Publications by authors named "Benjamin Alman"

156 Publications

Tumor propagating side population cells are a dynamic subpopulation in undifferentiated pleomorphic sarcoma.

JCI Insight 2021 Oct 7. Epub 2021 Oct 7.

Department of Orthopedic Surgery, Duke University School of Medicine, Durham, United States of America.

Sarcomas contain a subpopulation of tumor propagating cells (TPCs) with enhanced tumor-initiating and self-renewal properties. However, it is unclear whether the TPC phenotype in sarcomas is stable or a dynamic cell state that can derive from non-tumor propagating cells (non-TPCs). In this study, we utilized a mouse model of undifferentiated pleomorphic sarcoma (UPS) to trace the lineage relationship between sarcoma side population (SP) cells that are enriched for TPCs and non-side population (non-SP) cells. By co-transplanting SP and non-SP cells expressing different endogenous fluorescent reporters, we show that non-SP cells can give rise to SP cells with enhanced tumor propagating potential in-vivo. Lineage trajectory analysis using single-cell RNA sequencing from SP and non-SP cells supports the notion that non-SP cells can assume the SP cell phenotype de novo. To test the effect of eradicating SP cells on tumor growth and self-renewal, we generated mouse sarcomas in which the Diphtheria Toxin Receptor (DTR) is expressed in the SP cells and their progeny. Ablation of the SP population using diphtheria toxin (DT) did not impede tumor growth or self-renewal. Together, we show that sarcoma SP is a dynamic cell state and targeting TPCs alone is insufficient to eliminate tumor progression.
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http://dx.doi.org/10.1172/jci.insight.148768DOI Listing
October 2021

Growth Modulation by Stimulating the Growth Plate: A Pilot Study.

Ultrasound Med Biol 2021 08 17;47(8):2339-2345. Epub 2021 May 17.

Department of Orthopaedics, Duke University Medical Center, Durham, NC, USA. Electronic address:

This study investigates the ability of low-intensity pulsed ultrasound (LIPUS) or direct injection of recombinant growth hormone (rGH) to stimulate local growth of long bones. In a randomized controlled animal trial, healthy immature rabbits were allocated to 1 of the following 4 conditions: epiphyseal rGH periosteal injection, transdermal LIPUS, saline periosteal injection, or no treatment. New bone deposition was labeled with calcein at days 1 and 18, and microscopic measurements of growth were conducted by blinded observers. Statistically significant differences in growth were observed between the LIPUS and rGH stimulated legs compared with contralateral control legs (35% p = 0.04 and 41% p = 0.04, respectively); whereas no difference was observed between the 4 control groups (p = 0.37). There was no evidence of physeal bar formation, suggesting that direct injection of rGH and application of LIPUS around the distal femoral physis in rabbits may have a positive effect on microscopic growth without short-term adverse sequelae.
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http://dx.doi.org/10.1016/j.ultrasmedbio.2021.03.036DOI Listing
August 2021

Mutant IDH and non-mutant chondrosarcomas display distinct cellular metabolomes.

Cancer Metab 2021 Mar 24;9(1):13. Epub 2021 Mar 24.

Department of Orthopaedic Surgery, Duke University, 311 Trent, Durham, NC, 27710, USA.

Background: Majority of chondrosarcomas are associated with a number of genetic alterations, including somatic mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 genes, but the downstream effects of these mutated enzymes on cellular metabolism and tumor energetics are unknown. As IDH mutations are likely to be involved in malignant transformation of chondrosarcomas, we aimed to exploit metabolomic changes in IDH mutant and non-mutant chondrosarcomas.

Methods: Here, we profiled over 69 metabolites in 17 patient-derived xenografts by targeted mass spectrometry to determine if metabolomic differences exist in mutant IDH1, mutant IDH2, and non-mutant chondrosarcomas. UMAP (Uniform Manifold Approximation and Projection) analysis was performed on our dataset to examine potential similarities that may exist between each chondrosarcoma based on genotype.

Results: UMAP revealed that mutant IDH chondrosarcomas possess a distinct metabolic profile compared with non-mutant chondrosarcomas. More specifically, our targeted metabolomics study revealed large-scale differences in organic acid intermediates of the tricarboxylic acid (TCA) cycle, amino acids, and specific acylcarnitines in chondrosarcomas. Lactate and late TCA cycle intermediates were elevated in mutant IDH chondrosarcomas, suggestive of increased glycolytic metabolism and possible anaplerotic influx to the TCA cycle. A broad elevation of amino acids was found in mutant IDH chondrosarcomas. A few acylcarnitines of varying carbon chain lengths were also elevated in mutant IDH chondrosarcomas, but with minimal clustering in accordance with tumor genotype. Analysis of previously published gene expression profiling revealed increased expression of several metabolism genes in mutant IDH chondrosarcomas, which also correlated to patient survival.

Conclusions: Overall, our findings suggest that IDH mutations induce global metabolic changes in chondrosarcomas and shed light on deranged metabolic pathways.
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http://dx.doi.org/10.1186/s40170-021-00247-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7992867PMC
March 2021

Monocyte/Macrophage Lineage Cells From Fetal Erythromyeloid Progenitors Orchestrate Bone Remodeling and Repair.

Front Cell Dev Biol 2021 4;9:622035. Epub 2021 Feb 4.

Department of Orthopaedic Surgery, Duke University School of Medicine, Durham, NC, United States.

A third of the population sustains a bone fracture, and the pace of fracture healing slows with age. The slower pace of repair is responsible for the increased morbidity in older individuals who sustain a fracture. Bone healing progresses through overlapping phases, initiated by cells of the monocyte/macrophage lineage. The repair process ends with remodeling. This last phase is controlled by osteoclasts, which are bone-specific multinucleated cells also of the monocyte/macrophage lineage. The slower rate of healing in aging can be rejuvenated by macrophages from young animals, and secreted proteins from macrophage regulate undifferentiated mesenchymal cells to become bone-forming osteoblasts. Macrophages can derive from fetal erythromyeloid progenitors or from adult hematopoietic progenitors. Recent studies show that fetal erythromyeloid progenitors are responsible for the osteoclasts that form the space in bone for hematopoiesis and the fetal osteoclast precursors reside in the spleen postnatally, traveling through the blood to participate in fracture repair. Differences in secreted proteins between macrophages from old and young animals regulate the efficiency of osteoblast differentiation from undifferentiated mesenchymal precursor cells. Interestingly, during the remodeling phase osteoclasts can form from the fusion between monocyte/macrophage lineage cells from the fetal and postnatal precursor populations. Data from single cell RNA sequencing identifies specific markers for populations derived from the different precursor populations, a finding that can be used in future studies. Here, we review the diversity of macrophages and osteoclasts, and discuss recent finding about their developmental origin and functions, which provides novel insights into their roles in bone homeostasis and repair.
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http://dx.doi.org/10.3389/fcell.2021.622035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7889961PMC
February 2021

Enchondromatosis and Growth Plate Development.

Curr Osteoporos Rep 2021 02 11;19(1):40-49. Epub 2020 Dec 11.

Department of Orthopaedic Surgery, Duke University School of Medicine, Durham, NC, 27710, USA.

Purpose Of Review: Enchondroma is a common cartilage benign tumor that develops from dysregulation of chondrocyte terminal differentiation during growth plate development. Here we provide an overview of recent progress in understanding causative mutations for enchondroma, dysregulated signaling and metabolic pathways in enchondroma, and the progression from enchondroma to malignant chondrosarcoma.

Recent Findings: Several signaling pathways that regulate chondrocyte differentiation are dysregulated in enchondromas. Somatic mutations in the metabolic enzymes isocitrate dehydrogenase 1 and 2 (IDH1/2) are the most common findings in enchondromas. Mechanisms including metabolic regulation, epigenetic regulation, and altered signaling pathways play a role in enchondroma formation and progression. Multiple pathways regulate growth plate development in a coordinated manner. Deregulation of the process can result in chondrocytes failing to undergo differentiation and the development of enchondroma.
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http://dx.doi.org/10.1007/s11914-020-00639-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7935756PMC
February 2021

Parabiosis: Assessing the Effects of Circulating Cells and Factors on the Skeleton.

Methods Mol Biol 2021 ;2230:105-113

Department of Orthopaedic Surgery, Duke Molecular Physiology Institute, Duke University, Durham, NC, USA.

The circulatory system carries within it numerous types of cells, proteins, and other factors that are able to influence the local biology of tissues. Within this chapter, we present a protocol for parabiosis, a surgical model which results in shared circulation between two mice. Such chimeras have recently been used to probe the impact of age-associated changes in the circulation on skeletal, muscular, and neural biology. In conjunction with transgenic mouse models, parabiosis can be used as a tool to investigate the effects of specific factors on local tissues. Here we discuss our adaptation of this surgical procedure including technique details, pitfalls, and suggestions for optimization.
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http://dx.doi.org/10.1007/978-1-0716-1028-2_7DOI Listing
March 2021

The Calpain Gene is Correlated With Metal-on-Metal Hip Replacement Failures.

J Arthroplasty 2021 01 30;36(1):236-241.e3. Epub 2020 Jul 30.

Department of Orthopaedic Surgery, Duke University Medical Center, Durham, NC.

Background: Metal-on-metal (MOM) total hip arthroplasty is associated with unacceptable failure rates secondary to metal ion reactions. Efforts to identify which patients will go on to failure have been limited; recently, there has been a suggestion for a potential genetic basis for the increased risk of revision in MOM hip replacements (MOMHRs). The purpose of this study is to determine whether certain immunologic genotypes are predictive of the need for revision in patients with MOM total hip implants.

Methods: This is a case-control study of all patients undergoing primary MOMHR between September 2002 and January 2012 with a minimum of 5-year follow-up. Our investigational "case" cohort was comprised of patients who underwent revision for MOMHR for a reason other than infection. A single-nucleotide polymorphism (SNP) array analysis was performed to identify a potential genetic basis for failure.

Results: Thirty-two patients (15 case and 17 control) were included in our analysis. All patients in the revision group had a chief complain of pain; revision patients were more likely to have a posterior approach (P = .01) and larger head size (P = .04) than nonrevision patients. No patient or implant characteristics were independently associated with revision in a multivariate analysis. Patients with SNP kgp9316441 were identified as having an increased odds of revision for MOM failure (P < .001).

Conclusion: This study identified an SNP, kgp9316441, encoding proteins associated with inflammation and macrophage activation. This SNP was associated with significantly increased odds of revision for MOMHR. Future studies are warranted to validate this gene target both in vitro and in vivo.

Level Of Evidence: III.
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http://dx.doi.org/10.1016/j.arth.2020.07.054DOI Listing
January 2021

Yolk-sac-derived macrophages progressively expand in the mouse kidney with age.

Elife 2020 04 17;9. Epub 2020 Apr 17.

Division of Nephrology, Department of Medicine, Duke University School of Medicine, Durham, United States.

Renal macrophages represent a highly heterogeneous and specialized population of myeloid cells with mixed developmental origins from the yolk-sac and hematopoietic stem cells (HSC). They promote both injury and repair by regulating inflammation, angiogenesis, and tissue remodeling. Recent reports highlight differential roles for ontogenically distinct renal macrophage populations in disease. However, little is known about how these populations change over time in normal, uninjured kidneys. Prior reports demonstrated a high proportion of HSC-derived macrophages in the young adult kidney. Unexpectedly, using genetic fate-mapping and parabiosis studies, we found that yolk-sac-derived macrophages progressively expand in number with age and become a major contributor to the renal macrophage population in older mice. This chronological shift in macrophage composition involves local cellular proliferation and recruitment from circulating progenitors and may contribute to the distinct immune responses, limited reparative capacity, and increased disease susceptibility of kidneys in the elderly population.
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http://dx.doi.org/10.7554/eLife.51756DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7205460PMC
April 2020

Erythromyeloid progenitors give rise to a population of osteoclasts that contribute to bone homeostasis and repair.

Nat Cell Biol 2020 01 6;22(1):49-59. Epub 2020 Jan 6.

Department of Orthopaedic Surgery and Regeneration Next Initiative, Duke University, Durham, NC, USA.

Osteoclasts are multinucleated cells of the monocyte/macrophage lineage that degrade bone. Here, we used lineage tracing studies-labelling cells expressing Cx3cr1, Csf1r or Flt3-to identify the precursors of osteoclasts in mice. We identified an erythromyeloid progenitor (EMP)-derived osteoclast precursor population. Yolk-sac macrophages of EMP origin produced neonatal osteoclasts that can create a space for postnatal bone marrow haematopoiesis. Furthermore, EMPs gave rise to long-lasting osteoclast precursors that contributed to postnatal bone remodelling in both physiological and pathological settings. Our single-cell RNA-sequencing data showed that EMP-derived osteoclast precursors arose independently of the haematopoietic stem cell (HSC) lineage and the data from fate tracking of EMP and HSC lineages indicated the possibility of cell-cell fusion between these two lineages. Cx3cr1 yolk-sac macrophage descendants resided in the adult spleen, and parabiosis experiments showed that these cells migrated through the bloodstream to the remodelled bone after injury.
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http://dx.doi.org/10.1038/s41556-019-0437-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6953622PMC
January 2020

Complications After Pediatric Hip Fractures: Evaluation and Management.

J Am Acad Orthop Surg 2020 Jan;28(1):10-19

From the Department of Orthopaedic Surgery, Duke University Medical Center, Durham, NC.

Pediatric proximal femur fractures are high-energy injuries with predictable and morbid complications. Osteonecrosis of the femoral head is the most common complication with identified risk factors including fracture type, patient's age, degree of displacement, timing to reduction, and stability of fixation. Additional complications include nonunion, coxa vara, and premature physeal arrest. The mainstay of treatment for traumatic pediatric osteonecrosis is hip preservation with total hip arthroplasty being reserved as a salvage procedure. An anatomic fracture reduction and a biomechanically stable construct are critical to prevent both nonunion and osteonecrosis. This review will look at the individual surgical interventions for the management of the associated complications of pediatric proximal femur fractures.
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http://dx.doi.org/10.5435/JAAOS-D-17-00689DOI Listing
January 2020

Tracing Tumor Evolution in Sarcoma Reveals Clonal Origin of Advanced Metastasis.

Cell Rep 2019 09;28(11):2837-2850.e5

Department of Orthopedic Surgery, Duke University School of Medicine, Durham, NC, USA; Department of Cell Biology, Duke University School of Medicine, Durham, NC, USA; Duke Cancer Institute, Duke University, Durham, NC, USA; Regeneration Next Initiative, Duke University, Durham, NC, USA; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada. Electronic address:

Cellular heterogeneity is frequently observed in cancer, but the biological significance of heterogeneous tumor clones is not well defined. Using multicolor reporters and CRISPR-Cas9 barcoding, we trace clonal dynamics in a mouse model of sarcoma. We show that primary tumor growth is associated with a reduction in clonal heterogeneity. Local recurrence of tumors following surgery or radiation therapy is driven by multiple clones. In contrast, advanced metastasis to the lungs is driven by clonal selection of a single metastatic clone (MC). Using RNA sequencing (RNA-seq) and in vivo assays, we identify candidate suppressors of metastasis, namely, Rasd1, Reck, and Aldh1a2. These genes are downregulated in MCs of the primary tumors prior to the formation of metastases. Overexpression of these suppressors of metastasis impair the ability of sarcoma cells to colonize the lungs. Overall, this study reveals clonal dynamics during each step of tumor progression, from initiation to growth, recurrence, and distant metastasis.
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http://dx.doi.org/10.1016/j.celrep.2019.08.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6750751PMC
September 2019

Pharmacologic targeting of β-catenin improves fracture healing in old mice.

Sci Rep 2019 06 21;9(1):9005. Epub 2019 Jun 21.

Department of Orthopaedic Surgery, Duke University, Durham, NC, USA.

β-catenin protein needs to be precisely regulated for effective fracture repair. The pace of fracture healing slows with age, associated with a transient increase in β-catenin during the initial phase of the repair process. Here we examined the ability of pharmacologic agents that target β-catenin to improve the quality of fracture repair in old mice. 20 month old mice were treated with Nefopam or the tankyrase inhibitor XAV939 after a tibia fracture. Fractures were examined 21 days later by micro-CT and histology, and 28 days later using mechanical testing. Daily treatment with Nefopam for three or seven days but not ten days improved the amount of bone present at the fracture site, inhibited β-catenin protein level, and increased colony forming units osteoblastic from bone marrow cells. At 28 days, treatment increased the work to fracture of the injured tibia. XAV939 had a more modest effect on β-catenin protein, colony forming units osteoblastic, and the amount of bone at the fracture site. This data supports the notion that high levels of β-catenin in the early phase of fracture healing in old animals slows osteogenesis, and suggests a pharmacologic approach that targets β-catenin to improve fracture repair in the elderly.
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http://dx.doi.org/10.1038/s41598-019-45339-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6588693PMC
June 2019

Intracellular cholesterol biosynthesis in enchondroma and chondrosarcoma.

JCI Insight 2019 04 30;5. Epub 2019 Apr 30.

Department of Cell Biology and.

Enchondroma and chondrosarcoma are the most common benign and malignant cartilaginous neoplasms. Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) are present in the majority of these tumors. We performed RNA-seq analysis on chondrocytes from Col2a1Cre;Idh1LSL/+ animals and found that genes implied in cholesterol synthesis pathway were significantly upregulated in the mutant chondrocytes. We examined the phenotypic effect of inhibiting intracellular cholesterol biosynthesis on enchondroma formation by conditionally deleting SCAP (sterol regulatory element-binding protein cleavage-activating protein), a protein activating intracellular cholesterol synthesis, in IDH1 mutant mice. We found fewer enchondromas in animals lacking SCAP. Furthermore, in chondrosarcomas, pharmacological inhibition of intracellular cholesterol synthesis significantly reduced chondrosarcoma cell viability in vitro and suppressed tumor growth in vivo. Taken together, these data suggest that intracellular cholesterol synthesis is a potential therapeutic target for enchondromas and chondrosarcomas.
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http://dx.doi.org/10.1172/jci.insight.127232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6629100PMC
April 2019

Unique and overlapping GLI1 and GLI2 transcriptional targets in neoplastic chondrocytes.

PLoS One 2019 29;14(1):e0211333. Epub 2019 Jan 29.

Department of Orthopaedic Surgery, Duke University, Durham, North Carolina, United States of America.

Excessive Hedgehog (Hh) signaling in chondrocytes is sufficient to cause formation of enchondroma-like lesions which can progress to chondrosarcoma. To elucidate potential underlying mechanisms, we identified GLI1 and GLI2 target genes in human chondrosarcoma. Using chromatin immunoprecipitation (ChIP) sequencing and microarray data, in silico analyses were conducted to identify and characterize unique and overlapping GLI1 and GLI2 binding regions in neoplastic chondrocytes. After overlaying microarray data from human chondrosarcoma, 204 upregulated and 106 downregulated genes were identified as Hh-responsive Gli binding targets. After overlaying published Gli ChIP-on-chip data from mouse, 48 genes were identified as potential direct downstream targets of Hedgehog signaling with shared GLI binding regions in evolutionarily conserved DNA elements. Among these was BMP2, pointing to potential cross-talk between TGF beta signaling and Hh signaling. Our identification of potential target genes that are unique and common to GLI1 and GLI2 in neoplastic chondrocytes contributes to elucidating potential pathways through which Hh signaling impacts cartilage tumor biology.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0211333PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6350985PMC
October 2019

Macrophage cells secrete factors including LRP1 that orchestrate the rejuvenation of bone repair in mice.

Nat Commun 2018 12 5;9(1):5191. Epub 2018 Dec 5.

Department of Orthopaedic Surgery, Duke University, Durham, 27710, USA.

The pace of repair declines with age and, while exposure to a young circulation can rejuvenate fracture repair, the cell types and factors responsible for rejuvenation are unknown. Here we report that young macrophage cells produce factors that promote osteoblast differentiation of old bone marrow stromal cells. Heterochronic parabiosis exploiting young mice in which macrophages can be depleted and fractionated bone marrow transplantation experiments show that young macrophages rejuvenate fracture repair, and old macrophage cells slow healing in young mice. Proteomic analysis of the secretomes identify differential proteins secreted between old and young macrophages, such as low-density lipoprotein receptor-related protein 1 (Lrp1). Lrp1 is produced by young cells, and depleting Lrp1 abrogates the ability to rejuvenate fracture repair, while treating old mice with recombinant Lrp1 improves fracture healing. Macrophages and proteins they secrete orchestrate the fracture repair process, and young cells produce proteins that rejuvenate fracture repair in mice.
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http://dx.doi.org/10.1038/s41467-018-07666-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6281653PMC
December 2018

Intracellular biosynthesis of lipids and cholesterol by Scap and Insig in mesenchymal cells regulates long bone growth and chondrocyte homeostasis.

Development 2018 07 9;145(13). Epub 2018 Jul 9.

Department of Orthopaedic Surgery and Regeneration Next Initiative, Duke University, Durham, NC 27710, USA

During enchondral ossification, mesenchymal cells express genes regulating the intracellular biosynthesis of cholesterol and lipids. Here, we have investigated conditional deletion of or of and (Scap inhibits intracellular biosynthesis and Insig proteins activate intracellular biosynthesis). Mesenchymal condensation and chondrogenesis was disrupted in mice lacking in mesenchymal progenitors, whereas mice lacking the Insig genes in mesenchymal progenitors had short limbs, but normal chondrogenesis. Mice lacking in chondrocytes showed severe dwarfism, with ectopic hypertrophic cells, whereas deletion of Insig genes in chondrocytes caused a mild dwarfism and shortening of the hypertrophic zone. studies showed that intracellular cholesterol in chondrocytes can derive from exogenous and endogenous sources, but that exogenous sources cannot completely overcome the phenotypic effect of deficiency. Genes encoding cholesterol biosynthetic proteins are regulated by Hedgehog (Hh) signaling, and Hh signaling is also regulated by intracellular cholesterol in chondrocytes, suggesting a feedback loop in chondrocyte differentiation. Precise regulation of intracellular biosynthesis is required for chondrocyte homeostasis and long bone growth, and these data support pharmacological modulation of cholesterol biosynthesis as a therapy for select cartilage pathologies.
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http://dx.doi.org/10.1242/dev.162396DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6053657PMC
July 2018

The Role of the Immune Cells in Fracture Healing.

Curr Osteoporos Rep 2018 04;16(2):138-145

Department of Orthopaedic Surgery, Duke Molecular Physiology Institute, Duke University, DUMC 104775, 300 North Duke Street, Durham, NC, 27701, USA.

Purpose Of Review: Bone fracture healing is a complex physiological process relying on numerous cell types and signals. Inflammatory factors secreted by immune cells help to control recruitment, proliferation, differentiation, and activation of hematopoietic and mesenchymal cells. Within this review we will discuss the functional role of immune cells as it pertains to bone fracture healing. In doing so, we will outline the cytokines secreted and their effects within the healing fracture callus.

Recent Findings: Macrophages have been found to play an important role in fracture healing. These immune cells signal to other cells of the fracture callus, modulating bone healing. Cytokines and cellular signals within fracture healing continue to be studied. The findings from this work have helped to reinforce the importance of osteoimmunity in bone fracture healing. Owing to these efforts, immunomodulation is emerging as a potential therapeutic target to improve bone fracture healing.
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http://dx.doi.org/10.1007/s11914-018-0423-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5866272PMC
April 2018

Diagnosis and management of Duchenne muscular dystrophy, part 2: respiratory, cardiac, bone health, and orthopaedic management.

Lancet Neurol 2018 04 3;17(4):347-361. Epub 2018 Feb 3.

Division of Endocrinology and Metabolism, Children's Hospital of Eastern Ontario, and University of Ottawa, Ottawa, ON, Canada.

A coordinated, multidisciplinary approach to care is essential for optimum management of the primary manifestations and secondary complications of Duchenne muscular dystrophy (DMD). Contemporary care has been shaped by the availability of more sensitive diagnostic techniques and the earlier use of therapeutic interventions, which have the potential to improve patients' duration and quality of life. In part 2 of this update of the DMD care considerations, we present the latest recommendations for respiratory, cardiac, bone health and osteoporosis, and orthopaedic and surgical management for boys and men with DMD. Additionally, we provide guidance on cardiac management for female carriers of a disease-causing mutation. The new care considerations acknowledge the effects of long-term glucocorticoid use on the natural history of DMD, and the need for care guidance across the lifespan as patients live longer. The management of DMD looks set to change substantially as new genetic and molecular therapies become available.
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http://dx.doi.org/10.1016/S1474-4422(18)30025-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5889091PMC
April 2018

Eight-year outcomes of a competency-based residency training program in orthopedic surgery.

Med Teach 2018 10 18;40(10):1042-1054. Epub 2018 Jan 18.

a Division of Orthopaedic Surgery, Department of Surgery, Faculty of Medicine , University of Toronto , Toronto , ON , Canada.

The Division of Orthopaedic Surgery at the University of Toronto implemented a pilot residency training program that used a competency-based framework in July of 2009. The competency-based curriculum (CBC) deployed an innovative, modularized approach that dramatically intensified both the structured learning elements and the assessment processes. This paper discusses the initial curriculum design of the CBC pilot program; the refinement of the curriculum using curriculum mapping that allowed for efficiencies in educational delivery; details of evaluating resident competence; feedback from external reviews by accrediting bodies; and trainee and program outcomes for the first eight years of the program's implementation. Feedback from the residents, the faculty, and the postgraduate residency training accreditation bodies on the CBC has been positive and suggests that the essential framework of the program may provide a valuable tool to other programs that are contemplating embarking on transition to competency-based education. While the goal of the program was not to shorten training per se, efficiencies gained through a modular, competency-based program have resulted in shortened time to completion of residency training for some learners.
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http://dx.doi.org/10.1080/0142159X.2017.1421751DOI Listing
October 2018

A Metabolomics Pilot Study on Desmoid Tumors and Novel Drug Candidates.

Sci Rep 2018 01 12;8(1):584. Epub 2018 Jan 12.

Developmental & Stem Cell Biology Program, Hospital for Sick Children, Toronto, ON, Canada.

Desmoid tumors (aggressive fibromatosis) are locally invasive soft tissue tumors that lack the ability to metastasize. There are no directed therapies or standard treatment plan, and chemotherapeutics, radiation, and surgery often have temporary effects. The majority of desmoid tumors are related to T41A and S45F mutations of the beta-catenin encoding gene (CTNNB1). Using broad spectrum metabolomics, differences were investigated between paired normal fibroblast and desmoid tumor cells from affected patients. There were differences identified, also, in the metabolomics profiles associated with the two beta-catenin mutations, T41A and S45F. Ongoing drug screening has identified currently available compounds which inhibited desmoid tumor cellular growth by more than 50% but did not affect normal fibroblast proliferation. Two drugs were investigated in this study, and Dasatinib and FAK Inhibitor 14 treatments resulted in unique metabolomics profiles for the normal fibroblast and desmoid tumor cells, in addition to the T41A and S45F. The biochemical pathways that differentiated the cell lines were aminoacyl-tRNA biosynthesis in mitochondria and cytoplasm and signal transduction amino acid-dependent mTORC1 activation. This study provides preliminary understanding of the metabolic differences of paired normal and desmoid tumors cells, their response to desmoid tumor therapeutics, and new pathways to target for therapy.
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http://dx.doi.org/10.1038/s41598-017-18921-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5766559PMC
January 2018

Effects of chondroitin sulfate proteoglycan 4 (NG2/CSPG4) on soft-tissue sarcoma growth depend on tumor developmental stage.

J Biol Chem 2018 02 1;293(7):2466-2475. Epub 2017 Dec 1.

From the Department of Orthopaedic Surgery and RegenerationNext Initiative and

Sarcomas, and the mesenchymal precursor cells from which they arise, express chondroitin sulfate proteoglycan 4 (NG2/CSPG4). However, NG2/CSPG4's function and its capacity to serve as a therapeutic target in this tumor type are unknown. Here, we used cells from human tumors and a genetically engineered autochthonous mouse model of soft-tissue sarcomas (STSs) to determine NG2/CSPG4's role in STS initiation and growth. Inhibiting NG2/CSPG4 expression in established murine and human STSs decreased tumor volume by almost two-thirds and cell proliferation rate by 50%. NG2/CSPG4 antibody immunotherapy in human sarcomas established as xenografts in mice similarly decreased tumor volume, and expression of a lentivirus blocking expression inhibited tumor cell proliferation and increased the latency of engraftment. Gene profiling showed that deletion altered the expression of genes regulating cell proliferation and apoptosis. Surprisingly, deletion at the time of tumor initiation resulted in larger tumors. Gene expression profiling indicated substantial down-regulation of insulin-like growth factor binding protein () genes when is depleted at tumor initiation, but not when is depleted after tumor initiation. Such differences may have clinical significance, as therapeutic targeting of a signaling pathway such as NG2/CSPG4 may have different effects on cell behavior with tumor progression. NG2/CSPG4 depletion has divergent effects, depending on the developmental stage of sarcoma. In established tumors, IGF signaling is active, and NG2 inhibition targets cell proliferation and apoptosis.
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http://dx.doi.org/10.1074/jbc.M117.805051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5818183PMC
February 2018

Summary of the first inaugural joint meeting of the International Consortium for scoliosis genetics and the International Consortium for vertebral anomalies and scoliosis, March 16-18, 2017, Dallas, Texas.

Am J Med Genet A 2018 01 21;176(1):253-256. Epub 2017 Nov 21.

Texas Scottish Rite Hospital for Children, Dallas, Texas.

Scoliosis represents the most common musculoskeletal disorder in children and affects approximately 3% of the world population. Scoliosis is separated into two major phenotypic classifications: congenital and idiopathic. Idiopathic scoliosis is defined as a curvature of the spine of 10° or greater visualized on plane radiograph and does not have associated vertebral malformations (VM). "Congenital" scoliosis (CS) due to malformations in vertebrae is frequently associated with other birth defects. Recently, significant advances have been made in understanding the genetic basis of both conditions. There is evidence that both conditions are etiologically related. A 2-day conference entitled "Genomic Approaches to Understanding and Treating Scoliosis" was held at Scottish Rite Hospital for Children in Dallas, Texas, to synergize research in this field. This first combined, multidisciplinary conference featured international scoliosis researchers in basic and clinical sciences. A major outcome of the conference advancing scoliosis research was the proposal and subsequent vote in favor of merging the International Consortium for Vertebral Anomalies and Scoliosis (ICVAS) and International Consortium for Scoliosis Genetics (ICSG) into a single entity called International Consortium for Spinal Genetics, Development, and Disease (ICSGDD). The ICSGDD is proposed to meet annually as a forum to synergize multidisciplinary spine deformity research.
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http://dx.doi.org/10.1002/ajmg.a.38550DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6525596PMC
January 2018

The Fourth Year of Medical School: Time for Reassessment: AOA Critical Issues.

J Bone Joint Surg Am 2017 Jul;99(13):e72

1Duke University, Durham, North Carolina 2Thomas Jefferson University, Philadelphia, Pennsylvania 3Medical University of South Carolina, Charleston, South Carolina.

Most U.S. medical schools follow the 4-year model, consisting of 2 preclinical years, core clinical experience, and a fourth year intended to permit students to increase clinical competency, to explore specialty areas, and to transition to residency. Although the design and delivery of Years 1 through 3 have evolved to meet new challenges and expectations, the structure of Year 4 remains largely unchanged. For most students considering a career in orthopaedics, Year 4 is a series of elective rotations in which educational objectives become secondary to interviewing for residency programs. Most accreditation bodies recognize the importance of attainment of competency over the duration of medical school as the goal of educating physicians, and thus, there is a growing interest in reexamining the traditional medical school curriculum with the goal of integrating the final phases of undergraduate education and the first phases of postgraduate education.A literature search was undertaken to identify publications on the duration of medical education. Pilot approaches to competency-based integration of undergraduate medical school and postgraduate training in orthopaedic surgery were reviewed.There have been few data suggesting that 4 years of medical education is superior to shorter-duration programs. Three approaches to competency-based integration of undergraduate medical school and postgraduate training are presented. Their goal is to use student and faculty time more effectively. Each approach offers the opportunity to lower the cost and to decrease the time required for Board Certification in Orthopaedic Surgery. Two approaches shorten the entire duration of medical school and graduate training by using various proportions of the fourth year to begin residency, and one approach expands the duration of orthopaedic training by starting in the fourth year of medical school and including training equivalent to a fellowship program into the residency experience.The effectiveness of such programs will form the basis for revisions to the current orthopaedic training paradigm, resulting in a more effective, efficient, and integrated orthopaedic training curriculum.
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http://dx.doi.org/10.2106/JBJS.16.01094DOI Listing
July 2017

Pharmacologically targeting beta-catenin for NF1 associated deficiencies in fracture repair.

Bone 2017 05 22;98:31-36. Epub 2017 Feb 22.

Department of Orthopaedic Surgery, Duke University, Durham, USA. Electronic address:

Patients with Neurofibromatosis type 1 display delayed fracture healing and the increased deposition of fibrous tissue at the fracture site. Severe cases can lead to non-union and even congenital pseudarthrosis. Neurofibromatosis type 1 is caused by a mutation in the NF1 gene and mice lacking the Nf1 gene show a fracture repair phenotype similar to that seen in patients. Tissue from the fracture site of patients with Neurofibromatosis type 1 and from mice deficient in the Nf1 gene both show elevated levels of β-catenin protein and activation of β-catenin mediated signaling. Constitutively elevated β-catenin leads to a delayed and fibrous fracture repair process, and (RS)-5-methyl-1-phenyl-1,3,4,6-tetrahydro-2,5-benzoxazocine (Nefopam, a centrally-acting, non-narcotic analgesic agent) inhibits β-catenin mediated signaling during skin wound repair. Here we investigate Nefopam's potential as a modulator of bone repair in mice deficient in Nf1. Mice were treated with Nefopam and investigated for bone fracture repair. Bone marrow stromal cells flushed from the long bones of unfractured mice were treated with Nefopam and investigated for osteogenic potential. Treatment with Nefopam was able to lower the β-catenin level and the Axin2 transcript level in the fracture calluses of Nf1 deficient mice. Cultures from the bone marrow of Nf1 mice had significantly lower osteoblastic colonies and mineralized nodules, which was increased when cells were cultured in the presence of Nefopam. Fracture calluses were harvested and analyzed 14days and 21days after injury. Nf1 calluses had less bone, less cartilage, and higher fibrous tissue content than control calluses. Treatment with Nefopam increased the bone and cartilage content and decreased the fibrous tissue content in Nf1 calluses. These findings present a potential treatment for patients with Neurofibromatosis 1 in the context of bone repair. Since Nefopam is already in use in patient care, it could be rapidly translated to the clinical setting.
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http://dx.doi.org/10.1016/j.bone.2017.02.012DOI Listing
May 2017

Phenotype Determines Nanoparticle Uptake by Human Macrophages from Liver and Blood.

ACS Nano 2017 03 17;11(3):2428-2443. Epub 2017 Jan 17.

Multi Organ Transplant Program, Toronto General Research Institute, University Health Network , 200 Elizabeth Street, Toronto, Ontario, Canada M5G 2C4.

A significant challenge to delivering therapeutic doses of nanoparticles to targeted disease sites is the fact that most nanoparticles become trapped in the liver. Liver-resident macrophages, or Kupffer cells, are key cells in the hepatic sequestration of nanoparticles. However, the precise role that the macrophage phenotype plays in nanoparticle uptake is unknown. Here, we show that the human macrophage phenotype modulates hard nanoparticle uptake. Using gold nanoparticles, we examined uptake by human monocyte-derived macrophages that had been driven to a "regulatory" M2 phenotype or an "inflammatory" M1 phenotype and found that M2-type macrophages preferentially take up nanoparticles, with a clear hierarchy among the subtypes (M2c > M2 > M2a > M2b > M1). We also found that stimuli such as LPS/IFN-γ rather than with more "regulatory" stimuli such as TGF-β/IL-10 reduce per cell macrophage nanoparticle uptake by an average of 40%. Primary human Kupffer cells were found to display heterogeneous expression of M1 and M2 markers, and Kupffer cells expressing higher levels of M2 markers (CD163) take up significantly more nanoparticles than Kupffer cells expressing lower levels of surface CD163. Our results demonstrate that hepatic inflammatory microenvironments should be considered when studying liver sequestration of nanoparticles, and that modifying the hepatic microenvironment might offer a tool for enhancing or decreasing this sequestration. Our findings also suggest that models examining the nanoparticle/macrophage interaction should include studies with primary tissue macrophages.
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http://dx.doi.org/10.1021/acsnano.6b06245DOI Listing
March 2017

Mechanism of hard-nanomaterial clearance by the liver.

Nat Mater 2016 11 15;15(11):1212-1221. Epub 2016 Aug 15.

Institute of Biomaterials and Biomedical Engineering, University of Toronto, Rosebrugh Building, Room 407, 164 College Street, Toronto, Ontario M5S 3G9, Canada.

The liver and spleen are major biological barriers to translating nanomedicines because they sequester the majority of administered nanomaterials and prevent delivery to diseased tissue. Here we examined the blood clearance mechanism of administered hard nanomaterials in relation to blood flow dynamics, organ microarchitecture and cellular phenotype. We found that nanomaterial velocity reduces 1,000-fold as they enter and traverse the liver, leading to 7.5 times more nanomaterial interaction with hepatic cells relative to peripheral cells. In the liver, Kupffer cells (84.8 ± 6.4%), hepatic B cells (81.5 ± 9.3%) and liver sinusoidal endothelial cells (64.6 ± 13.7%) interacted with administered PEGylated quantum dots, but splenic macrophages took up less material (25.4 ± 10.1%) due to differences in phenotype. The uptake patterns were similar for two other nanomaterial types and five different surface chemistries. Potential new strategies to overcome off-target nanomaterial accumulation may involve manipulating intra-organ flow dynamics and modulating the cellular phenotype to alter hepatic cell interactions.
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http://dx.doi.org/10.1038/nmat4718DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5132626PMC
November 2016

Mesenchymal Tumors Can Derive from Ng2/Cspg4-Expressing Pericytes with β-Catenin Modulating the Neoplastic Phenotype.

Cell Rep 2016 07 14;16(4):917-927. Epub 2016 Jul 14.

Developmental and Stem Cell Biology Program, The Hospital for Sick Children, Toronto, ON M5G1X8, Canada; Department of Orthopaedic Surgery, Duke University, Durham, NC 27710, USA. Electronic address:

The cell of origin for most mesenchymal tumors is unclear. One cell type that contributes to this lineages is the pericyte, a cell expressing Ng2/Cspg4. Using lineage tracing, we demonstrated that bone and soft tissue sarcomas driven by the deletion of the Trp53 tumor suppressor, or desmoid tumors driven by a mutation in Apc, can derive from cells expressing Ng2/Cspg4. Deletion of the Trp53 tumor suppressor gene in these cells resulted in the bone and soft tissue sarcomas that closely resemble human sarcomas, while stabilizing β-catenin in this same cell type caused desmoid tumors. Comparing expression between Ng2/Cspg4-expressing pericytes lacking Trp53 and sarcomas that arose from deletion of Trp53 showed inhibition of β-catenin signaling in the sarcomas. Activation of β-catenin inhibited the formation and growth of sarcomas. Thus, pericytes can be a cell of origin for mesenchymal tumors, and β-catenin dysregulation plays an important role in the neoplastic phenotype.
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http://dx.doi.org/10.1016/j.celrep.2016.06.058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4963269PMC
July 2016

β-Catenin modulation in neurofibromatosis type 1 bone repair: therapeutic implications.

FASEB J 2016 09 15;30(9):3227-37. Epub 2016 Jun 15.

Division of Developmental and Stem Cell Biology, University of Toronto, Toronto, Ontario, Canada; Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada; Department of Orthopaedic Surgery, Duke University, Durham, North Carolina, USA

Tibial pseudarthrosis causes substantial morbidity in patients with neurofibromatosis type 1 (NF1). We studied tibial pseudarthrosis tissue from patients with NF1 and found elevated levels of β-catenin compared to unaffected bone. To elucidate the role of β-catenin in fracture healing, we used a surgically induced tibial fracture model in conditional knockout (KO) Nfl (Nf1(flox/flox)) mice. When treated with a Cre-expressing adenovirus (Ad-Cre), there was a localized knockdown of Nf1 in the healing fracture and a subsequent development of a fibrous pseudarthrosis. Consistent with human data, elevated β-catenin levels were found in the murine fracture sites. The increased fibrous tissue at the fracture site was rescued by local treatment with a Wingless-type MMTV integration site (Wnt) antagonist, Dickkopf-1 (Dkk1). The murine pseudarthrosis phenotype was also rescued by conditional β-catenin gene inactivation. The number of colony-forming unit osteoblasts (CFU-Os), a surrogate marker of undifferentiated mesenchymal cells able to differentiate to osteoblasts, correlated with the capacity to form bone at the fracture site. Our findings indicate that the protein level of β-catenin must be precisely regulated for normal osteoblast differentiation. An up-regulation of β-catenin in NF1 causes a shift away from osteoblastic differentiation resulting in a pseudarthrosis in vivo These results support the notion that pharmacological modulation of β-catenin can be used to treat pseudarthrosis in patients with NF1.-Ghadakzadeh, S., Kannu, P., Whetstone, H., Howard A., Alman, B. A. β-catenin modulation in neurofibromatosis type 1 bone repair: therapeutic implications.
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http://dx.doi.org/10.1096/fj.201500190RRDOI Listing
September 2016

Hedgehog inhibits β-catenin activity in synovial joint development and osteoarthritis.

J Clin Invest 2016 05 28;126(5):1649-63. Epub 2016 Mar 28.

Both the WNT/β-catenin and hedgehog signaling pathways are important in the regulation of limb development, chondrocyte differentiation, and degeneration of articular cartilage in osteoarthritis (OA). It is not clear how these signaling pathways interact in interzone cell differentiation and synovial joint morphogenesis. Here, we determined that constitutive activation of hedgehog signaling specifically within interzone cells induces joint morphological changes by selectively inhibiting β-catenin-induced Fgf18 expression. Stabilization of β-catenin or treatment with FGF18 rescued hedgehog-induced phenotypes. Hedgehog signaling induced expression of a dominant negative isoform of TCF7L2 (dnTCF7L2) in interzone progeny, which may account for the selective regulation of β-catenin target genes observed. Knockdown of TCF7L2 isoforms in mouse chondrocytes rescued hedgehog signaling-induced Fgf18 downregulation, while overexpression of the human dnTCF7L2 orthologue (dnTCF4) in human chondrocytes promoted the expression of catabolic enzymes associated with OA. Similarly, expression of dnTCF4 in human chondrocytes positively correlated with the aggrecanase ADAMTS4. Consistent with our developmental findings, activation of β-catenin also attenuated hedgehog-induced or surgically induced articular cartilage degeneration in mouse models of OA. Thus, our results demonstrate that hedgehog inhibits selective β-catenin target gene expression to direct interzone progeny fates and articular cartilage development and disease. Moreover, agents that increase β-catenin activity have the potential to therapeutically attenuate articular cartilage degeneration as part of OA.
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http://dx.doi.org/10.1172/JCI80205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4855923PMC
May 2016
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