Osteoarthritis (OA) is the most common degenerative joint disease and a major cause of pain and disability in adult individuals. The etiology of OA includes joint injury, obesity, aging, and heredity. However, the detailed molecular mechanisms of OA initiation and progression remain poorly understood and, currently, there are no interventions available to restore degraded cartilage or decelerate disease progression. The diathrodial joint is a complicated organ and its function is to bear weight, perform physical activity and exhibit a joint-specific range of motion during movement. During OA development, the entire joint organ is affected, including articular cartilage, subchondral bone, synovial tissue and meniscus. A full understanding of the pathological mechanism of OA development relies on the discovery of the interplaying mechanisms among different OA symptoms, including articular cartilage degradation, osteophyte formation, subchondral sclerosis and synovial hyperplasia, and the signaling pathway(s) controlling these pathological processes.

Osteoarthritis: Towards better treatment through understanding disease mechanisms

A better understanding of the molecular mechanisms underpinning osteoarthritis should enable the development of new treatment strategies. In a review article, Di Chen from the Rush University Medical Center in Chicago, USA, and colleagues discuss the causes of this common degenerative joint disease, which include injury, obesity, aging, and genetics, and the various techniques used to elucidate the biochemical changes implicated in osteoarthritis-associated pain and disability. These include studying the disease in a range of mouse models, and investigating human cells and tissue in the laboratory. Even though significant progress has been made in recent years, many questions remain about the pathological processes involved in the initiation and progression of osteoarthritis. Tackling these unknowns could lead to interventions that restore degraded cartilage or slow down disease development.

Cre/loxP technology has been widely used to study cell type-specific functions of genes. Proper interpretation of such data critically depends on a clear understanding of the tissue specificity of Cre expression. The Dmp1-Cre mouse, expressing Cre from a 14-kb DNA fragment of the mouse Dmp1 gene, has become a common tool for studying gene function in osteocytes, but the presumed cell specificity is yet to be fully established. By using the Ai9 reporter line that expresses a red fluorescent protein upon Cre recombination, we find that in 2-month-old mice, Dmp1-Cre targets not only osteocytes within the bone matrix but also osteoblasts on the bone surface and preosteoblasts at the metaphyseal chondro-osseous junction. In the bone marrow, Cre activity is evident in certain stromal cells adjacent to the blood vessels, but not in adipocytes. Outside the skeleton, Dmp1-Cre marks not only the skeletal muscle fibers, certain cells in the cerebellum and the hindbrain but also gastric and intestinal mesenchymal cells that express Pdgfra. Confirming the utility of Dmp1-Cre in the gastrointestinal mesenchyme, deletion of Bmpr1a with Dmp1-Cre causes numerous large polyps along the gastrointestinal tract, consistent with prior work involving inhibition of BMP signaling. Thus, caution needs to be exercised when using Dmp1-Cre because it targets not only the osteoblast lineage at an earlier stage than previously appreciated, but also a number of non-skeletal cell types.

Genetics: Caution concerning specificity of gene deletion system

A gene deletion system designed to target bone cells has been shown to have more widespread effects than originally thought. This research tool was designed to target osteocytes, the bone cells entombed in bone matrix. Some evidence has suggested, however, that it can also affect other cells involved in bone development, and cells in bone marrow, muscle and the brain. To investigate further, Fanxin Long from Washington University School of Medicine, USA, and colleagues created mice in which activity of the gene deletion system led to fluorescent labeling of affected cells. They not only confirmed the more widespread effects that had previously been suggested, but revealed previously unseen effects on cells in the gut, highlighting the need for caution when interpreting the effects of gene deletions made with this system.

Tight-skin (TSK) mice are commonly used as an animal model to study the pathogenesis of Marfan syndrome (MFS), but little is known of their skeletal phenotype and in particular of the development of the spinal deformities, common in MFS. Here we examined growth of the axial skeletons of TSK and wild-type(B6) mice during their period of rapid growth. The whole bodies of mice, 4–12 weeks of age, were scanned after sacrifice, by micro-computed tomography (microCT). We reconstructed three-dimensional models of the spine and ribs, and measured vertebral body heights and rib lengths using the Mac-based image-processing software “OsiriX”. Although the TSK mice were smaller than the B6 mice at 4 weeks, they experienced an early growth spurt and by 8 weeks the height, but not the width, of the vertebral body was significantly greater in the TSK mice than the B6 mice. Measurement of the angles of scoliotic and kyphotic curves post-mortem in the mice was problematic, hence we measured changes that develop in skeletal elements in these disorders. As a marker of kyphosis, we measured anterior wedging of the vertebral bodies; as a marker for scoliosis we measured asymmetries in rib length. We found, unlike in the B6 mice where the pattern was diffuse, wedging in TSK mice was directly related to spinal level and peaked steeply at the thoracolumbar junction. There was also significant asymmetry in length of the ribs in the TSK mice, but not in the B6 mice. The TSK mice thus appear to exhibit spinal deformities seen in MFS and could be a useful model for gaining understanding of the mechanisms of development of scoliosis and kyphosis in this disorder.

Marfan syndrome: A mouse model for spinal development

Studying mice with a connective tissue mutation may clarify how the spinal deformities seen in Marfan syndrome (MFS) develop. In MFS, mutation of a key connective tissue protein leads to long limbs and kyphosis (forward rounding) and scoliosis (sideways twisting) of the spine. TSK mice have a similar mutation and have been used to investigate other aspects of MFS, but it was unclear whether they could be used to study spinal development. Jill Urban at the University of Oxford in the UK and coworkers compared the development of TSK and healthy mice, using CT scans and 3D modeling. TSK mice showed strongly wedge-shaped vertebrae and longer left ribs, signs of kyphosis and scoliosis, respectively. Studying their spinal development could help clarify the mechanisms underlying the spinal deformities in MFS and similar disorders, ultimately helping to improve treatment.

This randomized prospective study aimed to evaluate the clinical outcome of denosumab treatment alone and in combination with teriparatide in treatment-naive postmenopausal Japanese female patients with osteoporosis. Thirty patients were randomly assigned to two groups: (1) denosumab group (denosumab alone, n=13); and (2) combination group (denosumab+teriparatide, n=17). Serum bone-specific alkaline phosphatase (BAP), serum tartrate-resistant acid phosphatase (TRACP)-5b, urinary cross-linked N-terminal telopeptides of type I collagen (NTX), and bone mineral density (BMD) of L1–4 lumbar vertebrae (L-BMD) and bilateral total hips (H-BMD) were determined at the first visit and at various time points up to 24 months post-treatment to determine percentage changes. Serum TRACP-5b and urinary NTX were equally suppressed in both groups and maintained at low levels, with slight increases at 12, 18 and 24 months. BAP was significantly decreased in both groups from 4 to 24 months, with significant differences between the groups at 4, 8 and 15 months (P<0.05). L-BMD was significantly increased at most time points in both groups, with a significant difference between the combination group and denosumab group at 24 months (17.2% increase versus 9.6% increase; P<0.05). There was no significant difference in H-BMD between the two groups, although the levels tended to be higher in the combination group than in the denosumab group (9.5% increase versus 5.6% increase). These findings suggest that denosumab+teriparatide combination therapy may represent an important treatment for primary osteoporotic patients at high risk of vertebral fracture.

A two-year trial has confirmed the benefits of using two osteoporosis treatments in combination in Japanese postmenopausal women. Yukio Nakamura from the Shinshu University School of Medicine, Japan, and colleagues tested denosumab, a synthetic antibody that inhibits bone resorption, alone and in combination with teriparatide, a synthetic form of parathyroid hormone which promotes bone formation, in 30 women. In patients who received the combination therapy, hip and lumbar bone mineral density increased to a greater extent than in patients who received denosumab alone. Previous studies had yielded similar results, but included patients who had already received bisphosphates, the standard first-line therapy for osteoporosis, which might have influenced the effects of denosumab and teriparatide. The new trial identifies the combination therapy as an important treatment option for patients with severe osteoporosis.

Irisin is a polypeptide hormone derived from the proteolytic cleavage of fibronectin-type III domain-containing 5 (FNDC5) protein. Once released to circulation upon exercise or cold exposure, irisin stimulates browning of white adipose tissue (WAT) and uncoupling protein 1 (UCP1) expression, leading to an increase in total body energy expenditure by augmented UCP1-mediated thermogenesis. It is currently unknown whether irisin is secreted by bone upon exercise or whether it regulates bone metabolism in vivo. In this study, we found that 2 weeks of voluntary wheel-running exercise induced high levels of FNDC5 messenger RNA as well as FNDC5/irisin protein expression in murine bone tissues. Increased immunoreactivity due to exercise-induced FNDC5/irisin expression was detected in different regions of exercised femoral bones, including growth plate, trabecular bone, cortical bone, articular cartilage, and bone–tendon interface. Exercise also increased expression of osteogenic markers in bone and that of UCP1 in WAT, and led to bodyweight loss. Irisin intraperitoneal (IP) administration resulted in increased trabecular and cortical bone thickness and osteoblasts numbers, and concurrently induced UCP1 expression in subcutaneous WAT. Lentiviral FNDC5 IP administration increased cortical bone thickness. In vitro studies in bone cells revealed irisin increases osteoblastogenesis and mineralization, and inhibits receptor activator of nuclear factor-kB ligand (RANKL)-induced osteoclastogenesis. Taken together, our findings show that voluntary exercise increases irisin production in bone, and that an increase in circulating irisin levels enhances osteogenesis in mice.

Endocrinology: 'Exercise hormone' promotes bone formation

A fat-burning hormone that is released during exercise also enhances bone formation in mice. A team led by Jake Chen and Qisheng Tu from Tufts University School of Dental Medicine in Boston, USA, examined the expression of genes and proteins involved in producing irisin, the so-called “exercise hormone”, in mice allowed two weeks of voluntary wheel running and in a control group kept under normal cage conditions. They found higher levels of irisin and associated factors in the bone tissue of exercised mice. The researchers also injected mice with irisin or viruses engineered to express the hormone and observed increases in bone thickness. Experiments with bone cell lines showed that irisin induced bone-forming cells and inhibited bone-absorbing cells. The findings illustrate the complex interplay between exercise, muscle, bone and fat tissues.

Fibrogenesis imperfecta ossium is a rare disorder of bone usually characterized by marked osteopenia and associated with variable osteoporosis and osteosclerosis, changing over time. Histological examination shows that newly formed collagen is abnormal, lacking birefringence when examined by polarized light. The case presented demonstrates these features and, in addition, a previously undocumented finding of a persistent marked reduction of the serum C3 and C4. Osteoblasts established in culture from a bone biopsy showed abnormal morphology on electron microscopy and increased proliferation when cultured with benzoylbenzoyl-ATP and 1,25-dihydroxyvitamin D, contrasting with findings in normal osteoblasts in culture. A gene microarray study showed marked upregulation of the messenger RNA (mRNA) for G-protein-coupled receptor 128 (GPR 128), an orphan receptor of unknown function and also of osteoprotegerin in the patient’s osteoblasts in culture. When normal osteoblasts were cultured with the patient’s serum, there was marked upregulation of the mRNA for aquaporin 1. A single pathogenetic factor to account for the features of this disorder has not been defined, but the unique findings described here may facilitate more definitive investigation of the abnormal bone cell function.

Bone disorders: Getting a better handle on collagen deficiency

The identification of biological anomalies in a patient may soon facilitate the diagnosis and treatment of fibrogenesis imperfecta ossium. In this rare but debilitating disorder, the dense fibrous collagen network, which imparts tensile strength to bone, progressively degrades, giving rise to brittle tissue. However, the mechanisms involved in this disorder remain unclear, which leads to misdiagnosis and inadequate therapeutic options. To close this knowledge gap, Phillip Clifton-Bligh and co-workers from the University of Sydney, Australia, evaluated the disorder at multiple stages. Blood tests of patients revealed reduced levels in serum C3 and C4, two proteins associated with the immune system. Moreover, osteoblasts extracted from affected bone exhibited unusual proliferation, protein production and gene expression. These findings lay the foundation for deeper studies of abnormal bone cell function.

Familial hypocalciuric hypercalcemia (FHH) is caused by inactivating mutations in the calcium-sensing receptor (CaSR) gene. The loss of function of CaSR presents with rickets as the predominant skeletal abnormality in mice, but is rarely reported in humans. Here we report a case of a 16-year-old boy with FHH who presented with skeletal manifestations of rickets. To identify the possible pathogenic mutation, the patient was evaluated clinically, biochemically, and radiographically. The patient and his family members were screened for genetic mutations. Physical examination revealed a pigeon breast deformity and X-ray examinations showed epiphyseal broadening, both of which indicate rickets. Biochemical tests also showed increased parathyroid hormone (PTH), 1,25-dihydroxyvitamin D, and elevated ionized calcium. Based on these results, a diagnosis of FHH was suspected. Sequence analysis of the patient’s CaSR gene revealed a new missense mutation (c.2279T>A) in exon 7, leading to the damaging amino change (p.I760N) in the mature CaSR protein, confirming the diagnosis of FHH. Moreover, the skeletal abnormities may be related to but not limited to vitamin D abnormity. Elevated PTH levels and a rapid skeletal growth period in adolescence may have also contributed. Our study revealed that rickets-like features have a tendency to present atypically in FHH patients who have a mild vitamin D deficiency, and that CaSR mutations may have a partial role in the pathogenesis of skeletal deformities.

Calcium metabolism: Rickets a rare symptom of genetic disease

A report of rickets in a teenage boy has shown that the condition can be a symptom of a genetic disorder that causes calcium imbalances. Familial hypocalciuric hypercalcemia (FHH) is caused by a mutation in the calcium-sensing receptor gene (CASR) and leads to high blood calcium levels and low urine levels. Patients are usually asymptomatic, but mice with the mutation develop skeletal problems. Here, Weibo Xia and colleagues at the Peking Union Medical College Hospital, China, describe the case of a 16-year-old boy who presented with rickets. He was subsequently diagnosed with FHH because he was found to have a previously unseen mutation in the CASR gene. The patient’s clinical profile led the authors to conclude that rickets might be a rare symptom of FHH in patients who also have a mild vitamin D deficiency.

To investigate whether the administration frequency of parathyroid hormone (PTH) is associated with the development of cortical porosity, this study established 15 dosage regimens of teriparatide [human PTH(1–34), TPTD] with four distinct concentrations and four distinct administration frequencies of TPTD to 16-week-old ovariectomized rats. Our analyses demonstrated that the bone mineral density, mechanical properties, and bone turnover were associated with the total amount of TPTD administered. Our observations further revealed that the cortical porosity was markedly developed as a result of an increased administration frequency with a lower concentration of total TPTD administration in our setting, although the highest concentration also induced cortical porosity. Deconvolution fluorescence tiling imaging on calcein-labeled undecalcified bone sections also demonstrated the development of cortical porosity to be closely associated with the bone site where periosteal bone formation took place. This site-specific cortical porosity involved intracortical bone resorption and an increased number and proximity of osteocytic lacunae, occasionally causing fused lacunae. Taken together, these findings suggested the involvement of local distinctions in the rate of bone growth that may be related to the site-specific mechanical properties in the development of cortical porosity induced by frequent and/or high doses of TPTD.

Bone microarchitecture: Different hormone regimens affect bone porosity

The effect on bone porosity of a drug that promotes bone growth depends on the dosing and treatment frequency. A team in Japan led by Tadahiro Iimura from Ehime University and Ryoko Takao-Kawabata from Asahi Kasei Pharma Corporation injected female rats that had had their ovaries removed to mimic the effects of menopause with 15 different regimens of a synthetic form of the human parathyroid hormone. The regimens comprised four different concentrations of the drug and four distinct administration frequencies. The researchers found that more frequent treatment schedules and higher hormone doses created more porous, fragile vertebrae that were prone to fractures. These changes in bone microarchitecture were more pronounced in the front part of the vertebrae than in the back. The findings could help inform clinical dosing regimens for post-menopausal women receiving synthetic parathyroid hormone.

Leucine-rich repeat kinase 1 (LRRK1) plays a critical role in regulating cytoskeletal organization, osteoclast activity, and bone resorption with little effect on bone formation parameters. Deficiency of Lrrk1 in mice causes a severe osteopetrosis in the metaphysis of the long bones and vertebrae bones, which makes LRRK1 an attractive alternative drug target for the treatment of osteoporosis and other high-turnover bone diseases. This review summarizes recent advances on the functions of the Lrrk1-related family members, Lrrk1 deficiency-induced skeletal phenotypes, LRRK1 structure–function, potential biological substrates and interacting proteins, and the mechanisms of LRRK1 action in osteoclasts.

Bone resorption: Enzyme regulates cells responsible for bone breakdown

An enzyme called LRRK1 plays a critical role in regulating bone resorption and provides an attractive drug target for treating bone diseases. In a review article, Weirong Xing and colleagues from the Jerry L. Pettis Memorial VA Medical Center in Loma Linda, California, USA, describe how researchers identified LRRK1 (leucine-rich repeat kinase 1) in a screen of genes involved in skeletal development. The authors summarize recent discoveries on the functions of LRRK1 and related proteins, including the mechanisms by which LRRK1 modulates the activity of bone-absorbing osteoclast cells. They explain how mice that lack the gene encoding LRRK1 develop a severe bone disease known as osteopetrosis in which bones harden and become denser as a consequence of osteoclast dysfunction. Drugs that target LRRK1 activity could have therapeutic value in people with weakened bones.

Intervertebral disc (IVD) degeneration is the leading cause of disability with no disease-modifying treatment. IVD degeneration is associated with instable mechanical loading in the spine, but little is known about how mechanical stress regulates nucleus notochordal (NC) cells to maintain IVD homeostasis. Here we report that mechanical stress can result in excessive integrin α_vβ₆-mediated activation of transforming growth factor beta (TGFβ), decreased NC cell vacuoles, and increased matrix proteoglycan production, and results in degenerative disc disease (DDD). Knockout of TGFβ type II receptor (TβRII) or integrin α_v in the NC cells inhibited functional activity of postnatal NC cells and also resulted in DDD under mechanical loading. Administration of RGD peptide, TGFβ, and α_vβ₆-neutralizing antibodies attenuated IVD degeneration. Thus, integrin-mediated activation of TGFβ plays a critical role in mechanical signaling transduction to regulate IVD cell function and homeostasis. Manipulation of this signaling pathway may be a potential therapeutic target to modify DDD.

Spinal health: Halting disc degeneration

Treatments targeting a signalling molecule may help to prevent intervertebral disk (IVD) degeneration. As IVDs lose water and shrink, they become ineffective cushions, leading to back pain. This degeneration is a leading cause of disability, and no effective treatments are currently available. Unstable loading of spinal disks is known to be involved, but little is known about the underlying molecular mechanisms. Xu Cao at Johns Hopkins University in Maryland and an international team of coworkers investigated the cell-level events leading to IVD degeneration using different rodent models. They found that unstable loading led to high levels of the signalling molecule transforming growth factor beta (TGFβ), accelerating changes in disk cells and inducing IVD degeneration. Administration of an antibody reduced TGFβ levels and slowed deterioration, suggesting a potential therapeutic target for treatment of IVD degeneration.

Ultrasound could be a fast and cost-effective means of assessing joint changes in mouse models of posttraumatic osteoarthritis (PTOA). Such models are essential for understanding the biology of this degenerative joint disease and developing new treatments, but noninvasive methods of evaluating disease activity are lacking. Because ultrasound can visualize both joint space volumes and blood flow in the joints, it could provide an alternative to microscopic examination of tissue, assuming it accurately reflects the pathological changes. To test this, Lianping Xing at Rochester Medical Center in New York, Yongjun Wang at Shanghai University of Traditional Chinese Medicine and colleagues surgically induced PTOA in the knees of mice and then assessed the animals at regular intervals using either ultrasound or tissue microscopy. The changes detected by ultrasound strongly correlated with synovial inflammation and cartilage damage. In addition, ultrasound provides a tool for longitudinally assessing the changes of joint tissue lesions in PTOA.

Stress during prenatal development is correlated with detrimental cognitive and behavioral outcomes in offspring. However, the long-term impact of prenatal stress (PS) and disrupted glucocorticoid signaling on bone mass and strength is not understood. In contrast, the detrimental effect of lead (Pb) on skeletal health is well documented. As stress and Pb act on common biological targets via glucocorticoid signaling pathways and co-occur in the environment, this study first sought to assess the combined effect of stress and Pb on bone quality in association with alterations in glucocorticoid signaling. Bone parameters were evaluated using microCT, histomorphometry, and strength determination in 8-month-old male mouse offspring subjected to PS on gestational days 16 and 17, lifetime Pb exposure (100 p.p.m. Pb in drinking water), or to both. Pb reduced trabecular bone mass and, when combined with PS, Pb unmasked an exaggerated decrement in bone mass and tensile strength. Next, to characterize a mechanism of glucocorticoid effect on bone, prednisolone was implanted subcutaneously (controlled-release pellet, 5 mg·kg⁻¹ per day) in 5-month-old mice that decreased osteoblastic activity and increased sclerostin and leptin levels. Furthermore, the synthetic glucocorticoid dexamethasone alters the anabolic Wnt signaling pathway. The Wnt pathway inhibitor sclerostin has several glucocorticoid response elements, and dexamethasone administration to osteoblastic cells induces sclerostin expression. Dexamethasone treatment of isolated bone marrow cells decreased bone nodule formation, whereas removal of sclerostin protected against this decrement in mineralization. Collectively, these findings suggest that bone loss associated with steroid-induced osteoporosis is a consequence of sclerostin-mediated restriction of Wnt signaling, which may mechanistically facilitate glucocorticoid toxicity in bone.

Bone quality: Lead, stress and hormones

Research has provided new insight into the mechanism by which lead exposure and prenatal stress detrimentally affect bones. The adverse effects of lead exposure on bones are well documented, and evidence suggests that prenatal stress has a similar impact. The effects of both are thought to result from an excess of hormones called glucocorticoids, but the molecular pathways are unclear. J Edward Puzas from the University of Rochester, USA, and colleagues studied the effects of combined lead exposure and prenatal stress in mice. The combination reduced bone mass and strength, and affected bone development. The researchers found that the mechanism involved glucocorticoid-mediated increases in levels of sclerostin, a protein that blocks a signaling pathway important for bone formation. The finding suggests that sclerostin could be a target for treatments to improve bone health.

Tissue engineering has become a promising strategy for repairing damaged cartilage and bone tissue. Among the scaffolds for tissue-engineering applications, injectable hydrogels have demonstrated great potential for use as three-dimensional cell culture scaffolds in cartilage and bone tissue engineering, owing to their high water content, similarity to the natural extracellular matrix (ECM), porous framework for cell transplantation and proliferation, minimal invasive properties, and ability to match irregular defects. In this review, we describe the selection of appropriate biomaterials and fabrication methods to prepare novel injectable hydrogels for cartilage and bone tissue engineering. In addition, the biology of cartilage and the bony ECM is also summarized. Finally, future perspectives for injectable hydrogels in cartilage and bone tissue engineering are discussed.

Tissue engineering: Optimizing injectable hydrogels to improve bone repair

A review of injectable hydrogels highlights their potential for bone engineering, but stresses the need to optimize their fabrication. Tissue engineering requires a scaffold that encourages cells to grow on it; hydrogels, which are polymeric materials that contain large amounts of water, can form such scaffolds and can be injected, thereby avoiding surgery and easily filling irregularly shaped bone defects. Nongyue He from Southeast University, Nanjing, China and colleagues have reviewed the materials and techniques available to make injectable hydrogels. They conclude that natural materials are biocompatible but lack strength, whereas synthetic materials are strong but not biocompatible. Similarly, physical fabrication is simple but produces hydrogels with low strength, whereas chemical fabrication yields strong hydrogels that are not biocompatible. New approaches and integration of existing methods are needed to produce an injectable hydrogel with ideal properties.

Bone morphogenetic proteins (Bmp) regulate growth of different types of bone tissue via different mechanisms. Although Bmp have long been known to control formation of bone and cartilage, the exact mechanisms remain poorly understood. Fanxin Long at the Washington University School of Medicine in St. Louis, USA, and coworkers had previously shown that deleting a particular Bmp receptor causes formation of too much cancellous bone, the spongy, metabolically active tissue in the middle of bones, and not enough periosteal bone, the outermost layer. Long and coworkers investigated the role of Bmp signaling using genetically modified mice and discovered that Bmp regulates formation of the two types of bone tissue via two different mechanisms. These results improve our understanding of the signaling mechanisms that regulate bone growth.

Cartilage tissue engineering based on biomimetic scaffolds has become a rapidly developing strategy for repairing cartilage defects. In this study, a biphasic CAN-PAC hydrogel for osteochondral defect (OCD) regeneration was fabricated based on the density difference between the two layers via a thermally reactive, rapid cross-linking method. The upper hydrogel was cross-linked by CSMA and NIPAm, and the lower hydrogel was composed of PECDA, AAm and PEGDA. The interface between the two layers was first grafted by the physical cross-linking of calcium gluconate and alginate, followed by the chemical cross-linking of the carbon-carbon double bonds in the other components. The pore sizes of the upper and lower hydrogels were ~187.4 and ~112.6 μm, respectively. The moduli of the upper and lower hydrogels were ~0.065 and ~0.261 MPa. This prepared bilayer hydrogel exhibited the characteristics of mimetic composition, mimetic structure and mimetic stiffness, which provided a microenvironment for sustaining cell attachment and viability. Meanwhile, the biodegradability and biocompatibility of the CAN-PAC hydrogel were examined in vivo. Furthermore, an osteochondral defect model was developed in rabbits, and the bilayer hydrogels were implanted into the defect. The regenerated tissues in the bilayer hydrogel group exhibited new translucent cartilage and repaired subchondral bone, indicating that the hydrogel can enhance the repair of osteochondral defects.

Biomaterials: Dual-layered hydrogel aids bone and cartilage repair

A dual-layered polymer hydrogel could help to treat injuries to cartilage and bone. Yunfeng Lin and colleagues from Sichuan University in Chengdu, China, developed a relatively simple recipe for synthesizing a biphasic hydrogel with an upper layer that mirrors the properties of articular cartilage and a lower one that resembles subchondral bone. In cell culture, the upper and lower layers maintained the viability of cartilage cells and bone-forming osteoblast cells, respectively. The hydrogel broke down with minimal inflammation when implanted inside rats, demonstrating its biodegradability and biocompatibility. Experiments in rabbits with leg injuries showed that the hydrogel served as a temporary scaffold to enhance regeneration before being replaced by native tissue. The researchers suggest that this or similar dual-layered hydrogels could be used in the future application of bone and cartilage tissue engineering to people.

The activation of M1 macrophages can be achieved by stimulating them with lipopolysaccharide (LPS) and interferon-γ (IFN-γ). However, M1 can be found under physiological conditions without any pathological stimuli. This study aimed to understand the involvement of RANKL-induced M1 macrophages in bone formation compared with pathologically induced macrophages. Fischer rats were used to investigate macrophage distribution in normal and injured femoral condyles in vivo. Bone marrow-derived macrophages (BMDMs) were activated with LPS+IFN-γ and RANKL to achieve M1 activation in vitro. Gene expression related to inflammation, osteoclastogenesis, angiogenesis, and migration was determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and fluorescence-activated cell sorting (FACS). Tissue macrophages showed distinct expression patterns at different bone regions. RANKL was found in close proximity to inducible nitric oxide synthase-positive (iNOS+) cells in vivo, suggesting an association between RANKL expression and iNOS+ cells, especially in trabecular bone. RANKL-induced macrophages showed a different cytokine secretion profile compared with pathologically induced macrophages. Both osteoclasts and M1 macrophages peaked on day 7 during bone healing. RANKL could trigger M1-like macrophages with properties that were different from those of LPS+IFN-γ-induced macrophages. These RANKL-activated M1 macrophages were actively involved in bone formation.

Immunology: White blood cells crucial for bone healing

White blood cells induced by physiological rather than pathological stimuli have a crucial role in bone healing. Yinghong Zhou and colleagues from the Queensland University of Technology in Brisbane, Australia, analyzed the distribution of white blood cells known as macrophages in rat femurs. They found the anti-inflammatory M2 type of macrophages in the bone marrow and the pro-inflammatory M1 macrophages at the bone surface. Alongside the M1 macrophages, they also found high levels of a bone-remodeling protein called RANKL, which is essential for bone remodeling. They then treated macrophages in cell culture with either RANKL or proteins associated with bacterial infection, and found that only the RANKL-treated M1 macrophages were actively involved in bone repair, a finding that could be useful in treating bone disorders.

Bone is an endocrine organ involved in modulating glucose homeostasis. The role of the bone formation marker osteocalcin (OCN) in predicting diabetes was reported, but with conflicting results. No study has explored the association between baseline bone resorption activity and incident diabetes or prediabetes during follow-up. Our objective was to examine the relationship between the baseline bone resorption marker crosslinked C-telopeptide of type I collagen (CTX) and glycemic dysregulation after 4 years. This longitudinal study was conducted in a university teaching hospital. A total of 195 normal glucose tolerant (NGT) women at baseline were invited for follow-up. The incidence of diabetes and prediabetes (collectively defined as dysglycemia) was recorded. A total of 128 individuals completed the 4-year study. The overall conversion rate from NGT to dysglycemia was 31.3%. The incidence of dysglycemia was lowest in the middle tertile [16.3% (95% confidence interval (CI), 6.8%–30.7%)] compared with the lower [31.0% (95% CI, 17.2%–46.1%)] and upper [46.5% (95% CI, 31.2%–62.6%)] tertiles of CTX, with a significant difference seen between the middle and upper tertiles (P=0.002 5). After adjusting for multiple confounding variables, the upper tertile of baseline CTX was associated with an increased risk of incident dysglycemia, with an odds ratio of 7.09 (95% CI, 1.73–28.99) when the middle tertile was the reference. Osteoclasts actively regulate glucose homeostasis in a biphasic model that moderately enhanced bone resorption marker CTX at baseline provides protective effects against the deterioration of glucose metabolism, whereas an overactive osteoclastic function contributes to an increased risk of subsequent dysglycemia.

Biomarkers: CTX is a potential marker for diabetes

Measuring levels of a biomarker commonly used to determine bone health shows promise as a predictor for diabetes risk. Disruptions to bone regeneration and resorption may alter glucose homeostasis, possibly aiding the development of diabetes. Ting-ting Liu at Shanghai Jiao Tong University School of Medicine, China, and co-workers examined levels of the bone resorption marker CTX to determine if they correlate with the onset of diabetes. The team analyzed samples from 128 patients over a period of 4 years, during which 31% of the cohort developed abnormal glucose levels (dysglycemia). The correlation between CTX and dysglycemia was U-shaped, meaning that patients with elevated or low CTX had an increased risk of developing prediabetes or diabetes. Mid-range CTX levels indicated a healthier outlook. Larger-scale studies will help to clarify whether CTX is a viable marker of diabetes.

To evaluate the differences in outcomes of treatment with denosumab alone or denosumab combined with vitamin D and calcium supplementation in patients with primary osteoporosis. Patients were split into a denosumab monotherapy group (18 cases) or a denosumab plus vitamin D supplementation group (combination group; 23 cases). We measured serum bone alkaline phosphatase (BAP), tartrate-resistant acid phosphatase (TRACP)-5b and urinary N-terminal telopeptide of type-I collagen (NTX) at baseline, 1 week, as well as at 1 month and 2, 4, 8 and 12 months. We also measured bone mineral density (BMD) of L1–4 lumbar vertebrae (L)-BMD and bilateral hips (H)-BMD at baseline and at 4, 8 and 12 months. There was no significant difference in patient background. TRACP-5b and urinary NTX were significantly suppressed in both groups from 1 week to 12 months (except at 12 months for NTX). In the combination group, TRACP-5b was significantly decreased compared with the denosumab monotherapy group at 2 and 4 months (P<0.05). BAP was significantly suppressed in both groups at 2–12 months. L-BMD significantly increased at 8 and 12 months (8.9%) in the combination group and at 4, 8 and 12 months (6.0%) in the denosumab monotherapy group, compared with those before treatment. H-BMD was significantly increased in the combination group (3.6%) compared with the denosumab group (1.2%) at 12 months (P<0.05). Compared with denosumab monotherapy, combination therapy of denosumab with vitamin D and calcium stopped the decrease in calcium caused by denosumab, inhibited bone metabolism to a greater extent, and increased BMD (especially at the hips).

Osteoporosis: Vitamin D and calcium augment drug treatment

Vitamin D and calcium supplements help make an osteoporosis drug called denosumab safer and more effective. Researchers in Japan led by Yukio Nakamura of Shinshu University School of Medicine ran a year-long clinical trial in which 18 patients with primary osteoporosis received denosumab alone while another 23 patients received denosumab plus vitamin D tablets and calcium. Denosumab helps treat osteoporosis by blocking the RANKL protein involved in promoting the formation of bone-resorbing cells. Since RANKL also regulates calcium levels, one side effect of denosumab treatment is the risk of low blood calcium. The researchers found that the combination therapy worked better than denosumab alone at inhibiting bone resorption and increasing bone mineral density (especially in the hips). The combination also helped keep blood calcium levels in a healthier range.

Neutralizing CSF1 in vivo completely prevents ovariectomy (OVX)-induced bone loss in mice. There are two isoforms of CSF1, soluble (sCSF1), and membrane-bound (mCSF1), but their individual biological functions are unclear. It had been previously reported that mCSF1 knockout (K/O) and wild type (Wt) female mice experience the same degree of bone loss following OVX. In Wt mice the expression of sCSF1 was elevated fourfold in skeletal tissue following OVX while expression of mCSF1 was unchanged. To examine the role of sCSF1 in OVX-induced bone loss, mice were engineered in which sCSF1 was not expressed but expression of mCSF1 was unaffected (sCSF1 K/O). Isoform-specific reverse transcription PCR confirmed the absence of transcripts for sCSF1 in bone tissue isolated from these animals and no circulating CSF1 was detected by ELISA. Surprisingly, there were no significant differences in bone mineral density (BMD) between sCSF1 K/O mice and Wt controls as assessed by dual-energy X-ray absorptiometry and micro-CT. However, one month after OVX, femoral, spinal and total BMD had declined by 11.2%, 8.9%, and 8.7% respectively in OVX-Wt animals as compared to Sham-OVX. In contrast OVX sCSF1 K/O mice showed changes of +0.1%, −2.4%, and +2.3% at the same 3 sites compared to Sham-OVX sCSF1 K/O mice. These data indicate important non-redundant functions for the two isoforms of CSF1 and suggest that sCSF1, but not mCSF1, plays a key role in estrogen-deficiency bone loss.

Bone physiology: Colony-stimulating factor in estrogen-induced bone loss

Only one of the two forms of colony-stimulating factor 1 (CSF1) plays a role in estrogen-deficiency bone loss. CSF1, a protein responsible for white cell proliferation, is required for the development of cells that break down bone tissue. The two forms of CSF1 are soluble (sCSF1) and membrane-bound. A team headed by Gang-Qing Yao at Yale University School of Medicine, New Haven, USA, investigated the relative importance of these forms in mediating bone breakdown. The authors examined the role of sCSF1 in bone loss induced by estrogen deficiency (through ovary removal) in mice. Mice in which sCSF1 expression was blocked but membrane-bound CSF1 unaffected showed no bone loss, indicating that sCSF1 plays a key role in estrogen-deficiency bone loss. Coupled with work by other groups reporting a role for sCSF1 in inflammatory arthritis, the current work suggests that sCSF may be involved in several skeletal disorders.

Vitamin D co-regulates cell proliferation, differentiation and apoptosis in numerous tissues, including cancers. The known anti-proliferative and pro-apoptotic actions of the active metabolite of vitamin D, 1,25-dihydroxy-vitamin D [1,25(OH)₂D] are mediated through binding to the vitamin D receptor (VDR). Here, we report on the unexpected finding that stable knockdown of VDR expression in the human breast and prostate cancer cell lines, MDA-MB-231 and PC3, strongly induces cell apoptosis and inhibits cell proliferation in vitro. Implantation of these VDR knockdown cells into the mammary fat pad (MDA-MB-231), subcutaneously (PC3) or intra-tibially (both cell lines) in immune-incompetent nude mice resulted in reduced tumor growth associated with increased apoptosis and reduced cell proliferation compared with controls. These growth-retarding effects of VDR knockdown occur in the presence and absence of vitamin D and are independent of whether cells were grown in bone or soft tissues. Transcriptome analysis of VDR knockdown and non-target control cell lines demonstrated that loss of the VDR was associated with significant attenuation in the Wnt/β-catenin signaling pathway. In particular, cytoplasmic and nuclear β-catenin protein levels were reduced with a corresponding downregulation of downstream genes such as Axin2, Cyclin D1, interleukin-6 (IL-6), and IL-8. Stabilization of β-catenin using the GSK-3β inhibitor BIO partly reversed the growth-retarding effects of VDR knockdown. Our results indicate that the unliganded VDR possesses hitherto unknown functions to promote breast and prostate cancer growth, which appear to be operational not only within but also outside the bone environment. These novel functions contrast with the known anti-proliferative nuclear actions of the liganded VDR and may represent targets for new diagnostic and therapeutic approaches in breast and prostate cancer.

Cancer: Blocked receptor for vitamin D shrinks bone tumors

Blockade of the cellular receptor for vitamin D may help shrink tumors that have spread to bone tissue. Markus Seibel from the ANZAC Research Institute in Sydney, Australia, and colleagues experimentally lowered the expression of the gene encoding the vitamin D receptor (VDR) in both human breast and prostate cancer cell lines. These altered cells had a reduced growth rate and an elevated death rate, both in cell culture and when implanted into mice, whether they were inserted into the breast, under the skin, or into the bone. These growth-retarding effects occurred whether vitamin D was present or not. Activation of VDR with vitamin D was previously shown to have similar effects, suggesting that either blocking VDR or activating it can lead to the same outcome: reduced tumor growth, including tumors that have spread to bone.

Correction to: Bone Research (2017) 5, 17003; doi:10.1038/boneres.2017.3; published online 14 March 2017 The author’s name Weirong Xing (abbreviated name: W Xing) was misspelled as “Weirong R Xing” in this paper when it was published. The name has been corrected in the online version of this article. The publisher regrets the error.

Pregnancy represents a dynamic period with physical and physiological changes in both the mother and her developing fetus. The dramatic 2–3 fold increase in the active hormone 1,25(OH)₂D concentrations during the early weeks of pregnancy despite minimal increased calcium demands during that time of gestation and which are sustained throughout pregnancy in both the mother and fetus suggests an immunomodulatory role in preventing fetal rejection by the mother. While there have been numerous observational studies that support the premise of vitamin D's role in maintaining maternal and fetal well-being, until recently, there have been few randomized clinical trials with vitamin D supplementation. One has to exhibit caution, however, even with RCTs, whose results can be problematic when analyzed on an intent-to-treat basis and when there is high non-adherence to protocol (as if often the case), thereby diluting the potential good or harm of a given treatment at higher doses. As such, a biomarker of a drug or in this case “vitamin” or pre-prohormone is better served. For these reasons, the effect of vitamin D therapies using the biomarker circulating 25(OH)D is a far better indicator of true “effect.” When pregnancy outcomes are analyzed using the biomarker 25(OH)D instead of treatment dose, there are notable differences in maternal and fetal outcomes across diverse racial/ethnic groups, with improved health in those women who attain a circulating 25(OH)D concentration of at least 100 nmol·L⁻¹ (40 ng·mL⁻¹). Because an important issue is the timing or initiation of vitamin D treatment/supplementation, and given the potential effect of vitamin D on placental gene expression and its effects on inflammation within the placenta, it appears crucial to start vitamin D treatment before placentation (and trophoblast invasion); however, this question remains unanswered. Additional work is needed to decipher the vitamin D requirements of pregnant women and the optimal timing of supplementation, taking into account a variety of lifestyles, body types, baseline vitamin D status, and maternal and fetal vitamin D receptor (VDR) and vitamin D binding protein (VDBP) genotypes. Determining the role of vitamin D in nonclassical, immune pathways continues to be a challenge that once answered will substantiate recommendations and public health policies.

Vitamin D: supplementation urged for pregnant women

Larger amounts of vitamin D are needed during pregnancy than are currently recommended assert two experts. In a review article, Bruce Hollis and Carol Wagner from the Medical University of South Carolina in Charleston, USA, argue that current guidelines regarding vitamin D levels during pregnancy fail to take into account the latest research showing that vitamin D supplementation can help protect both the mother from pregnancy-related complications and the developing fetus from autoimmune disorders. The authors describe how, apart from its usual function as a calcium-regulating factor, vitamin D during pregnancy primarily helps maintain a proper immune balance. They argue that a lack of vitamin D during pregnancy or the early months of infancy may be responsible for certain diseases in later life, including asthma and multiple sclerosis.

Type 2 diabetes (T2D) is associated with systemic abnormal bone remodeling and bone loss. Meanwhile, abnormal subchondral bone remodeling induces cartilage degradation, resulting in osteoarthritis (OA). Accordingly, we investigated alterations in subchondral bone remodeling, microstructure and strength in knees from T2D patients and their association with cartilage degradation. Tibial plateaus were collected from knee OA patients undergoing total knee arthroplasty and divided into non-diabetic (n=70) and diabetes (n=51) groups. Tibial plateaus were also collected from cadaver donors (n=20) and used as controls. Subchondral bone microstructure was assessed using micro-computed tomography. Bone strength was evaluated by micro-finite-element analysis. Cartilage degradation was estimated using histology. The expression of tartrate-resistant acidic phosphatase (TRAP), osterix, and osteocalcin were calculated using immunohistochemistry. Osteoarthritis Research Society International (OARSI) scores of lateral tibial plateau did not differ between non-diabetic and diabetes groups, while higher OARSI scores on medial side were detected in diabetes group. Lower bone volume fraction and trabecular number and higher structure model index were found on both sides in diabetes group. These microstructural alterations translated into lower elastic modulus in diabetes group. Moreover, diabetes group had a larger number of TRAP⁺ osteoclasts and lower number of Osterix⁺ osteoprogenitors and Osteocalcin⁺ osteoblasts. T2D knees are characterized by abnormal subchondral bone remodeling and microstructural and mechanical impairments, which were associated with exacerbated cartilage degradation. In regions with intact cartilage the underlying bone still had abnormal remodeling in diabetes group, suggesting that abnormal bone remodeling may contribute to the early pathogenesis of T2D-associated knee OA.

Osteoarthritis: Diabetic knees show bone and cartilage deficiencies

Abnormal bone remodeling may contribute to the early development of knee osteoarthritis associated with type 2 diabetes (T2D). A group headed by William Lu at the University of Hong Kong investigated changes in subchondral bone remodeling (metabolism of bone below the cartilage in a joint), microstructure, and strength in knees from T2D patients. The authors collected the upper surfaces of shin bones from diabetic and non-diabetic patients undergoing total knee replacement. They found that the knees of diabetic patients showed abnormal bone remodeling as well as microstructural and mechanical deficiencies, which were associated with cartilage deterioration. In these patients, regions with intact cartilage displayed abnormal remodeling of the underlying bone. The authors concluded that T2D patients undergo abnormal subchondral bone remodeling, which may facilitate the development of knee osteoarthritis.

Osteoporosis is a common disease that affects patient quality of life, especially among the elderly population. Although inflammation contributes significantly to osteoporosis, the underlying mechanism is unclear. In this study, we found that tumor necrosis factor (TNF)-α, an inflammatory environment mimic, inhibits osteogenesis of bone mesenchymal stem cells (BMSCs), induces miR-146a and decreases Smad4. Moreover, overexpression of miR-146a inhibited the osteogenic ability of BMSCs, whereas blocking miR-146a partially rescued the osteogenesis deficiency under TNF-α treatment. Molecularly, miR-146a decreased Smad4 expression at the protein level by binding to an element located in the Smad4 3′-untranslated region, and restoration of Smad4 reversed the inhibitory effects of miR-146a on osteogenesis. Together, our results showed that the inflammatory environment mimic TNF-α inhibits osteogenesis via upregulation of miR-146a and subsequent downregulation of Smad4, thus suggesting that therapeutic manipulation of miR-146a maybe a potential strategy to improve osteogenesis in the context of osteoporosis.

Osteoporosis: Explaining the link with inflammation

A molecular mechanism that blocks bone cell formation as a result of inflammation suggests a new therapeutic strategy in osteoporosis. Inflammation is known to contribute to osteoporosis, but the underlying mechanism is unknown. Jiali Tan from the Sun Yat-sen University, Guangzhou, China, and colleagues suspected involvement of the small RNA molecule miR-146a, as it is involved in bone cell production and is targeted by the pro-inflammatory protein TNF-α. The researchers tested their hypothesis by treating mouse bone stem cells with TNF-α, and found that levels of miR-146a in the cells were increased. This increase led to a decrease in levels of another protein, Smad4, which is essential for bone cell development. The findings suggest that manipulation of miR-146a levels in bone stem cells could encourage bone growth in osteoporosis.

To evaluate the long-term consequence of repetitive mild traumatic brain injury (mTBI) on bone, mTBI was induced in 10-week-old female C57BL/6J mice using a weight drop model, once per day for 4 consecutive days at different drop heights (0.5, 1 and 1.5 m) and the skeletal phenotype was evaluated at different time points after the impact. In vivo micro-CT (μ-CT) analysis of the tibial metaphysis at 2, 8 and 12 weeks after the impact revealed a 5%–32% reduction in trabecular bone mass. Histomorphometric analyses showed a reduced bone formation rate in the secondary spongiosa of 1.5 m impacted mice at 12 weeks post impact. Apparent modulus (bone strength), was reduced by 30% (P<0.05) at the proximal tibial metaphysis in the 1.5 m drop height group at 2 and 8 weeks post impact. Ex vivo μ-CT analysis of the fifth lumbar vertebra revealed a significant reduction in trabecular bone mass at 12 weeks of age in all three drop height groups. Serum levels of osteocalcin were decreased by 22%, 15%, and 19% in the 0.5, 1.0 and 1.5 m drop height groups, respectively, at 2 weeks post impact. Serum IGF-I levels were reduced by 18%–32% in mTBI mice compared to contro1 mice at 2 weeks post impact. Serum osteocalcin and IGF-I levels correlated with trabecular BV/TV (r ²=0.14 and 0.16, P<0.05). In conclusion, repetitive mTBI exerts significant negative effects on the trabecular bone microarchitecture and bone mechanical properties by influencing osteoblast function via reduced endocrine IGF-I actions.

Brain injury: Consequences for bone growth

Mild traumatic brain injury (mTBI) can impair bone growth by disrupting growth hormone production. These injuries can be sustained during sporting events or military service, and can have significant long-term negative effects on other parts of the body. They are known to disrupt the brain centers responsible for hormone production, and researchers are beginning to explore the resulting health consequences. Subburaman Mohan at the Musculoskeletal Disease Center in Loma Linda, USA, and coworkers hypothesized that mTBI would negatively affect bone growth, and tested this by experimentally inducing mTBI in mice, and then measuring bone microarchitecture using microCT scans. They found that mice with mTBI showed significantly lower bone mass and strength. Unraveling the multiple effects of mTBIs may facilitate development of improved treatments that reduce the long-term adverse health consequences.

AFF1 and AFF4 belong to the AFF (AF4/FMR2) family of proteins, which function as scaffolding proteins linking two different transcription elongation factors, positive elongation factor b (P-TEFb) and ELL1/2, in super elongation complexes (SECs). Both AFF1 and AFF4 regulate gene transcription through elongation and chromatin remodeling. However, their function in the osteogenic differentiation of mesenchymal stem cells (MSCs) is unknown. In this study, we show that small interfering RNA (siRNA)-mediated depletion of AFF1 in human MSCs leads to increased alkaline phosphatase (ALP) activity, enhanced mineralization and upregulated expression of osteogenic-related genes. On the contrary, depletion of AFF4 significantly inhibits the osteogenic potential of MSCs. In addition, we confirm that overexpression of AFF1 and AFF4 differentially affects osteogenic differentiation in vitro and MSC-mediated bone formation in vivo. Mechanistically, we find that AFF1 regulates the expression of DKK1 via binding to its promoter region. Depletion of DKK1 in HA-AFF1-overexpressing MSCs abrogates the impairment of osteogenic differentiation. Moreover, we detect that AFF4 is enriched in the promoter region of ID1. AFF4 knockdown blunts the BRE luciferase activity, SP7 expression and ALP activity induced by BMP2 treatment. In conclusion, our data indicate that AFF1 and AFF4 differentially regulate the osteogenic differentiation of human MSCs.

Bone development: Related proteins affect stem cells differently

Two proteins from the same gene-regulating family have been shown to have opposite effects on the development of stem cells into bone cells. AFF1 and AFF4 both regulate the expression of genes involved in the development of adult stem cells into bone cells, but the exact roles of the two proteins were unclear. Quan Yuan from Sichuan University, China, and colleagues investigated their roles by manipulating the levels of each in cultured human adult stem cells. Depletion of AFF1 encouraged bone cell development, whereas its overexpression impaired development. By contrast, depletion of AFF4 impaired bone cell development, whereas its overexpression encouraged development. The findings reveal the critical importance of AFF1 and AFF4 in bone cell development, and clarify the nature of their roles.

Postmenopausal osteoporosis (PMO) is a prevalent metabolic bone disease characterized by bone loss and structural destruction, which increases the risk of fracture in postmenopausal women. Owing to the high morbidity and serious complications of PMO, many efforts have been devoted to its prophylaxis and treatment. The intestinal microbiota is the complex community of microorganisms colonizing the gastrointestinal tract. Probiotics, which are dietary or medical supplements consisting of beneficial intestinal bacteria, work in concert with endogenous intestinal microorganisms to maintain host health. Recent studies have revealed that bone loss in PMO is closely related to host immunity, which is influenced by the intestinal microbiota. The curative effects of probiotics on metabolic bone diseases have also been demonstrated. The effects of the intestinal microbiota on bone metabolism suggest a promising target for PMO management. This review seeks to summarize the critical effects of the intestinal microbiota and probiotics on PMO, with a focus on the molecular mechanisms underlying the pathogenic relationship between bacteria and host, and to define the possible treatment options.

Osteoporosis: Go with the gut

Probiotics may improve treatment of postmenopausal osteoporosis (PMO). In PMO, decreased estrogen levels weaken bones and increase the risk of fracture, usually of the hip, femur, or spine. Complications can be serious, and better prophylactic and treatment options are needed. The intestinal microbial community is known to affect bone formation, suggesting a new treatment avenue. Xuedong Zhou of Sichuan University, Chengdu, China and co-workers reviewed the evidence that probiotics might help combat PMO. They report that in PMO, low estrogen levels decrease microbial diversity and weaken the intestinal lining, opening the door for pathogens and triggering immune responses associated with bone loss. Treatment with probiotics ameliorates these effects, restoring intestinal diversity, normalizing immune responses, and increasing calcium absorption and production of estrogen-like compounds. The authors conclude that probiotics are a promising supplementary treatment for PMO.

Adipose-derived stromal cells (ASCs) have gained great attention in regenerative medicine. Progress in our understanding of adult neovascularization further suggests the potential of ASCs in promoting vascular regeneration, although the specific cues that stimulate their angiogenic behavior remain controversial. In this study, we established a three-dimensional (3D) angiogenesis model by co-culturing ASCs and endothelial cells (ECs) in collagen gel and found that ASC-EC-instructed angiogenesis was regulated by the canonical Wnt pathway. Furthermore, the angiogenesis that occurred in implants collected after injections of our collagen gel-based 3D angiogenesis model into nude mice was confirmed to be functional and also regulated by the canonical Wnt pathway. Wnt regulation of angiogenesis involving changes in vessel length, vessel density, vessel sprout, and connection numbers occurred in our system. Wnt signaling was then shown to regulate ASC-mediated paracrine signaling during angiogenesis through the nuclear translocation of β-catenin after its cytoplasmic accumulation in both ASCs and ECs. This translocation enhanced the expression of nuclear co-factor Lef-1 and cyclin D1 and activated the angiogenic transcription of vascular endothelial growth factor A (VEGFA), basic fibroblast growth factor (bFGF), and insulin-like growth factor 1 (IGF-1). The angiogenesis process in the 3D collagen model appeared to follow canonical Wnt signaling, and this model can help us understand the importance of the canonical Wnt pathway in the use of ASCs in vascular regeneration.

Blood vessel development: 3D culture models implicate key signaling pathway

Stem cells found in fat help promote new blood vessel formation by activating an evolutionarily conserved pathway crucial to development. Yunfeng Lin and colleagues from Sichuan University in Chengdu, China, cultured connective tissue cells (stromal cells), which are rich in stem cells, from mouse fat, with endothelial cells from mouse brain in a collagen gel, creating three-dimensional (3D) cellular models of blood vessel development. By applying different chemicals that either activate or repress proteins involved in the Wnt pathway, the researchers showed that Wnt signals from the stromal cells affected blood vessel length, density and sprouting, and did so by promoting the movement of an important regulatory protein into the cell nucleus. The blood vessels formed in these 3D models proved functional when implanted into mice, highlighting the potential of this system for studying vascular regeneration.

Multi-functional nanoshuttles for remotely targeted and on-demand delivery of therapeutic molecules and imaging to defined tissues and organs hold great potentials in personalized medicine, including precise early diagnosis, efficient prevention and therapy without toxicity. Yet, in spite of 25 years of research, there are still no such shuttles available. To this end, we have designed magnetic and gold nanoparticles (NP)-embedded silica nanoshuttles (MGNSs) with nanopores on their surface. Fluorescently labeled Doxorubicin (DOX), a cancer drug, was loaded in the MGNSs as a payload. DOX loaded MGNSs were encapsulated in heat and pH sensitive polymer P(NIPAM-co-MAA) to enable controlled release of the payload. Magnetically-guided transport of MGNSs was examined in: (a) a glass capillary tube to simulate their delivery via blood vessels; and (b) porous hydrogels to simulate their transport in composite human tissues, including bone, cartilage, tendon, muscles and blood–brain barrier (BBB). The viscoelastic properties of hydrogels were examined by atomic force microscopy (AFM). Cellular uptake of DOX-loaded MGNSs and the subsequent pH and temperature-mediated release were demonstrated in differentiated human neurons derived from induced pluripotent stem cells (iPSCs) as well as epithelial HeLa cells. The presence of embedded iron and gold NPs in silica shells and polymer-coating are supported by SEM and TEM. Fluorescence spectroscopy and microscopy documented DOX loading in the MGNSs. Time-dependent transport of MGNSs guided by an external magnetic field was observed in both glass capillary tubes and in the porous hydrogel. AFM results affirmed that the stiffness of the hydrogels model the rigidity range from soft tissues to bone. pH and temperature-dependent drug release analysis showed stimuli responsive and gradual drug release. Cells’ viability MTT assays showed that MGNSs are non-toxic. The cell death from on-demand DOX release was observed in both neurons and epithelial cells even though the drug release efficiency was higher in neurons. Therefore, development of smart nanoshuttles have significant translational potential for controlled delivery of theranostics’ payloads and precisely guided transport in specified tissues and organs (for example, bone, cartilage, tendon, bone marrow, heart, lung, liver, kidney, and brain) for highly efficient personalized medicine applications.

Targeted therapeutics: Using nanoparticle assemblies as drug carriers

Magnetic and gold nanoparticles embedded in silica nanospheres (MGNSs) can be used for remotely targeted delivery of therapeutic molecules. Despite the great potential of controlled delivery of drugs and active biomolecules to target cells, extensive research has not led to satisfactory results. A team headed by Ratneshwar Lal at University of California, San Diego, has now succeeded in developing MGNSs as a next-generation multifunctional delivery system. The authors demonstrated the effectiveness of MGNSs for controlled, highly efficient drug uptake and release using human cell culture. MGNSs show great promise for cell-specific controlled drug-release systems. The use of a magnetic field offers potential for magnetic guidance of MGNSs to precisely target specific anatomic locations, in particular hard-to-reach sites in the skeleton.

Calcium phosphate cements (CPCs) are frequently used to repair bone defects. Since their discovery in the 1980s, extensive research has been conducted to improve their properties, and emerging evidence supports their increased application in bone tissue engineering. Much effort has been made to enhance the biological performance of CPCs, including their biocompatibility, osteoconductivity, osteoinductivity, biodegradability, bioactivity, and interactions with cells. This review article focuses on the major recent developments in CPCs, including 3D printing, injectability, stem cell delivery, growth factor and drug delivery, and pre-vascularization of CPC scaffolds via co-culture and tri-culture techniques to enhance angiogenesis and osteogenesis.

Regenerative medicine: Smart cement could scaffold living bone

A natural component of bone is showing promise as a scaffold on which to build a new generation of living implants to correct bone defects. Bone injuries are often repaired using calcium phosphate cement (CPC), an inorganic component of bone. In recent years, interest has grown in using CPC as a scaffold for building new, living bone tissue, rather than just as a structural material. CPC attaches to bone, and allows cells to attach, migrate and proliferate on its surface. The recent incorporation of stem cells, drugs and growth factors into CPC scaffolds should further enhance their ability to regenerate bone, write Hockin HK Xu at the University of Maryland in Baltimore, USA, and colleagues in this review. Recent animal studies have suggested that such scaffolds can form a blood supply, important for correcting large bone defects.

Bone biomaterials play a vital role in bone repair by providing the necessary substrate for cell adhesion, proliferation, and differentiation and by modulating cell activity and function. In past decades, extensive efforts have been devoted to developing bone biomaterials with a focus on the following issues: (1) developing ideal biomaterials with a combination of suitable biological and mechanical properties; (2) constructing a cell microenvironment with pores ranging in size from nanoscale to submicro- and microscale; and (3) inducing the oriented differentiation of stem cells for artificial-to-biological transformation. Here we present a comprehensive review of the state of the art of bone biomaterials and their interactions with stem cells. Typical bone biomaterials that have been developed, including bioactive ceramics, biodegradable polymers, and biodegradable metals, are reviewed, with an emphasis on their characteristics and applications. The necessary porous structure of bone biomaterials for the cell microenvironment is discussed, along with the corresponding fabrication methods. Additionally, the promising seed stem cells for bone repair are summarized, and their interaction mechanisms with bone biomaterials are discussed in detail. Special attention has been paid to the signaling pathways involved in the focal adhesion and osteogenic differentiation of stem cells on bone biomaterials. Finally, achievements regarding bone biomaterials are summarized, and future research directions are proposed.

Bone repair: Building better bone grafts

A review of bone repair biomaterials highlights the best materials available and examines their interactions with stem cells. Bone structure is complex, with a crystalline outer layer for strength, and a spongy interior where blood flows and new red blood cells are made. Developing strong, biocompatible materials that mimic this structure and promote new bone growth is challenging. Cijun Shuai at Central South University in China and co-workers have published a review summarizing recent advances and indicating promising research directions. The review highlights materials that: provide the best combination of strength and flexibility; contain pores of many sizes from nano- to microscale, similar to bone; biodegrade at the same rate as new bone grows; and provide an optimal environment for stem cells to attach and then differentiate into bone cells.