein production in human adipose-derived mesenchymal stem cells on three-dimensional polycaprolactone (PCL) scaffolds responds to GDF5 or FGF2.

PURPOSE: The poor healing potential of intra-articular ligament injuries drives a need for the development of novel, viable 'neo-ligament' alternatives. Ex vivo approaches combining stem cell engineering, 3-dimensional biocompatible scaffold design and enhancement of biological and biomechanical functionality via the introduction of key growth factors and morphogens, represent a promising solution to ligament regeneration. METHODS: We investigated growth, differentiation and extracellular matrix (ECM) protein production of human adipose-derived mesenchymal stem/stromal cells (MSCs), cultured in 5% human platelet lysate (PL) and seeded on three-dimensional polycaprolactone (PCL) scaffolds, in response to the connective-tissue related ligands fibroblast growth factor 2 (basic) (FGF2) and growth and differentiation factor-5 (GDF5). Phenotypic alterations of MSCs under different biological conditions were examined using cell viability assays, real time qPCR analysis of total RNA, as well as immunofluorescence microscopy. RESULTS: Phenotypic conversion of MSCs into ECM producing fibroblastic cells proceeds spontaneously in the presence of human platelet lysate. Administration of FGF2 and/or GDF5 enhances production of mRNAs for several ECM proteins including Collagen types I and III, as well as Tenomodulin (e.g., COL1A1, TNMD), but not Tenascin-C (TNC). Differences in the in situ deposition of ECM proteins Collagen type III and Tenascin-C were validated by immunofluorescence microscopy. SUMMARY: Treatment of MSCs with FGF2 and GDF5 was not synergistic and occasionally antagonistic for ECM production. Our results suggest that GDF5 alone enhances the conversion of MSCs to fibroblastic cells possessing a phenotype consistent with that of connective-tissue fibroblasts.

Expression of the ectodomain-releasing protease ADAM17 is directly regulated by the osteosarcoma and bone-related transcription factor RUNX2.

Osteoblast differentiation is controlled by transcription factor RUNX2 which temporally activates or represses several bone-related genes, including those encoding extracellular matrix proteins or factors that control cell-cell, and cell-matrix interactions. Cell-cell communication in the many skeletal pericellular micro-niches is critical for bone development and involves paracrine secretion of growth factors and morphogens. This paracrine signaling is in part regulated by "A Disintegrin And Metalloproteinase" (ADAM) proteins. These cell membrane-associated metalloproteinases support proteolytic release ("shedding") of protein ectodomains residing at the cell surface. We analyzed microarray and RNA-sequencing data for Adam genes and show that Adam17, Adam10, and Adam9 are stimulated during BMP2 mediated induction of osteogenic differentiation and are robustly expressed in human osteoblastic cells. ADAM17, which was initially identified as a tumor necrosis factor alpha (TNFα) converting enzyme also called (TACE), regulates TNFα-signaling pathway, which inhibits osteoblast differentiation. We demonstrate that Adam17 expression is suppressed by RUNX2 during osteoblast differentiation through the proximal Adam17 promoter region (-0.4 kb) containing two functional RUNX2 binding motifs. Adam17 downregulation during osteoblast differentiation is paralleled by increased RUNX2 expression, cytoplasmic-nuclear translocation and enhanced binding to the Adam17 proximal promoter. Forced expression of Adam17 reduces Runx2 and Alpl expression, indicating that Adam17 may negatively modulate osteoblast differentiation. These findings suggest a novel regulatory mechanism involving a reciprocal Runx2-Adam17 negative feedback loop to regulate progression through osteoblast differentiation. Our results suggest that RUNX2 may control paracrine signaling through regulation of ectodomain shedding at the cell surface of osteoblasts by directly suppressing Adam17 expression.

Enhancer of zeste homolog 2 (Ezh2) controls bone formation and cell cycle progression during osteogenesis in mice.

Epigenetic mechanisms control skeletal development and osteoblast differentiation. Pharmacological inhibition of the histone 3 Lys-27 (H3K27) methyltransferase enhancer of zeste homolog 2 (EZH2) in WT mice enhances osteogenesis and stimulates bone formation. However, conditional genetic loss of Ezh2 early in the mesenchymal lineage (i.e. through excision via Prrx1 promoter-driven Cre) causes skeletal abnormalities due to patterning defects. Here, we addressed the key question of whether Ezh2 controls osteoblastogenesis at later developmental stages beyond patterning. We show that Ezh2 loss in committed pre-osteoblasts by Cre expression via the osterix/Sp7 promoter yields phenotypically normal mice. These Ezh2 conditional knock-out mice (Ezh2 cKO) have normal skull bones, clavicles, and long bones but exhibit increased bone marrow adiposity and reduced male body weight. Remarkably, in vivo Ezh2 loss results in a low trabecular bone phenotype in young mice as measured by micro-computed tomography and histomorphometry. Thus, Ezh2 affects bone formation stage-dependently. We further show that Ezh2 loss in bone marrow-derived mesenchymal cells suppresses osteogenic differentiation and impedes cell cycle progression as reflected by decreased metabolic activity, reduced cell numbers, and changes in cell cycle distribution and in expression of cell cycle markers. RNA-Seq analysis of Ezh2 cKO calvaria revealed that the cyclin-dependent kinase inhibitor Cdkn2a is the most prominent cell cycle target of Ezh2 Hence, genetic loss of Ezh2 in mouse pre-osteoblasts inhibits osteogenesis in part by inducing cell cycle changes. Our results suggest that Ezh2 serves a bifunctional role during bone formation by suppressing osteogenic lineage commitment while simultaneously facilitating proliferative expansion of osteoprogenitor cells.

Molecular characterization of physis tissue by RNA sequencing.

The physis is a well-established and anatomically distinct cartilaginous structure that is crucial for normal long-bone development and growth. Abnormalities in physis function are linked to growth plate disorders and other pediatric musculoskeletal diseases. Understanding the molecular pathways operative in the physis may permit development of regenerative therapies to complement surgically-based procedures that are the current standard of care for growth plate disorders. Here, we performed next generation RNA sequencing on mRNA isolated from human physis and other skeletal tissues (e.g., articular cartilage and bone; n = 7 for each tissue). We observed statistically significant enrichment of gene sets in the physis when compared to the other musculoskeletal tissues. Further analysis of these upregulated genes identified physis-specific networks of extracellular matrix proteins including collagens (COL2A1, COL6A1, COL9A1, COL14A1, COL16A1) and matrilins (MATN1, MATN2, MATN3), and signaling proteins in the WNT pathway (WNT10B, FZD1, FZD10, DKK2) or the FGF pathway (FGF10, FGFR4). Our results provide further insight into the gene expression networks that contribute to the physis' unique structural composition and regulatory signaling networks. Physis-specific expression profiles may guide ongoing initiatives in tissue engineering and cell-based therapies for treatment of growth plate disorders and growth modulation therapies. Furthermore, our findings provide new leads for therapeutic drug discovery that would permit future intervention through pharmacological rather than surgical strategies.

VEGF-mediated angiogenesis and vascularization of a fumarate-crosslinked polycaprolactone (PCLF) scaffold.

PURPOSE: Revascularization of natural and synthetic scaffolds is a critical part of the scaffold's incorporation and tissue ingrowth. Our goals were to create a biocompatible polymer scaffold with 3D-printing technology, capable of sustaining vascularization and tissue ingrowth. METHODS: We synthesized biodegradable polycaprolactone fumarate (PCLF) scaffolds to allow tissue ingrowth via large interconnected pores. The scaffolds were prepared with Poly(lactic-co-glycolic acid)(PLGA) microspheres seeded with or without different growth factors including VEGF,FGF-2, and/or BMP-2. Scaffolds were implanted into the subcutaneous tissues of rats before undergoing histologic and microCT angiographic analysis. RESULTS: At harvest after 12 weeks, scaffolds had tissue infiltrating into their pores without signs of scar tissue formation, fibrous capsule formation, or immune responses against PCLF. Histology for M1/M2 macrophage phenotypes confirmed that there were no overt signs of immune responses. Both microCT angiography and histologic analysis demonstrated marked tissue and vessel ingrowth throughout the pores traversing the body of the scaffolds. Scaffolds seeded with microspheres containing VEGF or VEGF with either BMP-2 or FGF-2 had significantly higher vascular ingrowth and vessel penetration than controls. All VEGF-augmented scaffolds were positive for Factor-VIII and exhibited collagen tissue infiltration throughout the pores. Furthermore, scaffolds with VEGF and BMP-2 had high levels of mineral deposition throughout the scaffold that are attributable to BMP-2. CONCLUSIONS: PCLF polymer scaffold can be utilized as a framework for vascular ingrowth and regeneration of multiple types of tissues. This novel scaffold material has promise in tissue regeneration across all types of tissues from soft tissue to bone.

Animal models for studying the etiology and treatment of low back pain.

Chronic 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.

RNA sequencing identifies gene regulatory networks controlling extracellular matrix synthesis in intervertebral disk tissues.

Degenerative 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.

Histone Deacetylase 3 Deletion in Mesenchymal Progenitor Cells Hinders Long Bone Development.

Long bone formation is a complex process that requires precise transcriptional control of gene expression programs in mesenchymal progenitor cells. Histone deacetylases (Hdacs) coordinate chromatin structure and gene expression by enzymatically removing acetyl groups from histones and other proteins. Hdac inhibitors are used clinically to manage mood disorders, cancers, and other conditions but are teratogenic to the developing skeleton and increase fracture risk in adults. In this study, the functions of Hdac3, one of the enzymes blocked by current Hdac inhibitor therapies, in skeletal mesenchymal progenitor cells were determined. Homozygous deletion of Hdac3 in Prrx1-expressing cells prevented limb lengthening, altered pathways associated with endochondral and intramembranous bone development, caused perinatal lethality, and slowed chondrocyte and osteoblast differentiation in vitro. Transcriptomic analysis revealed that Hdac3 regulates vastly different pathways in mesenchymal cells expressing the Prxx1-Cre driver than those expressing the Col2-CreERT driver. Notably, Fgf21 was elevated in Hdac3-CKOPrrx1 limbs as well as in chondrogenic cells exposed to Hdac3 inhibitors. Elevated expression of Mmp3 and Mmp10 transcripts was also observed. In conclusion, Hdac3 regulates distinct pathways in mesenchymal cell populations and is required for mesenchymal progenitor cell differentiation and long bone development. © 2017 American Society for Bone and Mineral Research.

Molecular Validation of Chondrogenic Differentiation and Hypoxia Responsiveness of Platelet-Lysate Expanded Adipose Tissue-Derived Human Mesenchymal Stromal Cells.

OBJECTIVE: To determine the optimal environmental conditions for chondrogenic differentiation of human adipose tissue-derived mesenchymal stromal/stem cells (AMSCs). In this investigation we specifically investigate the role of oxygen tension and 3-dimensional (3D) culture systems. DESIGN: Both AMSCs and primary human chondrocytes were cultured for 21 days in chondrogenic media under normoxic (21% oxygen) or hypoxic (2% oxygen) conditions using 2 distinct 3D culture methods (high-density pellets and poly-ε-caprolactone [PCL] scaffolds). Histologic analysis of chondro-pellets and the expression of chondrocyte-related genes as measured by reverse transcriptase quantitative polymerase chain reaction were used to evaluate the efficiency of differentiation. RESULTS: AMSCs are capable of expressing established cartilage markers including COL2A1, ACAN, and DCN when grown in chondrogenic differentiation media as determined by gene expression and histologic analysis of cartilage markers. Expression of several cartilage-related genes was enhanced by low oxygen tension, including ACAN and HAPLN1. The pellet culture environment also promoted the expression of hypoxia-inducible cartilage markers compared with cells grown on 3D scaffolds. CONCLUSIONS: Cell type-specific effects of low oxygen and 3D environments indicate that mesenchymal cell fate and differentiation potential is remarkably sensitive to oxygen. Genetic programming of AMSCs to a chondrocytic phenotype is effective under hypoxic conditions as evidenced by increased expression of cartilage-related biomarkers and biosynthesis of a glycosaminoglycan-positive matrix. Lower local oxygen levels within cartilage pellets may be a significant driver of chondrogenic differentiation.

Wnt/ß-Catenin Signaling Activates Expression of the Bone-Related Transcription Factor RUNX2 in Select Human Osteosarcoma Cell Types.

Osteosarcoma is the most common malignant bone tumor in children and adolescents. Metastasis and poor responsiveness to chemotherapy in osteosarcoma correlates with over-expression of the runt-related transcription factor RUNX2, which normally plays a key role in osteogenic lineage commitment, osteoblast differentiation, and bone formation. Furthermore, WNT/ß-catenin signaling is over-activated in osteosarcoma and promotes tumor progression. Importantly, the WNT/ß-catenin pathway normally activates RUNX2 gene expression during osteogenic lineage commitment. Therefore, we examined whether the WNT/ß-catenin pathway controls the tumor-related elevation of RUNX2 expression in osteosarcoma. We analyzed protein levels and nuclear localization of ß-catenin and RUNX2 in a panel of human osteosarcoma cell lines (SAOS, MG63, U2OS, HOS, G292, and 143B). In all six cell lines, ß-catenin and RUNX2 are expressed to different degrees and localized in the nucleus and/or cytoplasm. SAOS cells have the highest levels of RUNX2 protein that is localized in the nucleus, while MG63 cells have the lowest RUNX2 levels which is mostly localized in the cytoplasm. Levels of ß-catenin and RUNX2 protein are enhanced in HOS, G292, and 143B cells after treatment with the GSK3ß inhibitor SB216763. Furthermore, small interfering RNA (siRNA)-mediated depletion of ß-catenin inhibits RUNX2 expression in G292 cells. Thus, WNT/ß-catenin activation is required for RUNX2 expression in at least some osteosarcoma cell types, where RUNX2 is known to promote expression of metastasis related genes. J. Cell. Biochem. 118: 3662-3674, 2017. © 2017 Wiley Periodicals, Inc.

Chondrocyte Attachment, Proliferation, and Differentiation on Three-Dimensional Polycaprolactone Fumarate Scaffolds.

Current treatment options for cartilage injuries are limited. The goals of this study are to create a biodegradable polymer scaffold with the capabilities of sustaining chondrocyte growth and proliferation, enable cell-to-cell communication and tissue regeneration through large pores, and assess the biological augmentation of the scaffold capabilities using platelet lysate (PL). We synthesized biodegradable polycaprolactone fumarate (PCLF) scaffolds to allow cell-cell communication through large interconnected pores. Molds were printed using a three-dimensional printer and scaffolds synthesized through UV crosslinking. Culture medium included alpha modified Eagle's media with either 10% fetal bovine serum (FBS) or 5% PL, a mixture of platelet release products, after being seeded onto scaffolds through a dynamic bioreactor. Assays included cellular proliferation (MTS), toxicity and viability (live/dead immunostaining), differentiation (glycosaminoglycan [GAG], alkaline phosphatase [ALP], and total collagen), and immunostaining for chondrogenic markers collagen II and Sox 9 (with collagen I as a negative control). The large interconnected pores (500 and 750 µm) enable cell-to-cell communication and cellular infiltration into the scaffolds, as the cells remained viable and proliferated for 2 weeks. Chondrocytes cultured in PL showed increased rates of proliferation when compared with FBS. The chondrogenic markers GAG and total collagen contents increased over 2 weeks at each time point, whereas the osteogenic marker ALP did not significantly change. Immunostaining at 2 and 4 weeks for the expression of chondrogenic markers Collagen II and Sox 9 was increased when compared with control human fibroblasts. These results show that the PCLF polymer scaffold enables chondrocytes to attach, proliferate, and retain their chondrogenic phenotypes, demonstrating potential in chondrocyte engineering and cartilage regeneration.

Inhibiting DNA-PKCS radiosensitizes human osteosarcoma cells.

Osteosarcoma survival rate has not improved over the past three decades, and the debilitating side effects of the surgical treatment suggest the need for alternative local control approaches. Radiotherapy is largely ineffective in osteosarcoma, indicating a potential role for radiosensitizers. Blocking DNA repair, particularly by inhibiting the catalytic subunit of DNA-dependent protein kinase (DNA-PKCS), is an attractive option for the radiosensitization of osteosarcoma. In this study, the expression of DNA-PKCS in osteosarcoma tissue specimens and cell lines was examined. Moreover, the small molecule DNA-PKCS inhibitor, KU60648, was investigated as a radiosensitizing strategy for osteosarcoma cells in vitro. DNA-PKCS was consistently expressed in the osteosarcoma tissue specimens and cell lines studied. Additionally, KU60648 effectively sensitized two of those osteosarcoma cell lines (143B cells by 1.5-fold and U2OS cells by 2.5-fold). KU60648 co-treatment also altered cell cycle distribution and enhanced DNA damage. Cell accumulation at the G2/M transition point increased by 55% and 45%, while the percentage of cells with >20 γH2AX foci were enhanced by 59% and 107% for 143B and U2OS cells, respectively. These results indicate that the DNA-PKCS inhibitor, KU60648, is a promising radiosensitizing agent for osteosarcoma.

Safety Studies for Use of Adipose Tissue-Derived Mesenchymal Stromal/Stem Cells in a Rabbit Model for Osteoarthritis to Support a Phase I Clinical Trial.

Adipose-derived mesenchymal stem cells (AMSCs) offer potential as a therapeutic option for clinical applications in musculoskeletal regenerative medicine because of their immunomodulatory functions and capacity for trilineage differentiation. In preparation for a phase I clinical trial using AMSCs to treat patients with osteoarthritis, we carried out preclinical studies to assess the safety of human AMSCs within the intra-articular joint space. Culture-expanded human AMSCs grown in human platelet-lysate were delivered via intra-articular injections into normal healthy rabbit knees and knees at risk for the development of osteoarthritis after bilateral medial anterior hemimeniscectomy. Treatment outcomes and safety were evaluated by assessing the general health, function, and behavior of the animals. Joint tissues were analyzed by x-ray, magnetic resonance imaging, and histopathology. Intra-articular AMSC therapy was well tolerated in this study. We did not observe adverse systemic reactions, nor did we find evidence of damage to intra-articular joint tissues. Thus, the data generated in this study show a favorable safety profile for AMSCs within the joint space in support of a phase I clinical trial evaluating the clinical utility of AMSCs to treat osteoarthritis. Stem Cells Translational Medicine 2017;6:910-922.

Molecular landscape of arthrofibrosis: Microarray and bioinformatic analysis of the temporal expression of 380 genes during contracture genesis.

PURPOSE: Inflammatory changes are suspected in the pathophysiology of arthrofibrosis formation and require early molecular examination. Here, we assessed the hypothesis that early inflammatory genes are related to arthrofibrosis by ascertaining gene expression during the early stages of contracture genesis in an animal model. METHODS: Joint trauma was incited surgically in a cohort of rabbits (n=36) knees followed by immobilization in a model of contracture. Six groups of 6 rabbits were sacrificed at multiple time points (0, 6, 12, 24, 72h and 2weeks). Microarray expression and RT-qPCR profiling were performed to determine genes that are significantly up or downregulated. Bioinformatic analysis was carried out to understand which biological programs and functional groups of genes are modulated in arthrofibrosis. RESULTS: Gene expression profiling revealed a large number biologically relevant genes (>100) that are either upregulated or downregulated by at least a 1.5 fold (log2) during the first two weeks after joint injury during contracture development. Gene ontology analysis identified molecular pathways and programs that act during the course of fibrosis and joint contracture. Our main finding is that the development of contractures occur concomitant with modulation of genes mediating inflammatory responses, ECM remodeling and the epithelial-to-mesenchymal transition. CONCLUSION: The genesis of joint contracture reflects an imbalance between pro- and anti-fibrotic expression. Our study indicates that inflammatory genes may be involved in the process of contracture genesis and initiated at relatively early stages. Our findings also may inform clinical practice in the future by suggesting potential therapeutic targets in preventing the long-term development of arthrofibrosis.

Hypoxia-related microRNA-210 is a diagnostic marker for discriminating osteoblastoma and osteosarcoma.

Osteoblastoma is a benign bone tumor that can often be difficult to distinguish from malignant osteosarcoma. Because misdiagnosis can result in unfavorable clinical outcomes, we have investigated microRNAs as potential diagnostic biomarkers for distinguishing between these two tumor types. Next generation RNA sequencing was used as an expression screen to evaluate >2,000 microRNAs present in tissue derived from rare formalin fixed paraffin embedded (FFPE) archival tumor specimens. MicroRNAs displaying the greatest ability to discriminate between these two tumors were validated on an independent tumor set, using qPCR assays. Initial screening by RNA-seq identified four microRNA biomarker candidates. Expression of three miRNAs (miR-451a, miR-144-3p, miR-486-5p) was higher in osteoblastoma, while the miR-210 was elevated in osteosarcoma. Validation of these microRNAs on an independent data set of 22 tumor specimens by qPCR revealed that miR-210 is the most discriminating marker. This microRNA displays low levels of expression across all of the osteoblastoma specimens and robust expression in the majority of the osteosarcoma specimens. Application of these biomarkers to a clinical test case showed that these microRNA biomarkers permit re-classification of a misdiagnosed FFPE tumor sample from osteoblastoma to osteosarcoma. Our findings establish that the hypoxia-related miR-210 is a discriminatory marker that distinguishes between osteoblastoma and osteosarcoma. This discovery provides a complementary molecular approach to support pathological classification of two diagnostically challenging musculoskeletal tumors. Because miR-210 is linked to the cellular hypoxia response, its detection may be linked to well-established pro-angiogenic and metastatic roles of hypoxia in osteosarcomas and other tumor cell types. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1137-1146, 2017.