Osteoblast Progenitors Research Articles

Identification of Senescent Cells in the Bone Microenvironment.

Cellular senescence is a fundamental mechanism by which cells remain metabolically active yet cease dividing and undergo distinct phenotypic alterations, including upregulation of p16Ink4a , profound secretome changes, telomere shortening, and decondensation of pericentromeric satellite DNA. Because senescent cells accumulate in multiple tissues with aging, these cells and the dysfunctional factors they secrete, termed the senescence-associated secretory phenotype (SASP), are increasingly recognized as promising therapeutic targets to prevent age-related degenerative pathologies, including osteoporosis. However, the cell type(s) within the bone microenvironment that undergoes senescence with aging in vivo has remained poorly understood, largely because previous studies have focused on senescence in cultured cells. Thus in young (age 6 months) and old (age 24 months) mice, we measured senescence and SASP markers in vivo in highly enriched cell populations, all rapidly isolated from bone/marrow without in vitro culture. In both females and males, p16Ink4a expression by real-time quantitative polymerase chain reaction (rt-qPCR) was significantly higher with aging in B cells, T cells, myeloid cells, osteoblast progenitors, osteoblasts, and osteocytes. Further, in vivo quantification of senescence-associated distension of satellites (SADS), ie, large-scale unraveling of pericentromeric satellite DNA, revealed significantly more senescent osteocytes in old compared with young bone cortices (11% versus 2%, p < 0.001). In addition, primary osteocytes from old mice had sixfold more (p < 0.001) telomere dysfunction-induced foci (TIFs) than osteocytes from young mice. Corresponding with the age-associated accumulation of senescent osteocytes was significantly higher expression of multiple SASP markers in osteocytes from old versus young mice, several of which also showed dramatic age-associated upregulation in myeloid cells. These data show that with aging, a subset of cells of various lineages within the bone microenvironment become senescent, although senescent myeloid cells and senescent osteocytes predominantly develop the SASP. Given the critical roles of osteocytes in orchestrating bone remodeling, our findings suggest that senescent osteocytes and their SASP may contribute to age-related bone loss. © 2016 American Society for Bone and Mineral Research.

MicroRNAs regulate signaling pathways in osteogenic differentiation of mesenchymal stem cells (Review).

Osteogenesis is a complex multi-step process involving the differentiation of mesenchymal stem cells (MSCs) into osteoblast progenitor cells, preosteoblasts, osteoblasts and osteocytes, and the crosstalk between multiple cell types for the formation and remodeling of bone. The signaling regulatory networks during osteogenesis include various components, including growth factors, transcription factors, micro (mi)RNAs and effectors, a number of which form feedback loops controlling the balance of osteogenic differentiation by positive or negative regulation. miRNAs have been found to be important regulators of osteogenic signaling pathways in multiple aspects and multiple signaling pathways. The present review focusses on the progress in elucidating the role of miRNA in the osteogenesis signaling networks of MSCs as a substitute for bone implantation the the field of bone tissue engineering. In particular, the review classifies which miRNAs promote or suppress the osteogenic process, and summarizes which signaling pathway these miRNAs are involved in. Improvements in knowledge of the characteristics of miRNAs in osteogenesis provide an important step for their application in translational investigations of bone tissue engineering and bone disease.

Increased EPO Levels Are Associated With Bone Loss in Mice Lacking PHD2 in EPO-Producing Cells.

The main oxygen sensor hypoxia inducible factor (HIF) prolyl hydroxylase 2 (PHD2) is a critical regulator of tissue homeostasis during erythropoiesis, hematopoietic stem cell maintenance, and wound healing. Recent studies point toward a role for the PHD2-erythropoietin (EPO) axis in the modulation of bone remodeling, even though the studies produced conflicting results. Here, we used a number of mouse strains deficient of PHD2 in different cell types to address the role of PHD2 and its downstream targets HIF-1α and HIF-2α in bone remodeling. Mice deficient for PHD2 in several cell lineages, including EPO-producing cells, osteoblasts, and hematopoietic cells (CD68:cre-PHD2f/f ) displayed a severe reduction of bone density at the distal femur as well as the vertebral body due to impaired bone formation but not bone resorption. Importantly, using osteoblast-specific (Osx:cre-PHD2f/f ) and osteoclast-specific PHD2 knock-out mice (Vav:cre- PHD2f/f ), we show that this effect is independent of the loss of PHD2 in osteoblast and osteoclasts. Using different in vivo and in vitro approaches, we show here that this bone phenotype, including the suppression of bone formation, is directly linked to the stabilization of the α-subunit of HIF-2, and possibly to the subsequent moderate induction of serum EPO, which directly influenced the differentiation and mineralization of osteoblast progenitors resulting in lower bone density. Taken together, our data identify the PHD2:HIF-2α:EPO axis as a so far unknown regulator of osteohematology by controlling bone homeostasis. Further, these data suggest that patients treated with PHD inhibitors or EPO should be monitored with respect to their bone status. © 2016 American Society for Bone and Mineral Research.

Low bone mass and changes in the osteocyte network in mice lacking autophagy in the osteoblast lineage.

Autophagy maintains cell function and homeostasis by recycling intracellular components. This process is also required for morphological changes associated with maturation of some cell types. Osteoblasts are bone forming cells some of which become embedded in bone and differentiate into osteocytes. This transformation includes development of long cellular projections and a reduction in endoplasmic reticulum and mitochondria. We examined the role of autophagy in osteoblasts by deleting Atg7 using an Osterix1-Cre transgene, which causes recombination in osteoblast progenitors and their descendants. Mice lacking Atg7 in the entire osteoblast lineage had low bone mass and fractures associated with reduced numbers of osteoclasts and osteoblasts. Suppression of autophagy also reduced the amount of osteocyte cellular projections and led to retention of endoplasmic reticulum and mitochondria in osteocytes. These results demonstrate that autophagy in osteoblasts contributes to skeletal homeostasis and to the morphological changes associated with osteocyte formation.

A thermoresponsive polydiolcitrate-gelatin scaffold and delivery system mediates effective bone formation from BMP9-transduced mesenchymal stem cells

Successful bone tissue engineering requires at the minimum sufficient osteoblast progenitors, efficient osteoinductive factors, and biocompatible scaffolding materials. We previously demonstrated that bone morphogenetic protein 9 (BMP9) is one of the most potent factors in inducing osteogenic differentiation of mesenchymal stem cells (MSCs). Here, we investigated the potential use of a biodegradable citrate-based thermosensitive macromolecule, poly(polyethyleneglycol citrate-co-N-isopropylacrylamide) (PPCN) mixed with gelatin (PPCNG) as a scaffold for the delivery of BMP9-stimulated MSCs to promote localized bone formation. The addition of gelatin to PPCN effectively enhanced the cell adhesion and survival properties of MSCs entrapped within the gel in 3D culture. Using the BMP9-transduced MSC line immortalized mouse embryonic fibroblasts (iMEFs), we found that PPCNG facilitated BMP9-induced osteogenic differentiation of iMEFs in vivo and promoted the formation of well-ossified and vascularized trabecular bone-like structures in a mouse model of ectopic bone formation. Histologic evaluation revealed that vascularization of the bony masses retrieved from the iMEFs + PPCNG group was significantly more pronounced than that of the direct cell injection group. Accordingly, vascular endothelial growth factor (VEGF) expression was shown to be significantly higher in the bony masses recovered from the iMEFs + PPCNG group. Taken together, our results suggest that PPCNG may serve as a novel biodegradable and injectable scaffold and carrier for gene and cell-based bone tissue engineering.

PELA microspheres with encapsulated arginine–chitosan/pBMP-2 nanoparticles induce pBMP-2 controlled-release, transfected osteoblastic progenitor cells, and promoted osteogenic differentiation

Repair of the bone injury remains a challenge in clinical practices. Recent progress in tissue engineering and therapeutic gene delivery systems have led to promising new strategies for successful acceleration of bone repair process. The aim of this study was to create a controlled-release system to slowly release the arginine–chitosan/plasmid DNA nanoparticles encoding BMP-2 gene (Arg-CS/pBMP-2 NPs), efficiently transfect osteoblastic progenitor cells, secrete functional BMP-2 protein, and promote osteogenic differentiation. In this study, chitosan was conjugated with arginine to generate arginine–chitosan polymer (Arg-CS) for gene delivery. Mix the Arg-CS with pBMP-2 to condense pBMP-2 into nano-sized particles. In vitro transfection assays demonstrated that the transfection efficiency of Arg-CS/pBMP-2 nanoparticles and the expression level of BMP-2 was obviously exceed control groups. Further, PELA microspheres as the controlled-release carrier for the nanoparticles were used to encapsulate Arg-CS/pBMP-2 NPs. We demonstrated that the Arg-CS/pBMP-2 NPs could slowly release from the PELA microspheres at least for 42 d. During the co-culture with the PELA microspheres, the content of BMP-2 protein secreted by MC3T3-E1 reached the peak at 7 d. After 21d, the secretion of BMP-2 protein still maintain a higher level. The alkaline phosphatase activity, alizarin red staining, and osteogenesis-related gene expression by real-time quantitative PCR analysis all showed the PELA microspheres entrapping with Arg-CS/pBMP-2 NPs can obviously induce the osteogenic differentiation. The results indicated that the Arg-CS is a suitable gene vector which can promote the gene transfection. And the novel PELA microspheres-nanoparticle controlled-release system has potential clinical application in the future after further research.

Skeletal inflammation and attenuation of Wnt signaling, Wnt ligand expression, and bone formation in atherosclerotic ApoE-null mice.

ApoE-null (ApoE-KO) mice fed a high-fat diet (HFD) develop atherosclerosis, due in part to activation of vascular inflammation by oxidized low-density lipoprotein. Since bone loss also occurs in these mice, we used them to investigate the impact of oxidized lipids on bone homeostasis and to search for underlying pathogenic pathways. Four-month-old female ApoE-KO mice fed a HFD for three months exhibited increased levels of oxidized lipids in bone, as well as decreased femoral and vertebral trabecular and cortical bone mass, compared with ApoE-KO mice on normal diet. Despite HFD-induced increase in expression of Alox15, a lipoxygenase that oxidizes LDL and promotes atherogenesis, global deletion of this gene failed to ameliorate the skeletal impact of HFD. Osteoblast number and function were dramatically reduced in trabecular and cortical bone of HFD-fed mice, whereas osteoclast number was modestly reduced only in trabecular bone, indicating that an imbalance in favor of osteoclasts was responsible for HFD-induced bone loss. These changes were associated with decreased osteoblast progenitors and increased monocyte/macrophages in the bone marrow as well as increased expression of IL-1β, IL-6, and TNF. HFD also attenuated Wnt signaling as evidenced by reduced expression of Wnt target genes, and it decreased expression of pro-osteoblastogenic Wnt ligands. These results suggest that oxidized lipids decrease bone mass by increasing anti-osteoblastogenic inflammatory cytokines and decreasing pro-osteoblastogenic Wnt ligands.

VEGF stimulates intramembranous bone formation during craniofacial skeletal development

Deficiency of vascular endothelial growth factor A (VEGF) has been associated with severe craniofacial anomalies in both humans and mice. Cranial neural crest cell (NCC)-derived VEGF regulates proliferation, vascularization and ossification of cartilage and membranous bone. However, the function of VEGF derived from specific subpopulations of NCCs in controlling unique aspects of craniofacial morphogenesis is not clear. In this study a conditional knockdown strategy was used to genetically delete Vegfa expression in Osterix (Osx) and collagen II (Col2)-expressing NCC descendants. No major defects in calvaria and mandibular morphogenesis were observed upon knockdown of VEGF in the Col2+ cell population. In contrast, loss of VEGF in Osx+ osteoblast progenitor cells led to reduced ossification of calvarial and mandibular bones without affecting the formation of cartilage templates in newborn mice. The early stages of ossification in the developing jaw revealed decreased initial mineralization levels and a reduced thickness of the collagen I (Col1)-positive bone template upon loss of VEGF in Osx+ precursors. Increased numbers of proliferating cells were detected within the jaw mesenchyme of mutant embryos. Explant culture assays revealed that mandibular osteogenesis occurred independently of paracrine VEGF action and vascular development. Reduced VEGF expression in mandibles coincided with increased phospho-Smad1/5 (P-Smad1/5) levels and bone morphogenetic protein 2 (Bmp2) expression in the jaw mesenchyme. We conclude that VEGF derived from Osx+ osteoblast progenitor cells is required for optimal ossification of developing mandibular bones and modulates mechanisms controlling BMP-dependent specification and expansion of the jaw mesenchyme.

CCL20/CCR6 Signaling Regulates Bone Mass Accrual in Mice.

CCL20 is a member of the macrophage inflammatory protein family and is reported to signal monogamously through the receptor CCR6. Although studies have identified the genomic locations of both Ccl20 and Ccr6 as regions important for bone quality, the role of CCL20/CCR6 signaling in regulating bone mass is unknown. By micro-computed tomography (μCT) and histomorphometric analysis, we show that global loss of Ccr6 in mice significantly decreases trabecular bone mass coincident with reduced osteoblast numbers. Notably, CCL20 and CCR6 were co-expressed in osteoblast progenitors and levels increased during osteoblast differentiation, indicating the potential of CCL20/CCR6 signaling to influence osteoblasts through both autocrine and paracrine actions. With respect to autocrine effects, CCR6 was found to act as a functional G protein-coupled receptor in osteoblasts and although its loss did not appear to affect the number or proliferation rate of osteoblast progenitors, differentiation was significantly inhibited as evidenced by delays in osteoblast marker gene expression, alkaline phosphatase activity, and mineralization. In addition, CCL20 promoted osteoblast survival concordant with activation of the PI3K-AKT pathway. Beyond these potential autocrine effects, osteoblast-derived CCL20 stimulated the recruitment of macrophages and T cells, known facilitators of osteoblast differentiation and survival. Finally, we generated mice harboring a global deletion of Ccl20 and found that Ccl20(-/-) mice exhibit a reduction in bone mass similar to that observed in Ccr6(-/-) mice, confirming that this phenomenon is regulated by CCL20 rather than alternate CCR6 ligands. Collectively, these data indicate that CCL20/CCR6 signaling may play an important role in regulating bone mass accrual, potentially by modulating osteoblast maturation, survival, and the recruitment of osteoblast-supporting cells. © 2016 American Society for Bone and Mineral Research.

Systemic neutralization of TGF‐β attenuates osteoarthritis

Osteoarthritis (OA) is a major source of pain and disability worldwide with no effective medical therapy due to poor understanding of its pathogenesis. Transforming growth factor β (TGF-β) has been reported to play a role in subchondral bone pathology and articular cartilage degeneration during the progression of OA. In this study, we demonstrated that systemic use of a TGF-β-neutralizing antibody (1D11) attenuates OA progression by targeting subchondral bone pathological features in rodent OA models. Systemic administration of 1D11 preserves the subchondral bone microarchitecture, preventing articular cartilage degeneration by inhibition of excessive TGF-β activity, in both subchondral bone and the circulation. Moreover, the aberrant increases in the numbers of blood vessels, nestin(+) mesenchymal stromal/stem cells, and osterix(+) osteoblast progenitors were normalized by 1D11 systemic injection. Thus, systemic neutralization of excessive TGF-β ligands effectively prevented OA progression in animal models, with promising clinical implications for OA treatment.

Characterization of Cellular and Molecular Heterogeneity of Bone Marrow Stromal Cells.

Human bone marrow-derived stromal stem cells (hBMSC) exhibit multiple functions, including differentiation into skeletal cells (progenitor function), hematopoiesis support, and immune regulation (nonprogenitor function). We have previously demonstrated the presence of morphological and functional heterogeneity of hBMSC cultures. In the present study, we characterized in detail two hTERT-BMSC clonal cell populations termed here CL1 and CL2 that represent an opposing phenotype with respect to morphology, markers expression: alkaline phosphatase (ALP) and CD146, and ex vivo differentiation potential. CL1 differentiated readily to osteoblasts, adipocytes, and chondrocytes as shown by expression of lineage specific genes and proteins. Whole genome transcriptome profiling of CL1 versus CL2 revealed enrichment in CL1 of bone-, mineralization-, and skeletal muscle-related genes, for example, ALP, POSTN, IGFBP5 BMP4, and CXCL12. On the other hand, CL2 transcriptome was enriched in immune modulatory genes, for example, CD14, CD99, NOTCH3, CXCL6, CFB, and CFI. Furthermore, gene expression microarray analysis of osteoblast differentiated CL1 versus CL2 showed significant upregulation in CL1 of bone development and osteoblast differentiation genes which included several homeobox genes: TBX15, HOXA2 and HOXA10, and IGF1, FGFR3, BMP6, MCAM, ITGA10, IGFBP5, and ALP. siRNA-based downregulation of the ALP gene in CL1 impaired osteoblastic and adipocytic differentiation. Our studies demonstrate the existence of molecular and functional heterogeneity in cultured hBMSC. ALP can be employed to identify osteoblastic and adipocytic progenitor cells in the heterogeneous hBMSC cultures.

Effects of Fibronectin Coating on Bacterial and Osteoblast Progenitor Cells Adherence in a Co-culture Assay.

Bacterial adherence to the surface of implants functionalized with cell-adhesive biomolecules is a critical first step of infection development. This study was designed to determine how the immobilization of human plasmatic fibronectin (pFN) could impact bacterial and osteoblast cells interaction with the surface during concomitant exposition to the two cell-types. Calibrated suspensions of P. aeruginosa PAOI or S. aureus CIP4.83 bacteria and STRO-1+A osteoblast progenitor cells were mixed, co-seeded on glass coverslips coated or not with pFN and incubated at 37 °C. After 3 h of co-culture, the presence of bacteria did not modify the STRO-1+A cells adherence to glass. pFN coating significantly enhanced STRO-1+A cells, CIP4.83 and PAOI adherence to glass and bacterial interaction with STRO-1+A cells. Confocal laser scanning microscopy observations revealed that cells on the pFN-coated substrate exhibited a greater spreading, better organized network of cytoskeletal filaments, and an increased cellular FN expression than cells on the uncoated substrate. The use of fluorescently labeled pFN showed that adherent STRO-1+A cells were able to remodel and to concentrate coated pFN at the cells surface. Thus, the use of FN coating could increase the risk of bacterial adherence to the material surface, acting either directly onto the coating layer or indirectly on adherent osteoblastic cells. This may increase the infection risk in the presence of bacterial contamination.

Cell autonomous roles of Nedd4 in craniofacial bone formation

Nedd4 is an E3 ubiquitin ligase that has an essential role in craniofacial development. However, how and when Nedd4 controls skull formation is ill defined. Here we have used a collection of complementary genetic mouse models to dissect the cell-autonomous roles of Nedd4 in the formation of neural crest cell derived cranial bone. Removal of Nedd4 specifically from neural crest cells leads to profound craniofacial defects with marked reduction of cranial bone that was preceded by hypoplasia of bone forming osteoblasts. Removal of Nedd4 after differentiation of neural crest cells into progenitors of chondrocytes and osteoblasts also led to profound deficiency of craniofacial bone in the absence of cartilage defects. Notably, these skull malformations were conserved when Nedd4 was specifically removed from the osteoblast lineage after specification of osteoblast precursors from mesenchymal skeletal progenitors. We further show that absence of Nedd4 in pre-osteoblasts results in decreased cell proliferation and altered osteogenic differentiation. Taken together our data demonstrate a novel cell-autonomous role for Nedd4 in promoting expansion of the osteoblast progenitor pool to control craniofacial development. Nedd4 mutant mice therefore represent a unique mouse model of craniofacial anomalies that provide an ideal resource to explore the cell-intrinsic mechanisms of neural crest cells in craniofacial morphogenesis.

The Role of Adipocyte Lineage Cells in Myeloma Growth and Dissemination in Bone

Background: Multiple myeloma (MM) is hematologic malignancy of plasma cells that thrives in and progresses throughout the bone marrow microenvironment. The bone marrow is host to a variety of cell types, including bone marrow stromal cells and hematopoietic cells, as well as osteoblasts, osteoclasts and adipocytes. We and others have shown that MM cells not only alter the local bone microenvironment to support MM progression, but also modify distant bone sites through secretion of soluble factors before arrival of tumor cells. One critical alteration in bone is the shift of osteoblast progenitor cells from osteoblastogenesis towards adipogenesis. Whether and how these increased adipocyte lineage cells contribute to MM cells homing to and growth in bone are currently unknown. Both mature and pre-adipocytes have multiple endocrine functions such as cytokine and growth factor secretion. Thus, an increase in adipocyte lineage cells likely alters the bone microenvironment in favor of supporting MM. Here, we hypothesized that adipocytes and their precursors play an active role in MM progression that contributes to MM growth and dissemination throughout bone.Methods: Our hypothesis was tested using a co-culture system in which 3T3-L1 mouse pre-adipocytes or mature adipocytes were separated by a porous membrane from 5TGM1-luc mouse MM cells. This system allowed cross-talk by secreted molecules but not through direct cell-cell contact. After three days of co-culture, MM cells were collected for (i) intravenous (I.V.) tail-vein injections into syngeneic C57Bl/KaLwRiJ mice or (ii) protein collection for Western blot analyses. For in vivo experiments, tumor progression was tracked by bioluminescent luciferase imaging and total tumor burden was evaluated by IgG2bκ (a soluble marker of 5TGM1-luc MM cells) levels in mouse serum. In addition, conditioned medium (CM) was collected from either pre-adipocytes or mature adipocytes for MM cell migration assays or for analyses of soluble factors in the CM by cytokine/chemokine array.Results: I.V. injection of 5TGM1-luc MM cells into mice revealed that those previously co-cultured with pre-adipocytes more rapidly homed to bone and grew larger tumors compared to 5TGM1-luc MM cells cultured alone, whereas the MM cells cultured with mature adipocytes showed no significant increase in either bone homing or growth. Analysis of pre-adipocyte and mature adipocyte CM by cytokine/chemokine arrays demonstrated that pre-adipocytes secrete significantly more HGF, MCP-1, OPN and SDF-1α compared to mature adipocytes. Migration assays in which pre-adipocyte or mature adipocyte CM was used as a chemoattractant indicated that MM cells migrate significantly more towards both pre-adipocyte and mature adipocyte CM than fresh media. However, the pre-adipocyte CM exhibited significantly more chemoattraction than mature adipocyte CM. Furthermore, addition of an MCP-1 or SDF-1α neutralizing antibody to both pre-adipocyte CM and mature adipocyte CM resulted in significantly reduced migration of MM cells. However, pre-adipocyte CM required higher concentrations of antibodies than mature adipocyte CM, indicating a higher concentration of MCP-1 and SDF-1α in pre-adipocyte CM. MM cells also exhibited a significant dose-dependent migration towards recombinant pre-adipocyte factor-1 (Pref-1), a marker of pre-adipocytes that is down-regulated during adipogenesis. Finally, Western blots revealed that co-culture of MM cells with pre-adipocytes resulted in activation of β-catenin signaling, which is important for cell proliferation, survival and motility, in MM cells.Conclusions: These data indicate that adipocyte lineage cells play active but differentiation-dependent roles in MM progression, likely via the secretion of soluble factors. Both pre-adipocytes and mature adipocytes directly attract MM cells by secreting chemoattractants such as MCP-1 and SDF-1α. Interestingly, our data identify pre-adipocytes, and not mature adipocytes, as the main driver of the aggressive bone phenotype of MM cells. In sum, these data suggest that an increase in adipocyte lineage cells in the bone marrow at distant bone sites could feed back to MM cells and support MM dissemination to these distant bone sites. Studies to determine the intricacies of this novel role of pre-adipocytes in MM are currently ongoing. DisclosuresSuva:University of Arkansas for Medical Sciences: Employment.

Myeloma impairs mature osteoblast function but causes early expansion of osteo-progenitors: temporal changes in bone physiology and gene expression in the KMS12BM model.

Myeloma bone disease results from an uncoupling of osteoclastic resorption and osteoblastic bone formation, but early changes in osteogenic function remain poorly defined. We used the KMS12BM xenograft model to investigate cellular and molecular events at early and late stages of disease. Lytic lesions and changes in osteoblast and osteoclast numbers occur late (8 weeks), however, micro-computed tomography of femora revealed significant reduction in bone volume at earlier disease stages (3 weeks) when tumour burden is low. Calcein labelling demonstrated reduced mineralization and bone formation at 3 weeks, suggesting functional impairment despite preserved osteoblast numbers. Osteo-progenitors from compact bone increased early (1 week), but fell at 3 weeks and were profoundly suppressed by 8 weeks. Exposure of osteoblast progenitors to multiple myeloma (MM) cells in vitro induced cell cycling, suggesting a mechanistic basis for early expansion of osteo-progenitors. We observed temporal changes in chemokine, osteogenic and osteoclastogenic genes in the stromal compartment. Notably, an early rise in CCL3 may underlie functional changes in mature osteoblasts at 3 weeks. Our data indicate that MM has distinct effects on mature osteoblasts and immature osteo-progenitors. Our findings argue for early clinical intervention to prevent bone changes that ultimately lead to the development of osteolytic disease.

Estrogen and estrogen receptor alpha promotes malignancy and osteoblastic tumorigenesis in prostate cancer.

The role of estrogen signaling in regulating prostate tumorigenesis is relatively underexplored. Although, an increasing body of evidence has linked estrogen receptor beta (ERß) to prostate cancer, the function of estrogen receptor alpha (ERα) in prostate cancer is not very well studied. We have discovered a novel role of ERα in the pathogenesis of prostate tumors. Here, we show that prostate cancer cells express ERα and estrogen induces oncogenic properties in prostate cancer cells through ERα. Importantly, ERα knockdown in the human prostate cancer PacMetUT1 cells as well as pharmacological inhibition of ERα with ICI 182,780 inhibited osteoblastic lesion formation and lung metastasis in vivo. Co-culture of pre-osteoblasts with cancer cells showed a significant induction of osteogenic markers in the pre-osteoblasts, which was attenuated by knockdown of ERα in cancer cells suggesting that estrogen/ERα signaling promotes crosstalk between cancer and osteoblastic progenitors to stimulate osteoblastic tumorigenesis. These results suggest that ERα expression in prostate cancer cells is essential for osteoblastic lesion formation and lung metastasis. Thus, inhibition of ERα signaling in prostate cancer cells may be a novel therapeutic strategy to inhibit the osteoblastic lesion development as well as lung metastasis in patients with advanced prostate cancer.

Multicolor flow cytometry-based cellular phenotyping identifies osteoprogenitors and inflammatory cells in the osteoarthritic subchondral bone marrow compartment

The cellular component of subchondral bone is thought to be responsible for aberrant bone remodeling in osteoarthritis (OA). Direct phenotypical analysis of the cellular compartment is critical to better understand the OA disease process. This study provides proof-of-principle for flow cytometry-based phenotyping of isolated subchondral trabecular bone (STB) marrow cells without prior use of cell culture techniques. Tibial plateaus were obtained from OA patients undergoing total knee arthroplasty. Subchondral bone chips were digested with collagenase IA and single cell suspensions were directly phenotyped using flow cytometry. Cells were analyzed for the expression of alkaline phosphatase (ALP) as osteoblast/osteoprogenitor marker and monocyte/macrophage markers (CD14, CD68, HLA-DR, CD115). MTT staining revealed abundant viable cells in the bone marrow compartment of STB prior to digestion, which were efficiently released by collagenase. Within the CD45-negative cell fraction, approximately 20% of the cells were positive for the early osteoblast/osteoprogenitor marker ALP. Within the CD45+ hematopoietic cell fraction, the majority of cells were of monocytic origin (>80%) displaying strong surface expression of CD14. Discreet macrophage populations (CD14+/HLA-DR+/CD68+) and putative osteoclast progenitors (CD45+/HLA-DR-/CD115+) were unequivocally identified. Osteoblast, macrophage and osteoclast progenitor presence in the subchondral bone unit (SBU) was confirmed by (immuno)histochemical staining for osteocalcin, CD68 and tartrate-resistant acid phosphatase, respectively. Flow cytometric analysis is a valuable methodology to study the cellular compartment of STB marrow. This method provides a proof of principle that the whole resident cell population can be directly phenotypically characterized without the prior use of cell culture techniques.

Fibroblast growth factors 1 and 2 inhibit adipogenesis of human bone marrow stromal cells in 3D collagen gels

Multipotent human bone marrow stromal cells (hBMSCs) are the common progenitors of osteoblasts and adipocytes. A shift in hBMSC differentiation in favor of adipogenesis may contribute to the bone loss and marrow fat accumulation observed in aging and osteoporosis. Hence, the identification of factors modulating marrow adipogenesis is of great therapeutic interest. Fibroblast growth factors 1 (FGF1) and 2 (FGF2) play important roles in several cellular processes including differentiation. Their role in adipogenesis is, however, still unclear given the contradictory reports found in the literature. In this work, we investigated the effect of FGF signaling on hBMSC adipogenesis in a 3D collagen gel system to mimic the natural microenvironment. We successfully established adipogenic differentiation of hBMSC embedded in type I collagen gels. We found that exogenous FGF1 and FGF2 exerted an inhibitory effect on lipid droplet accumulation and gene expression of adipogenic markers, which was abolished by pharmacological blocking of FGF receptor (FGFR) signaling. FGF treatment also affected the expression of the matrix metalloproteinase 13 (MMP13) and the tissue inhibitor of metalloproteinases 1 (TIMP1), altering the MMP/TIMP balance, which modulates collagen processing and turnover. FGF1- and FGF2-mediated inhibition of differentiation was, however, not restricted to adipogenesis since FGF1 and FGF2 treatment also resulted in the inhibition of the osteogenic differentiation in collagen gels. We conclude that FGFR signaling inhibits the in vitro adipogenic commitment of hBMSCs, downregulating core differentiation markers and altering ECM composition.

Age-Related Changes in FGF-2, Fibroblast Growth Factor Receptors and β-Catenin Expression in Human Mesenchyme-Derived Progenitor Cells

FGF-2 stimulates preosteoblast replication, and knockout of the FGF-2 gene in mice resulted in osteopenia with age, associated with decreased Wnt-β-Catenin signaling. In addition, targeted expression of FGF-2 in osteoblast progenitors increased bone mass in mice via Wnt-β-Catenin signaling. We posited that diminution of the intrinsic proliferative capacity of human mesenchyme-derived progenitor cells (HMDPCs) with age is due in part to reduction in FGF-2. To test this hypothesis HMDPCs from young (27-38), middle aged (47-56), and old (65-76) female human subjects were isolated from bone discarded after orthopedic procedures. HMDPCs cultures were mostly homogeneous with greater than 90% mesenchymal progenitor cells, determined by fluorescence-activated cell sorting. There was a progressive decrease in FGF-2 and FGFR1 mRNA and protein in HMDPCs with age. Since FGF-2 activates β-catenin, which can enhance bone formation, we also assessed its age-related expression in HMDPCs. An age-related decrease in total-β-Catenin mRNA and protein expression was observed. However there were increased levels of p-β-Catenin and decreased levels of activated-β-Catenin in old HMDSCs. FGF-2 treatment increased FGFR1 and β-Catenin protein, reduced the level of p-β-Catenin and increased activated-β-Catenin in aged HMDPCs. In conclusion, reduction in FGF-2 expression could contribute to age-related impaired function of HMDPCs via modulation of Wnt-β-catenin signaling.

Editorial Introduction: Epigenomics and Prevalent Skeletal Disorders.

A common definition of epigenetics refers to the mechanisms that modify gene expression in a stable manner without changing DNA sequence. They include the methylation of cytosines, noncoding RNAs, and chromatin modifications. Epigenetic mechanisms are also understood as mediators of the interaction between environmental factors and the genetic background. Thus, epigenetics is likely to play an important role in the pathogenesis of the so called complex disorders, which result from the interaction of acquired and hereditary factors. Osteoporosis and osteoarthritis are the most prevalent complex disorders of the skeleton. Osteoporosis is characterized by a decrease in bone mass that results from an imbalance between the activity of the bone forming osteoblasts and the bone resorbing osteoclasts. On the other hand, cartilage degeneration is the hallmark of osteoarthritis. Secondary changes in the adjacent bone also contribute to the disease. These disorders represent an important burden in men and women over 50 years of age. This thematic issue includes a number of reviews about the role of epigenetic mechanisms in the biology of bone and cartilage cells and in the pathogenesis of osteoarthritis and osteoporosis. The reviews focus particularly on DNA methylation and microRNAs. Genetic and environmental factors determine the peak bone mass attained by the third decade of life, which is, in turn, a critical factor determining the risk of osteoporosis later in life. In line with this, in the first paper Wood et al. review the role of developmental factors in osteoporosis, and specifically the influence of the intrauterine environment. Then, Papaioannou focuses on the role of microRNAs in the development of the skeleton. Mesenchymal stem cells are the common progenitors of adypocytes, muscle cells, chondrocytes and osteoblasts. Therefore, an adequate pool of mesenshymal stem cells is critical to maintain the pool of skeletal forming cells. Perez-Campo et al. review how epigenetic mechanisms modulate the proliferation and differentiation of these important cells. In the next article, Reppe et al. focus on the involvement of epigenetic mechanisms, and particularly DNA methylation, in the regulation of the activity of differentiated bone cells. Several microRNAs appear to be involved in the homeostasis of cartilage. Therefore, they are likely important in the pathogenesis of osteoarthritis. This subject is reviewed by Li et al. Several studies in the last decade have shown that aging is associated with changes in the level of DNA methylation. In fact, with aging there is a trend for a decrease in cytosine methylation. However this phenomenon is not uniform throughout the genome. The role of the changes in DNA methylation in age-related disorders is reviewed by Sierra et al. In the next paper, den Hollander and Meulenbelt focus on the specific role of changes in DNA methylation in the pathogenesis of osteoarthritis. Then, I provide a brief overview of the current evidence about the involvement of epigenetic mechanisms in osteoporosis. Also, a framework to critically appraise studies of epigenetic epidemiology is proposed. The differences between genetic and epigenetic studies are highlighted. Finally, Nakasa et al. address the potential role of manipulating microRNA signals to enhance bone formation. They specifically consider the therapeutic tool when bone regeneration is needed. Overall, this thematic issue provides an update of the role of epigenetic mechanisms in skeletal biology and pathophysiology. New data in this emerging field are deepening our understanding the pathophysiology of complex disorders and in future years may lead to the discovery of new therapeutic targets for prevalent skeletal diseases.