Osteoporosis and consequent fracture are not limited to postmenopausal women. There is increasing attention being paid to osteoporosis in older men. Men suffer osteoporotic fractures about 10 years later in life than women, but life expectancy is increasing faster in men than women. Thus, men are living long enough to fracture, and when they do the consequences are greater than in women, with men having about twice the 1-year fatality rate after hip fracture, compared to women. Men at high risk for fracture include those men who have already had a fragility fracture, men on oral glucocorticoids or those men being treated for prostate cancer with androgen deprivation therapy. Beyond these high risk men, there are many other risk factors and secondary causes of osteoporosis in men. Evaluation includes careful history and physical examination to reveal potential secondary causes, including many medications, a short list of laboratory tests, and bone mineral density testing by dual energy X-ray absorptiometry (DXA) of spine and hip. Recently, international organizations have advocated a single normative database for interpreting DXA testing in men and women. The consequences of this change need to be determined. There are several choices of therapy for osteoporosis in men, with most fracture reduction estimation based on studies in women.

Osteoporosis: Older men are also at risk

The serious consequences of osteoporosis in men deserve greater attention from both patients and clinicians. Robert Adler from the McGuire Veterans Affairs Medical Center in Richmond, Virginia, USA, reviews the risk factors in men for osteoporotic bone fractures, a problem long thought to be mainly limited to postmenopausal women. The author discusses the ways in which the outcomes of the disease differ between the sexes. Men, for example, experience hip fractures on average a decade later in life than women but the one-year fatality rates from such breaks are about twice as high. Adler calls for more evaluations in older men by history, physical examination, and laboratory testing in addition to bone mineral density. This would improve the diagnosis and treatment of osteoporosis in this underserved but at-risk population.

Osteoarthritis (OA) is a common joint degenerative disease affecting the whole joint structure, including articular cartilage, subchondral bone and synovial tissue. Although extensive work has been done in recent years to explore the molecular mechanism underlying this disease, the pathogenesis of OA is still poorly understood and currently, there is no effective disease-modifying treatment for OA. Recently, both in vitro and in vivo studies suggest that confirmed (TGF-β)/SMAD pathway plays a critical role during OA development. This short review will focus on the function and signaling mechanisms of TGF-β/SMAD pathway in articular chondrocytes, mesenchymal progenitor cells of subchondral bone and synovial lining cells during OA development.

Osteoarthritis: Pathway to therapy

A regulatory pathway that plays a role in the development of osteoarthritis should be a priority target for treatment, say researchers. Osteoarthritis (OA) causes pain and reduced mobility in millions of people worldwide but the mechanisms that trigger and accelerate the disease are poorly understood. Di Chen and co-workers at the University of Rochester and Rush University in the US, reviewed recent papers that link the regulatory pathway of the protein TGF-β (transforming growth factor β) to the development of OA. They confirmed that TGF-β increases the volume of cells in cartilage, promotes the differentiation of cells that can synthesize bone and strengthens linings within joints. The researchers identify genes related to TGF-β that could serve as targets for OA therapy, and urge further research into three other regulatory pathways that interact with TGF-β.

Fibroblast growth factor (FGF)/fibroblast growth factor receptor (FGFR) signaling plays essential roles in bone development and diseases. Missense mutations in FGFs and FGFRs in humans can cause various congenital bone diseases, including chondrodysplasia syndromes, craniosynostosis syndromes and syndromes with dysregulated phosphate metabolism. FGF/FGFR signaling is also an important pathway involved in the maintenance of adult bone homeostasis. Multiple kinds of mouse models, mimicking human skeleton diseases caused by missense mutations in FGFs and FGFRs, have been established by knock-in/out and transgenic technologies. These genetically modified mice provide good models for studying the role of FGF/FGFR signaling in skeleton development and homeostasis. In this review, we summarize the mouse models of FGF signaling-related skeleton diseases and recent progresses regarding the molecular mechanisms, underlying the role of FGFs/FGFRs in the regulation of bone development and homeostasis. This review also provides a perspective view on future works to explore the roles of FGF signaling in skeletal development and homeostasis.

Bone disease: Mouse models offer new drug leads

Mouse models are revealing new insights into the roles of fibroblast growth factor (FGF) in human bone development and skeletal diseases. In a review article, Lin Chen and colleagues from the Third Military Medical University in Chongqing, China, highlight the many essential roles that FGFs, a family of growth factors, and their receptors play in the formation of a healthy skeleton. Mutations in FGF-associated genes can cause a range of congenital bone disorders. The authors discuss the ways in which scientists are genetically engineering mice to harbor mutations in a range of genes coding for FGFs and their receptors. These animal models are offering a window into the molecular mechanisms that underlie genetic skeletal diseases and providing attractive drug targets for treating many bone-related disorders.

Indian hedgehog (Ihh) is an essential signal that regulates endochondral bone development. We have previously shown that Wnt7b promotes osteoblast differentiation during mouse embryogenesis, and that its expression in the perichondrium is dependent on Ihh signaling. To test the hypothesis that Wnt7b may mediate some aspects of Ihh function during endochondral bone development, we activated Wnt7b expression from the R26-Wnt7b allele with Col2-Cre in the Ihh ^−/− mouse. Artificial expression of Wnt7b rescued vascularization of the hypertrophic cartilage in the Ihh ^−/− mouse, but failed to restore orthotopic osteoblast differentiation in the perichondrium. Similarly, Wnt7b did not recover Ihh-dependent perichondral bone formation in the Ihh ^−/− ; Gli3 ^−/− embryo. Interestingly, Wnt7b induced bone formation at the diaphyseal region of long bones in the absence of Ihh, possibly due to increased vascularization in the area. Thus, Ihh-dependent expression of Wnt7b in the perichondrium may contribute to vascularization of the hypertrophic cartilage during endochondral bone development.

Bone development: Bringing in the blood vessels

Recent work has clarified the roles of two regulatory proteins in the multi-step process of fetal bone development. Bones are generated through the gradual transformation of cartilage into bone, and the development of blood vessels within the cartilage is a key step in this process. The signaling protein Ihh induces multiple steps in bone development, such as bone cell maturation and blood vessel formation, but how its many effects are mediated has not been clear. A group led by Fanxin Long at Washington University School of Medicine, USA, showed that the regulatory protein Wnt7b could induce blood vessel formation in the cartilage of animals lacking Ihh. However, it could not carry out all the other functions of Ihh. The findings show how regulatory proteins work together to induce specific aspects of bone development.

The hypoxia inducible factors (Hifs) are evolutionarily conserved transcriptional factors that control homeostatic responses to low oxygen. In developing bone, Hif-1 generated signals induce angiogenesis necessary for osteoblast specification, but in mature bone, loss of Hif-1 in osteoblasts resulted in a more rapid accumulation of bone. These findings suggested that Hif-1 exerts distinct developmental functions and acts as a negative regulator of bone formation. To investigate the function of Hif-1α in osteoanabolic signaling, we assessed the effect of Hif-1α loss-of-function on bone formation in response to intermittent parathyroid hormone (PTH). Mice lacking Hif-1α in osteoblasts and osteocytes form more bone in response to PTH, likely through a larger increase in osteoblast activity and increased sensitivity to the hormone. Consistent with this effect, exposure of primary mouse osteoblasts to PTH resulted in the rapid induction of Hif-1α protein levels via a post-transcriptional mechanism. The enhanced anabolic response appears to result from the removal of Hif-1α-mediated suppression of β-catenin transcriptional activity. Together, these data indicate that Hif-1α functions in the mature skeleton to restrict osteoanabolic signaling. The availability of pharmacological agents that reduce Hif-1α function suggests the value in further exploration of this pathway to optimize the therapeutic benefits of PTH.

Osteoporosis: Novel target could improve treatment

US researchers have identified a novel target that could enhance the effects of parathyroid hormone (PTH) in the treatment of osteoporosis. A group led by Ryan Riddle at the Johns Hopkins University looked at factors that influence bone turnover at the cellular level in response to PTH. They found a rapid accumulation of bone occurred when they gave PTH to mutant mice lacking the regulatory protein hypoxia-inducible factor 1-alpha (Hif-1α). As bone matures, Hif-α interferes with cellular pathways which increase bone formation through its interaction with the protein β-catenin. The authors conclude that drugs that inhibit Hif-1α or impair the interaction of Hif-1α with β-catenin could be used to lower the therapeutic dose of PTH. This would increase bone accumulation and reduce the risk of fracture in patients with osteoporosis.

Lipoprotein receptor-related protein 6 (LRP6) plays a critical role in skeletal development and homeostasis in adults. However, the role of LRP6 in mesenchymal stem cells (MSCs), skeletal stem cells that give rise to osteoblastic lineage, is unknown. In this study, we generated mice lacking LRP6 expression specifically in nestin⁺ MSCs by crossing nestin-Cre mice with LRP6 ^flox mice and investigated the functional changes of bone marrow MSCs and skeletal alterations. Mice with LRP6 deletion in nestin⁺ cells demonstrated reductions in body weight and body length at 1 and 3 months of age. Bone architecture measured by microCT (µCT) showed a significant reduction in bone mass in both trabecular and cortical bone of homozygous and heterozygous LRP6 mutant mice. A dramatic reduction in the numbers of osteoblasts but much less significant reduction in the numbers of osteoclasts was observed in the mutant mice. Osterix⁺ osteoprogenitors and osteocalcin⁺ osteoblasts significantly reduced at the secondary spongiosa area, but only moderately decreased at the primary spongiosa area in mutant mice. Bone marrow MSCs from the mutant mice showed decreased colony forming, cell viability and cell proliferation. Thus, LRP6 in bone marrow MSCs is essential for their survival and proliferation, and therefore, is a key positive regulator for bone formation during skeletal growth and remodeling.

Bone formation: Controlling bone stem cells

A cell surface protein involved in bone formation is required for cell division and survival of skeletal stem cells. Lipoprotein receptor-related protein 6 (LRP6) is known to play an important role in the development and maintenance of mature bone cells but its role in skeletal stem cells had been less clear. A group led by Mei Wan at Johns Hopkins University School of Medicine in Baltimore, Maryland, USA, showed that mice that lacked LRP6 in skeletal stem cells were lighter and smaller than normal mice, and their bones were thinner. They had fewer mature bone cells, and their stem cells showed reduced survival and proliferation when compared with stem cells from normal mice. The findings suggest that LRP6 is important for bone growth during development, and bone maintenance during adulthood.

Although insulin-like growth factor-I (IGF-I) and estrogen signaling pathways have been shown to be involved in mediating the bone anabolic response to mechanical loading, it is not known whether these two signaling pathways crosstalk with each other in producing a skeletal response to mechanical loading. To test this, at 5 weeks of age, partial ovariectomy (pOVX) or a sham operation was performed on heterozygous IGF-I conditional knockout (H IGF-I KO) and control mice generated using a Cre-loxP approach. At 10 weeks of age, a 10 N axial load was applied on the right tibia of these mice for a period of 2 weeks and the left tibia was used as an internal non-non-loaded control. At the cortical site, partial estrogen loss reduced total volumetric bone mineral density (BMD) by 5% in control pOVX mice (P=0.05, one-way ANOVA), but not in the H IGF-I KO pOVX mice. At the trabecular site, bone volume/total volume (BV/TV) was reduced by 5%–6% in both control pOVX (P<0.05) and H IGF-I KO pOVX (P=0.05) mice. Two weeks of mechanical loading caused a 7%–8% and an 11%–13% (P<0.05 vs. non-loaded bones) increase in cortical BMD and cortical thickness (Ct.Th), respectively, in the control sham, control pOVX and H IGF-I KO sham groups. By contrast, the magnitude of cortical BMD (4%, P=0.13) and Ct.Th (6%, P<0.05) responses were reduced by 50% in the H IGF-I KO pOVX mice compared to the other three groups. The interaction between genotype and estrogen deficiency on the mechanical loading-induced cortical bone response was significant (P<0.05) by two-way ANOVA. Two weeks of axial loading caused similar increases in trabecular BV/TV (13%–17%) and thickness (17%–23%) in all four groups of mice. In conclusion, partial loss of both estrogen and IGF-I significantly reduced cortical but not the trabecular bone response to mechanical loading, providing in vivo evidence of the above crosstalk in mediating the bone response to loading.

Bone formation: Regulating responses to exercise

Mechanical strain on bones increases bone strength due to the activation and interaction of two bone growth-inducing regulatory pathways. Exercise prevents bone loss by putting mechanical strain on bones, which then causes an increase in bone thickness. Two regulatory pathways, one driven by estrogen and the other by insulin-like growth factor, are known to be involved in the growth of bones after exercise. A group led by Chandrasekhar Kesavan at the VA Loma Linda Healthcare System, USA, showed that in mice these two pathways augmented each other's ability to thicken the cortical bone tissue that surrounds bones but not the trabecular bone tissue found within bones. The findings suggest that maintaining both these pathways could be a key way to prevent the bone loss that occurs during aging.

Tendon–bone junctions (TBJs) are frequently injured, especially in athletic settings. Healing of TBJ injuries is slow and is often repaired with scar tissue formation that compromises normal function. This study explored the feasibility of using kartogenin (KGN), a biocompound, to enhance the healing of injured TBJs. We first determined the effects of KGN on the proliferation and chondrogenic differentiation of rabbit bone marrow stromal cells (BMSCs) and patellar tendon stem/progenitor cells (PTSCs) in vitro. KGN enhanced cell proliferation in both cell types in a concentration-dependent manner and induced chondrogenic differentiation of stem cells, as demonstrated by high expression levels of chondrogenic markers aggrecan, collagen II and Sox-9. Besides, KGN induced the formation of cartilage-like tissues in cell cultures, as observed through the staining of abundant proteoglycans, collagen II and osteocalcin. When injected into intact rat patellar tendons in vivo, KGN induced cartilage-like tissue formation in the injected area. Similarly, when KGN was injected into experimentally injured rat Achilles TBJs, wound healing in the TBJs was enhanced, as evidenced by the formation of extensive cartilage-like tissues. These results suggest that KGN may be used as an effective cell-free clinical therapy to enhance the healing of injured TBJs.

Tendon injury: Biocompound could promote healing of tendon-bone interface

The biocompound kartogenin (KGN) may help promote the healing of tendon-bone interfaces following an injury or anterior cruciate ligament (ACL) reconstruction. Jianying Zhang and James Wang from the University of Pittsburgh School of Medicine, USA, found that KGN improved the growth of two types of cells: those found in the bone marrow and those in tendons. The biocompound also accelerated the formation of cartilage-like tissues within cell cultures and promoted wound healing when injected into injured rat Achilles tendon-bone junctions. Hence, KGN may be used as an effective cell-free therapy to enhance the healing of injured tendon-bone junctions in clinical settings. KGN may also be used to enhance tendon-bone interface healing after ACL reconstruction by injecting the biocompound along with engineered tendon matrix during surgery.

Osteoporotic fractures are a major public health problem worldwide, but incidence varies greatly across racial groups and geographic regions. Recent work suggests that the incidence of osteoporotic fracture is rising among Asian populations. Studies comparing areal bone mineral density and fracture across races generally indicate lower bone mineral density in Asian individuals including the Chinese, but this does not reflect their relatively low risk of non-vertebral fractures. In contrast, the Chinese have relatively high vertebral fracture rates similar to that of Caucasians. The paradoxically low risk for some types of fractures among the Chinese despite their low areal bone mineral density has been elucidated in part by recent advances in skeletal imaging. New techniques for assessing bone quality non-invasively demonstrate that the Chinese compensate for smaller bone size by differences in hip geometry and microstructural skeletal organization. Studies evaluating factors influencing racial differences in bone remodeling, as well as bone acquisition and loss, may further elucidate racial variation in bone microstructure. Advances in understanding the microstructure of the Chinese skeleton have not only helped to explain the epidemiology of fracture in the Chinese, but may also provide insight into the epidemiology of fracture in other races as well.

Fracture: Imaging advances explain Chinese fracture risk

Advances in imaging technology have shed light on the reasons why the Chinese have different fracture risks compared to other populations. Marcella Walker and Elaine Cong from the New York Presbyterian Hospital reviewed the role new techniques for assessing bone quality have played in advancing our understanding of the epidemiology of fracture in the Chinese population. In particular, new imaging techniques have revealed that the Chinese skeleton has microstructural and mechanical advantages, explaining the lower risk of hip and non-vertebral fractures compared to other populations. The authors conclude that understanding structural and mechanical differences in skeletons between races is vital to understanding differences in bone strength and fracture risk between populations. Ongoing study of the Chinese skeleton will help to identify individuals at high risk of fracture across all populations.

Cone-beam computed tomography (CBCT) has been recently used to analyse trabecular bone structure around dental implants. To validate the use of CBCT for three-dimensional (3D) peri-implant trabecular bone morphometry by comparing it to two-dimensional (2D) histology, 36 alveolar bone samples (with implants n=27 vs. without implants n=9) from six mongrel dogs, were scanned ex vivo using a high-resolution (80 µm) CBCT. After scanning, all samples were decalcified and then sectioned into thin histological sections (∼6 μm) to obtain high contrast 2D images. By using CTAn imaging software, bone morphometric parameters including trabecular number (Tb.N), thickness (Tb.Th), separation (Tb.Sp) and bone volume fraction (BV/TV) were examined on both CBCT and corresponding histological images. Higher Tb.Th and Tb.Sp, lower BV/TV and Tb.N were found on CBCT images (P<0.001). Both measurements on the peri-implant trabecular bone structure showed moderate to high correlation (r=0.65–0.85). The Bland–Altman plots showed strongest agreement for Tb.Th followed by Tb.Sp, Tb.N and BV/TV, regardless of the presence of implants. The current findings support the assumption that peri-implant trabecular bone structures based on high-resolution CBCT measurements are representative for the underlying histological bone characteristics, indicating a potential clinical diagnostic use of CBCT-based peri-implant bone morphometric characterisation.

Bone quality: Three-dimensional scanning shows promise

Using an advanced approach of cone-beam computed tomography (CBCT) scanning and elaborate analysis to measure bone quality accurately and safely could help enhance the survival rate of dental implants. Traditionally, time-consuming microscopy is used to measure the internal structure and quality of bones. MRI and traditional CT scanning can be used prior to dental surgery but are associated with problems with metal artefacts and increased radiation doses. Now, Reinhilde Jacobs at the Department of Imaging and Pathology at the University of Leuven, Belgium, together with international colleagues, has conducted an analysis of 36 jawbone biopsies from dogs to compare traditional microscopic techniques with high-resolution, three-dimensional CBCT. The team found that CBCT was a potentially safe and useful clinical method for assessing the bone quality at dental implant sites, although further trials and testing are needed.

Heterogeneous nuclear ribonucleoprotein (hnRNP) C plays a key role in RNA processing but also exerts a dominant negative effect on responses to 1,25-dihydroxyvitamin D (1,25(OH)₂D) by functioning as a vitamin D response element-binding protein (VDRE-BP). hnRNPC acts a tetramer of hnRNPC1 (huC1) and hnRNPC2 (huC2), and organization of these subunits is critical to in vivo nucleic acid-binding. Overexpression of either huC1 or huC2 in human osteoblasts is sufficient to confer VDRE-BP suppression of 1,25(OH)₂D-mediated transcription. However, huC1 or huC2 alone did not suppress 1,25(OH)₂D-induced transcription in mouse osteoblastic cells. By contrast, overexpression of huC1 and huC2 in combination or transfection with a bone-specific polycistronic vector using a “self-cleaving” 2A peptide to co-express huC1/C2 suppressed 1,25D-mediated induction of osteoblast target gene expression. Structural diversity of hnRNPC between human/NWPs and mouse/rat/rabbit/dog was investigated by analysis of sequence variations within the hnRNP CLZ domain. The predicted loss of distal helical function in hnRNPC from lower species provides an explanation for the altered interaction between huC1/C2 and their mouse counterparts. These data provide new evidence of a role for hnRNPC1/C2 in 1,25(OH)₂D-driven gene expression, and further suggest that species-specific tetramerization is a crucial determinant of its actions as a regulator of VDR-directed transactivation.

Vitamin D: Species-specific effects of heterogeneous ribonucleoproteins in mice

Species-specific assembly of RNA-binding proteins determines the effects of vitamin D in osteoblasts (bone-building cells). Previous studies revealed that overexpression of human heterogeneous ribonucleoprotein (hnRNP) isoforms C1 and C2 together, or individually, in human osteoblasts inhibits vitamin D-induced gene expression, which is associated with vitamin D-resistant bone disease. Martin Hewison and John S. Adams at the University of California, Los Angeles, and colleagues recently discovered that overexpression of each human hnRNPC isoform individually in mouse osteoblasts was insufficient to recapitulate the vitamin D-resistant phenotype. To exert their effects, hnRNPC proteins form tetramers and this study identified a key amino acid in human hnRNPCs (asparagine 200) required for formation of functional tetramers. However, mouse hnRNPCs contain a serine at this position that prevents stabilization of inter-species tetramers, suggesting that species-specific tetramerization of hnRNPC is crucial for vitamin D-dependent gene expression in mouse osteoblasts.

Insufficient insulin production or action in diabetic states is associated with growth retardation and impaired bone healing, while the underling mechanisms are unknown. In this study, we sought to define the role of insulin signaling in the growth plate. Insulin treatment of embryonic metatarsal bones from wild-type mice increased chondrocyte proliferation. Mice lacking insulin receptor (IR) selectively in chondrocytes (CartIR ^−/−) had no discernable differences in total femoral length compared to control littermates. However, CartIR ^−/− mice exhibited an increase in chondrocyte numbers in the growth plate than that of the controls. Chondrocytes lacking IR had elevated insulin-like growth factor (IGF)-1R mRNA and protein levels. Subsequently, IGF-1 induced phosphorylation of Akt and ERK was enhanced, while this action was eliminated when the cells were treated with IGF-1R inhibitor Picropodophyllin. Deletion of the IR impaired chondrogenic differentiation, and the effect could not be restored by treatment of insulin, but partially rescued by IGF-1 treatment. Intriguingly, the size of hypertrophic chondrocytes was smaller in CartIR ^−/− mice when compared with that of the control littermates, which was associated with upregulation of tuberous sclerosis complex 2 (TSC2). These results suggest that deletion of the IR in chondrocytes sensitizes IGF-1R signaling and action, IR and IGF-1R coordinate to regulate the proliferation, differentiation and hypertrophy of growth plate chondrocytes.

Bone growth: The role of insulin

Insulin and insulin-like growth factors (IGFs) have independent, overlapping effects on bone growth. Insulin deficiency or insensitivity, caused by diseases such as diabetes, is known to impair bone growth but the mechanism remains unknown. An international team led by Chao Wan at the Chinese University of Hong Kong investigated how insulin affects chondrocytes, collagen-producing cells essential for building new bone, using genetically modified mice whose chondrocytes lacked insulin receptors (IRs). The mice had normal-length bones, and the IR-deficient chondrocytes showed increased production of and sensitivity to IGF-1, indicating that IGFs can compensate for reduced insulin. However, chondrocytes in the zone where new bone has yet to harden were small, indicating that insulin plays an important role in regulating chondrocyte size. These results will help clarify how diseases like diabetes impair bone growth and healing.

Locking plate fixation is being widely applied for fixation of forearm fractures and has many potential advantages, such as fixed angle fixation and improved construct stability, especially in osteoporotic bone. Biomechanical data comparing locking devices to commonly used Low Contact Dynamic Compression (LCDCP) plates for the fixation of forearm fractures has been lacking. The purpose of this study was to compare the fixation stability of a 3.5-mm unicortical locked plate with bicortical non-locked LCDCP plates. Six matched pairs of fresh frozen cadaveric forearms were randomly assigned to unicortical locked and bicortical unlocked groups. Non-destructive four-point bending and torsional test was performed on the ulna and radius separately, using a servohydraulic testing system to obtain construct stiffness of the intact specimens and specimens after osteotomy and plating. The specimens were then loaded to failure to test the fixation strength. The locked unicortical fixation showed significantly higher bending stiffness than the unlocked bicortical fixation, but with significantly lower stiffness and strength in torsion. Fixation strength was comparable between the two groups under bending, but significantly greater in the bicortical non-locked group under torsion. Findings from this study suggest that postoperative rehabilitation protocols may need modification to limit torsional loading in the early stage when using locked unicortical fixation. The study also points out the potential advantage of a hybrid fixation that combines locked unicortical and unlocked bicortical screws.

Fractures: Fixing forearm fractures

A new type of surgically implanted plate for fixation of broken bones is stronger and reduces bone damage from screws. The older screws pass straight through a bone, contacting the hard outer layer (cortex) on both sides (bicortical). Newer screws penetrate only one side of a bone (unicortical) and are held at the correct angle by their threaded heads. Unicortical screws are becoming more common but few data are available comparing the two types of screw. Steven Grindel from the Medical College of Wisconsin, USA and co-workers tested whether unicortical or bicortical screws provide better stability for fixation of broken forearms, using matched pairs of forearm bones from donated cadavers. The unicortical screws led to greater bone density following plate removal and were more resistant to bending but were weaker under twisting loads.

Osteoarthritis (OA) is a degenerative joint disease and a major cause of pain and disability in older adults. We have previously identified epidermal growth factor receptor (EGFR) signaling as an important regulator of cartilage matrix degradation during epiphyseal cartilage development. To study its function in OA progression, we performed surgical destabilization of the medial meniscus (DMM) to induce OA in two mouse models with reduced EGFR activity, one with genetic modification (Egfr ^Wa5/+mice) and the other one with pharmacological inhibition (gefitinib treatment). Histological analyses and scoring at 3 months post-surgery revealed increased cartilage destruction and accelerated OA progression in both mouse models. TUNEL staining demonstrated that EGFR signaling protects chondrocytes from OA-induced apoptosis, which was further confirmed in primary chondrocyte culture. Immunohistochemistry showed increased aggrecan degradation in these mouse models, which coincides with elevated amounts of ADAMTS5 and matrix metalloproteinase 13 (MMP13), the principle proteinases responsible for aggrecan degradation, in the articular cartilage after DMM surgery. Furthermore, hypoxia-inducible factor 2α (HIF2α), a critical catabolic transcription factor stimulating MMP13 expression during OA, was also upregulated in mice with reduced EGFR signaling. Taken together, our findings demonstrate a primarily protective role of EGFR during OA progression by regulating chondrocyte survival and cartilage degradation.

Arthritis: Protein protects against cartilage damage

A protein called epidermal growth factor receptor (EGFR) helps protect against cartilage destruction in mouse models of osteoarthritis. Ling Qin, from the University of Pennsylvania Perelman School of Medicine, USA, and colleagues in China and Canada induced the degenerative joint disease osteoarthritis by surgically destabilizing the meniscus in mouse joints. They reduced the levels of EGFR in mice either through genetic modification or with gefitinib, a cancer drug. Tissue analyses conducted three months after surgery revealed that chondrocytes, the cells that maintain healthy cartilage, were more severely damaged in mice with reduced levels of EGFR than in mice with normal levels. Elevated amounts of destructive proteinase enzymes contributed to the cartilage degradation, resulting in accelerated progression of osteoarthritis. The findings point to EGFR as a potential therapeutic target for osteoarthritis drug development.

Hispanic Americans of Caribbean origin are a fast-growing subset of the US population, but there are no studies on bone density, microstructure and biomechanical integrity in this minority group. In this study, we aimed to compare Caucasian and Caribbean Hispanic postmenopausal American women with respect to these characteristics. Thirty-three Caribbean Hispanics were age-matched to thirty-three Caucasian postmenopausal women. At the lumbar spine, the Hispanic women had significantly lower areal bone mineral density (aBMD). At the radius by high-resolution peripheral quantitative computed tomography (HR-pQCT), there were minimal differences between Hispanic and Caucasian women. At the tibia, Hispanic women had lower trabecular volumetric bone density and trabecular number, and higher trabecular separation. Individual trabecula segmentation (ITS) analyses indicated that at the tibia, Hispanic women not only had significantly lower bone volume fraction, but also had significantly lower rod bone volume fraction, plate trabecular number, rod trabecular number and lower plate–plate, plate–rod and rod–rod junction densities compared to Caucasian women. The differences in bone quantity and quality contributed to lower whole bone stiffness at the radius, and both whole bone and trabecular bone stiffness at the tibia in Hispanic women. In conclusion, Hispanic women had poorer bone mechanical and microarchitectural properties than Caucasian women, especially at the load-bearing distal tibia.

Bone structure: Caribbean Hispanic women have weaker bones

A study of American post-menopausal women has revealed weaker bones in Caribbean Hispanics than Caucasians, increasing the risk of fracture. Bone properties vary between ethnic groups but little is known about the bones of Caribbean Hispanic women, a growing population in the US. Led by Edward Guo and Elizabeth Shane at Columbia University, USA, researchers analyzed the mineral density and microstructure of the bones of 33 post-menopausal women in each ethnic group. They found that the Hispanic women had lower mineral density in the lower spine, while the microstructure of the tibia, or shinbone, showed differences that cause lower bone stiffness. This small-group study suggests that the population of Caribbean Hispanic women in the USA is at increased risk of post-menopausal fractures and that larger population-based studies are needed.

Tissue engineering is promising to meet the increasing need for bone regeneration. Nanostructured calcium phosphate (CaP) biomaterials/scaffolds are of special interest as they share chemical/crystallographic similarities to inorganic components of bone. Three applications of nano-CaP are discussed in this review: nanostructured calcium phosphate cement (CPC); nano-CaP composites; and nano-CaP coatings. The interactions between stem cells and nano-CaP are highlighted, including cell attachment, orientation/morphology, differentiation and in vivo bone regeneration. Several trends can be seen: (i) nano-CaP biomaterials support stem cell attachment/proliferation and induce osteogenic differentiation, in some cases even without osteogenic supplements; (ii) the influence of nano-CaP surface patterns on cell alignment is not prominent due to non-uniform distribution of nano-crystals; (iii) nano-CaP can achieve better bone regeneration than conventional CaP biomaterials; (iv) combining stem cells with nano-CaP accelerates bone regeneration, the effect of which can be further enhanced by growth factors; and (v) cell microencapsulation in nano-CaP scaffolds is promising for bone tissue engineering. These understandings would help researchers to further uncover the underlying mechanisms and interactions in nano-CaP stem cell constructs in vitro and in vivo, tailor nano-CaP composite construct design and stem cell type selection to enhance cell function and bone regeneration, and translate laboratory findings to clinical treatments.

Tissue engineering: New approach could replace traditional bone grafts

A new approach to regenerating bone has the potential to overtake traditional bone grafting techniques following fracture or trauma. Standard procedures for replacing bone tissue are hampered by the need to use stem cells from patients or donors, resulting in a need to develop new techniques. Hockin Xu and colleagues from the University of Maryland in Baltimore reviewed the use of nano-sized calcium phosphate (nano-CaP) biomaterials in bone repair. These bio-materials mimic the major inorganic component of bone and easily bond with neighbouring bone. They work by influencing stem cells to produce bone but the underlying mechanisms are not fully understood. The chemical composition, surface roughness, stiffness and size of the biomaterial are all thought to play a role. The researchers conclude that learning more about these mechanisms could help translate the findings into clinical treatments.

Toll-like receptor (TLR)-mediated inflammatory response could negatively affect bone metabolism. In this study, we determined how osteogenesis is regulated during inflammatory responses that are downstream of TLR signaling. Human primary osteoblasts were cultured in collagen gels. Pam3CSK4 (P3C) and Escherichia coli lipopolysaccharide (EcLPS) were used as TLR2 and TLR4 ligand respectively. Porphyromonas gingivalis LPS having TLR2 activity with either TLR4 agonism (Pg1690) or TLR4 antagonism (Pg1449) and mutant E. coli LPS (LPxE/LPxF/WSK) were used. IL-1β, SH2-containing inositol phosphatase-1 (SHIP1) that has regulatory roles in osteogenesis, alkaline phosphatase and mineralization were analyzed. 3α-Aminocholestane (3AC) was used to inhibit SHIP1. Our results suggest that osteoblasts stimulated by P3C, poorly induced IL-1β but strongly upregulated SHIP1 and enhanced osteogenic mediators. On the contrary, EcLPS significantly induced IL-1β and osteogenic mediators were not induced. While Pg1690 downmodulated osteogenic mediators, Pg1449 enhanced osteogenic responses, suggesting that TLR4 signaling annuls osteogenesis even with TLR2 activity. Interestingly, mutant E. coli LPS that induces weak inflammation upregulated osteogenesis, but SHIP1 was not induced. Moreover, inhibiting SHIP1 significantly upregulated TLR2-mediated inflammatory response and downmodulated osteogenesis. In conclusion, these results suggest that induction of weak inflammatory response through TLR2 (with SHIP1 activity) and mutant TLR4 ligands could enhance osteogenesis.

Bone formation: Targeting inflammatory response may prevent bone loss

US researchers have uncovered how the immune system interacts with the skeletal system to influence bone formation and turnover. Led by Manoj Muthukuru from West Virginia University in Morgantown, the researchers looked at how bone formation is regulated during inflammation. Chronic inflammatory conditions like arthritis can affect the precise interplay between cells responsible for bone formation (osteoblasts) and bone resorption (osteoclasts). Osteoblasts express proteins called Toll-like receptors (TLRs), which interact with the immune system to recognise microbial structures, but little is known about how this interaction affects bone formation. Analysing human osteoblasts, the researchers discovered that activation of TLR-2 contributed to an inflammatory response that enhanced bone-forming activities (osteogenesis). The authors conclude that targeting these inflammatory responses could be a novel therapeutic approach to boosting bone production in people with inflammatory bone disease.

A large body of literature suggests that bone metabolism is susceptible to the ill effects of reactive species that accumulate in the body and cause cellular dysfunction. One of the body’s front lines in defense against such damage is the transcription factor, Nrf2. This transcription factor regulates a plethora of antioxidant and cellular defense pathways to protect cells from such damage. Despite the breadth of knowledge of both the function of Nrf2 and the effects of reactive species in bone metabolism, the direct role of Nrf2 in skeletal biology has yet to be thoroughly examined. Thus, in the current study, we have examined the role of Nrf2 in postnatal bone metabolism in mice. Mice lacking Nrf2 (Nrf2^−/−) exhibited a marked deficit in postnatal bone acquisition, which was most severe at 3 weeks of age when osteoblast numbers were 12-fold less than observed in control animals. While primary osteoblasts from Nrf2^−/− mice functioned normally in vitro, the colony forming capacity of bone marrow stromal cells (BMSCs) from these mice was significantly reduced compared to controls. This defect could be rescued through treatment with the radical scavenger N-acetyl cysteine (NAC), suggesting that increased reactive species stress might impair early osteoblastogenesis in BMSCs and lead to the failure of bone acquisition observed in Nrf2^−/− animals. Taken together, these studies suggest Nrf2 represents a key pathway in regulating bone metabolism, which may provide future therapeutic targets to treat osteoporosis.

Bone development: Regulating reactive oxygen species

A protein that regulates reactive oxygen species (ROS) levels, Nrf2, is critical for healthy bone growth. ROS are natural byproducts of cell metabolism and their levels are tightly controlled by regulators such as Nrf2. Elevated levels damage cells and are known to impair bone growth. However, whether Nrf2 plays a protective role in bone metabolism remained unknown. Douglas DiGirolamo and Rajesh Thimmulappa and coworkers at Johns Hopkins University, USA, examined bone tissue in mice lacking Nrf2. The mice showed up to 45% reduced bone mass and a 12-fold decrease in the number of bone-building cells known as osteoblasts. The osteoblasts were normal but their progenitors, bone marrow stromal cells (BMSCs), were impaired, indicating that elevated ROS levels prevented BMSCs from forming enough osteoblasts to build healthy bone. This research may help to develop therapies for osteoporosis.

Screening gene function in vivo is a powerful approach to discover novel drug targets. We present high-throughput screening (HTS) data for 3 762 distinct global gene knockout (KO) mouse lines with viable adult homozygous mice generated using either gene-trap or homologous recombination technologies. Bone mass was determined from DEXA scans of male and female mice at 14 weeks of age and by microCT analyses of bones from male mice at 16 weeks of age. Wild-type (WT) cagemates/littermates were examined for each gene KO. Lethality was observed in an additional 850 KO lines. Since primary HTS are susceptible to false positive findings, additional cohorts of mice from KO lines with intriguing HTS bone data were examined. Aging, ovariectomy, histomorphometry and bone strength studies were performed and possible non-skeletal phenotypes were explored. Together, these screens identified multiple genes affecting bone mass: 23 previously reported genes (Calcr, Cebpb, Crtap, Dcstamp, Dkk1, Duoxa2, Enpp1, Fgf23, Kiss1/Kiss1r, Kl (Klotho), Lrp5, Mstn, Neo1, Npr2, Ostm1, Postn, Sfrp4, Slc30a5, Slc39a13, Sost, Sumf1, Src, Wnt10b), five novel genes extensively characterized (Cldn18, Fam20c, Lrrk1, Sgpl1, Wnt16), five novel genes with preliminary characterization (Agpat2, Rassf5, Slc10a7, Slc26a7, Slc30a10) and three novel undisclosed genes coding for potential osteoporosis drug targets.

Osteoporosis: Mouse screen reveals novel drug targets

A systematic analysis in mice has identified multiple genes affecting bone mass that could code for novel drug targets. Robert Brommage and his colleagues from Lexicon Pharmaceuticals in Texas, USA, scanned the bones f 3,776 mouse lines, each containing a global ‘knockout’ mutation that disables the function of a single gene. The researchers further examined bone architecture, strength and mineralization in any mice with signs of skeletal phenotypes. These analyses revealed dozens of genes that affected bone mass in some way, many of which were not previously known to impact skeletal health. Three of the newly identified genes were not disclosed. These genes code for enzymes or secreted proteins and, although their identity remains a secret, the authors suggest they could be potential therapeutic targets for treating osteoporosis.

IFT20 is the smallest member of the intraflagellar transport protein (IFT) complex B. It is involved in cilia formation. Studies of IFT20 have been confined to ciliated cells. Recently, IFT20 was found to be also expressed in non-ciliated T cells and have functions in immune synapse formation and signaling in vitro. However, how IFT20 regulates T-cell development and activation in vivo is still unknown. We deleted the IFT20 gene in early and later stages of T-cell development by crossing IFT20 ^flox/flox (IFT20 ^f/f ) mice with Lck-Cre and CD4-Cre transgenic mice, and investigated the role of IFT20 in T-cell maturation and in the development of T cell-mediated collagen-induced arthritis (CIA). We found that both Lck-Cre/IFT20 ^f/f and CD4-Cre/IFT20 ^f/f mice were indistinguishable from their wild-type littermates in body size, as well as in the morphology and weight of the spleen and thymus. However, the number of CD4- and CD8-positive cells was significantly lower in thymus and spleen in Lck-Cre/IFT20 ^f/f mice. Meanwhile, the incidence and severity of CIA symptoms were significantly decreased, and inflammation in the paw was significantly inhibited in Lck-Cre/IFT20 ^f/f mice compared to Lck-Cre/IFT20 ^+/+ littermates. Deletion IFT20 in more mature T cells of CD4-Cre/IFT20 ^f/f mice had only mild effects on the development of T cells and CIA. The expression of IL-1β, IL-6 and TGF-β1 were significantly downregulated in the paw of Lck-Cre/IFT20 ^f/f mice, but just slight decreased in CD4-Cre/IFT20 ^f/f mice. These results demonstrate that deletion of IFT20 in the early stage of T-cell development inhibited CIA development through regulating T-cell development and the expression of critical cytokines.

Arthritis: Understanding the role of T-cells

The severity of arthritis in mice is reduced by deletion of a protein in early stage T-cell differentiation. T-cells, a type of white blood cell, are known to mediate arthritis; however, the processes governing T-cell behavior in arthritis are unclear. Shuying Yang and co-workers at the University at Buffalo, SUNY have uncovered the complex role of IFT20 in T-cell differentiation by studying mice with collagen-induced arthritis (CIA). The team created two CIA mouse models: one model with IFT20 deletion at early stage T-cell differentiation, and a second model with deletion in mature T-cells. Loss of IFT20 led to a significant reduction in CIA symptoms in the first model, but not the second. The team concluded that IFT20 is an important regulator of T-cell differentiation, but does not noticeably affect mature T-cells.

Daily 20-μg and once-weekly 56.5-μg teriparatide (parathyroid hormone 1–34) treatment regimens increase bone mineral density (BMD) and prevent fractures, but changes in bone turnover markers differ between the two regimens. The aim of the present study was to explain changes in bone turnover markers using once-weekly teriparatide with a simulation model. Temporary increases in bone formation markers and subsequent decreases were observed during once-weekly teriparatide treatment for 72 weeks. These observations support the hypothesis that repeated weekly teriparatide administration stimulates bone remodeling, replacing old bone with new bone and leading to a reduction in the active remodeling surface. A simulation model was developed based on the iterative remodeling cycle that occurs on residual old bone. An increase in bone formation and a subsequent decrease were observed in the preliminary simulation. For each fitted time point, the predicted value was compared to the absolute values of the bone formation and resorption markers and lumbar BMD. The simulation model strongly matched actual changes in bone turnover markers and BMD. This simulation model indicates increased bone formation marker levels in the early stage and a subsequent decrease. It is therefore concluded that remodeling-based bone formation persisted during the entire treatment period with once-weekly teriparatide.

Osteoporosis: Remodeling bone

Simulating bone turnover marker changes seen during weekly parathyroid hormone treatment reveals bone remodeling during treatment. A human parathyroid hormone fragment called teriparatide is an approved treatment for osteoporosis, decreasing fracture risk and increasing bone density. Weekly treatment with teriparatide leads to a temporary increase in markers of bone formation and a decrease in markers of bone breakdown in the blood. Sakae Tanaka at the University of Tokyo generated a simulation model which matched the changes in bone turnover markers and bone mineral density over time. This suggests that teriparatide affects both the formation of new bone and the loss of old bone, a process called bone remodeling. The findings explain how treatment with teriparatide has beneficial effects on bone density and osteoporosis.

Fractures across the stages of chronic kidney disease (CKD) could be due to osteoporosis, some form of renal osteodystrophy defined by specific quantitative histomorphometry or chronic kidney disease–mineral and bone disorder (CKD–MBD). CKD–MBD is a systemic disease that links disorders of mineral and bone metabolism due to CKD to either one or all of the following: abnormalities of calcium, phosphorus, parathyroid hormone or vitamin D metabolism; abnormalities in bone turnover, mineralization, volume, linear growth or strength; or vascular or other soft-tissue calcification. Osteoporosis, as defined by the National Institutes of Health, may coexist with renal osteodystrophy or CKD–MBD. Differentiation among these disorders is required to manage correctly the correct disorder to reduce the risk of fractures. While the World Health Organization (WHO) bone mineral density (BMD) criteria for osteoporosis can be used in patients with stages 1–3 CKD, the disorders of bone turnover become so aberrant by stages 4 and 5 CKD that neither the WHO criteria nor the occurrence of a fragility fracture can be used for the diagnosis of osteoporosis. The diagnosis of osteoporosis in stages 4 and 5 CKD is one of the exclusion—excluding either renal osteodystrophy or CKD–MBD as the cause of low BMD or fragility fractures. Differentiations among the disorders of renal osteodystrophy, CKD–MBD or osteoporosis are dependent on the measurement of specific biochemical markers, including serum parathyroid hormone (PTH) and/or quantitative bone histomorphometry. Management of fractures in stages 1–3 CKD does not differ in persons with or without CKD with osteoporosis assuming that there is no evidence for CKD–MBD, clinically suspected by elevated PTH, hyperphosphatemia or fibroblast growth factor 23 due to CKD. Treatment of fractures in persons with osteoporosis and stages 4 and 5 CKD is not evidence-based, with the exception of post-hoc analysis suggesting efficacy and safety of specific osteoporosis therapies (alendronate, risedronate and denosumab) in stage 4 CKD. This review also discusses how to diagnose and manage fragility fractures across the five stages of CKD.

Fractures: due to osteoporosis or kidney disease?

Distinguishing between fractures related to kidney disease or osteoporosis is clinically challenging but important for treatment. Paul Miller from the Colorado Center for Bone Research in Lakewood, USA, reviewed the intimate biological relationships between the kidney and skeleton that can result in bone diseases that increase the risk of low-trauma fracture. Diagnosing whether fractures are related to chronic kidney disease (CKD) or osteoporosis is important as patients are managed differently. Diagnosis can be difficult because both diseases are characterized by low bone mineral density and low-trauma fractures. Analyzing bone structure and biomarkers of bone turnover should exclude osteoporosis in patients with severe CKD and low-trauma fractures. Although more information is needed on the use of approved osteoporosis drugs in CKD-related bone disease, drugs in development may offer more targeted therapy.