The mammalian/mechanistic target of rapamycin (mTOR) is a serine/threonine protein kinase that integrates inputs from nutrients and growth factors to control many fundamental cellular processes through two distinct protein complexes mTORC1 and mTORC2. Recent mouse genetic studies have established that mTOR pathways play important roles in regulating multiple aspects of skeletal development and homeostasis. In addition, mTORC1 has emerged as a common effector mediating the bone anabolic effect of Igf1, Wnt and Bmp. Dysregulation of mTORC1 could contribute to various skeletal diseases including osteoarthritis and osteoporosis. Here we review the current understanding of mTOR signaling in skeletal development and bone homeostasis, as well as in the maintenance of articular cartilage. We speculate that targeting mTOR signaling may be a valuable approach for treating skeletal diseases.

Skeletal development: Regulatory pathway offers drug target for bone disease

Drugs directed at a key cellular signaling pathway could prove useful for treating skeletal diseases. Jianquan Chen from Soochow University in Suzhou, China, and Fanxin Long from Washington University School of Medicine in St. Louis, Missouri, USA, provide an overview of how proteins involved in the mechanistic target of rapamycin (mTOR) signaling pathway sense and integrate a range of environmental cues to regulate bone and cartilage development. In particular, they review the differing roles of the two distinct mTOR-containing protein complexes, mTORC1 and mTORC2. Both seem to mediate bone formation and resorption but in different ways, with implications for how best to treat osteoarthritis, osteoporosis, and other degenerative skeletal diseases. The authors suggest that more specific mTOR inhibitors with minimal side effects are needed to help stimulate bone growth in these diseases.

Cancer metastasis to bone is a three-dimensional (3D), multistep, dynamic process that requires the sequential involvement of three microenvironments, namely, the primary tumour microenvironment, the circulation microenvironment and the bone microenvironment. Engineered 3D approaches allow for a vivid recapitulation of in vivo cancerous microenvironments in vitro, in which the biological behaviours of cancer cells can be assessed under different metastatic conditions. Therefore, modelling bone metastasis microenvironments with 3D cultures is imperative for advancing cancer research and anti-cancer treatment strategies. In this review, multicellular tumour spheroids and bioreactors, tissue engineering constructs and scaffolds, microfluidic systems and 3D bioprinting technology are discussed to explore the progression of the 3D engineering approaches used to model the three microenvironments of bone metastasis. We aim to provide new insights into cancer biology and advance the translation of new therapies for bone metastasis.

Oncology: Modelling bone metastasis in 3-dimensions

Better 3D models are needed to understand the entire process of bone cancer metastasis, if new treatments are to be developed. Bone metastasis is a major complication of several common cancers, yet its biology is poorly understood - in part, because conventional cell culture systems have failed to replicate the 3D microenvironment of the body, where cancer cells dynamically interact with healthy cells and the extracellular matrix. To address this, 3D models of the primary tumour microenvironment, circulation microenvironment and bone microenvironment are needed, suggest Han Qiao and Tingting Tang at Shanghai Jiao Tong University School of Medicine in China. They describe promising 3D approaches, including multicellular tumour spheroids, tissue engineering constructs, microfluidic systems, and 3D bioprinting, but stress that none of these currently recapitulates the entire process of metastasis in a single culture system.

Skeletal health relies on architectural integrity and sufficient bone mass, which are maintained through a tightly regulated equilibrium of bone resorption by osteoclasts and bone formation by osteoblasts. Genetic studies have linked the gene coding for low-density lipoprotein receptor-related protein1 (Lrp1) to bone traits but whether these associations are based on a causal molecular relationship is unknown. Here, we show that Lrp1 in osteoblasts is a novel regulator of osteoclast activity and bone mass. Mice lacking Lrp1 specifically in the osteoblast lineage displayed normal osteoblast function but severe osteoporosis due to highly increased osteoclast numbers and bone resorption. Osteoblast Lrp1 limited receptor activator of NF-κB ligand (RANKL) expression in vivo and in vitro through attenuation of platelet-derived growth factor (PDGF-BB) signaling. In co-culture, Lrp1-deficient osteoblasts stimulated osteoclastogenesis in a PDGFRβ-dependent manner and in vivo treatment with the PDGFR tyrosine kinase inhibitor imatinib mesylate limited RANKL production and led to complete remission of the osteoporotic phenotype. These results identify osteoblast Lrp1 as a key regulator of osteoblast-to-osteoclast communication and bone mass through a PDGF–RANKL signaling axis in osteoblasts and open perspectives to further explore the potential of PDGF signaling inhibitors in counteracting bone loss as well as to evaluate the importance of functional LRP1 gene variants in the control of bone mass in humans.

Osteoporosis: A novel path to strength

Maintaining strong bones critically depends on a receptor (Lrp1) for low-density lipoprotein. Bones are continually remodeled, with osteoblast cells adding new bone and osteoclast cells resorbing old bone. Imbalanced growth and resorption can lead to osteoporosis. Genetic studies had previously linked Lrp1 to bone health, but the nature of the link remained unknown. Andreas Niemeier at the University Medical Center Hamburg-Eppendorf in Germany and co-workers used model mice whose osteoblasts lacked Lrp1 to investigate how the receptor is involved in bone turnover. Lrp-1-deficient mice showed severe osteoporosis. They also showed high numbers of osteoclasts but normal numbers of osteoblasts, indicating that lack of the receptor caused increased bone resorption. Treatment of the mice with a drug related to Lrp1 restored bone strength. These results may help to identify new treatments for bone loss.

Parathyroid hormone (PTH) regulates bone remodeling by activating PTH type 1 receptor (PTH1R) in osteoblasts/osteocytes. Insulin-like growth factor type 1 (IGF-1) stimulates mesenchymal stem cell differentiation to osteoblasts. However, little is known about the signaling mechanisms that regulates the osteoblast-to-osteocyte transition. Here we report that PTH and IGF-I synergistically enhance osteoblast-to-osteocyte differentiation. We identified that a specific tyrosine residue, Y494, on the cytoplasmic domain of PTH1R can be phosphorylated by insulin-like growth factor type I receptor (IGF1R) in vitro. Phosphorylated PTH1R localized to the barbed ends of actin filaments and increased actin polymerization during morphological change of osteoblasts into osteocytes. Disruption of the phosphorylation site reduced actin polymerization and dendrite length. Mouse models with conditional ablation of PTH1R in osteoblasts demonstrated a reduction in the number of osteoctyes and dendrites per osteocyte, with complete overlap of PTH1R with phosphorylated-PTH1R positioning in osteocyte dendrites in wild-type mice. Thus, our findings reveal a novel signaling mechanism that enhances osteoblast-to-osteocyte transition by direct phosphorylation of PTH1R by IGF1R.

Bone formation: Hormone and growth factor work together

A key hormone and growth factor work together to help turn bone-forming cells into mature bone. Janet Crane and colleagues from Johns Hopkins University School of Medicine in Baltimore, Maryland, USA, tested the effects of parathyroid hormone (PTH) and insulin like-growth factor type 1 (IGF-1) signaling on the differentiation of bone-forming osteoblasts by modulating the activity of their receptors in genetically engineered mice. They found a specific part of the PTH type 1 receptor has a phosphate group added to it by the IGF-1 receptor. This chemical tagging leads to changes in the cytoskeleton of osteoblasts that enhance the formation of mature bone cells known as osteocytes. Mice without this PTH receptor had reduced numbers of osteocytes in their bone. The findings reveal a novel signaling mechanism behind this cellular transition during bone building.

The vast osteocytic network is believed to orchestrate bone metabolic activity in response to mechanical stimuli through production of sclerostin, RANKL, and osteoprotegerin (OPG). However, the mechanisms of osteocyte mechanotransduction remain poorly understood. We’ve previously shown that osteocyte mechanosensitivity is encoded through unique intracellular calcium (Ca²⁺) dynamics. Here, by simultaneously monitoring Ca²⁺ and actin dynamics in single cells exposed to fluid shear flow, we detected actin network contractions immediately upon onset of flow-induced Ca²⁺ transients, which were facilitated by smooth muscle myosin and further confirmed in native osteocytes ex vivo. Actomyosin contractions have been linked to the secretion of extracellular vesicles (EVs), and our studies demonstrate that mechanical stimulation upregulates EV production in osteocytes through immunostaining for the secretory vesicle marker Lysosomal-associated membrane protein 1 (LAMP1) and quantifying EV release in conditioned medium, both of which are blunted when Ca²⁺ signaling was inhibited by neomycin. Axial tibia compression was used to induce anabolic bone formation responses in mice, revealing upregulated LAMP1 and expected downregulation of sclerostin in vivo. This load-related increase in LAMP1 expression was inhibited in neomycin-injected mice compared to vehicle. Micro-computed tomography revealed significant load-related increases in both trabecular bone volume fraction and cortical thickness after two weeks of loading, which were blunted by neomycin treatment. In summary, we found mechanical stimulation of osteocytes activates Ca²⁺-dependent contractions and enhances the production and release of EVs containing bone regulatory proteins. Further, blocking Ca²⁺ signaling significantly attenuates adaptation to mechanical loading in vivo, suggesting a critical role for Ca²⁺-mediated signaling in bone adaptation.

How bone grows in response to loading

People gain bone in response to exercise and lose it during prolonged bedrest; now we’re closer to understanding how this happens.

Bone cells called osteocytes act as mechanical sensors, responding to changes in force by regulating the activity of bone-forming osteoblasts and bone- resorbing osteoclasts. X. Edward Guo at Columbia University in New York and colleagues had previously shown that osteocytes exhibit oscillations in intracellular calcium in response to mechanical stimulation, but the downstream effects of this had been unclear. Using multiple approaches, they have now shown that the cytoskeleton contracts in response to these oscillations, in turn triggering the production and release of extracellular vesicles containing bone-regulatory proteins.

When calcium signaling was blocked, vesicle production and release was blunted, and mice failed to show the normal increase in bone formation in response to mechanical loading.

Imbalances between bone formation and bone resorption, which can occur due to aging or sex hormone deprivation, result in decreased bone mass and an increased risk of fracture. Previous studies have suggested that the β-galactoside binding lectin, galectin-3, is involved in bone remodeling. We compared bone parameters of mice having null alleles of the galectin-3 gene (Lgals3-KO) with those of their wild-type littermates. Lgals3 deficiency increased cortical bone expansion at 36 weeks (wk) and preserved or enhanced bone mass in both male and female mutant mice. In addition, female Lgals3-KO mice were protected from age-related loss of trabecular bone. Histomorphometry and ex vivo primary cell differentiation assays showed increased osteoblastogenesis with little-to-no effect on osteoclastogenesis, suggesting the increased bone mass phenotype is primarily due to increased anabolism. Our study identifies galectin-3 as a negative regulator of bone formation and suggests that disruption of galectin-3 may be useful in preventing bone loss during aging.

Osteoporosis: A knockout for stronger bones

Researchers have identified a promising new drug target to reduce bone loss during aging, a protein called galectin-3. Bones undergo lifelong remodeling via resorption of old bone and generation of new bone. With aging, the balance tips towards resorption, weakening bones. Galectin-3 was known to be involved in bone remodeling and levels increased with age. Bart Williams and co-workers at the Van Andel Research Institute in Grand Rapids, USA, investigated whether the age-related increase in galectin-3 increased bone loss. Using mice lacking the gene encoding galectin-3, the researchers measured bone mass at different ages. In older mice, bone mass was preserved or even enhanced. Further investigation of bone cells showed the increase was probably due to increased bone formation, rather than decreased bone resorption. The researchers conclude that disrupting galectin-3 may help to prevent aging-related bone loss.

Genome-wide association studies (GWASs) have been instrumental in understanding complex phenotypic traits. However, they have rarely been used to understand lineage-specific pathways and functions that contribute to the trait. In this study, by integrating lineage-specific enhancers from mesenchymal and myeloid compartments with bone mineral density loci, we were able to segregate osteoblast- and osteoclast (OC)-specific functions. Specifically, in OCs, a PU.1-dependent transcription factor (TF) network was revealed. Deletion of PU.1 in OCs in mice resulted in severe osteopetrosis. Functional genomic analysis indicated PU.1 and MITF orchestrated a TF network essential for OC differentiation. Several of these TFs were regulated by cooperative binding of PU.1 with BRD4 to form superenhancers. Further, PU.1 is essential for conformational changes in the superenhancer region of Nfatc1. In summary, our study demonstrates that combining GWASs with genome-wide binding studies and model organisms could decipher lineage-specific pathways contributing to complex disease states.

Genetics: Cellular fate dictates osteoporosis risk

Genetic variation in non-coding regions of DNA could raise osteoporosis risk by affecting osteoclast differentiation. Osteoporosis occurs when the normal process of bone remodeling by osteoblasts and osteoclasts falls out of balance. Genome-wide association studies (GWAS) have identified numerous single nucleotide polymorphisms (SNPs) associated with osteoporosis, but how these affect specific cell types was unclear. Sudarshana Sharma and Michael Ostrowski at the Medical University of South Carolina and colleagues wondered if variations in non-coding ‘enhancer’ regions of DNA, might shed light on the molecular underpinnings of osteoporosis. So, they overlaid SNPs associated with reduced bone mineral density onto enhancers in mesenchymal and myeloid cells—the precursors of osteoblasts and osteoclasts—identifying a transcription factor network in myeloid cells that drives the differentiation of osteoclasts. When this was disrupted in mice, severe defects in osteoclast differentiation and function resulted.

Osteoporosis is a frequent complication of chronic inflammatory diseases and increases in the pro-inflammatory cytokines make an important contribution to bone loss by promoting bone resorption and impairing bone formation. Omentin-1 is a newly identified adipocytokine that has anti-inflammatory effects, but little is known about the role of omentin-1 in inflammatory osteoporosis. Here we generated global omentin-1 knockout (omentin-1 ^−/−) mice and demonstrated that depletion of omentin-1 induces inflammatory bone loss-like phenotypes in mice, as defined by abnormally elevated pro-inflammatory cytokines, increased osteoclast formation and bone tissue destruction, as well as impaired osteogenic activities. Using an inflammatory cell model induced by tumor necrosis factor-α (TNF-α), we determined that recombinant omentin-1 reduces the production of pro-inflammatory factors in the TNF-α-activated macrophages, and suppresses their anti-osteoblastic and pro-osteoclastic abilities. In the magnesium silicate-induced inflammatory osteoporosis mouse model, the systemic administration of adenoviral-delivered omentin-1 significantly protects from osteoporotic bone loss and inflammation. Our study suggests that omentin-1 can be used as a promising therapeutic agent for the prevention or treatment of inflammatory bone diseases by downregulating the pro-inflammatory cytokines.

Osteoporosis: Fat-secreted protein guards against bone loss in mice

An anti-inflammatory molecule produced by fat tissue helps protect against bone loss in mouse model of osteoporosis. Researchers from China’s Central South University in Changsha, led by Hui Xie, examined whether omentin-1, a protein secreted by fat tissue recently implicated in the development of obesity and diabetes, might play a role in osteoporosis as well. In mice engineered to lack omentin-1, they observed more bone tissue destruction and increased numbers of bone-resorbing osteoclast cells than in normal, healthy animals. The researchers also administered exogenous omentin-1 to mice either with an overactive inflammatory molecule or experimentally induced to develop osteoporosis. In both cases, they found that the protein suppressed inflammation and guarded against bone loss. Omentin-1 thus provides promising therapeutic agent for preventing or treating inflammatory bone diseases such as osteoporosis.

Carboxyl terminus of Hsp70-interacting protein (CHIP or STUB1) is an E3 ligase and regulates the stability of several proteins which are involved in different cellular functions. Our previous studies demonstrated that Chip deficient mice display bone loss phenotype due to increased osteoclast formation through enhancing TRAF6 activity in osteoclasts. In this study we provide novel evidence about the function of CHIP. We found that osteoblast differentiation and bone formation were also decreased in Chip KO mice. In bone marrow stromal (BMS) cells derived from Chip ^−/− mice, expression of a panel of osteoblast marker genes was significantly decreased. ALP activity and mineralized bone matrix formation were also reduced in Chip-deficient BMS cells. We also found that in addition to the regulation of TRAF6, CHIP also inhibits TNFα-induced NF-κB signaling through promoting TRAF2 and TRAF5 degradation. Specific deletion of Chip in BMS cells downregulated expression of osteoblast marker genes which could be reversed by the addition of NF-κB inhibitor. These results demonstrate that the osteopenic phenotype observed in Chip ^−/− mice was due to the combination of increased osteoclast formation and decreased osteoblast differentiation. Taken together, our findings indicate a significant role of CHIP in bone remodeling.

Bone remodeling: protein promise for bone loss disorders

A protein involved in maintaining balance in bone formation may prove a useful target for treating bone loss-associated diseases. Bones continuously undergo formation and resorption, but certain diseases can interfere with this remodeling process, leading to loss of bone mass. Previous studies by Di Chen at Rush University Medical Center in Chicago, US, and co-workers found that mice without a key protein called CHIP display increased osteoclast formation and abnormally high levels of bone resorption. Further investigations by Chen’s team now indicate that CHIP deficiency also results in reduced bone formation and loss of mass. They found that CHIP regulates a family of proteins called TRAF—the overexpression of which disturbs the precise balance of osteoclast/osteoblast bone cell formation. CHIP may provide a target for drug development for bone loss diseases.

Osteosarcoma is the most common primary bone sarcoma that mostly occurs in young adults. The causes of osteosarcoma are heterogeneous and still not fully understood. Identification of novel, important oncogenic factors in osteosarcoma and development of better, effective therapeutic approaches are in urgent need for better treatment of osteosarcoma patients. In this study, we uncovered that the oncogene MYC is significantly upregulated in metastastic osteosarcoma samples. In addition, high MYC expression is associated with poor survival of osteosarcoma patients. Analysis of MYC targets in osteosarcoma revealed that most of the osteosarcoma super enhancer genes are bound by MYC. Treatment of osteosarcoma cells with super enhancer inhibitors THZ1 and JQ1 effectively suppresses the proliferation, migration, and invasion of osteosarcoma cells. Mechanistically, THZ1 treatment suppresses a large group of super enhancer containing MYC target genes including CDK6 and TGFB2. These findings revealed that the MYC-driven super enhancer signaling is crucial for the osteosarcoma tumorigenesis and targeting the MYC/super enhancer axis represents as a promising therapeutic strategy for treatment of osteosarcoma patients.

Bone cancer: Disease-promoting DNA regions revealed as therapeutic targets

Insight into a molecular pathway involved in an aggressive bone cancer has suggested a new approach to its treatment. Osteosarcoma usually develops in growing bone tissue, but often spreads to other organs, consequently reducing survival. The molecular mechanisms behind osteosarcoma are not fully understood. A team led by Shuibin Lin from Sun Yat-sen University investigated the role of MYC, a gene that is important in other cancers. They found that increased expression of MYC is associated with the spread of osteosarcoma and a poor prognosis because the protein product of MYC activates many super-enhancers, regions of DNA that increase the expression of cancer-related genes. Inhibition of the super-enhancers suppressed growth and spreading of osteosarcoma in cultured cells and a mouse model, suggesting a novel therapeutic approach to osteosarcoma.

Chondrocytes and osteoblasts differentiate from a common mesenchymal precursor, the osteochondroprogenitor (OCP), and help build the vertebrate skeleton. The signaling pathways that control lineage commitment for OCPs are incompletely understood. We asked whether the ubiquitously expressed protein-tyrosine phosphatase SHP2 (encoded by Ptpn11) affects skeletal lineage commitment by conditionally deleting Ptpn11 in mouse limb and head mesenchyme using “Cre-loxP”-mediated gene excision. SHP2-deficient mice have increased cartilage mass and deficient ossification, suggesting that SHP2-deficient OCPs become chondrocytes and not osteoblasts. Consistent with these observations, the expression of the master chondrogenic transcription factor SOX9 and its target genes Acan, Col2a1, and Col10a1 were increased in SHP2-deficient chondrocytes, as revealed by gene expression arrays, qRT-PCR, in situ hybridization, and immunostaining. Mechanistic studies demonstrate that SHP2 regulates OCP fate determination via the phosphorylation and SUMOylation of SOX9, mediated at least in part via the PKA signaling pathway. Our data indicate that SHP2 is critical for skeletal cell lineage differentiation and could thus be a pharmacologic target for bone and cartilage regeneration.

Chondrogenesis: SHP2 regulates skeletal cell fate

The enzyme SHP2 is critical for the differentiation of skeletal cells and could be a pharmacological target for bone and cartilage regeneration. Osteo-chondroprogenitor cells arise from stem cells in the bone marrow and can differentiate into either osteoblasts (bone-producing cells) or chondrocytes (cartilage cells). A team headed by Wentian Yang at Brown University, Rhode Island found that mice deficient in SHP2 had increased cartilage mass and decreased ossification. As a result of SHP2 deficiency, abnormal cartilage growth developed at sites where mineralized bone would normally form. The team’s findings suggest that SHP2-deficient osteo-chondroprogenitor cells become chondrocytes, not osteoblasts. The authors believe that the ability to therapeutically manipulate cell fate by regulating SHP2 activity offers a new means of controlling cartilage formation in patients with various cartilage-related disorders, including tumors and osteoarthritis.

Discriminating sterile inflammation from infection, especially in cases of aseptic loosening versus an actual prosthetic joint infection, is challenging and has significant treatment implications. Our goal was to evaluate a novel human monoclonal antibody (mAb) probe directed against the Gram-positive bacterial surface molecule lipoteichoic acid (LTA). Specificity and affinity were assessed in vitro. We then radiolabeled the anti-LTA mAb and evaluated its effectiveness as a diagnostic imaging tool for detecting infection via immunoPET imaging in an in vivo mouse model of prosthetic joint infection (PJI). In vitro and ex vivo binding of the anti-LTA mAb to pathogenic bacteria was measured with Octet, ELISA, and flow cytometry. The in vivo PJI mouse model was assessed using traditional imaging modalities, including positron emission tomography (PET) with [¹⁸F]FDG and [¹⁸F]NaF as well as X-ray computed tomography (CT), before being evaluated with the zirconium-89-labeled antibody specific for LTA ([⁸⁹Zr]SAC55). The anti-LTA mAb exhibited specific binding in vitro to LTA-expressing bacteria. Results from imaging showed that our model could reliably simulate infection at the surgical site by bioluminescent imaging, conventional PET tracer imaging, and bone morphological changes by CT. One day following injection of both the radiolabeled anti-LTA and isotype control antibodies, the anti-LTA antibody demonstrated significantly greater (P < 0.05) uptake at S. aureus-infected prosthesis sites over either the same antibody at sterile prosthesis sites or of control non-specific antibody at infected prosthesis sites. Taken together, the radiolabeled anti-LTA mAb, [⁸⁹Zr]SAC55, may serve as a valuable diagnostic molecular imaging probe to help distinguish between sterile inflammation and infection in the setting of PJI. Future studies are needed to determine whether these findings will translate to human PJI.

Joint replacements: Distinguishing infection from inflammation

A new imaging technique distinguishes bacterial infection at the site of joint implants from less-serious postoperative inflammation, saving patients from unnecessary and invasive treatments. Daniel Thorek of Johns Hopkins University School of Medicine and colleagues used an antibody that binds to lipoteichoic acid on the cell wall of Staphylococcus bacteria to detect infection at joint implant sites. The antibody was labeled with a radioactive agent and injected into mice that simulated infection of a knee replacement site. A PET scan conducted 1 day after antibody injection showed that it gathered at the infected joint significantly more than it did at the uninfected implant sites in other mice. This method could improve the diagnosis of joint implant infection, which necessitates removal of the prosthetic and all infected tissues, followed by prolonged antibiotic therapy.

Multiple regulatory mechanisms control osteoblast differentiation and function to ensure unperturbed skeletal formation and remodeling. In this study we identify histone lysine-specific demethylase 1(LSD1/KDM1A) as a key epigenetic regulator of osteoblast differentiation. Knockdown of LSD1 promoted osteoblast differentiation of human mesenchymal stem cells (hMSCs) in vitro and mice lacking LSD1 in mesenchymal cells displayed increased bone mass secondary to accelerated osteoblast differentiation. Mechanistic in vitro studies revealed that LSD1 epigenetically regulates the expression of WNT7B and BMP2. LSD1 deficiency resulted in increased BMP2 and WNT7B expression in osteoblasts and enhanced bone formation, while downregulation of WNT7B- and BMP2-related signaling using genetic mouse model or small-molecule inhibitors attenuated bone phenotype in vivo. Furthermore, the LSD1 inhibitor tranylcypromine (TCP) could increase bone mass in mice. These data identify LSD1 as a novel regulator of osteoblast activity and suggest LSD1 inhibition as a potential therapeutic target for treatment of osteoporosis.

Epigenetics: Taking the brakes off bone formation

A molecular brake controlling the differentiation of bone-forming osteoblasts has been identified, which could provide a new therapeutic target for osteoporosis. LSD1 regulates the expression of various genes by interfering with the ability of the cellular machinery to bind to DNA and transcribe it. A previous study had suggested that inhibiting LSD1 in stem cells from human fat promoted the formation of new osteoblasts. To further investigate its role in bone development, Weiguo Zou at the Shanghai Institute of Biochemistry and Cell Biology and colleagues engineered mice which lacked LSD1 in their mesenchymal cells–precursors of bone, muscle and fat cells—the animals had increased numbers of osteoblasts and greater bone mass. Further experiments revealed that blocking LSD1 resulted in increased expression of two osteoblast-stimulating factors, although it may also influence bone resorbing osteoclasts.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that primarily affects the lining of the synovial joints and is associated with progressive disability, premature death, and socioeconomic burdens. A better understanding of how the pathological mechanisms drive the deterioration of RA progress in individuals is urgently required in order to develop therapies that will effectively treat patients at each stage of the disease progress. Here we dissect the etiology and pathology at specific stages: (i) triggering, (ii) maturation, (iii) targeting, and (iv) fulminant stage, concomitant with hyperplastic synovium, cartilage damage, bone erosion, and systemic consequences. Modern pharmacologic therapies (including conventional, biological, and novel potential small molecule disease-modifying anti-rheumatic drugs) remain the mainstay of RA treatment and there has been significant progress toward achieving disease remission without joint deformity. Despite this, a significant proportion of RA patients do not effectively respond to the current therapies and thus new drugs are urgently required. This review discusses recent advances of our  understanding of RA pathogenesis, disease modifying drugs, and provides perspectives on next generation therapeutics for RA.

Rheumatoid Arthritis: A preventable disease?

The preclinical stages of rheumatoid arthritis (RA) represent a golden window for the development of therapies which could someday prevent the onset of clinical disease. The autoimmune processes underpinning RA usually begin many years before symptoms such as joint pain and stiffness emerge. Recent studies have identified some of the key cellular players driving these processes and begun to unpick how genetic and environmental risk factors combine to trigger them; they also suggest the existence of several distinct subtypes of RA, which require further exploration. Jiake Xu at the University of Western Australia in Perth and colleagues review current treatment strategies for RA and how such insights could ultimately lead to the earlier diagnosis of RA - as well as providing new opportunities for drug treatment and prevention through behavioral changes in high-risk individuals.

The skeleton is a dynamic organ that is constantly remodeled. Proteins secreted from bone cells, namely osteoblasts, osteocytes, and osteoclasts exert regulation on osteoblastogenesis, osteclastogenesis, and angiogenesis in a paracrine manner. Osteoblasts secrete a range of different molecules including RANKL/OPG, M-CSF, SEMA3A, WNT5A, and WNT16 that regulate osteoclastogenesis. Osteoblasts also produce VEGFA that stimulates osteoblastogenesis and angiogenesis. Osteocytes produce sclerostin (SOST) that inhibits osteoblast differentiation and promotes osteoclast differentiation. Osteoclasts secrete factors including BMP6, CTHRC1, EFNB2, S1P, WNT10B, SEMA4D, and CT-1 that act on osteoblasts and osteocytes, and thereby influenceaA osteogenesis. Osteoclast precursors produce the angiogenic factor PDGF-BB to promote the formation of Type H vessels, which then stimulate osteoblastogenesis. Besides, the evidences over the past decades show that at least three hormones or “osteokines” from bone cells have endocrine functions. FGF23 is produced by osteoblasts and osteocytes and can regulate phosphate metabolism. Osteocalcin (OCN) secreted by osteoblasts regulates systemic glucose and energy metabolism, reproduction, and cognition. Lipocalin-2 (LCN2) is secreted by osteoblasts and can influence energy metabolism by suppressing appetite in the brain. We review the recent progresses in the paracrine and endocrine functions of the secretory proteins of osteoblasts, osteocytes, and osteoclasts, revealing connections of the skeleton with other tissues and providing added insights into the pathogenesis of degenerative diseases affecting multiple organs and the drug discovery process.

Bone Metabolism: Functions of secretory proteins from bone cells

Proteins secreted from bone cells (osteoblasts, osteocytes, and osteoclasts) regulate the formation of osteoblasts, osteoclasts, and new blood vessels in a paracrine (local hormonal action) manner. The human skeleton is constantly being modeled, and that remodeling involves the removal of old or damaged bone by osteoclasts and its subsequent replacement with new bone by osteoblasts. Osteocytes inhibit osteoblast differentiation and promote osteoclast differentiation. A team headed by Weiguo Zou of the Shanghai Institute of Biochemistry and Cell Biology, University of Chinese Academy of Sciences, reviewed recent progress that has been made regarding the hormonal functions of secretory proteins from osteoblasts, osteocytes, and osteoclasts. The authors highlight the growing awareness of how bone functions as both a paracrine and endocrine (distant hormonal action) organ, which is of great importance in developing treatments for metabolic disorders and degenerative diseases.

Dysregulated Wnt signaling is associated with the pathogenesis of cancers, fibrosis, and vascular diseases. Inhibition of Wnt signaling has shown efficacy in various pre-clinical models of these disorders. One of the key challenges in developing targeted anti-cancer drugs is to balance efficacy with on-target toxicity. Given the crucial role Wnts play in the differentiation of osteoblasts and osteoclasts, acute inhibition of Wnt signaling is likely to affect bone homeostasis. In this study, we evaluated the skeletal effect of small molecule inhibitor of an o-acyl transferase porcupine (PORCN) that prevents Wnt signaling by blocking the secretion of all Wnts. Micro-computed tomography and histomorphometric evaluation revealed that the bones of mice treated with two structurally distinct PORCN inhibitors LGK974 and ETC-1922159 (ETC-159) had loss-of-bone volume and density within 4 weeks of exposure. This decreased bone mass was associated with a significant increase in adipocytes within the bone marrow. Notably, simultaneous administration of a clinically approved anti-resorptive, alendronate, a member of the bisphosphonate family, mitigated loss-of-bone mass seen upon ETC-159 treatment by regulating activity of osteoclasts and blocking accumulation of bone marrow adipocytes. Our results support the addition of bone protective agents when treating patients with PORCN inhibitors. Mitigation of bone toxicity can extend the therapeutic utility of Wnt pathway inhibitors.

Bone loss: Drug combination reduces toxicity of cancer treatment

Potential bone loss caused by cancer drugs could be mitigated by administering an existing osteoporosis drug. Over-activation of the Wnt signaling pathway, which helps maintain healthy tissues and bone development, is often found in cancer. Scientists are trialing cancer drugs that block a key enzyme PORCN and therefore inhibit Wnt signaling, but these drugs may also adversely affect patients’ bone structure. David Virshup at Duke-NUS Medical School in Singapore and Bart Williams at the Van Andel Research Institute in Michigan, US, and co-workers found that mice treated with PORCN -inhibiting cancer drugs lost bone volume and density within four weeks of exposure. The team then combined the cancer drug with another drug, alendronate, which is already used to treat osteoporosis. This combination targeted aberrant Wnt signaling and limited bone toxicity in the mice.

YAP (yes-associated protein) is a transcriptional factor that is negatively regulated by Hippo pathway, a conserved pathway for the development and size control of multiple organs. The exact function of YAP in bone homeostasis remains controversial. Here we provide evidence for YAP’s function in promoting osteogenesis, suppressing adipogenesis, and thus maintaining bone homeostasis. YAP is selectively expressed in osteoblast (OB)-lineage cells. Conditionally knocking out Yap in the OB lineage in mice reduces cell proliferation and OB differentiation and increases adipocyte formation, resulting in a trabecular bone loss. Mechanistically, YAP interacts with β-catenin and is necessary for maintenance of nuclear β-catenin level and Wnt/β-catenin signaling. Expression of β-catenin in YAP-deficient BMSCs (bone marrow stromal cells) diminishes the osteogenesis deficit. These results thus identify YAP-β-catenin as an important pathway for osteogenesis during adult bone remodeling and uncover a mechanism underlying YAP regulation of bone homeostasis.

Bone homeostasis: Genes play a balancing act

A key regulatory gene both promotes bone formation and suppresses production of fat-storing bone marrow cells, thus supporting the key process of bone remodeling. As adults, our bones are in a constant state of flux, constantly being dissolved and rebuilt to maintain a steady state known as homeostasis. An international team led by Wen-Cheng Xiong, of Case Western Reserve University, Cleveland, Ohio, studied the role of the gene ‘yes-associated protein’ (YAP), a transcription factor (gene controlling the activity of other genes). Using mice, they found that YAP is essential for osteogenesis, and prevents adipocyte (fat-storing cell) formation. Without a functional YAP gene, mice experienced a loss of a softer type of bone known as trabecular bone. The molecular pathways controlled by YAP may thus be important for bone remodeling in vertebrates.

There is a worldwide epidemic of skeletal diseases causing not only a public health issue but also accounting for a sizable portion of healthcare expenditures. The vertebrate skeleton is known to be formed by mesenchymal cells condensing into tissue elements (patterning phase) followed by their differentiation into cartilage (chondrocytes) or bone (osteoblasts) cells within the condensations. During the growth and remodeling phase, bone is formed directly via intramembranous ossification or through a cartilage to bone conversion via endochondral ossification routes. The canonical pathway of the endochondral bone formation process involves apoptosis of hypertrophic chondrocytes followed by vascular invasion that brings in osteoclast precursors to remove cartilage and osteoblast precursors to form bone. However, there is now an emerging role for chondrocyte-to-osteoblast transdifferentiation in the endochondral ossification process. Although the concept of “transdifferentiation” per se is not recent, new data using a variety of techniques to follow the fate of chondrocytes in different bones during embryonic and post-natal growth as well as during fracture repair in adults have identified three different models for chondrocyte-to-osteoblast transdifferentiation (direct transdifferentiation, dedifferentiation to redifferentiation, and chondrocyte to osteogenic precursor). This review focuses on the emerging models of chondrocyte-to-osteoblast transdifferentiation and their implications for the treatment of skeletal diseases as well as the possible signaling pathways that contribute to chondrocyte-to-osteoblast transdifferentiation processes.

Cell biology: When cartilage becomes bone

A basic principal of cell differentiation is that cells become increasingly specialized until they reach a fixed terminal state - yet recent studies suggest that mature cells can, and do, change into new types. Such ‘transdifferentiation’ seems to occur in many different tissues, but in this review, Patrick Aghajanian and Subburaman Mohan at VA Lorna Linda Healthcare System focus on the transition of cartilage to bone. Although a similar transition is well-known to occur during fetal development and fracture healing, in both cases, mesenchymal stem cells brought in by invading blood vessels were thought to differentiate into bone-producing cells. However, recent studies suggest that cartilage cells can themselves transdifferentiate into bone cells. A better understanding of this process could lead to new therapies to boost fracture healing and tackle bone-wasting disorders.

Free fatty acids (FFAs), which are elevated with metabolic syndrome, are considered the principal offender exerting lipotoxicity. Few previous studies have reported a causal relationship between FFAs and osteoarthritis pathogenesis. However, the molecular mechanism by which FFAs exert lipotoxicity and induce osteoarthritis remains largely unknown. We here observed that oleate at the usual clinical range does not exert lipotoxicity while oleate at high pathological ranges exerted lipotoxicity through apoptosis in articular chondrocytes. By investigating the differential effect of oleate at toxic and nontoxic concentrations, we revealed that lipid droplet (LD) accumulation confers articular chondrocytes, the resistance to lipotoxicity. Using high fat diet-induced osteoarthritis models and articular chondrocytes treated with oleate alone or oleate plus palmitate, we demonstrated that articular chondrocytes gain resistance to lipotoxicity through protein kinase casein kinase 2 (PKCK2)—six-transmembrane protein of prostate 2 (STAMP2)—and fat-specific protein 27 (FSP27)-mediated LD accumulation. We further observed that the exertion of FFAs-induced lipotoxicity was correlated with the increased concentration of cellular FFAs freed from LDs, whether FFAs are saturated or not. In conclusion, PKCK2/STAMP2/FSP27-mediated sequestration of FFAs in LD rescues osteoarthritic chondrocytes. PKCK2/STAMP2/FSP27 should be considered for interventions against metabolic OA.

Oil droplets protect cartilage from toxic fatty acids

Cartilage tissue deals with the stress of exposure to free fatty acids by sequestering the toxic molecules into sub-cellular oil droplets. Young Hyun Yoo from Dong-A University College of Medicine in Busan, South Korea, and coworkers exposed rat cartilage cells to increasing levels of a fatty acid called oleate, a by-product of fat metabolism, and observed that the accumulation of oil droplets conferred resistance to oleate-induced toxicity. In these rat cells and in experiments involving mouse models of osteoarthritis fed a high-fat diet, the researchers then identified three of the protective proteins needed for cartilage tissue to properly quarantine fatty acids into oil droplets. Those proteins — and their connected regulatory networks — could now serve as drug targets for treating metabolic syndrome-associated osteoarthritis.

Degenerative disc disease (DDD) is associated with intervertebral disc degeneration of spinal instability. Here, we report that the cilia of nucleus pulposus (NP) cells mediate mechanotransduction to maintain anabolic activity in the discs. We found that mechanical stress promotes transport of parathyroid hormone 1 receptor (PTH1R) to the cilia and enhances parathyroid hormone (PTH) signaling in NP cells. PTH induces transcription of integrin α_vβ₆ to activate the transforming growth factor (TGF)-β-connective tissue growth factor (CCN2)-matrix proteins signaling cascade. Intermittent injection of PTH (iPTH) effectively attenuates disc degeneration of aged mice by direct signaling through NP cells, specifically improving intervertebral disc height and volume by increasing levels of TGF-β activity, CCN2, and aggrecan. PTH1R is expressed in both mouse and human NP cells. Importantly, knockout PTH1R or cilia in the NP cells results in significant disc degeneration and blunts the effect of PTH on attenuation of aged discs. Thus, mechanical stress-induced transport of PTH1R to the cilia enhances PTH signaling, which helps maintain intervertebral disc homeostasis, particularly during aging, indicating therapeutic potential of iPTH for DDD.

Degenerative disc disease: hormone signaling keeps intervertebral core in balance

Sensory structures found in the jelly-like space between spinal discs release a hormone that helps preserve back health in aging mice. Xu Cao from Johns Hopkins University in Baltimore, Maryland, USA, and colleagues observed that levels of a critical growth factor declined in the space between adjacent vertebrae as mice aged, and that injecting a naturally occurring hormone that activates this growth factor could attenuate disc degeneration in older animals. The researchers showed, in response to mechanical stresses, receptor proteins that respond to this hormone relocate themselves to particular sensory organelles known as cilia that found within cells of the intervertebral core. That results in elevated hormone signaling—and drugs designed to have the same effect could help treat degenerative disc disease, one of the most common causes of chronic back pain.

Low-density lipoprotein receptor–related protein 6 (LRP6) is a co-receptor for Wnt signaling and can be recruited by multiple growth factors/hormones to their receptors facilitating intracellular signaling activation. The ligands that bind directly to LRP6 have not been identified. Here, we report that bioactive oxidized phospholipids (oxPLs) are native ligands of LRP6, but not the closely related LRP5. oxPLs are products of lipid oxidation involving in pathological conditions such as hyperlipidemia, atherosclerosis, and inflammation. We found that cell surface LRP6 in bone marrow mesenchymal stromal cells (MSCs) decreased rapidly in response to increased oxPLs in marrow microenvironment. LRP6 directly bound and mediated the uptake of oxPLs by MSCs. oxPL-LRP6 binding induced LRP6 endocytosis through a clathrin-mediated pathway, decreasing responses of MSCs to osteogenic factors and diminishing osteoblast differentiation ability. Thus, LRP6 functions as a receptor and molecular target of oxPLs for their adverse effect on MSCs, revealing a potential mechanism underlying atherosclerosis-associated bone loss.

Bone loss: revealing a molecular cause of ‘numb’ stem cells

A constituent of oxidized ‘bad cholesterol’ blocks essential signaling processes leading to pathogenic bone loss. LRP6 is a crucial receptor involved in multiple physiological processes, including bone loss; however, direct pathogenic modulators of the receptor have yet to be elucidated. Now, John Hopkins University School of Medicine’s Mei Wan, with a US and Chinese research team, has discovered that oxPL, a byproduct of the oxidization of cholesterol carrier LDL, binds directly to LRP6 and causes its removal from the surface of bone marrow stem cells. As a result, these stem cells are unable to sense the external signaling molecules that drive bone growth. oxPLs are products of diseases such as hyperlipidemia and atherosclerosis, and this paper helps to reveal their pathogenesis and offers potential targets for therapeutic interventions.

Male Igfbp2−/− mice have a significant reduction in bone mass and administration of a peptide that contains the insulin-like growth factor binding protein-2(IGFBP-2) receptor-binding domain stimulates bone formation in these animals. Female Igfbp2−/− mice do not have this phenotype but following ovariectomy (OVX) lose more bone than OVX wild-type mice. This suggests that in the absence of estrogen, IGFBP-2 is required to maintain bone mass. Therefore these studies were undertaken to determine if this peptide could stimulate bone acquisition in OVX rats. OVX rats were divided into seven treatment groups: sham animals, OVX animals, OVX animals receiving a control scrambled peptide, or one of three doses of the active peptide termed PEG-HBD-1 (0.7, 2, and 6 mg·kg^-1) and an OVX group receiving parathyroid hormone (PTH) (50 µg·kg^-1 per day). The peptides were administered for 8 weeks. DXA revealed a significant reduction in femoral and tibial areal bone mineral density (aBMD) after OVX, whereas treatment with the high-dose peptide increased aBMD by 6.2% ± 2.4% (P < 0.01) compared to control peptide; similar to the increase noted with PTH (5.6% ± 3.0%, P < 0.01). Similar increases were noted with two lower doses of the peptide (3.8% ± 1.5%, P < 0.05 for low dose; 3.1% ± 1.6%, P = 0.07 for middle dose). Micro CT showed that the OVX control peptide animals had reductions of 41% and 64% in femoral trabecular BV/TV and trabecular number, respectively. All three doses of the peptide increased bone volume/total volume (BV/TV) significantly, while the low and middle doses increased trabecular number. Cortical BV/TV and thickness at the midshaft increased significantly with each dose of peptide (18.9% ± 9.8%, P < 0.01 and 14.2% ± 7.9%, P < 0.01 for low dose; 23.7% ± 10.7%, P < 0.001 and 15.8% ± 6.1%, P < 0.001 for middle dose; 19.0% ± 6.9%, P < 0.01 and 16.2% ± 9.7%, P < 0.001 for high dose) and with PTH (25.8% ± 9.2%, P < 0.001 and 19.4% ± 8.8%, P < 0.001). Histomorphometry showed that the lowest dose of peptide stimulated BV/TV, trabecular thickness, mineral apposition rate (MAR), bone formation rate/bone surface (BFR/BS), number of osteoblasts/bone perimeter (N.ob/B.pm), and decreased osteoclast surface/bone perimeter (Oc.S/B.Pm). The highest dose stimulated each of these parameters except MAR and BFR/BS. Thus, the heparin-binding domain receptor region of IGFBP-2 accounts for its anabolic activity in bone. Importantly, this peptide enhances bone mass in estrogen-deficient animals.

Drug discovery: peptide boost for bone mass

An experimental peptide stimulates bone acquisition in female rats who have had their ovaries removed, raising the prospect a new drug for osteoporosis.

IGFBP-2 is an insulin-like growth factor (IGF) binding protein, which regulates the amount of IGF-I and II that are transported out of the blood and are available to influence the growth and proliferation of bone-producing osteoblasts. Previous studies have suggested that IGFBP-2 is required to maintain bone mass in the absence of estrogen, and that a 13 amino acid peptide (HBD1) from the core of the protein could provide a substitute for it.

In this study, David Clemmons at the University of North Carolina at Chapel Hill and his colleagues demonstrate that injecting the peptide into ovariectomized female rats prompts significant increases in bone mass, whereas control animals lost bone.

The R-spondin family of proteins are Wnt agonists, and the complete embryonic disruption of Rspo2 results in skeletal developmental defects that recapitulate the phenotype observed with Lrp5/6 deficiency. Previous work has shown that R-spondin-2 (Rspo2, RSPO2) is both highly expressed in Wnt-stimulated pre-osteoblasts and its overexpression induces osteoblast differentiation in the same cells, supporting its putative role as a positive autocrine regulator of osteoblastogenesis. However, the role of Rspo2 in regulating osteoblastogenesis and bone formation in postnatal bone has not been explored. Here we show that limb-bud progenitor cells from Rspo2 knockout mice undergo reduced mineralization during osteoblastogenesis in vitro and have a corresponding alteration in their osteogenic gene expression profile. We also generated the first Rspo2 conditional knockout (Rspo2^floxed) mouse and disrupted Rspo2 expression in osteoblast-lineage cells by crossing to the Osteocalcin-Cre mouse line (Ocn-Cre + Rspo2^f/f). Ocn-Cre + Rspo2^f/f male and female mice at 1, 3, and 6 months were examined. Ocn-Cre + Rspo2^f/f mice are decreased in overall body size compared to their control littermates and have decreased bone mass. Histomorphometric analysis of 1-month-old mice revealed a similar number of osteoblasts and mineralizing surface per bone surface with a simultaneous decrease in mineral apposition and bone formation rates. Consistent with this observation, serum osteocalcin in 3-month-old Ocn-Cre + Rspo2^f/f was reduced, and bone marrow-mesenchymal stem cells from Ocn-Cre + Rspo2^f/f mice undergo less mineralization in vitro. Finally, gene expression analysis and immunohistochemistry of mature bone shows reduced beta-catenin signaling in Ocn-Cre + Rspo2^f/f. Overall, RSPO2 reduces osteoblastogenesis and mineralization, leading to reduced bone mass.

Bone Physiology: R-spondin-2 reduces mineralization, leading to decreased bone mass

A loss of R-spondin-2 reduces osteoblastogenesis (production of osteoblasts, the cells from which bone develops) and mineralization, thereby leading to decreased bone mass in adults. R-spondin-2 is one of a family of four proteins that are expressed in the developing mouse limb as well as other tissues; each R-spondin family member exerts a different functional effect. R-spondins clearly influence several aspects of skeletal biology, but their specific roles—especially in postnatal bone—remained to be elucidated. A team headed by Kurt Hankenson at the University of Michigan Medical School investigated the role of R-spondin-2 in osteoblastogenesis, both in vitro and in vivo, using a mouse model. For the first time, the team was able to demonstrate that R-spondin-2 promotes osteoblastogenesis, bone development, and consequent bone mass growth in adult mice.

Bone tissue engineering is an exciting approach to directly repair bone defects or engineer bone tissue for transplantation. Biomaterials play a pivotal role in providing a template and extracellular environment to support regenerative cells and promote tissue regeneration. A variety of signaling cues have been identified to regulate cellular activity, tissue development, and the healing process. Numerous studies and trials have shown the promise of tissue engineering, but successful translations of bone tissue engineering research into clinical applications have been limited, due in part to a lack of optimal delivery systems for these signals. Biomedical engineers are therefore highly motivated to develop biomimetic drug delivery systems, which benefit from mimicking signaling molecule release or presentation by the native extracellular matrix during development or the natural healing process. Engineered biomimetic drug delivery systems aim to provide control over the location, timing, and release kinetics of the signal molecules according to the drug’s physiochemical properties and specific biological mechanisms. This article reviews biomimetic strategies in signaling delivery for bone tissue engineering, with a focus on delivery systems rather than specific molecules. Both fundamental considerations and specific design strategies are discussed with examples of recent research progress, demonstrating the significance and potential of biomimetic delivery systems for bone tissue engineering.

Biomechanics: Drug delivery systems in bone tissue engineering

Bone tissue engineering offers exciting possibilities for repairing bone defects or regenerating bone tissue for transplantation, and biomimetic drug delivery systems (DDSs) hold promise in providing an environment to support the tissue regeneration. Biomimetic DDSs mimic the release of signaling molecules during development or in the natural healing process, and a team headed by Peter Ma at the University of Michigan in the United States conducted a review of the biomimetic strategies that have been adopted for bone tissue engineering. Various DDSs have been developed that mimic the natural healing or development processes, and they provide controlled drug release. The authors conclude that integrating DDSs with bone implants or scaffolds (which stimulate the growth of bone cells on their surface) could lead to advanced tissue engineering therapy for repairing bone defects.

There is currently no effective medical treatment for temporomandibular joint osteoarthritis (TMJ-OA) due to a limited understanding of its pathogenesis. This study was undertaken to investigate the key role of transforming growth factor-β (TGF-β) signalling in the cartilage and subchondral bone of the TMJ using a temporomandibular joint disorder (TMD) rat model, an ageing mouse model and a Camurati–Engelmann disease (CED) mouse model. In the three animal models, the subchondral bone phenotypes in the mandibular condyles were evaluated by µCT, and changes in TMJ condyles were examined by TRAP staining and immunohistochemical analysis of Osterix and p-Smad2/3. Condyle degradation was confirmed by Safranin O staining, the Mankin and OARSI scoring systems and type X collagen (Col X), p-Smad2/3a and Osterix immunohistochemical analyses. We found apparent histological phenotypes of TMJ-OA in the TMD, ageing and CED animal models, with abnormal activation of TGF-β signalling in the condylar cartilage and subchondral bone. Moreover, inhibition of TGF-β receptor I attenuated TMJ-OA progression in the TMD models. Therefore, aberrant activation of TGF-β signalling could be a key player in TMJ-OA development.

Osteoarthritis: growth factor signalling drives degeneration of jaw joint

Blocking the activity of a critical growth factor could help treat degenerative disease of the jaw joint, according to experiments in three rodent models. Xuedong Zhou from Sichuan University in Chengdu, China, examined the cartilage and adjoining layer of bone found at the ends of the jawbone in a rat model of temporomandibular joint disorder and in two related mouse models. In all three, the researchers observed tissue abnormalities consistent with what’s seen in humans with osteoarthritis of the jaw joint, a condition with no effective therapeutic options. They showed that transforming growth factor-β, a master regulatory protein, displayed aberrant signalling patterns in these tissues and that blocking this protein’s receptor with a drug attenuated the disease process. The findings help explain what drives jaw joint osteoarthritis — and point to a strategy for treating it.

Mutations in the liver/bone/kidney alkaline phosphatase (Alpl) gene cause hypophosphatasia (HPP) and early-onset bone dysplasia, suggesting that this gene is a key factor in human bone development. However, how and where Alpl acts in bone ageing is largely unknown. Here, we determined that ablation of Alpl induces prototypical premature bone ageing characteristics, including bone mass loss and marrow fat gain coupled with elevated expression of p16^INK4A (p16) and p53 due to senescence and impaired differentiation in mesenchymal stem cells (MSCs). Mechanistically, Alpl deficiency in MSCs enhances ATP release and reduces ATP hydrolysis. Then, the excessive extracellular ATP is, in turn, internalized by MSCs and causes an elevation in the intracellular ATP level, which consequently inactivates the AMPKα pathway and contributes to the cell fate switch of MSCs. Reactivating AMPKα by metformin treatment successfully prevents premature bone ageing in Alpl ^+/- mice by improving the function of endogenous MSCs. These results identify a previously unknown role of Alpl in the regulation of ATP-mediated AMPKα alterations that maintain MSC stemness and prevent bone ageing and show that metformin offers a potential therapeutic option.

Cell biology: diabetes drug prevents bone aging

The diabetes drug metformin restores the ability of stem cells to grow and differentiate into bone-producing osteoblasts, preventing bone aging. The alkaline phosphatase gene (Alpl) has been implicated in abnormal bone and tooth development, but its role in bone aging was unclear. The protein it encodes is enriched in the cell membrane of mesenchymal stem cells (MSCs) – precursors of bone-producing osteoblasts – and is involved in the metabolism of ATP, which is implicated in MSC differentiation. Here, Yan Lin at the Xi’an Institute of Tissue Engineering and Regenerative Medicine in China and colleagues demonstrate that Alpl deficiency contributes to bone aging by boosting levels of extracellular ATP. When internalized, this triggers the AMPKα pathway and impairs MSCs’ ability to grow and differentiate. Treatment with metformin reactivates this pathway and prevents premature bone aging.

Signal transduction between different organs is crucial in the normal development of the human body. As an important medium for signal communication, exosomes can transfer important information, such as microRNAs (miRNAs), from donors to receptors. MiRNAs are known to fine-tune a variety of biological processes, including maxillofacial development; however, the underlying mechanism remains largely unknown. In the present study, transient apoptosis was found to be due to the expression of a miniature swine maxillofacial-specific miRNA, ssc-mir-133b. Upregulation of ssc-mir-133b resulted in robust apoptosis in primary dental mesenchymal cells in the maxillofacial region. Cell leukemia myeloid 1 (Mcl-1) was verified as the functional target, which triggered further downstream activation of endogenous mitochondria-related apoptotic processes during tooth development. More importantly, mandible exosomes were responsible for the initial apoptosis signal. An animal study demonstrated that ectopic expression of ssc-mir-133b resulted in failed tooth formation after 12 weeks of subcutaneous transplantation in nude mice. The tooth germ developed abnormally without the indispensable exosomal signals from the mandible.

Tooth development: special delivery from the mandible

The delivery of the small regulatory molecule microRNA-133b via extracellular vesicles released from the lower jaw is required for tooth formation in pigs and mice. Several microRNAs have been implicated in tooth development, but their precise roles are poorly understood. Songlin Wang at Capital Medical University, China, and colleagues found that microRNA-133b causes temporary cell death at sites of molar development by reducing the levels of the pro-survival protein myeloid cell leukemia-1. Moreover, they showed that microRNA-133b is delivered from the lower jaw in exosomes and that interrupting this signal prevents tooth development. These findings highlight the importance of cross-talk between jaw and tooth tissue for normal development and reveal a possible mechanism for the prevention and treatment of abnormal tooth formation.

Calvarial bones are connected by fibrous sutures. These sutures provide a niche environment that includes mesenchymal stem cells (MSCs), osteoblasts, and osteoclasts, which help maintain calvarial bone homeostasis and repair. Abnormal function of osteogenic cells or diminished MSCs within the cranial suture can lead to skull defects, such as craniosynostosis. Despite the important function of each of these cell types within the cranial suture, we have limited knowledge about the role that crosstalk between them may play in regulating calvarial bone homeostasis and injury repair. Here we show that suture MSCs give rise to osteoprogenitors that show active bone morphogenetic protein (BMP) signalling and depend on BMP-mediated Indian hedgehog (IHH) signalling to balance osteogenesis and osteoclastogenesis activity. IHH signalling and receptor activator of nuclear factor kappa-Β ligand (RANKL) may function synergistically to promote the differentiation and resorption activity of osteoclasts. Loss of Bmpr1a in MSCs leads to downregulation of hedgehog (Hh) signalling and diminished cranial sutures. Significantly, activation of Hh signalling partially restores suture morphology in Bmpr1a mutant mice, suggesting the functional importance of BMP-mediated Hh signalling in regulating suture tissue homeostasis. Furthermore, there is an increased number of CD200+ cells in Bmpr1a mutant mice, which may also contribute to the inhibited osteoclast activity in the sutures of mutant mice. Finally, suture MSCs require BMP-mediated Hh signalling during the repair of calvarial bone defects after injury. Collectively, our studies reveal the molecular and cellular mechanisms governing cell–cell interactions within the cranial suture that regulate calvarial bone homeostasis and repair.

Skull bone remodeling: Cranial suture cross-talk

Understanding the signaling mechanisms regulating cells in cranial sutures could help develop strategies for repairing skull defects or fractures. Little is known about how osteoblasts, osteoclasts and mesenchymal stem cells (MSCs) in cranial sutures regulate the homeostasis and repair of skull bones. Yang Chai at the University of Southern California, United States, and colleagues show that preventing the expression of bone morphogenetic protein receptor type IA (Bmpr1a) in MSCs leads to defective cranial sutures in which osteogenic activity is increased and osteoclast activity is reduced. Stimulating the Hedgehog signaling pathway not only partially rescued the defective sutures but also promoted skull bone healing after injury in Bmpr1a mutant mice, highlighting the importance of BMP-mediated Hedgehog signaling for balancing skull bone formation and resorption.

Hard tissue repair and regeneration cost hundreds of billions of dollars annually worldwide, and the need has substantially increased as the population has aged. Hard tissues include bone and tooth structures that contain calcium phosphate minerals. Smart biomaterial-based tissue engineering and regenerative medicine methods have the exciting potential to meet this urgent need. Smart biomaterials and constructs refer to biomaterials and constructs that possess instructive/inductive or triggering/stimulating effects on cells and tissues by engineering the material’s responsiveness to internal or external stimuli or have intelligently tailored properties and functions that can promote tissue repair and regeneration. The smart material-based approaches include smart scaffolds and stem cell constructs for bone tissue engineering; smart drug delivery systems to enhance bone regeneration; smart dental resins that respond to pH to protect tooth structures; smart pH-sensitive dental materials to selectively inhibit acid-producing bacteria; smart polymers to modulate biofilm species away from a pathogenic composition and shift towards a healthy composition; and smart materials to suppress biofilms and avoid drug resistance. These smart biomaterials can not only deliver and guide stem cells to improve tissue regeneration and deliver drugs and bioactive agents with spatially and temporarily controlled releases but can also modulate/suppress biofilms and combat infections in wound sites. The new generation of smart biomaterials provides exciting potential and is a promising opportunity to substantially enhance hard tissue engineering and regenerative medicine efficacy.

Bone repair: a new generation of smart biomaterials

Smart biomaterials that are able to instruct bone repair can overcome some of the shortcomings of bone grafting and meet the growing need for hard tissue regeneration in ageing populations. Hockin Xu at the University of Maryland in Baltimore, United States, and colleagues review recent advances in the development of smart biomaterials for repairing and regenerating damaged bones and teeth. They highlight scaffolds that closely mimic natural bone tissue and immunomodulatory biomaterials that can prevent infection and promote cell survival for guiding and enhancing bone regeneration. Furthermore, scaffolds with shape-memory capability and materials that provide tailored spatio-temporal delivery of drugs or bioactive agents in response to internal or external stimuli, hold great promise not only for bone tissue engineering but also for dentistry.

The adaptor protein NUMB is involved in asymmetric division and cell fate determination and recognized as an antagonist of Notch. Previous studies have proved that Notch activation in osteoblasts contributes to a high bone mass. In this study,  however, an osteopenic phenotype was found in 9-week-old mice using osteoblastic specific Col1a1–2.3-Cre to ablate both Numb and its homologue Numbl . The trabecular bone mass decreased dramatically while the cortical bone mass was unaffected. Here, the Notch signal was not activated, while the tensin homologue deleted on human chromosome 10 (PTEN), which dephosphorylates phosphatidylinositide 3-kinases, was elevated, attenuating protein kinase B (Akt). The ubiquitination assay revealed that NUMB may physiologically promote PTEN ubiquitination in the presence of neural precursor cell-expressed developmentally downregulated protein 4–1. In addition, the deficiency of Numb/Numbl also activated the Hedgehog pathway through GLI1. This process was found to improve the ratio of the receptor activator of nuclear factor-kB ligand to osteoprotegerin, which enhanced the differentiation of osteoclasts and bone resorption . In conclusion, this study provides an insight into  new functons of   NUMB and NUMBL on bone homeostasis.

Homeostasis: Protein regulators of bone mass

The related proteins NUMB and NUMBL maintain the survival of bone-generating osteoblast cells. NUMB was previously recognized to antagonize Notch signaling pathway ; In this study, it observes  that genetically altered mice, unable to express the two proteins, suffered from degraded bone quality. This suggests that the two proteins play a more complex, nuanced role in the process of bone mass maintenance. The team’s studies showed that NUMB and NUMBL suppression inhibits a signaling pathway important to skeletal development and protein synthesis in osteoblasts, though raise that further investigations are essential to elucidate the exact mechanistic action of these proteins. The authors of this study suggest that NUMB constitutes a potential target for therapies targeting bone loss and reduced bone strength in patients with osteoporosis.

How osteoblast cells are induced is a central question for understanding skeletal formation. Abnormal osteoblast differentiation leads to a broad range of devastating craniofacial diseases. Here we have investigated intramembranous ossification during cranial bone development in mouse models of skeletal genetic diseases that exhibit craniofacial bone defects. The GNAS gene encodes Gα_s that transduces GPCR signaling. GNAS activation or loss-of-function mutations in humans cause fibrous dysplasia (FD) or progressive osseous heteroplasia (POH) that shows craniofacial hyperostosis or craniosynostosis, respectively. We find here that, while Hh ligand-dependent Hh signaling is essential for endochondral ossification, it is dispensable for intramembranous ossification, where Gα_s regulates Hh signaling in a ligand-independent manner. We further show that Gα_s controls intramembranous ossification by regulating both Hh and Wnt/β-catenin signaling. In addition, Gα_s activation in the developing cranial bone leads to reduced ossification but increased cartilage presence due to reduced cartilage dissolution, not cell fate switch. Small molecule inhibitors of Hh and Wnt signaling can effectively ameliorate cranial bone phenotypes in mice caused by loss or gain of Gnas function mutations, respectively. Our work shows that studies of genetic diseases provide invaluable insights in both pathological bone defects and normal bone development, understanding both leads to better diagnosis and therapeutic treatment of bone diseases.

Skull formation: Finding new avenues

Investigating two genetic diseases of skull formation revealed underlying similarities that may help improve treatments. During skull development, growth of too much or too little bone leads to deformation of the head or face, with potentially devastating medical consequences. To improve our understanding of the fine-scale processes that control bone deposition, Yingzi Yang at the Harvard School of Dental Medicine and co-workers investigated how bone formation occurs in progressive osseous heteroplasia (POH) and fibrous dysplasia (FD), which cause excessive and insufficient bone formation, respectively, during skull development. Using POH and FD model mice, they identified a single molecule underlying both diseases. Pinpointing this molecule allowed the researchers to identify drugs, previously developed for other diseases, that could help treat POH and FD; initial tests showed improved bone formation in both POH and FD model mice.

RANKL signaling is essential for osteoclastogenesis. Its role in osteoblastic differentiation and bone formation is unknown. Here we demonstrate that RANK is expressed at an early stage of bone marrow mesenchymal stem cells (BMSCs) during osteogenic differentiation in both mice and human and decreased rapidly. RANKL signaling inhibits osteogenesis by promoting β-catenin degradation and inhibiting its synthesis. In contrast, RANKL signaling has no significant effects on adipogenesis of BMSCs. Interestingly, conditional knockout of rank in BMSCs with Prx1-Cre mice leads to a higher bone mass and increased trabecular bone formation independent of osteoclasts. In addition, rank ^flox/flox: Prx1-Cre mice show resistance to ovariectomy-(OVX) induced bone loss. Thus, our results reveal that RANKL signaling regulates both osteoclasts and osteoblasts by inhibition of osteogenic differentiation of BMSCs and promotion of osteoclastogenesis.

Developmental Genetics: A dual-use gene regulates bone formation and breakdown

Researchers in China have shown that a gene known for breaking bones down is also involved in making new bone. Bones are constantly repaired and reshaped by cells which break down bone tissue, osteoclasts, and those which create new bone, osteoblasts. The RANKL gene is known to play an important role in osteoclast development, but Jiacan Su of the Second Military Medical University has shown that it is also important for osteoblasts. Su’s team detected high expression of RANKL and its receptor, RANK, in bone marrow stem cells, and the levels decreased as the stem cells differentiated into osteoblasts. Artificially increasing RANK expression decreased osteoblast differentiation, while reducing its expression increased osteoblast differentiation and bone mass. By showing that RANKL regulates osteoblasts as well as osteoclasts, these findings open new avenues for understanding bones development.

Exosomes are a heterogeneous group of cell-derived membranous structures, which mediate crosstalk interaction between cells. Recent studies have revealed a close relationship between exosomes and bone homeostasis. It is suggested that bone cells can spontaneously secret exosomes containing proteins, lipids and nucleic acids, which then to regulate osteoclastogenesis and osteogenesis. However, the network of regulatory activities of exosomes in bone homeostasis as well as their therapeutic potential in bone injury remain largely unknown. This review will detail and discuss the characteristics of exosomes, the regulatory activities of exosomes in bone homeostasis as well as the clinical potential of exosomes in bone injury.

Development: Exosomes guide bone development and offer clinical promise

Vesicles known as exosomes may prove to be valuable clinical tools once their function is clarified. Exosomes were discovered in the 1980s but not observed in bone tissue until 2003. Minghao Zheng of the University of Western Australia, together with colleagues elsewhere, has reviewed the biology of exosomes, their role in maintaining bones, and their potential clinical uses. Exosomes carry lipids, proteins, and nucleic acids between cells. They are released by every type of bone cell, with the role of each exosome determined by its specific contents. Exosome-mediated crosstalk is involved in regulating bone remodeling, and exosomes have also been implicated in myelomas. Recent work has shown that exosome treatment can improve fracture healing. The authors conclude that a better understanding of the role of exosomes in bone homeostasis will unlock their significant clinical potential.

With the incidence of different bone diseases increasing, effective therapies are needed that coordinate a combination of various technologies and biological materials. Bone tissue engineering has also been considered as a promising strategy to repair various bone defects. Therefore, different biological materials that can promote stem cell proliferation, migration, and osteoblastic differentiation to accelerate bone tissue regeneration and repair have also become the focus of research in multiple fields. Stem cell therapy, biomaterial scaffolds, and biological growth factors have shown potential for bone tissue engineering; however, off-target effects and cytotoxicity have limited their clinical use. The application of nucleic acids (deoxyribonucleic acid or ribonucleic acid) and nucleic acid analogs (peptide nucleic acids or locked nucleic acids), which are designed based on foreign genes or with special structures, can be taken up by target cells to exert different effects such as modulating protein expression, replacing a missing gene, or targeting specific gens or proteins. Due to some drawbacks, nucleic acids and nucleic acid analogs are combined with various delivery systems to exert enhanced effects, but current studies of these molecules have not yet satisfied clinical requirements. In-depth studies of nucleic acid or nucleic acid analog delivery systems have been performed, with a particular focus on bone tissue regeneration and repair. In this review, we mainly introduce delivery systems for nucleic acids and nucleic acid analogs and their applications in bone repair and regeneration. At the same time, the application of conventional scaffold materials for the delivery of nucleic acids and nucleic acid analogs is also discussed.

Bone Regeneration: Nucleic acids and analogs in bone repair

Used with an appropriate delivery system, nucleic acids and nucleic acid analogs have excellent potential for bone repair and regeneration. Owing to various challenges with bone tissue regeneration, current research is largely focused on gene therapy, which employs genes to treat or prevent disease, and such new materials as nucleic acids (DNA and RNA) and nucleic acid analogs (compounds structurally similar to naturally occurring nucleic acids). A team headed by Yunfeng Lin at Sichuan University, China conducted a review of delivery systems for nucleic acids and nucleic acid analogs and their application in bone repair and regeneration. The authors identified the use of biomaterial scaffolds (which mimic living tissue) as one of the most important research areas for gene therapy, and that strategy has proven effective with all types of bone regeneration and repair.

TGF-β 1–3 are unique multi-functional growth factors that are only expressed in mammals, and mainly secreted and stored as a latent complex in the extracellular matrix (ECM). The biological functions of TGF-β in adults can only be delivered after ligand activation, mostly in response to environmental perturbations. Although involved in multiple biological and pathological processes of the human body, the exact roles of TGF-β in maintaining stem cells and tissue homeostasis have not been well-documented until recent advances, which delineate their functions in a given context. Our recent findings, along with data reported by others, have clearly shown that temporal and spatial activation of TGF-β is involved in the recruitment of stem/progenitor cell participation in tissue regeneration/remodeling process, whereas sustained abnormalities in TGF-β ligand activation, regardless of genetic or environmental origin, will inevitably disrupt the normal physiology and lead to pathobiology of major diseases. Modulation of TGF-β signaling with different approaches has proven effective pre-clinically in the treatment of multiple pathologies such as sclerosis/fibrosis, tumor metastasis, osteoarthritis, and immune disorders. Thus, further elucidation of the mechanisms by which TGF-β is activated in different tissues/organs and how targeted cells respond in a context-dependent way can likely be translated with clinical benefits in the management of a broad range of diseases with the involvement of TGF-β.

Growth factor: Activation in health and disease

Targeting a critical growth factor involved in bone and other tissue remodeling could help treat osteoarthritis and other skeletal disorders. A team led by Zhou Xuedong from Sichuan University in Chengdu, China, and Xu Cao from the Johns Hopkins University School of Medicine in Baltimore, Maryland, USA, review the ways in which temporal and spatial activation of transforming growth factor-β (TGF-β), a multi-functional signaling molecule, are needed for proper tissue development and regulation of stem cells throughout the body. Looking at the skeletal system in particular, the researchers discuss how TGF-β controls the balance between bone resorption and bone formation. Faulty TGF-β signaling can lead to numerous bone-associated disorders, including rare genetic diseases and metastatic cancers. The authors also summarize clinical efforts to modulate TGF-β with drugs for the treatment of osteoarthritis and other conditions.