Osteogenesis imperfecta (OI) comprises a group of heritable connective tissue disorders generally defined by recurrent fractures, low bone mass, short stature and skeletal fragility. Beyond the skeletal complications of OI, many patients also report intolerance to physical activity, fatigue and muscle weakness. Indeed, recent studies have demonstrated that skeletal muscle is also negatively affected by OI, both directly and indirectly. Given the well-established interdependence of bone and skeletal muscle in both physiology and pathophysiology and the observations of skeletal muscle pathology in patients with OI, we investigated the therapeutic potential of simultaneous anabolic targeting of both bone and skeletal muscle using a soluble activin receptor 2B (ACVR2B) in a mouse model of type III OI (oim). Treatment of 12-week-old oim mice with ACVR2B for 4 weeks resulted in significant increases in both bone and muscle that were similar to those observed in healthy, wild-type littermates. This proof of concept study provides encouraging evidence for a holistic approach to treating the deleterious consequences of OI in the musculoskeletal system.

Brittle bone disease: Treating bone and muscle

A protein known to increase bone and muscle mass may herald a new approach to treating brittle bone disease. Many patients with the congenital bone disease osteogenesis imperfecta (OI), also known as brittle bone disease, report fatigue, muscle weakness and intolerance to physical activity. The activin signaling pathway is well known for its ability to regulate bone and skeletal muscle mass. Led by Douglas DiGirolamo from the Johns Hopkins University and Emily Germain-Lee from the Kennedy Krieger Institute/Johns Hopkins University in Baltimore, the researchers treated bone and skeletal muscle in mice with one of the most severe forms of OI (type III) with the soluble activin receptor 2B protein. Four weeks of treatment increased bone and muscle mass to a similar extent to that observed in healthy littermates. The findings offer a promising holistic approach to treating the effects of OI on the musculoskeletal system.

To improve the osteogenic property of bone repairing materials and to accelerate bone healing are major tasks in bone biomaterials research. The objective of this study was to investigate if the mechanical force could be used to accelerate bone formation in a bony defect in vivo. The calcium sulfate cement was implanted into the left distal femoral epiphyses surgically in 16 rats. The half of rats were subjected to external mechanical force via treadmill exercise, the exercise started at day 7 postoperatively for 30 consecutive days and at a constant speed 8 m·min⁻¹ for 45 min·day⁻¹, while the rest served as a control. The rats were scanned four times longitudinally after surgery using microcomputed tomography and newly formed bone was evaluated. After sacrificing, the femurs had biomechanical test of three-point bending and histological analysis. The results showed that bone healing under mechanical force were better than the control with residual defect areas of 0.64±0.19 mm² and 1.78±0.39 mm² (P<0.001), and the ultimate loads to failure under mechanical force were 69.56±4.74 N, stronger than the control with ultimate loads to failure of 59.17±7.48 N (P=0.039). This suggests that the mechanical force might be used to improve new bone formation and potentially offer a clinical strategy to accelerate bone healing.

Bone repair: Mechanical force enhances bone healing

When damaged bone that has been repaired with cement is put under mechanical stress, healing is enhanced, report Chinese researchers. Previous evidence showed that mechanical force has a positive effect on bone formation. Zong-Ping Luo and colleagues from Soochow University in Suzhou therefore investigated whether external mechanical force could be used to accelerate bone healing in damaged rat femur bones implanted with bone-repairing material. Results showed that rats that ran on a treadmill for 45 minutes a day had a significant reduction in the defective area of bone at 30 days compared with a control group of rats that did not exercise. External mechanical force could be used to promote the bone-forming properties of bone-repairing materials and could be used as an effective non-invasive way of accelerating bone healing, concluded the authors.

Parathyroid hormone (PTH) secretion is characterized by an ultradian rhythm with tonic and pulsatile components. In healthy subjects, the majority of PTH is secreted in tonic fashion, whereas approximately 30% is secreted in low-amplitude and high-frequency bursts occurring every 10–20 min, superimposed on tonic secretion. Changes in the ultradian PTH secretion were shown to occur in patients with primary and secondary osteoporosis, with skeletal effects depending on the reciprocal modifications of pulsatile and tonic components. Indeed, pathophysiology of spontaneous PTH secretion remains an area potentially suitable to be explored, particularly in those conditions such as secondary forms of osteoporosis, in which conventional biochemical and densitometric parameters may not always give reliable diagnostic and therapeutic indications. This review will highlight the literature data supporting the hypothesis that changes of ultradian PTH secretion may be correlated with skeletal fragility in primary and secondary osteoporosis.

Osteoporosis: Altered hormone secretion may influence skeletal fragility

Further research is needed into changes in hormone secretion which may be associated with increased bone fragility in osteoporosis. Parathyroid hormone (PTH) is secreted by the parathyroid gland, and regulates levels of calcium in blood and bones. In young healthy individuals, around 70% of PTH secretion is continuous (to maintain a baseline level), while 30% of secretion is pulsatile – the hormone is released in short bursts, every 10–20 minutes. In a review of recent research, Andrea Guistina and co-workers at the University of Brescia, Italy, suggest that alterations to this normal pattern of secretion may exacerbate bone fragility, particularly in forms of osteoporosis triggered by hormonal alterations. The team found only a few studies linking PTH with bone disease progression, primarily because of difficulties in assessing spontaneous PTH secretion accurately.

Modern warfare has caused a large number of severe extremity injuries, many of which become infected. In more recent conflicts, a pattern of co-infection with Acinetobacter baumannii and methicillin-resistant Staphylococcus aureus has emerged. We attempted to recreate this pattern in an animal model to evaluate the role of vascularity in contaminated open fractures. Historically, it has been observed that infected bones frequently appear hypovascular, but vascularity in association with bone infection has not been examined in animal models. Adult rats underwent femur fracture and muscle crush injury followed by stabilization and bacterial contamination with A. baumannii complex and methicillin-resistant Staphylococcus aureus. Vascularity and perfusion were assessed by microCT angiography and SPECT scanning, respectively, at 1, 2 and 4 weeks after injury. Quantitative bacterial cultures were also obtained. Multi-bacterial infections were successfully created, with methicillin-resistant S. aureus predominating. There was overall increase in blood flow to injured limbs that was markedly greater in bacteria-inoculated limbs. Vessel volume was greater in the infected group. Quadriceps atrophy was seen in both groups, but was greater in the infected group. In this animal model, infected open fractures had greater perfusion and vascularity than non-infected limbs.

Open fractures: Investigating infection and recovery

A rodent model for studying extreme injuries such as open fractures and crushing of bones gives insight into wound infection and recovery. Severe bone injuries sustained in warfare are susceptible to infection from bacterial pathogens such as Acinetobacter baumannii (ABC) and methicillin-resistant Staphylococcus aureus (MRSA). Little is known about the process of recovery from such injuries. Shawn Gilbert and co-workers at the University of Alabama at Birmingham, USA, created a rodent model to investigate the role of blood vessel structures in the bone during contaminated injuries. The researchers compared two groups of injured rats, uninfected and infected with ABC and MRSA, and monitored blood vessel behavior and blood flow in the injured limbs. Results indicate that infected open fractures experienced greater blood flow and quicker recovery of blood vessel structures than non-infected injuries.

Severe burn injury triggers the body's nonspecific adaptive responses to acute insult, including the systemic inflammatory and stress responses, as well as the sympathetic response to immobilization. These responses trigger inflammatory bone resorption followed by glucocorticoid-induced apoptosis of osteoblasts and probably osteocytes. Because these patients are catabolic, they suffer concomitant muscle wasting and negative nitrogen balance. The use of anabolic agents such as recombinant human growth hormone and oxandrolone results in improved bone mineral content and muscle strength after approximately 1 year. Use of bisphosphonates within the first 10 days of a severe burn completely blocks the resorptive bone loss and has the added advantage of appearing to preserve muscle protein from excessive breakdown. The mechanism for the protective effect on muscle is not currently known. However, if the effect of bisphosphonates on muscle can be confirmed, it raises the possibility that bone communicates with muscle.

Osteoporosis: Bone may communicate with muscle

Bisphosphonates, drugs commonly used to treat bone loss, may also protect muscle which would be good news for osteoporosis patients. In a review article, Gordon Klein from the University of Texas Medical Branch looked at bone loss and muscle breakdown following severe burns. Studies in children showed that the use of growth hormone or oxandrolone, an anabolic steroid, after severe burns can improve bone mineral content and muscle strength after one year. However, administering bisphosphonates within the first ten days after a severe burn stopped bone loss and appeared to preserve muscle protein from excessive breakdown. If the effects of these drugs on muscle are confirmed it raises the possibillity that bone communicates with muscle. Patients with osteoporosis and muscle loss would benefit if treatment for bone loss also resulted in preservation of muscle mass.

Age related defect of the osteogenic differentiation of mesenchymal stem cells (MSCs) plays a key role in osteoporosis. Mechanical loading is one of the most important physical stimuli for osteoblast differentiation. Here, we compared the osteogenic potential of MSCs from young and adult rats under three rounds of 2 h of cyclic stretch of 2.5% elongation at 1 Hz on 3 consecutive days. Cyclic stretch induced a significant osteogenic differentiation of MSCs from young rats, while a compromised osteogenesis in MSCs from the adult rats. Accordingly, there were much more reactive oxygen species (ROS) production in adult MSCs under cyclic stretch compared to young MSCs. Moreover, ROS scavenger N-acetylcysteine rescued the osteogenic differentiation of adult MSCs under cyclic stretch. Gene expression analysis revealed that superoxide dismutase 1 (SOD1) was significantly downregulated in those MSCs from adult rats. In summary, our data suggest that reduced SOD1 may result in excessive ROS production in adult MSCs under cyclic stretch, and thus manipulation of the MSCs from the adult donors with antioxidant would improve their osteogenic ability.

Osteoporosis: Anti-oxidants could prevent age-related bone loss

Anti-oxidants could prevent age-related bone loss and protect against osteoporosis, researchers from China say. Stem cells play a key role in bone formation, but aging makes them less effective at producing bone, increasing the risk of osteoporosis. Looking at the possible mechanisms behind age-related osteoporosis Jiali Tan (Sun Yat-sen University) and Wei Kuang (Guangzhou General Hospital of Guangzhou Military Command) and colleagues focused on the effect on stem cells of chemically reactive molecules containing oxygen (ROS), which are known to inhibit bone formation. They found that reduced levels of a key antioxidant enzyme led to a higher level of ROS in adult rats than younger rats. These findings suggest that increasing the antioxidant properties of adult stem cells would help bone regeneration, which could be of clinical importance for treating age-related osteoporosis.

Transforming growth factor-beta (TGF-β)/bone morphogenetic protein (BMP) plays a fundamental role in the regulation of bone organogenesis through the activation of receptor serine/threonine kinases. Perturbations of TGF-β/BMP activity are almost invariably linked to a wide variety of clinical outcomes, i.e., skeletal, extra skeletal anomalies, autoimmune, cancer, and cardiovascular diseases. Phosphorylation of TGF-β (I/II) or BMP receptors activates intracellular downstream Smads, the transducer of TGF-β/BMP signals. This signaling is modulated by various factors and pathways, including transcription factor Runx2. The signaling network in skeletal development and bone formation is overwhelmingly complex and highly time and space specific. Additive, positive, negative, or synergistic effects are observed when TGF-β/BMP interacts with the pathways of MAPK, Wnt, Hedgehog (Hh), Notch, Akt/mTOR, and miRNA to regulate the effects of BMP-induced signaling in bone dynamics. Accumulating evidence indicates that Runx2 is the key integrator, whereas Hh is a possible modulator, miRNAs are regulators, and β-catenin is a mediator/regulator within the extensive intracellular network. This review focuses on the activation of BMP signaling and interaction with other regulatory components and pathways highlighting the molecular mechanisms regarding TGF-β/BMP function and regulation that could allow understanding the complexity of bone tissue dynamics.

Bone formation: Still much to learn about its complexities

How bone morphogenetic protein (BMP), discovered in 1965, interacts within a complex network to form bone is not yet clearly understood. Disturbances in BMP activity are linked to a wide range of autoimmune diseases, cancers and skeletal diseases including fibrodysplasia ossificans progressiva. In their review Md. Shaifur Rahman from Tissue Banking and Biomaterial Research Unit, Atomic Energy Research Establishment, Dahka, Bangladesh, and his colleagues focus on the structure of BMP and its receptors, and the pathways it uses to regulate and stimulate bone growth. A large number of factors and targets have now been identified for the BMP pathways. This has led to the discovery of a complex interactive network that researchers are still trying to understand. Knowing more about how the pathways affect the transformation of bone-forming cells at different developmental stages would help to develop potential therapies for bone-related diseases.

The mechanical environment is known to influence fracture healing. We speculated that connexin43 (Cx43) gap junctions, which impact skeletal homeostasis, fracture healing and the osteogenic response to mechanical load, may play a role in mediating the response of the healing bone to mechanical strain. Here, we used an established rat fracture model, which uses a 2 mm osteotomy gap stabilized by an external fixator, to examine the impact of various cyclical axial loading protocols (2%, 10%, and 30% strain) on osteotomy healing. We examined the presence of Cx43 in the osteotomy-healing environment and assessed how mechanical strain modulates Cx43 expression patterns in the callus. We demonstrated that increased cyclical axial strain results in increased radiographic and histologic bone formation. In addition, we show by immunohistochemistry that Cx43 is abundantly expressed in the healing callus, with the expression most robust in samples exposed to increased cyclical axial strain. These data are consistent with the concept that an increase in Cx43 expression by mechanical load may be part of the mechanisms by which mechanical forces enhances fracture healing.

Bone fractures: Why movement aids healing

Low-level mechanical strain on bone fractures enhances the expression of a protein which triggers new bone formation and accelerates healing. The protein connexin43 (Cx43) aids intercellular communication in bone, and is abundantly expressed in both newly forming and mature bones. Scientists have long understood that some degree of movement and mechanical strain can aid the healing of fractures, but the precise reasons were unclear. Joseph Stains and co-workers at the University of Maryland, USA, conducted a series of experiments on rats to examine Cx43 expression in response to low-level mechanical strain. The team found that incremental mechanical loading of up to 30% strain on fractured bone led to a corresponding increase in Cx43 expression. The increased protein levels directly influenced bone formation around the fractures, leading to quicker, more effective healing.

Osteocytes, the most abundant bone cells, form an interconnected network in the lacunar-canalicular pore system (LCS) buried within the mineralized matrix, which allows osteocytes to obtain nutrients from the blood supply, sense external mechanical signals, and communicate among themselves and with other cells on bone surfaces. In this study, we examined key features of the LCS network including the topological parameter and the detailed structure of individual connections and their variations in cortical and cancellous compartments, at different ages, and in two disease conditions with altered mechanosensing (perlecan deficiency and diabetes). LCS network showed both topological stability, in terms of conservation of connectivity among osteocyte lacunae (similar to the “nodes” in a computer network), and considerable variability the pericellular annular fluid gap surrounding lacunae and canaliculi (similar to the “bandwidth” of individual links in a computer network). Age, in the range of our study (15–32 weeks), affected only the pericellular fluid annulus in cortical bone but not in cancellous bone. Diabetes impacted the spacing of the lacunae, while the perlecan deficiency had a profound influence on the pericellular fluid annulus. The LCS network features play important roles in osteocyte signaling and regulation of bone growth and adaptation.

Bone structure: A network of mechano-sensing bone cells

Researchers in the US have investigated the impact of disease and aging on the complex network that houses mechanical sensitive bone cells. The interconnected network of spaces and channels within mineralized bone allows bone cells to obtain nutrients from the blood supply and sense external mechanical signals. Identifying changes in this network is important for understanding how bone grows and adapts. Researchers led by Liyun Wang at the University of Delaware, Newark, analysed bone from old and young mice, and bones from mice affected by diabetes or deficiency in the proteoglycan perlecan. They identified network features that are not changed or altered with bone site, age, diabetes, and perlecan deficiency. Their findings help understand how bone cells communicate and the role of bone fluid flow in bone s response to mechanical forces.

Annulus fibrosus (AF) tissue engineering has recently received increasing attention as a treatment for intervertebral disc (IVD) degeneration; however, such engineering remains challenging because of the remarkable complexity of AF tissue. In order to engineer a functional AF replacement, the fabrication of cell-scaffold constructs that mimic the cellular, biochemical and structural features of native AF tissue is critical. In this study, we fabricated aligned fibrous polyurethane scaffolds using an electrospinning technique and used them for culturing AF-derived stem/progenitor cells (AFSCs). Random fibrous scaffolds, also prepared via electrospinning, were used as a control. We compared the morphology, proliferation, gene expression and matrix production of AFSCs on aligned scaffolds and random scaffolds. There was no apparent difference in the attachment or proliferation of cells cultured on aligned scaffolds and random scaffolds. However, compared to cells on random scaffolds, the AFSCs on aligned scaffolds were more elongated and better aligned, and they exhibited higher gene expression and matrix production of collagen-I and aggrecan. The gene expression and protein production of collagen-II did not appear to differ between the two groups. Together, these findings indicate that aligned fibrous scaffolds may provide a favourable microenvironment for the differentiation of AFSCs into cells similar to outer AF cells, which predominantly produce collagen-I matrix.

Back pain: Hope for regeneration of disc tissue

Scientists have discovered a way of growing stem cells from discs in the spine that may have the potential to treat degenerative back pain. Intervertebral discs (IVDs) are cushions between the vertebrae and act as the spine's shock-absorbing system. Weakening of the annulus fibrosus (AF), the tough exterior that protects the soft material at the centre of the disc, is a common cause of chronic low back pain. Current treatments are limited and engineering AF tissue has received attention as a way of regenerating IVDs. Exploring this technique, a team led by Bin Li from Soochow University in Jiangsu, China, cultured AF-derived stem cells from rabbits. They discovered that an aligned fibrous scaffold provided a favorable environment for AF stem cells to grow into cells that produced disc-strengthening collagen.

^99mTc-Methylene diphosphonate (^99mTc-MDP) is widely used in clinical settings to detect bone abnormalities. However, the mechanism of ^99mTc-MDP uptake in bone is not well elucidated. In this study, we utilized a mouse tibia injury model, single-photon emission computed tomography (gamma scintigraphy or SPECT), ex vivo micro-computed tomography, and histology to monitor ^99mTc-MDP uptake in injury sites during skeletal healing. In an ex vivo culture system, calvarial cells were differentiated into osteoblasts with osteogenic medium, pulsed with ^99mTc-MDP at different time points, and quantitated for ^99mTc-MDP uptake with a gamma counter. We demonstrated that ^99mTc-MDP uptake in the injury sites corresponded to osteoblast generation in those sites throughout the healing process. The ^99mTc-MDP uptake within the injury sites peaked on day 7 post-injury, while the injury sites were occupied by mature osteoblasts also starting from day 7. ^99mTc-MDP uptake started to decrease 14 days post-surgery, when we observed the highest level of bony tissue in the injury sites. We also found that ^99mTc-MDP uptake was associated with osteoblast maturation and mineralization in vitro. This study provides direct and biological evidence for ^99mTc-MDP uptake in osteoblasts during bone healing in vivo and in vitro.

MDP: A traditional method to monitor bone healing and osteoblasts

An imaging procedure to detect bone abnormalities and monitor the progress of bone repair has been further validated by research in the USA. The compound ^99mTc-methylene diphosphonate (^99mTc-MDP) contains a radioactive isotope of the element technetium, allowing its uptake into bone and subsequent imaging using computed tomography. This technique is widely used in clinical settings but the relationship of uptake to bone cell development is not well characterized. Bart Willams and co-workers at the Van Andel Research Institute in Michigan and the University of California, USA, studied the uptake of ^99mTc-MDP in the fractured bones of mice during healing. They found a close correlation between the incorporation of ^99mTc-MDP and the development and mineralization of bone-forming cells. This confirms and helps explain the validity of using ^99mTc-MDP to monitor bone abnormalities and healing in humans.

The bone marrow contains a heterogeneous milieu of cells, including macrophages, which are key cellular mediators for resolving infection and inflammation. Macrophages are most well known for their ability to phagocytose foreign bodies or apoptotic cells to maintain homeostasis; however, little is known about their function in the bone microenvironment. In the current study, we investigated the in vitro interaction of murine macrophages and bone marrow stromal cells (BMSCs), with focus on the juxtacrine induction of IL-6 signaling and the resultant effect on BMSC migration and growth. The juxtacrine interaction of primary mouse macrophages and BMSCs activated IL-6 signaling in the co-cultures, which subsequently enhanced BMSC migration and increased BMSC numbers. BMSCs and macrophages harvested from IL-6 knockout mice revealed that IL-6 signaling was essential for enhancement of BMSC migration and increased BMSC numbers via juxtacrine interactions. BMSCs were the main contributor of IL-6 signaling, and hence activation of the IL-6/gp130/STAT3 pathway. Meanwhile, macrophage derived IL-6 remained important for the overall production of IL-6 protein in the co-cultures. Taken together, these findings show the function of macrophages as co-inducers of migration and growth of BMSCs, which could directly influence bone formation and turnover.

Bone formation: Immune cell interactions promote growth

Immune cells in the bone marrow promote bone formation via interactions with other cells, with implications for bone healing and disease. This insight comes from work by Laurie K McCauley and colleagues at the Michigan School of Dentistry, USA, that focused on macrophages, a type of immune cell that destroys pathogens and dead cells. The researchers showed that the physical interactions of mouse macrophages with other cells in the bone marrow, known as bone marrow stromal cells, stimulated production of interleukin-6, a signaling molecule that is involved in bone formation. The increased production of interleukin-6 promoted growth and migration of the bone marrow stromal cells, thereby promoting bone formation. The findings suggest that macrophages are important for stimulation of bone growth during healing of fractures and could be important in understanding bone diseases such as osteoporosis.

Mesenchymal stem cell (MSC)-based treatments have shown promise for improving tendon healing and repair. MSCs have the potential to differentiate into multiple lineages in response to select chemical and physical stimuli, including into tenocytes. Cell elongation and cytoskeletal tension have been shown to be instrumental to the process of MSC differentiation. Previous studies have shown that inhibition of stress fiber formation leads MSCs to default toward an adipogenic lineage, which suggests that stress fibers are required for MSCs to sense the environmental factors that can induce differentiation into tenocytes. As the Rho/ROCK signal transduction pathway plays a critical role in both stress fiber formation and in cell sensation, we examined whether the activation of this pathway was required when inducing MSC tendon differentiation using rope-like silk scaffolds. To accomplish this, we employed a loss-of-function approach by knocking out ROCK, actin and myosin (two other components of the pathway) using the specific inhibitors Y-27632, Latrunculin A and blebbistatin, respectively. We demonstrated that independently disrupting the cytoskeleton and the Rho/ROCK pathway abolished the expression of tendon differentiation markers and led to a loss of spindle morphology. Together, these studies suggest that the tension that is generated by MSC elongation is essential for MSC teno-differentiation and that the Rho/ROCK pathway is a critical mediator of tendon differentiation on rope-like silk scaffolds.

Tendon formation: elongation drives stem cell differentiation

The shape change caused by the elongation of adult stem cells leads the stem cells themselves to commit to tendon differentiation. Working with mesenchymal stem cells, which can be isolated from various tissues, including bone marrow, fat and tendons, a team led by Dr. Hui B. Sun from the Albert Einstein College of Medicine in New York, USA, cultured the cells on a rope-like silk scaffold that allows for attachment, elongation, and growth. The stem cells changed shape to conform to the structure of the silk fibers, triggering biophysical alterations that stimulated the cells to differentiate toward tendon cells. The researchers used various drug inhibitors to demonstrate that the Rho/ROCK regulatory pathway is a critical mediator of this tension-induced transformation. A better understanding of this process could aid in the development of new stem cell-based therapies for tendon regeneration and repair.

Multiple growth factors (e.g., BMP2, TGF-β1, FGF2) and isolated genes have been shown to improve osteoblastic proliferation and mineralization, advancing bone tissue engineering. Among these factors, both polydopamine (PDA) and dopamine (DA) monomer have recently been reported to increase osteoblast proliferation and mineralization in vitro. Although a well-characterized neurotransmitter, DA’s role in the bone is unknown. We hypothesize that DA can directly act on osteoblasts, and examined whether osteoblasts express DA receptors that respond to exogenous DA. mRNAs and protein cell lysates were obtained from MC3T3-E1 cells during osteogenic differentiation phase. Reverse transcription polymerase chain reaction and western blot analysis were used to examine the expression of DA receptors, D1–D5. Dose-response effect and time course of DA treatment on cell proliferation, mineralization, and osteogenic differentiation were investigated at pre-determined days. Real-time PCR was performed to investigate whether DA affects osteogenic gene expression (ALP, BSP, OC, OSX, RUNX2, and Collagen1a2) with or without receptor antagonists (SCH233390 and GR103691). Two-way ANOVA was used for statistical analysis. All five DA receptors (D1, D2, D3, D4, and D5) mRNAs and proteins were expressed in MC3T3-E1 cells. DA treatment increased cell proliferation for up to 7 days (P < 0.05). Osteogenic mineralization was significantly greater in the DA-treated group than control group (P < 0.05). Finally, expression of all the osteogenic genes was inhibited by DA receptor antagonists for D1, D3, and D5. Our findings suggest that MC3T3-E1 osteoblasts express functional DA receptors that enhance proliferation and mineralization. PDA is not biologically inert and has important implications in orthopedic applications. Furthermore, osteoblast differentiation might be regulated by the nervous system, presumably during bone development, remodeling, or repair.

Bone formation: Neurotransmitters may boost bone cell division

Brain chemicals called neurotransmitters may play an important role in bone formation, new research from the US suggests. The role of the neurotransmitter dopamine in bone is largely unknown but recent research has suggested that bone cells may respond to neurotransmitters. Dr Ching-Chang Ko from the University of North Carolina and colleagues discovered adding dopamine to bone cells from mice stimulated cell division for up to seven days. Mineralization of the cells, an indication of bone formation, was also greater in the cells treated with dopamine than in untreated cells. The researchers suggest that dopamine can enhance bone regeneration. Using dopamine, potentially in conjunction with bone scaffolds in tissue engineering, could lead to a novel way of growing bone for use in orthopedic applications.

RBPjk-dependent Notch signaling regulates both the onset of chondrocyte hypertrophy and the progression to terminal chondrocyte maturation during endochondral ossification. It has been suggested that Notch signaling can regulate Sox9 transcription, although how this occurs at the molecular level in chondrocytes and whether this transcriptional regulation mediates Notch control of chondrocyte hypertrophy and cartilage development is unknown or controversial. Here we have provided conclusive genetic evidence linking RBPjk-dependent Notch signaling to the regulation of Sox9 expression and chondrocyte hypertrophy by examining tissue-specific Rbpjk mutant (Prx1Cre;Rbpjk ^f/f ), Rbpjk mutant/Sox9 haploinsufficient (Prx1Cre;Rbpjk ^f/f ;Sox9 ^f/+ ), and control embryos for alterations in SOX9 expression and chondrocyte hypertrophy during cartilage development. These studies demonstrate that Notch signaling regulates the onset of chondrocyte maturation in a SOX9-dependent manner, while Notch-mediated regulation of terminal chondrocyte maturation likely functions independently of SOX9. Furthermore, our in vitro molecular analyses of the Sox9 promoter and Notch-mediated regulation of Sox9 gene expression in chondrogenic cells identified the ability of Notch to induce Sox9 expression directly in the acute setting, but suppresses Sox9 transcription with prolonged Notch signaling that requires protein synthesis of secondary effectors.

Cartilage and bone formation: Knowledge of molecular mechanisms taken up a Notch

A study of cartilage and bone maturation has taken us a step closer to understanding the molecular mechanisms involved and how they malfunction in disease. Research led by Matthew Hilton from Duke University School of Medicine, US, investigated how a signaling pathway called Notch regulates the protein SOX9, which is itself a key regulator of cartilage and bone development. Some evidence suggests that SOX9 regulation is mediated by a protein called RBPjk, which alters gene expression in response to Notch signaling. By manipulating expression of SOX9 and RBPjk in mice and cultured cells, the researchers confirmed that Notch signaling regulates SOX9, but showed that RBPjk is not directly involved. Instead, proteins that are made in response to Notch signaling seem to be required. Further studies that identify these proteins will provide a greater understanding of cartilage and bone development.

The skeleton is a common site of cancer metastasis. Notably high incidences of bone lesions are found for breast, prostate, and renal carcinoma. Malignant bone tumors result in significant patient morbidity. Identification of these lesions is a critical step to accurately stratify patients, guide treatment course, monitor disease progression, and evaluate response to therapy. Diagnosis of cancer in the skeleton typically relies on indirect bone-targeted radiotracer uptake at sites of active bone remodeling. In this manuscript, we discuss established and emerging tools and techniques for detection of bone lesions, quantification of skeletal tumor burden, and current clinical challenges.

Cancer: Imaging the spread into bone

Methods are emerging to improve the imaging of skeletal metastasis, in which cancer spreads into bone. Detecting skeletal metastasis is vital for evaluating and treating primary cancers and their complications. This detection is critical for such common cancers as breast, prostate, thyroid, kidney and lung cancer. Daniel Thorek of John Hopkins University School of Medicine in Baltimore, USA and colleagues in New York and Sweden, have reviewed the use, challenges and limitations of existing and emerging imaging techniques. Scans that detect the uptake of various radioactive compounds are the most commonly used methods, but results can be complicated by changes in bone that are not related to cancer. Researchers are developing targeted imaging agents that are incorporated selectively into specific types of cancer cells. Semi-automated and whole-body image systems are also being refined.

Osteocytes reside as three-dimensionally (3D) networked cells in the lacunocanalicular structure of bones and regulate bone and mineral homeostasis. Despite of their important regulatory roles, in vitro studies of osteocytes have been challenging because: (1) current cell lines do not sufficiently represent the phenotypic features of mature osteocytes and (2) primary cells rapidly differentiate to osteoblasts upon isolation. In this study, we used a 3D perfusion culture approach to: (1) construct the 3D cellular network of primary murine osteocytes by biomimetic assembly with microbeads and (2) reproduce ex vivo the phenotype of primary murine osteocytes, for the first time to our best knowledge. In order to enable 3D construction with a sufficient number of viable cells, we used a proliferated osteoblastic population of healthy cells outgrown from digested bone chips. The diameter of microbeads was controlled to: (1) distribute and entrap cells within the interstitial spaces between the microbeads and (2) maintain average cell-to-cell distance to be about 19 µm. The entrapped cells formed a 3D cellular network by extending and connecting their processes through openings between the microbeads. Also, with increasing culture time, the entrapped cells exhibited the characteristic gene expressions (SOST and FGF23) and nonproliferative behavior of mature osteocytes. In contrast, 2D-cultured cells continued their osteoblastic differentiation and proliferation. This 3D biomimetic approach is expected to provide a new means of: (1) studying flow-induced shear stress on the mechanotransduction function of primary osteocytes, (2) studying physiological functions of 3D-networked osteocytes with in vitro convenience, and (3) developing clinically relevant human bone disease models.

Tissue engineering: Reproducing bone cell networks in three dimensions

Growing three-dimensional networks from bone cells (osteocytes) is now possible using microbeads as templates. Osteocyte structural cells play crucial roles in regulating bone and mineral balance in the body, but scientists lack suitable models to study their intricate physiological processes.WooLee and coworkers from the Stevens Institute of Technology in New Jersey, USA, harvested cells from mice and generated the osteocyte networks by perfusing calcium phosphate beads. Confined in a culture chamber, the beads formed a porous structure exhibiting evenly distributed spaces that entrapped the cells. The star-shaped cells extended and connected with each other through the spaces between the beads. Unlike two-dimensional cell cultures, the interconnected cells expressed genes typical of mature osteocytes and did not proliferate. This tissue is expected to clarify osteocyte function and provide clinical models for bone diseases.

Spinal cord injury (SCI)-induced bone loss represents the most severe osteoporosis with no effective treatment. Past animal studies have focused primarily on long bones at the acute stage using adolescent rodents. To mimic chronic SCI in human patients, we performed a comprehensive analysis of long-term structural and mechanical changes in axial and appendicular bones in adult rats after SCI. In this experiment, 4-month-old Fischer 344 male rats received a clinically relevant T13 contusion injury. Sixteen weeks later, sublesional femurs, tibiae, and L4 vertebrae, supralesional humeri, and blood were collected from these rats and additional non-surgery rats for micro-computed tomography (µCT), micro-finite element, histology, and serum biochemical analyses. At trabecular sites, extreme losses of bone structure and mechanical competence were detected in the metaphysis of sublesional long bones after SCI, while the subchondral part of the same bones showed much milder damage. Marked reductions in bone mass and strength were also observed in sublesional L4 vertebrae but not in supralesional humeri. At cortical sites, SCI induced structural and strength damage in both sub- and supralesional long bones. These changes were accompanied by diminished osteoblast number and activity and increased osteoclast number and activity. Taken together, our study revealed site-specific effects of SCI on bone and demonstrated sustained inhibition of bone formation and elevation of bone resorption at the chronic stage of SCI.

Spinal cord injury: long-term bone loss

Spinal cord injury (SCI) in rats has a chronic detrimental effect on skeletal structure with severe bone loss in the lower half of the body. Injury to the spinal cord can cause multiple long-term problems including severe osteoporosis, for which there is no effective treatment. Ling Qin at the University of Pennsylvania, USA, and co-workers conducted a long-term study of chronic SCI in Fischer 344 rats. They found a low level of bone formation and a high level of bone resorption at 16 weeks following SCI. This resulted in considerable bone loss and structural damage in the rat legs, particularly in spongy bone. The vertebrae below the SCI site also suffered bone loss, with more limited damage in the upper half of the body. The team recommend anabolic treatments that stimulate new bone formation to promote bone health in SCI patients.

The worldwide incidence of bone disorders and conditions has been increasing. Bone is a nanomaterials composed of organic (mainly collagen) and inorganic (mainly nano-hydroxyapatite) components, with a hierarchical structure ranging from nanoscale to macroscale. In consideration of the serious limitation in traditional therapies, nanomaterials provide some new strategy in bone regeneration. Nanostructured scaffolds provide a closer structural support approximation to native bone architecture for the cells and regulate cell proliferation, differentiation, and migration, which results in the formation of functional tissues. In this article, we focused on reviewing the classification and design of nanostructured materials and nanocarrier materials for bone regeneration, their cell interaction properties, and their application in bone tissue engineering and regeneration. Furthermore, some new challenges about the future research on the application of nanomaterials for bone regeneration are described in the conclusion and perspectives part.

Nanomaterials: bone repairs

Biocompatible nanomaterials that mimic live tissues are expected to transform bone repair and regeneration. Bone comprises organic collagen fibers and calcium phosphate crystals assembled into a complex architecture of a porous core surrounded by a compact shell. The growing incidence of age-related degenerative bone diseases such as infections, osteoporosis, and tumors has led to intensive research on artificial tissue scaffolds and substitutes to support bone regeneration, and nanomaterials have emerged as promising substances. Yun-Feng Lin and coworkers from Sichuan University, China, have reviewed the development of nanostructured materials and their application to bone engineering and regeneration. These nanomaterials interact readily with cells, providing scaffolds that stimulate stem cell proliferation, differentiation, and migration. They also promote repair by assisting the formation of the matrix surrounding the cells.

Osteoclasts (OCs) seeded on bone slices either drill round pits or dig long trenches. Whereas pits correspond to intermittent resorption, trenches correspond to continuous and faster resorption and require a distinct assembly of the resorption apparatus. It is unknown whether the distinction between pits and trenches has any biological relevance. Using OCs prepared from different blood donors, we found that female OCs achieved increased resorption mainly through pit formation, whereas male OCs did so through trench formation. Trench formation went along with high collagenolytic activity and high cathepsin K (CatK) expression, thereby allowing deeper demineralization. A specific CatK inhibitor abrogated the generation of trenches, while still allowing the generation of pits. OCs obtained from bone marrow were more prone to generate trenches than those obtained from blood. Scanning electron microscopy of bone surfaces eroded in vivo showed trenches and pits of similar size as those made by OCs in culture. We conclude that the distinction between trench- and pit-forming OCs is relevant to the differences among OCs from different skeletal sites, different individuals, including gender, and results from differences in collagenolytic power. This indicates a biological relevance and highlights the importance of discriminating between pits and trenches when assessing resorption.

Bone resorption: Digging further into bone degradation

Danish researchers have shown that the mechanism used by bone-degrading cells (osteoclasts) is determined by their source. Bone degradation by osteoclasts helps growth and repair but, in excess, can cause bone disease. Osteoclasts dig either circular pits or long trenches into bone. Trenches cause faster degradation, but the full significance of the two mechanisms is unknown. Kent Søe and Jean-Marie Delaissé, from the University of Southern Denmark and colleagues examined degradation by osteoclasts from different sources. Osteoclasts generated from bone marrow dug a greater proportion of trenches than osteoclasts generated from blood. Osteoclasts from men dug more trenches than pits, whereas the opposite was true of osteoclasts from women. Inhibiting the enzyme cathepsin K reversed the bias for trenches that was seen with osteoclasts from bone marrow and men, and could lead to a therapy for bone disease.

There are still controversies about the roles of microRNA-26a (miR-26a) in human malignancies, as it is a tumor suppressor in breast cancer, gastric cancer, and hepatocellular carcinoma, but is an oncogene in glioma and cholangiocarcinoma. Until now, the function of miR-26a in osteosarcoma remains largely elusive. Here, we found that miR-26a was downregualted in osteosarcoma tissues. Using in vitro and in vivo assays, we confirmed that miR-26a could inhibit the abilities of in vitro proliferation and suppress in vivo tumor growth in mouse model. Furthermore, we identified insulin-like growth factor 1 (IGF-1) as a novel and direct target of miR-26a and revealed that miR-26a exerted its tumor-suppressor function, at least in part, by inhibiting IGF-1 expression. These findings contribute to our understanding of the functions of miR-26a in osteosarcoma.

Bone cancer: small RNA offers drug target

A small regulatory molecule called microRNA-26a suppresses bone cancer by blocking the expression of a growth factor. MicroRNA-26a seems to play a different role in different kinds of cancer: it inhibits tumors of the breast, stomach, and liver, but promotes proliferation in cancers of the brain and bile ducts. Xinyu Tan and colleagues from the Third Affiliated Hospital of Southern Medical University in Guangzhou, China, examined the function of microRNA-26a in osteosarcoma, a type of bone cancer. Using cell lines and mouse models, they found that microRNA-26a serves as a tumor suppressor in this disease, and works, at least in part, by inhibiting a growth factor called insulin-like growth factor 1. These results offer a window into the molecular mechanism of osteosarcoma development, and suggest that boosting levels of microRNA-26a could help treat patients.