Inescapable foot shock induces a PTSD-like phenotype and negatively impacts adult murine bone.

Post-traumatic stress disorder (PTSD) is associated with osteopenia, osteoporosis and increased fracture risk in the clinical population. Yet, the development of preclinical models to study PTSD-induced bone loss remains limited. In this study, we present a previously unreported model of PTSD in adult female C57BL/6 mice, by employing inescapable foot shock and social isolation, that demonstrates high face and construct validity. A subset of mice exposed to this paradigm (i.e. PTSD mice) display long-term alterations in behavioral and inflammatory indices. Using three-dimensional morphometric calculations, cyclic reference point indentation (cRPI) testing and histological analyses, we find that PTSD mice exhibit loss of trabecular bone, altered bone material quality, and aberrant changes in bone tissue architecture and cellular activity. This adult murine model of PTSD exhibits clinically relevant changes in bone physiology and provides a valuable tool for investigating the cellular and molecular mechanisms underlying PTSD-induced bone loss.

From Stem to Sternum: The Role of Shp2 in the Skeleton.

Src homology-2 domain-containing phosphatase 2 (SHP2) is a ubiquitously expressed phosphatase that is vital for skeletal development and maintenance of chondrocytes, osteoblasts, and osteoclasts. Study of SHP2 function in small animal models has led to insights in phenotypes observed in SHP2-mutant human disease, such as Noonan syndrome. In recent years, allosteric SHP2 inhibitors have been developed to specifically target the protein in neoplastic processes. These inhibitors are highly specific and have great potential for disease modulation in cancer and other pathologies, including bone disorders. In this review, we discuss the importance of SHP2 and related signaling pathways (e.g., Ras/MEK/ERK, JAK/STAT, PI3K/Akt) in skeletal development. We review rodent models of pathologic processes caused by germline mutations that activate SHP2 enzymatic activity, with a focus on the skeletal phenotype seen in these patients. Finally, we discuss SHP2 inhibitors in development and their potential for disease modulation in these genetic diseases, particularly as it relates to the skeleton.

SPARC production by bone marrow-derived cells contributes to myocardial fibrosis in pressure overload.

In human heart failure and in murine hearts with left-ventricular pressure overload (LVPO), increases in fibrosis are associated with increases in myocardial stiffness. Secreted protein acidic and rich in cysteine (SPARC) is shown to be necessary for both cardiac fibrosis and increases in myocardial stiffness in response to LVPO; however, cellular sources of cardiac SPARC are incompletely defined. Irradiation and bone marrow transfer were undertaken to test the hypothesis that SPARC expression by bone marrow-derived cells is an important mediator of fibrosis in LVPO. In recipient SPARC-null mice transplanted with donor wild-type (WT) bone marrow and subjected to LVPO, levels of fibrosis similar to that of WT mice were found despite the lack of SPARC expression by resident cells. In recipient WT mice with donor SPARC-null bone marrow, significantly less fibrosis versus that of WT mice was found despite the expression of SPARC by resident cells. Increases in myocardial stiffness followed a similar pattern to that of collagen deposition. Myocardial macrophages were significantly reduced in SPARC-null mice with LVPO versus that of WT mice. Recipient SPARC-null mice transplanted with donor WT bone marrow exhibited an increase in cardiac macrophages versus that of SPARC-null LVPO and donor WT mice with recipient SPARC-null bone marrow. Expression of vascular cellular adhesion molecule (VCAM), a previously identified binding partner of SPARC, was assessed in all groups and with the exception of WT mice, increases in VCAM immunoreactivity with LVPO were observed. However, no differences in VCAM expression between bone marrow transplant groups were noted. In conclusion, SPARC expression by bone marrow-derived cells was critical for fibrotic deposition of collagen and influenced the expansion of myocardial macrophages in response to LVPO.NEW & NOTEWORTHY Myocardial fibrosis and the resultant increases in LV and myocardial stiffness represent pivotal consequences of chronic pressure overload (PO). In this study, a murine model of cardiac fibrosis induced by PO was used to demonstrate a critical function of SPARC in bone marrow-derived cells that drives cardiac fibrosis and increases in cardiac macrophages.

Murine Aseptic Surgical Model of Femoral Atrophic Nonunion.

Although bone repair is typically an efficient process, an inadequate healing response can occur, with approximately 5-20% of fractures developing nonunion. Even with improved healing strategies and external fixation devices, overall rate of nonunion has not been significantly reduced, particularly for atrophic nonunion. Atrophic nonunion is characterized by sparse or no callus formation and is difficult to treat clinically, resulting in long-term pain and functional limitation. Reliable preclinical models are needed to study the pathophysiology of atrophic nonunion to create better treatment options. The MouseNail kit (RISystem, Landquart, Switzerland) provides a highly standardized approach in which stabilized segmental bone defects are achieved through interlocked intramedullary nailing. However, reliably performing this surgery is technically challenging, particularly while maintaining strict asepsis. Skilled and aseptic surgical execution is important and necessary because it ensures optimal animal welfare and reproducibility. Therefore, the aim of this paper is to describe:•Novel modifications to the MouseNail kit that allow for: 1) a completely aseptic surgical environment, including description of a hanging limb orthopedic aseptic preparation and 2) a reduction in fracture gap size necessary for induction of atrophic nonunion.•Pre- to post-operative recommendations to facilitate successful performance of murine orthopedic survival surgery.

Effects of Neurological Disorders on Bone Health.

Neurological diseases, particularly in the context of aging, have serious impacts on quality of life and can negatively affect bone health. The brain-bone axis is critically important for skeletal metabolism, sensory innervation, and endocrine cross-talk between these organs. This review discusses current evidence for the cellular and molecular mechanisms by which various neurological disease categories, including autoimmune, developmental, dementia-related, movement, neuromuscular, stroke, trauma, and psychological, impart changes in bone homeostasis and mass, as well as fracture risk. Likewise, how bone may affect neurological function is discussed. Gaining a better understanding of brain-bone interactions, particularly in patients with underlying neurological disorders, may lead to development of novel therapies and discovery of shared risk factors, as well as highlight the need for broad, whole-health clinical approaches toward treatment.

Teaching Surgical Model Development in Research by Using Situated Learning and Instructional Scaffolding.

Resources detailing the scope, details, and duration for teaching and learning surgical model development in research are poorly described. Situated learning and instructional scaffolding are useful skill-building tools. Herein, we discuss educational theory in the context of a training paradigm for surgical researchers, using our experience with a nonunion femoral fracture model as an example. Stages of learning include cognitive, associative, and autonomous stages. In surgical training, the cognitive stage involves the acquisition of basic knowledge, including anatomy, surgical approach, instrumentation, and suturing, which can be taught by using books, videos, skeletons, and cadavers. To these basic skills, the associative stage adds advanced techniques-including anesthesia, asepsis, hemostasis, and the full surgical procedure-through mentored nonsurvival surgical experiences. After a mentor has assured competence, trainees perform supervised and then independent survival surgeries to complete the autonomous stage. Through these stages, instructional scaffolding is applied in the context of a situated learning environment in which trainees learn in a layered approach through their own experiences. Thus, the proposed training paradigm is structured to teach trainees how to think and act as surgeons so they can adapt and grow, rather than only to ensure technical competency in a specific model. Development and mastery of complex surgical models may require as long as 6 mo to achieve optimal outcomes, depending on the preexisting skill of the research surgeons, technical difficulty, and the stage of model evolution.

Impacts of Psychological Stress on Osteoporosis: Clinical Implications and Treatment Interactions.

The significant biochemical and physiological effects of psychological stress are beginning to be recognized as exacerbating common diseases, including osteoporosis. This review discusses the current evidence for psychological stress-associated mental health disorders as risk factors for osteoporosis, the mechanisms that may link these conditions, and potential implications for treatment. Traditional, alternative, and adjunctive therapies are discussed. This review is not intended to provide therapeutic recommendations, but, rather, the goal of this review is to delineate potential interactions of psychological stress and osteoporosis and to highlight potential multi-system implications of pharmacological interventions. Review of the current literature identifies several potentially overlapping mechanistic pathways that may be of interest (e.g., glucocorticoid signaling, insulin-like growth factor signaling, serotonin signaling) for further basic and clinical research. Current literature also supports the potential for cross-effects of therapeutics for osteoporosis and mental health disorders. While studies examining a direct link between osteoporosis and chronic psychological stress are limited, the studies reviewed herein suggest that a multi-factorial, personalized approach should be considered for improved patient outcomes in populations experiencing psychological stress, particularly those at high-risk for development of osteoporosis.

Identification of circulating murine CD34+OCN+ cells.

BACKGROUND AIMS: Previous studies identified a circulating human osteoblastic population that expressed osteocalcin (OCN), increased following fracture and pubertal growth, and formed mineralized colonies in vitro and bone in vivo. A subpopulation expressed CD34, a hematopoietic/endothelial marker. These findings led to our hypothesis that hematopoietic-derived CD34+OCN+ cells exist in the circulation of mice and are modulated after fracture. METHODS: Flow cytometry was used to identify CD34+OCN+ cells in male B6.SJL-PtprcaPepcb/BoyJ and Vav-Cre/mTmG (VavR) mice. Non-stabilized tibial fractures were created by three-point bend. Fractures were longitudinally imaged by micro-computed tomography, and immunofluorescent staining was used to evaluate CD34+OCN+ cells within fracture callus. AMD3100 (10 mg/kg) was injected subcutaneously for 3 days and the CD34+OCN+ population was evaluated by flow cytometry. RESULTS: Circulating CD34+OCN+ cells were identified in mice and confirmed to be of hematopoietic origin (CD45+; Vav1+) using two mouse models. Both circulating and bone marrow-derived CD34+OCN+ cells peaked three weeks post-non-stabilized tibial fracture, suggesting association with cartilage callus transition to bone and early mineralization. Co-expression of CD34 and OCN in the fracture callus at two weeks post-fracture was observed. By three weeks, there was 2.1-fold increase in number of CD34+OCN+ cells, and these were observed throughout the fracture callus. AMD3100 altered CD34+OCN+ cell levels in peripheral blood and bone marrow. DISCUSSION: Together, these data demonstrate a murine CD34+OCN+ circulating population that may be directly involved in fracture repair. Future studies will molecularly characterize CD34+OCN+ cells, determine mechanisms regulating their contribution, and examine if their number correlates with improved fracture healing outcomes.

Hematopoietic stem cell-derived cancer-associated fibroblasts are novel contributors to the pro-tumorigenic microenvironment.

Targeting the tumor microenvironment is critical toward improving the effectiveness of cancer therapeutics. Cancer-associated fibroblasts (CAFs) are one of the most abundant cell types of the tumor microenvironment, playing an important role in tumor progression. Multiple origins for CAFs have been proposed including resident fibroblasts, adipocytes, and bone marrow. Our laboratory previously identified a novel hematopoietic stem cell (HSC) origin for CAFs; however, the functional roles of HSC-derived CAFs (HSC-CAFs) in tumor progression have not yet been examined. To test the hypothesis that HSC-CAFs promote tumor progression through contribution to extracellular matrix (ECM) and paracrine production of pro-angiogenic factors, we developed a method to isolate HSC-CAFs. HSC-CAFs were profiled on the basis of their expression of hematopoietic and fibroblastic markers in two murine tumor models. Profiling revealed production of factors associated with ECM deposition and remodeling. Functional in vivo studies showed that co-injection of HSC-CAFs with tumor cells resulted in increased tumor growth rate and significantly larger tumors than tumor cells alone. Immunohistochemical studies revealed increased blood vessel density with co-injection, demonstrating a role for HSC-CAFs in tumor vascularization. Mechanistic in vitro studies indicated that HSC-CAFs play a role in producing vascular endothelial growth factor A and transforming growth factor-ß1 in endothelial tube formation and patterning. In vitro and in vivo findings suggest that HSC-CAFs are a critical component of the tumor microenvironment and suggest that targeting the novel HSC-CAF may be a promising therapeutic strategy.

Hematopoietic stem cell-derived adipocytes and fibroblasts in the tumor microenvironment.

The tumor microenvironment (TME) is complex and constantly evolving. This is due, in part, to the crosstalk between tumor cells and the multiple cell types that comprise the TME, which results in a heterogeneous population of tumor cells and TME cells. This review will focus on two stromal cell types, the cancer-associated adipocyte (CAA) and the cancer-associated fibroblast (CAF). In the clinic, the presence of CAAs and CAFs in the TME translates to poor prognosis in multiple tumor types. CAAs and CAFs have an activated phenotype and produce growth factors, inflammatory factors, cytokines, chemokines, extracellular matrix components, and proteases in an accelerated and aberrant fashion. Through this activated state, CAAs and CAFs remodel the TME, thereby driving all aspects of tumor progression, including tumor growth and survival, chemoresistance, tumor vascularization, tumor invasion, and tumor cell metastasis. Similarities in the tumor-promoting functions of CAAs and CAFs suggest that a multipronged therapeutic approach may be necessary to achieve maximal impact on disease. While CAAs and CAFs are thought to arise from tissues adjacent to the tumor, multiple alternative origins for CAAs and CAFs have recently been identified. Recent studies from our lab and others suggest that the hematopoietic stem cell, through the myeloid lineage, may serve as a progenitor for CAAs and CAFs. We hypothesize that the multiple origins of CAAs and CAFs may contribute to the heterogeneity seen in the TME. Thus, a better understanding of the origin of CAAs and CAFs, how this origin impacts their functions in the TME, and the temporal participation of uniquely originating TME cells may lead to novel or improved anti-tumor therapeutics.