Impact of Fluid Flow Shear Stress on Osteoblast Differentiation and Cross-Talk with Articular Chondrocytes.

Bone cells, in particular osteoblasts, are capable of communication with each other during bone growth and homeostasis. More recently it has become clear that they also communicate with other cell-types; including chondrocytes in articular cartilage. One way that this process is facilitated is by interstitial fluid movement within the pericellular and extracellular matrices. This stimulus is also an important mechanical signal in skeletal tissues, and is known to generate shear stresses at the micron-scale (known as fluid flow shear stresses (FFSS)). The primary aim of this study was to develop and characterize an in vitro bone-cartilage crosstalk system, to examine the effect of FFSS on these cell types. Specifically, we evaluated the response of osteoblasts and chondrocytes to FFSS and the effect of FFSS-induced soluble factors from the former, on the latter. This system will ultimately be used to help us understand the role of subchondral bone damage in articular cartilage degeneration. We also carried out a comparison of responses between cell lines and primary murine cells in this work. Our findings demonstrate that primary cells produce a more reliable and reproducible response to FFSS. Furthermore we found that at lower magnitudes , direct FFSS produces anabolic responses in both chondrocytes and osteoblasts, whereas higher levels produce more catabolic responses. Finally we show that exposure to osteoblast-derived factors in conditioned media experiments produced similarly catabolic changes in primary chondrocytes.

Bone Microdamage in Acute Knee Injury.

PURPOSE OF REVIEW: This review summarizes what is known about how bone tissue responds to microdamage, and how this applies to the subchondral region. This has significant relevance to acute joint injury, and is related to the occurrence of bone marrow lesions (BMLs) which are seen by MRI in 80% of acute knee joint injuries. Here, we review what is known about these phenomena (microcracks and BMLs) in the literature and discuss potential mechanisms by which they may be linked. RECENT FINDINGS: The recent findings in this field have shown that microcracks in bone initiate targeted remodeling via RANKL expression in osteocytes. Other work has shown that subchondral microcracks co-localize with BMLs as viewed by MRI. Finally, BMLs are associated with pain and structural joint degeneration. This paper demonstrates that subchondral microcracks likely occur during acute joint injury, and are closely linked to BML that are seem by clinical MRI and thus are potentially involved in the subsequent joint degeneration that occurs after injury.

In Vivo Osteocyte Mechanotransduction: Recent Developments and Future Directions.

PURPOSE OF REVIEW: Mechanical loading is an essential stimulus for skeletal tissues. Osteocytes are primarily responsible for sensing mechanical stimuli in bone and for orchestrating subsequent responses. This is critical for maintaining homeostasis, and responding to injury/disease. The osteocyte mechanotransduction pathway, and the downstream effects it mediates, is highly complex. In vivo models have proved invaluable in understanding this process. This review summarizes the commonly used models, as well as more recently developed ones, and describes how they are used to address emerging questions in the field. RECENT FINDINGS: Minimally invasive animal models can be used to determine mechanisms of osteocyte mechanotransduction, at the cell and molecular level, while simultaneously reducing potentially confounding responses such as inflammation/wound-healing. The details of osteocyte mechanotransduction in bone are gradually becoming clearer. In vivo model systems are a key tool in pursing this question. Advances in this field are explored and discussed in this review.

Diffusion tensor imaging of articular cartilage at 3T correlates with histology and biomechanics in a mechanical injury model.

PURPOSE: We establish a mechanical injury model for articular cartilage to assess the sensitivity of diffusion tensor imaging (DTI) in detecting cartilage damage early in time. Mechanical injury provides a more realistic model of cartilage degradation compared with commonly used enzymatic degradation. METHODS: Nine cartilage-on-bone samples were obtained from patients undergoing knee replacement. The 3 Tesla DTI (0.18 × 0.18 × 1 mm3 ) was performed before, 1 week, and 2 weeks after (zero, mild, and severe) injury, with a clinical radial spin-echo DTI (RAISED) sequence used in our hospital. We performed stress-relaxation tests and used a quasilinear-viscoelastic (QLV) model to characterize cartilage mechanical properties. Serial histology sections were dyed with Safranin-O and given an OARSI grade. We then correlated the changes in DTI parameters with the changes in QLV-parameters and OARSI grades. RESULTS: After severe injury the mean diffusivity increased after 1 and 2 weeks, whereas the fractional anisotropy decreased after 2 weeks (P < 0.05). The QLV-parameters and OARSI grades of the severe injury group differed from the baseline with statistical significance. The changes in mean diffusivity across all the samples correlated with the changes in the OARSI grade (r = 0.72) and QLV-parameters (r = -0.75). CONCLUSION: DTI is sensitive in tracking early changes after mechanical injury, and its changes correlate with changes in biomechanics and histology. Magn Reson Med 78:69-78, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

DMP-1-mediated Ghr gene recombination compromises skeletal development and impairs skeletal response to intermittent PTH.

Bone minerals are acquired during growth and are key determinants of adult skeletal health. During puberty, the serum levels of growth hormone (GH) and its downstream effector IGF-1 increase and play critical roles in bone acquisition. The goal of the current study was to determine how bone cells integrate signals from the GH/IGF-1 to enhance skeletal mineralization and strength during pubertal growth. Osteocytes, the most abundant bone cells, were shown to orchestrate bone modeling during growth. We used dentin matrix protein (Dmp)-1-mediated Ghr knockout (DMP-GHRKO) mice to address the role of the GH/IGF axis in osteocytes. We found that DMP-GHRKO did not affect linear growth but compromised overall bone accrual. DMP-GHRKO mice exhibited reduced serum inorganic phosphate and parathyroid hormone (PTH) levels and decreased bone formation indices and were associated with an impaired response to intermittent PTH treatment. Using an osteocyte-like cell line along with in vivo studies, we found that PTH sensitized the response of bone to GH by increasing Janus kinase-2 and IGF-1R protein levels. We concluded that endogenously secreted PTH and GHR signaling in bone are necessary to establish radial bone growth and optimize mineral acquisition during growth.

Osteocytes: master orchestrators of bone.

Osteocytes comprise the overwhelming majority of cells in bone and are its only true "permanent" resident cell population. In recent years, conceptual and technological advances on many fronts have helped to clarify the role osteocytes play in skeletal metabolism and the mechanisms they use to perform them. The osteocyte is now recognized as a major orchestrator of skeletal activity, capable of sensing and integrating mechanical and chemical signals from their environment to regulate both bone formation and resorption. Recent studies have established that the mechanisms osteocytes use to sense stimuli and regulate effector cells (e.g., osteoblasts and osteoclasts) are directly coupled to the environment they inhabit-entombed within the mineralized matrix of bone and connected to each other in multicellular networks. Communication within these networks is both direct (via cell-cell contacts at gap junctions) and indirect (via paracrine signaling by secreted signals). Moreover, the movement of paracrine signals is dependent on the movement of both solutes and fluid through the space immediately surrounding the osteocytes (i.e., the lacunar-canalicular system). Finally, recent studies have also shown that the regulatory capabilities of osteocytes extend beyond bone to include a role in the endocrine control of systemic phosphate metabolism. This review will discuss how a highly productive combination of experimental and theoretical approaches has managed to unearth these unique features of osteocytes and bring to light novel insights into the regulatory mechanisms operating in bone.

Tissue engineering approaches for dental pulp regeneration: The development of novel bioactive materials using pharmacological epigenetic inhibitors.

The drive for minimally invasive endodontic treatment strategies has shifted focus from technically complex and destructive root canal treatments towards more conservative vital pulp treatment. However, novel approaches to maintaining dental pulp vitality after disease or trauma will require the development of innovative, biologically-driven regenerative medicine strategies. For example, cell-homing and cell-based therapies have recently been developed in vitro and trialled in preclinical models to study dental pulp regeneration. These approaches utilise natural and synthetic scaffolds that can deliver a range of bioactive pharmacological epigenetic modulators (HDACis, DNMTis, and ncRNAs), which are cost-effective and easily applied to stimulate pulp tissue regrowth. Unfortunately, many biological factors hinder the clinical development of regenerative therapies, including a lack of blood supply and poor infection control in the necrotic root canal system. Additional challenges include a need for clinically relevant models and manufacturing challenges such as scalability, cost concerns, and regulatory issues. This review will describe the current state of bioactive-biomaterial/scaffold-based engineering strategies to stimulate dentine-pulp regeneration, explicitly focusing on epigenetic modulators and therapeutic pharmacological inhibition. It will highlight the components of dental pulp regenerative approaches, describe their current limitations, and offer suggestions for the effective translation of novel epigenetic-laden bioactive materials for innovative therapeutics.

Toxin for Treating Raynaud Conditions in Hands (The TORCH Study): A Systematic Review and Meta-analysis.

Background: Raynaud disease of the hands is a complex disorder resulting in inappropriate constriction and/or insufficient dilation in microcirculation. There is an emerging role for botulinum toxin type A (BTX-A) in the treatment armamentarium for refractory Raynaud disease. The aim of this systematic review was to critically evaluate the management of primary and secondary Raynaud disease treated with BTX-A intervention. Methods: We performed a Preferred Reporting Items for Systematic Reviews and Meta-Analyses-compliant systematic review of clinical studies assessing treatment of primary or secondary Raynaud disease with BTX-A by searching Ovid MEDLINE and Embase databases from inception to first August 2023. The review protocol was prospectively registered on the PROSPERO database (CRD42022312253). Results: Our search strategy identified 288 research articles, of which 18 studies [four randomized controlled trials (RCTs), two non-RCTs, five case series, and seven retrospective cohort studies] were eligible for analysis. Meta-analysis demonstrated that the probability of pain visual analog scale score improvement with BTX-A intervention was 81.95% [95% confidence interval (74.12-87.81) P = 0.19, heterogeneity I 2 = 26%] and probability of digital ulcer healing was 79.37% [95% confidence interval (62.45-89.9) P = 0.02, heterogeneity I 2 = 56%]. Conclusions: Delivery of BTX-A to digital vessels in the hand may be an effective management strategy for primary and secondary Raynaud disease. A definitive, appropriately-powered RCT with objective functional and patient-reported outcome measures is required to accurately assess and quantify the efficacy of BTX-A in Raynaud disease of the hands.

Reproducible Synthesis of Biocompatible Albumin Nanoparticles Designed for Intra-articular Administration of Celecoxib to Treat Osteoarthritis.

Osteoarthritis (OA) is the most common form of arthritis, with intra-articular (IA) delivery of therapeutics being the current best option to treat pain and inflammation. However, IA delivery is challenging due to the rapid clearance of therapeutics from the joint and the need for repeated injections. Thus, there is a need for long-acting delivery systems that increase the drug retention time in joints with the capacity to penetrate OA cartilage. As pharmaceutical utility also demands that this is achieved using biocompatible materials that provide colloidal stability, our aim was to develop a nanoparticle (NP) delivery system loaded with the COX-2 inhibitor celecoxib that can meet these criteria. We devised a reproducible and economical method to synthesize the colloidally stable albumin NPs loaded with celecoxib without the use of any of the following conditions: high temperatures at which albumin denaturation occurs, polymer coatings, oils, Class 1/2 solvents, and chemical protein cross-linkers. The spherical NP suspensions were biocompatible, monodisperse with average diameters of 72 nm (ideal for OA cartilage penetration), and they were stable over 6 months at 4 °C. Moreover, the NPs loaded celecoxib at higher levels than those required for the therapeutic response in arthritic joints. For these reasons, they are the first of their kind. Labeled NPs were internalized by primary human articular chondrocytes cultured from the knee joints of OA patients. The NPs reduced the concentration of inflammatory mediator prostaglandin E2 released by the primaries, an indication of retained bioactivity following NP synthesis. Similar results were observed in lipopolysaccharide-stimulated human THP-1 monocytes. The IA administration of these NPs is expected to avoid side-effects associated with oral administration of celecoxib and to maintain a high local concentration in the knee joint over a sustained period. They are now ready for evaluation by IA administration in animal models of OA.

Osteocyte signaling in bone.

Osteocytes, the cells residing within the bone matrix and comprising 90% to 95% of the all bone cells, have long been considered quiescent bystander cells compared to the osteoblasts and osteoclasts whose activities cause bone gain and loss, and whose dysfunction lead to growth defects and osteoporosis. However, recent studies show that osteocytes play a crucial, central role in regulating the dynamic nature of bone in all its diverse functions. Osteocytes are now known to be the principal sensors for mechanical loading of bone. They produce the soluble factors that regulate the onset of both bone formation and resorption. Osteocytes regulate local mineral deposition and chemistry at the bone matrix level, and they also function as endocrine cells producing factors that target distant organs such as the kidney to regulate phosphate transport. Osteocytes appear to be the major local orchestrator of many of bone's functions.

Mitochondria in Injury, Inflammation and Disease of Articular Skeletal Joints.

Inflammation is an important biological response to tissue damage caused by injury, with a crucial role in initiating and controlling the healing process. However, dysregulation of the process can also be a major contributor to tissue damage. Related to this, although mitochondria are typically thought of in terms of energy production, it has recently become clear that these important organelles also orchestrate the inflammatory response via multiple mechanisms. Dysregulated inflammation is a well-recognised problem in skeletal joint diseases, such as rheumatoid arthritis. Interestingly osteoarthritis (OA), despite traditionally being known as a 'non-inflammatory arthritis', now appears to involve an element of chronic inflammation. OA is considered an umbrella term for a family of diseases stemming from a range of aetiologies (age, obesity etc.), but all with a common presentation. One particular OA sub-set called Post-Traumatic OA (PTOA) results from acute mechanical injury to the joint. Whether the initial mechanical tissue damage, or the subsequent inflammatory response drives disease, is currently unclear. In the former case; mechanobiological properties of cells/tissues in the joint are a crucial consideration. Many such cell-types have been shown to be exquisitely sensitive to their mechanical environment, which can alter their mitochondrial and cellular function. For example, in bone and cartilage cells fluid-flow induced shear stresses can modulate cytoskeletal dynamics and gene expression profiles. More recently, immune cells were shown to be highly sensitive to hydrostatic pressure. In each of these cases mitochondria were central to these responses. In terms of acute inflammation, mitochondria may have a pivotal role in linking joint tissue injury with chronic disease. These processes could involve the immune cells recruited to the joint, native/resident joint cells that have been damaged, or both. Taken together, these observations suggest that mitochondria are likely to play an important role in linking acute joint tissue injury, inflammation, and long-term chronic joint degeneration - and that the process involves mechanobiological factors. In this review, we will explore the links between mechanobiology, mitochondrial function, inflammation/tissue-damage in joint injury and disease. We will also explore some emerging mitochondrial therapeutics and their potential for application in PTOA.

The Effect of Fluid Flow Shear Stress and Substrate Stiffness on Yes-Associated Protein (YAP) Activity and Osteogenesis in Murine Osteosarcoma Cells.

Osteosarcoma (OS) is an aggressive bone cancer originating in the mesenchymal lineage. Prognosis for metastatic disease is poor, with a mortality rate of approximately 40%; OS is an aggressive disease for which new treatments are needed. All bone cells are sensitive to their mechanical/physical surroundings and changes in these surroundings can affect their behavior. However, it is not well understood how OS cells specifically respond to fluid movement, or substrate stiffness-two stimuli of relevance in the tumor microenvironment. We used cells from spontaneous OS tumors in a mouse engineered to have a bone-specific knockout of pRb-1 and p53 in the osteoblast lineage. We silenced Sox2 (which regulates YAP) and tested the effect of fluid flow shear stress (FFSS) and substrate stiffness on YAP expression/activity-which was significantly reduced by loss of Sox2, but that effect was reversed by FFSS but not by substrate stiffness. Osteogenic gene expression was also reduced in the absence of Sox2 but again this was reversed by FFSS and remained largely unaffected by substrate stiffness. Thus we described the effect of two distinct stimuli on the mechanosensory and osteogenic profiles of OS cells. Taken together, these data suggest that modulation of fluid movement through, or stiffness levels within, OS tumors could represent a novel consideration in the development of new treatments to prevent their progression.

Innate Rhythms: Clocks at the Center of Monocyte and Macrophage Function.

The circadian cycle allows organisms to track external time of day and predict/respond to changes in the external environment. In higher order organisms, circadian rhythmicity is a central feature of innate and adaptive immunity. We focus on the role of the molecular clock and circadian rhythmicity specifically in monocytes and macrophages of the innate immune system. These cells display rhythmicity in their internal functions, such as metabolism and inflammatory mediator production as well as their external functions in pathogen sensing, phagocytosis, and migration. These inflammatory mediators are of clinical interest as many are therapeutic targets in inflammatory disease such as cardiovascular disease, diabetes, and rheumatoid arthritis. Moreover, circadian rhythm disruption is closely linked with increased prevalence of these conditions. Therefore, understanding the mechanisms by which circadian disruption affects monocyte/macrophage function will provide insights into novel therapeutic opportunities for these chronic inflammatory diseases.

Intraarticular injection of liposomal adenosine reduces cartilage damage in established murine and rat models of osteoarthritis.

Osteoarthritis (OA) affects nearly 10% of the population of the United States and other industrialized countries and, at present, short of surgical joint replacement, there is no therapy available that can reverse the progression of the disease. Adenosine, acting at its A2A receptor (A2AR), is a critical autocrine factor for maintenance of cartilage homeostasis and here we report that injection of liposomal suspensions of either adenosine or a selective A2AR agonist, CGS21680, significantly reduced OA cartilage damage in a murine model of obesity-induced OA. The same treatment also improved swelling and preserved cartilage in the affected knees in a rat model of established post-traumatic OA (PTOA). Differential expression analysis of mRNA from chondrocytes harvested from knees of rats with PTOA treated with liposomal A2AR agonist revealed downregulation of genes associated with matrix degradation and upregulation of genes associated with cell proliferation as compared to liposomes alone. Studies in vitro and in affected joints demonstrated that A2AR ligation increased the nuclear P-SMAD2/3/P-SMAD1/5/8 ratio, a change associated with repression of terminal chondrocyte differentiation. These results strongly suggest that targeting the A2AR is an effective approach to treat OA.

Variation of trabecular microarchitectural parameters in cranial, caudal and mid-vertebral regions of the ovine L3 vertebra.

The lumbar vertebrae are major load-bearing structures within the spinal column. The current understanding of the microstructure of these bodies and their full role in load-bearing is incomplete. There is a need to develop our understanding of these issues to improve fracture prediction in musculoskeletal diseases such as osteoporosis. The lumbar vertebrae consist primarily of trabecular bone enclosed in a thin cortical shell, but little is known about how microstructural parameters vary within these structures, particularly in relation to the trabecular compartment. The specific aim of this study was to use micro-computed tomography to characterize the trabecular microarchitecture of the ovine L3 vertebra in cranial, mid-vertebra and caudal regions. The L3 vertebra was obtained from skeletally mature ewes (n = 18) more than 4 years old. Three-dimensional reconstructions of three pre-defined regions were obtained and microarchitectural parameters were calculated. Whereas there was no difference in bone volume fraction or structural model index between regions, trabecular number, thickness, spacing, connectivity density, degree of anisotropy and bone mineral density all displayed significant regional variations. The observed differences were consistent with the biomechanical hypothesis that in vivo loads are distributed differently at the endplates compared with the mid-vertebra. Thus, a more integrative approach combining biomechanical theory and anatomical features may improve fracture risk assessment in the future.

Immunometabolism around the Clock.

Almost every cell has a molecular clock, which controls gene expression on a 24-h cycle, providing circadian rhythmicity. An example of a circadian behaviour common to most organisms is the feeding/fasting cycle, which shapes whole-body metabolism. However, the exact mechanisms by which the clock controls cellular metabolism have only recently become clear. The molecular clock and related metabolic pathways are also key drivers of immunity. Thus, a natural convergence of circadian biology, metabolism, and immunology has emerged to form a new field that we term 'circadian immunometabolism'. Expanding our understanding of this field will provide insights into chronic conditions such as obesity, cancer, diabetes, cardiovascular disease, and arthritis.

The effects of increased intracortical remodeling on microcrack behaviour in compact bone.

The behaviour of microdamage in bone is related to its microstructural features and thus has an important role in tissue structural properties. However, it is not known how cracks behave in areas of increased intracortical remodeling. More remodeling creates wider variation in the properties of the primary microstructural features of cortical bone, namely osteons. This situation may occur after treatment involving parathyroid hormone or events such as menopause/ovariectomy. High turnover was modeled in this study by using ovariectomy (OVX) to induce surgical menopause in sheep. We hypothesized that osteon age would influence microcrack behaviour during propagation. Five fluorochrome dyes were administered intravenously at different time-points over 12 months post-OVX to label remodeling sites and all animals were then euthanized. Compact bone specimens (2x2x36 mm) were harvested from the right metatarsal. Samples were cyclically loaded to failure and then histological analyses were carried out. Cracks were categorized by length into three groups; short (<100 mum), intermediate (100-300 mum) and long (>300 mum). Numerical crack density (Cr.Dn) of long cracks was greater in controls compared with OVX. Controls also displayed a higher crack surface density (Cr.S.Dn) compared with OVX (p<0.05). The behaviour of short cracks did not differ between old and new osteons, but intermediate and long cracks preferentially stopped at newer osteons compared with older ones (p<0.05). This mechanism may have an important role in terms of prolonging fatigue life. We conclude that recently formed secondary osteons have a unique influence on propagating microcracks compared with older osteons. Therefore localized remodeling levels should be considered when studying microcrack behaviour in bone.

Heterogeneous linear elastic trabecular bone modelling using micro-CT attenuation data and experimentally measured heterogeneous tissue properties.

High-resolution voxel-based finite element software, such as FEEBE developed at the NCBES, is widely used for studying trabecular bone at the micro-scale. A new approach to determine heterogeneous bone tissue material properties for computational models was proposed in this study. The specimen-specific range of tissue moduli across strut width was determined from nanoindentation testing. This range was mapped directly using linear interpolation to that specimen's micro-computed tomography (microCT) grey value range as input material properties for finite element analysis. The method was applied to cuboid trabecular bone samples taken from eight, 4-year-old (skeletally mature) ovine L5 vertebrae. Before undergoing experimental uniaxial compression tests, the samples were microCT scanned and 30 microm resolution finite element models were generated. The linear elastic finite element models were compressed to 1% strain. This material property assignment method for computational models accurately reproduced the experimentally determined apparent modulus and concentrations of stress at locations of failure.