Artesunate inhibits RANKL‐induced osteoclastogenesis and bone resorption in vitro and prevents LPS‐induced bone loss in vivo

Osteoclasts are multinuclear giant cells responsible for bone resorption in lytic bone diseases such as osteoporosis, arthritis, periodontitis, and bone tumors. Due to the severe side‐effects caused by the currently available drugs, a continuous search for novel bone‐protective therapies is essential. Artesunate (Art), the water‐soluble derivative of artemisinin has been investigated owing to its anti‐malarial properties. However, its effects in osteoclastogenesis have not yet been reported. In this study, Art was shown to inhibit the nuclear factor‐κB ligand (RANKL)‐induced osteoclastogenesis, the mRNA expression of osteoclastic‐specific genes, and resorption pit formation in a dose‐dependent manner in primary bone marrow‐derived macrophages cells (BMMs). Furthermore, Art markedly blocked the RANKL‐induced osteoclastogenesis by attenuating the degradation of IκB and phosphorylation of NF‐κB p65. Consistent with the in vitro results, Art inhibited lipopolysaccharide (LPS)‐induced bone resorption by suppressing the osteoclastogenesis. Together our data demonstrated that Art inhibits RANKL‐induced osteoclastogenesis by suppressing the NF‐κB signaling pathway and that it is a promising agent for the treatment of osteolytic diseases.     

Three‐dimensional intravital imaging in bone research

Intravital imaging has emerged as a novel and efficient tool for visualization of in situ dynamics of cellular behaviors and cell‐microenvironment interactions in live animals, based on desirable microscopy techniques featuring high resolutions, deep imaging and low phototoxicity. Intravital imaging, especially based on multi‐photon microscopy, has been used in bone research for dynamics visualization of a variety of physiological and pathological events at the cellular level, such as bone remodeling, hematopoiesis, immune responses and cancer development, thus, providing guidance for elucidating novel cellular mechanisms in bone biology as well as guidance for new therapies. This review is aimed at interpreting development and advantages of intravital imaging in bone research, and related representative discoveries concerning bone matrices, vessels, and various cells types involved in bone physiologies and pathologies. Finally, current limitations, further refinement, and extended application of intravital imaging in bone research are concluded.      

Tendon‐to‐bone attachment: From development to maturity

The attachment between tendon and bone occurs across a complex transitional tissue that minimizes stress concentrations and allows for load transfer between muscles and skeleton. This unique tissue cannot be reconstructed following injury, leading to high incidence of recurrent failure and stressing the need for new clinical approaches. This review describes the current understanding of the development and function of the attachment site between tendon and bone. The embryonic attachment unit, namely, the tip of the tendon and the bone eminence into which it is inserted, was recently shown to develop modularly from a unique population of Sox9‐ and Scx‐positive cells, which are distinct from tendon fibroblasts and chondrocytes. The fate and differentiation of these cells is regulated by transforming growth factor beta and bone morphogenetic protein signaling, respectively. Muscle loads are then necessary for the tissue to mature and mineralize. Mineralization of the attachment unit, which occurs postnatally at most sites, is largely controlled by an Indian hedgehog/parathyroid hormone‐related protein feedback loop. A number of fundamental questions regarding the development of this remarkable attachment system require further study. These relate to the signaling mechanism that facilitates the formation of an interface with a gradient of cellular and extracellular phenotypes, as well as to the interactions between tendon and bone at the point of attachment. Birth Defects Research (Part C) 102:101–112, 2014. © 2014 Wiley Periodicals, Inc.     

Recent advances in gene‐enhanced bone tissue engineering

The loss of bone tissue represents a critical clinical condition that is frequently faced by surgeons. Substantial progress has been made in the area of bone research, providing insight into the biology of bone under physiological and pathological conditions, as well as tools for the stimulation of bone regeneration. The present review discusses recent advances in the field of gene‐enhanced bone tissue engineering. Gene transfer strategies have emerged as highly effective tissue engineering approaches for supporting the repair of the musculoskeletal system. By contrast to treatment with recombinant proteins, genetically engineered cells can release growth factors at the site of injury over extended periods of time. Of particular interest are the expedited technologies that can be applied during a single surgical procedure in a cost‐effective manner, allowing translation from bench to bedside. Several promising methods based on the intra‐operative genetic manipulation of autologous cells or tissue fragments have been developed in preclinical studies. Moreover, gene therapy for bone regeneration has entered the clinical stage with clinical trials for the repair of alveolar bone. Current trends in gene‐enhanced bone engineering are also discussed with respect to the movement of the field towards expedited, translational approaches. It is possible that gene‐enhanced bone tissue engineering will become a clinical reality within the next few years.     

MicroRNA‐31a‐5p from aging BMSCs links bone formation and resorption in the aged bone marrow microenvironment

The alteration of age‐related molecules in the bone marrow microenvironment is one of the driving forces in osteoporosis. These molecules inhibit bone formation and promote bone resorption by regulating osteoblastic and osteoclastic activity, contributing to age‐related bone loss. Here, we observed that the level of microRNA‐31a‐5p (miR‐31a‐5p) was significantly increased in bone marrow stromal cells (BMSCs) from aged rats, and these BMSCs demonstrated increased adipogenesis and aging phenotypes as well as decreased osteogenesis and stemness. We used the gain‐of‐function and knockdown approach to delineate the roles of miR‐31a‐5p in osteogenic differentiation by assessing the decrease of special AT‐rich sequence‐binding protein 2 (SATB2) levels and the aging of BMSCs by regulating the decline of E2F2 and recruiting senescence‐associated heterochromatin foci (SAHF). Notably, expression of miR‐31a‐5p, which promotes osteoclastogenesis and bone resorption, was markedly higher in BMSCs‐derived exosomes from aged rats compared to those from young rats, and suppression of exosomal miR‐31a‐5p inhibited the differentiation and function of osteoclasts, as shown by elevated RhoA activity. Moreover, using antagomiR‐31a‐5p, we observed that, in the bone marrow microenvironment, inhibition of miR‐31a‐5p prevented bone loss and decreased the osteoclastic activity of aged rats. Collectively, our results reveal that miR‐31a‐5p acts as a key modulator in the age‐related bone marrow microenvironment by influencing osteoblastic and osteoclastic differentiation and that it may be a potential therapeutic target for age‐related osteoporosis.     

Elastin‐like‐polypeptide based fusion proteins for osteogenic factor delivery in bone healing

Modern treatments of bone injuries and diseases are becoming increasingly dependent on the usage of growth factors to stimulate bone growth. Bone morphogenetic protein‐2 (BMP‐2), a potent osteogenic inductive protein, exhibits promising results in treatment models, but recently has had its practical efficacy questioned due to the lack of local retention, ectopic bone formation, and potentially lethal inflammation. Where a new delivery technique of the BMP‐2 is necessary, here we demonstrate the viability of an elastin‐like peptide (ELP) fusion protein containing BMP‐2 for delivery of the BMP‐2. This fusion protein retains the performance characteristics of both the BMP‐2 and ELP. The fusion protein was found to induce osteogenic differentiation of mesenchymal stem cells as evidenced by the production of alkaline phosphatase and extracellular calcium deposits in response to treatment by the fusion protein. Retention of the ELPs inverse phase transition property has allowed for expression of the fusion protein within a bacterial host (such as Escherichia coli) and easy and rapid purification using inverse transition cycling. The fusion protein formed self‐aggregating nanoparticles at human‐body temperature. The data collected suggests the viability of these fusion protein nanoparticles as a dosage‐efficient and location‐precise noncytotoxic delivery vehicle for BMP‐2 in bone treatment. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1029–1037, 2016     

Technical note: False catastrophic age‐at‐death profiles in commingled bone deposits

Age‐at‐death profiles obtained using the minimum number of individuals (MNI) for mass deposits of commingled human remains may be biased by over‐representation of subadult individuals. A computer simulation designed in the R environment has shown that this effect may lead to misinterpretation of such samples even in cases where the completeness rate is relatively high. The simulation demonstrates that the use of the Most Likely Number of Individuals (MLNI) substantially reduces this bias. Am J Phys Anthropol 152:554–557, 2013. © 2013 Wiley Periodicals, Inc.     

Accumulation of apoptosis‐insensitive human bone marrow‐mesenchymal stromal cells after long‐term expansion

Cells undergo replicative senescence during in vitro expansion, which is induced by the accumulation of cellular damage caused by excessive reactive oxygen species. In this study, we investigated whether long‐term‐cultured human bone marrow mesenchymal stromal cells (MSCs) are insensitive to apoptotic stimulation. To examine this, we established replicative senescent cells from long‐term cultures of human bone marrow MSCs. Senescent cells were identified based on declining population doublings, increased expression of senescence markers p16 and p53 and increased senescence‐associated β‐gal activity. In cell viability assays, replicative senescent MSCs in late passages (i.e. 15–19 passages) resisted damage induced by oxidative stress more than those in early passages did (i.e. 7–10 passages). This resistance occurred via caspase‐9 and caspase‐3 rather than via caspase‐8. The senescent cells are gradually accumulated during long‐term expansion. The oxidative stress‐sensitive proteins ataxia‐telangiectasia mutated and p53 were phosphorylated, and the expression of apoptosis molecules Bax increased, and Bcl‐2 decreased in early passage MSCs; however, the expression of the apoptotic molecules did less change in response to apoptotic stimulation in late‐passage MSCs, suggesting that the intrinsic apoptotic signalling pathway was not induced by oxidative stress in long‐term‐cultured MSCs. Based on these results, we propose that some replicative senescent cells may avoid apoptosis signalling via impairment of signalling molecules and accumulation during long‐term expansion. Copyright © 2016 John Wiley & Sons, Ltd.     

Reconstruction of tissue‐engineered bone with bone marrow mesenchymal stem cells and partially deproteinized bone in vitro

We have explored the optimal seeding density and timing for transplantation of the tissue‐engineered bone with BMMSCs (bone marrow mesenchymal stem cells) and PDPB (partially deproteinized bone) in vitro. Rabbit BMMSCs of different densities were seeded into PDPB generated from fresh pig vertebrates to reconstruct tissue‐engineered bone in vitro. Adhesion and proliferation of BMMSCs were analysed by MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide] assay from which growth curves of BMMSCs on the PDPB materials were generated. The data show that BMMSCs began to adhere to PDPB after 24 h of primary culture, all groups reaching peak growth on the 6th day, after which the value of A decreased gradually and reached a plateau phase. The optimal BMMSCs seeding density of 5×10⁶/ml achieved an excellent adhesion and proliferation activity on PDPB. In summary, the best cell seeding density of constructing tissue‐engineered bone with BMMSCs in vitro is 5×10⁶/ml, the optimal timing to transplant is the 6th day.