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BM adipocytes (BMAd) are a unique cell population derived from BM mesenchymal progenitors and marrow adipogenic lineage precursors. Although they have long been considered to be a space filler within bone cavities, recent studies have revealed important physiological roles in hematopoiesis and bone metabolism. To date, the approaches used to study BMAd function have been confounded by contributions by nonmarrow adipocytes or by BM stromal cells. To address this gap in the field, we have developed a BMAd-specific Cre mouse model to deplete BMAds by expression of diphtheria toxin A (DTA) or by deletion of peroxisome proliferator-activated receptor gamma (Pparg). We found that DTA-induced loss of BMAds results in decreased hematopoietic stem and progenitor cell numbers and increased bone mass in BMAd-enriched locations, including the distal tibiae and caudal vertebrae. Elevated bone mass appears to be secondary to enhanced endosteal bone formation, suggesting a local effect caused by depletion of BMAd. Augmented bone formation with BMAd depletion protects mice from bone loss induced by caloric restriction or ovariectomy, and it facilitates the bone-healing process after fracture. Finally, ablation of Pparg also reduces BMAd numbers and largely recapitulates high-bone mass phenotypes observed with DTA-induced BMAd depletion.

The implantation of non-biological materials, including scaffolds for tissue engineering, ubiquitously leads to a foreign body response (FBR). We recently reported that this response negatively impacts fibroblasts encapsulated within a synthetic hydrogel and in turn leads to a more severe FBR, suggesting a cross-talk between encapsulated cells and inflammatory cells. Given the promise of mesenchymal stem cells (MSCs) in tissue engineering and recent evidence of their immunomodulatory properties, we hypothesized that MSCs encapsulated within poly(ethylene glycol) (PEG) hydrogels will attenuate the FBR. In vitro, murine MSCs encapsulated within PEG hydrogels attenuated classically activated primary murine macrophages by reducing gene expression and protein secretion of pro-inflammatory cytokines, most notably tumor necrosis factor-α. Using a COX2 inhibitor, prostaglandin E2 (PGE2) was identified as a mediator of MSC immunomodulation of macrophages. In vivo, hydrogels laden with MSCs, osteogenically differentiating MSCs, or no cells were implanted subcutaneously into C57BL/6 mice for 28 days to assess the impact of MSCs on the fibrotic response of the FBR. The presence of encapsulated MSCs reduced fibrous capsule thickness compared to acellular hydrogels, but this effect diminished with osteogenic differentiation. The use of MSCs prior to differentiation in tissue engineering may therefore serve as a dynamic approach, through continuous cross-talk between MSCs and the inflammatory cells, to modulate macrophage activation and attenuate the FBR to implanted synthetic scaffolds thus improving the long-term tissue engineering outcome., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Objectives: Morbidity associated with geriatric fractures may be attributed, in part, to compromised mesenchymal stem cell (MSC) function within the fracture callus. The Notch signaling pathway is important for the healing of nonskeletal tissues in an age-dependent manner, but the effect of Notch on age-dependent fracture healing and MSC dysfunction has not been substantiated. The objective of this study was to examine Notch signaling in MSCs obtained from young and geriatric mice., Methods: Marrow-derived MSCs were harvested from the femora of 5- and 25-month-old C57BL/6 mice. We assessed in vivo MSC number using CFU-F, proliferation using an Alamar Blue assay, osteoblast differentiation by Alizarin Red S staining, and adipogenic differentiation using Oil Red O staining. Notch receptor and ligand expression was assessed using quantitative PCR, and Notch signaling was assessed by evaluating Notch target gene expression (Hey and HES) under basal conditions and when cells were plated to Jagged-1 ligand., Results: MSC from geriatric mice exhibit reduced MSC number (CFU-F), proliferation, adipogenesis, and inconsistent osteogenesis. The highest expressed Notch receptor is Notch 2, and the highest expressed ligand is Jagged-1, but there were no differences in ligand and receptor gene expression between young and old MSCs. Interestingly, geriatric MSCs show decreased basal Notch signaling activity but are fully responsive to Jagged-1 stimulation., Conclusions: These data suggest that therapeutic targeting of Notch signaling should be explored in clinical therapies to improve geriatric fracture healing.

Nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) is an essential transcription factor for adipocyte differentiation. In mesenchymal stem cells, PPARγ has been assumed to play a negative role in osteoblastic differentiation, by working in an adipogenesis dependent manner, due to the reciprocal relationship between osteoblast and adipocyte differentiation. However, the direct role of PPARγ in osteoblast function is not fully understood, due in part to inadequate model systems. Here, we describe an adenoviral-mediated PPARγ knockout system in which suppression of PPARγ in mesenchymal stem cells enhanced osteoblast differentiation and inhibited adipogenesis in vitro. Consistent with this in vitro observation, lipoatrophic A-ZIP/F1 mice, which do not form adipocytes, displayed a phenotype in which both cortical and trabecular bone was significantly increased compared with wild-type mice. We next developed an inducible osteoblast-targeted PPARγ knockout (Osx Cre/flox- PPARγ) mouse to determine the direct role of PPARγ in bone formation. Data from both in vitro cultures of mesenchymal stem cells and in vivo µCT analysis of bones suggest that suppression of PPARγ activity in osteoblasts significantly increased osteoblast differentiation and trabecular number. Endogenous PPARγ in mesenchymal stem cells and osteoblasts strongly inhibited Akt/mammalian target of rapamycin (mTOR)/p70S6k activity and led to decreased osteoblastic differentiation. Therefore, we conclude that PPARγ modulates osteoblast differentiation and bone formation through both direct and indirect mechanisms. The direct mode, as shown here, involves PPARγ regulation of the mTOR pathway, while the indirect pathway is dependent on the regulation of adipogenesis., (© AlphaMed Press.)

It is often difficult to decouple the relative importance of different factors in regulating MSC differentiation. Genetically modified mice provide model systems whereby some variables can be manipulated while others are kept constant. Fracture repair in thrombospondin-2 (TSP2)-null mice is characterized by reduced endochondral ossification and enhanced intramembranous bone formation. The proposed mechanism for this shift in MSC fate is that increased vascular density and hence oxygen availability in TSP2-null mice regulates differentiation. However, TSP2 is multifunctional and regulates other aspects of the regenerative cascade, such as MSC proliferation. The objective of this study is to use a previously developed computational model of tissue differentiation, in which substrate stiffness and oxygen tension regulate stem cell differentiation, to simulate potential mechanisms which may drive alterations in MSC fate in TSP2-null mice. Four models (increased cell proliferation, increased numbers of MSCs in the marrow decreased cellular oxygen consumption, and an initially stiffer callus) were not predictive of experimental observations in TSP2-null mice. In contrast, increasing the rate of angiogenic progression led to a prediction of greater intramembranous ossification, diminished endochondral ossification, and a reduced region of hypoxia in the fracture callus similar to that quantified experimentally by the immunohistochemical detection of pimonidazole adducts that develop with hypoxia. This study therefore provides further support for the hypothesis that oxygen availability during early fracture healing is a key regulator of MSC bipotential differentiation, and furthermore, it highlights the advantages of integrating computational models with genetically modified mouse studies for further elucidating mechanisms regulating stem cell fate., (Copyright © 2013 Orthopaedic Research Society.)

JAG1, the gene for the Jagged-1 ligand (Jag1) in the Notch signaling pathway, is variably mutated in Alagille Syndrome (ALGS). ALGS patients have skeletal defects, and additionally JAG1 has been shown to be associated with low bone mass through genome-wide association studies. Plating human osteoblast precursors (human mesenchymal stem cells-hMSCs) on Jag1 is sufficient to induce osteoblast differentiation; however, exposure of mouse MSC (mMSC) to Jag1 actually inhibits osteoblastogenesis. Overexpression of the notch-2 intracellular domain (NICD2) is sufficient to mimic the effect of Jag1 on hMSC osteoblastogenesis, while blocking Notch signaling with a γ-secretase inhibitor or with dominant-negative mastermind inhibits Jag1-induced hMSC osteoblastogenesis. In pursuit of interacting signaling pathways, we discovered that treatment with a protein kinase C δ (PKCδ) inhibitor abrogates Jag1-induced hMSC osteoblastogenesis. Jag1 results in rapid PKCδ nuclear translocation and kinase activation. Furthermore, Jag1 stimulates the physical interaction of PKCδ with NICD. Collectively, these results suggest that Jag1 induces hMSC osteoblast differentiation through canonical Notch signaling and requires concomitant PKCδ signaling. This research also demonstrates potential deficiencies in using mouse models to study ALGS bone abnormalities., (Copyright © 2013 AlphaMed Press.)

Age-related osteoporosis is characterized by a decrease in bone-forming capacity mediated by defects in the number and function of osteoblasts. An important cellular mechanism that may in part explain osteoblast dysfunction that occurs with aging is senescence of mesenchymal progenitor cells (MPCs). In the telomere-based Wrn(-/-) Terc(-/-) model of accelerated aging, the osteoporotic phenotype of these mice is also associated with a major decline in MPC differentiation into osteoblasts. To investigate the role of MPC aging as a cell-autonomous mechanism in senile bone loss, transplantation of young wild-type whole bone marrow into Wrn(-/-) Terc(-/-) mutants was performed and the ability of engrafted cells to differentiate into cells of the osteoblast lineage was assessed. We found that whole bone marrow transplantation in Wrn(-/-) Terc(-/-) mice resulted in functional engraftment of MPCs up to 42 weeks, which was accompanied by a survival advantage as well as delays in microarchitectural features of skeletal aging., (Copyright © 2012 AlphaMed Press.)

The transcription factor osterix (Sp7) is essential for osteoblastogenesis and bone formation in mice. Genome wide association studies have demonstrated that osterix is associated with bone mineral density in humans; however, the molecular significance of osterix in human osteoblast differentiation is poorly described. In this study we have characterized the role of osterix in human mesenchymal progenitor cell (hMSC) differentiation. We first analyzed temporal microarray data of primary hMSC treated with bone morphogenetic protein-6 (BMP6) using clustering to identify genes that are associated with osterix expression. Osterix clusters with a set of osteoblast-associated extracellular matrix (ECM) genes, including bone sialoprotein (BSP) and a novel set of proteoglycans, osteomodulin (OMD), osteoglycin, and asporin. Maximum expression of these genes is dependent upon both the concentration and duration of BMP6 exposure. Next we overexpressed and repressed osterix in primary hMSC using retrovirus. The enforced expression of osterix had relatively minor effects on osteoblastic gene expression independent of exogenous BMP6. However, in the presence of BMP6, osterix overexpression enhanced expression of the aforementioned ECM genes. Additionally, osterix overexpression enhanced BMP6 induced osteoblast mineralization, while inhibiting hMSC proliferation. Conversely, osterix knockdown maintained hMSC in an immature state by decreasing expression of these ECM genes and decreasing mineralization and hMSC proliferation. Overexpression of the osterix regulated gene OMD with retrovirus promoted mineralization of hMSC. These results suggest that osterix is necessary, but not sufficient for hMSC osteoblast differentiation. Osterix regulates the expression of a set of ECM proteins which are involved in terminal osteoblast differentiation., (Copyright © 2011 Wiley Periodicals, Inc.)

Clinical trials utilizing bone marrow-derived mesenchymal stem cell (BM-MSC) therapies show promise for treating a variety of pathologic conditions. Paramount to optimization of such cell-based therapies is a thorough understanding of MSC biology. Despite the tremendous potential that exists for the clinical use of canine BM-MSCs in veterinary medicine, as well as in preclinical studies for human medicine, relatively little information exists regarding basic biological properties of the cells. In this study, we compared the importance of donor characteristics (age and harvest site) and ex vivo expansion on canine BM-MSC frequency (CFU-f) and differentiation potential. Advancing age was found to have a negative effect on CFU-f as well as osteogenic potential. Site of harvest was also found to have significant effects on MSC properties. MSCs obtained from the humerus were found at the lowest frequency and were least osteogenic compared to those harvested from the tibia, femur, and ilium. Osteogenic potential diminished significantly by the third passage. These results suggest important donor parameters and culture effects to consider in translational studies examining MSC-based regenerative medical strategies.

Objective: To evaluate effects of apheresis on mesenchymal stem cells (MSCs) and compare those MSCs with MSCs obtained from adipose tissue or bone marrow (BM)., Sample Population: Samples obtained from 6 adult horses., Procedures: Samples of blood from a peripheral vein, adipose tissue, and BM aspirate were obtained from each horse. Samples were processed via apheresis of blood and techniques reported elsewhere for adipose tissue and BM. Cultures were maintained until adherence and subsequently were subjected to differentiation protocols to evaluate adipogenic, osteoblastogenic, and chondrogenic potential., Results: Apheresis product had a significantly higher mononuclear percentage, higher platelet count, and lower RBC count, compared with values for peripheral blood. No cell adherence to the tissue culture plates was detected for the apheresis product. Adherence was detected for 6 of 6 adipose-derived and 4 of 6 BM-derived samples. Variations in efficiency were detected for differentiation of adipose- and BM-derived cells into adipocytes, chondrocytes, and osteoblasts., Conclusions and Clinical Relevance: Apheresis was able to concentrate mononuclear cells and reduce RBC contamination. However, the apheresis product was unable to adhere to the tissue culture plates. In matched horses, adipose- and BM-derived MSCs were capable of producing lipids, glycosaminoglycan, and mineral. The BM was vastly superior to adipose tissue as a source of MSCs with osteoblastogenic potential in matched horses. Additional studies will be necessary to optimize apheresis techniques for horses before peripheral blood can be considered a suitable source for multipotential cells for use in cell-based treatments.

Leptin functions through a well-documented central neuroendocrine pathway to regulate bone mass. However, the ability of leptin to modulate bone mass through a peripheral mechanism has been debated due to conflicting in vitro results and lack of sufficient in vivo models. We utilized mice with LoxP sites introduced into the long-form leptin receptor (ObRb) gene to determine how leptin regulates mesenchymal progenitor cell (MPC) differentiation and osteoblast function in vitro and in vivo. Rapid phosphorylation of Stat3 after leptin treatment of bone marrow stromal cells (BMSCs) from mice with conditional deletion of ObRb in macrophages (LysM(Cre+F/F)) confirmed expression of functional leptin receptors by BMSCs. Adenovirus-Cre mediated disruption of ObRb in primary stromal cells decreased mineralization and increased adipogenesis. In contrast, BMSCs harvested from leptin-signaling deficient Ob/Ob or Db/Db mice showed increased mineralization. To determine the physiologic relevance of these differences, mice with cell-specific deletion of ObRb in mesenchymal precursors (3.6(Cre+F/F)) or osteoblasts (2.3(Cre+F/F)) were generated. Although the 2.3(Cre+F/F) mice were grossly normal, the 3.6(Cre+F/F) mice displayed mild obesity that was not attributed to food intake. Femurs of 3.6(Cre+F/F) animals showed a 58%-61.9% increase in trabecular bone volume and a 65.5%-74% increase in bone mineral density. Cortical volume and mineral content were also increased 18%-22%. Primary 3.6(Cre+F/F) BMSCs recapitulated the high mineralization phenotype of Ob/Ob and Db/Db BMSCs. We conclude that leptin may have multiple peripheral roles depending on the differentiation state of MPC. Leptin (a) helps maintain MPCs in an undifferentiated state and (b) promotes mineralization of more differentiated osteoblasts.

Differentiation of marrow-derived mesenchymal progenitors to either the osteoblast or adipocyte lineage is reciprocally regulated. Factors that promote osteoblastogenesis inhibit adipogenesis, while adipogenic factors are inhibitory to osteoblast differentiation. Heparin, a soluble glycosaminoglycan, inhibits bone formation in vivo and osteoblast cell differentiation and function in vitro, and has been shown to promote adipocyte differentiation. To elucidate the role that heparin plays in the adipogenic induction of murine mesenchymal progenitors, we studied immortalized marrow stromal cells (IM-MSC), the MSC cell line, ST2, and 3T3L1 pre-adipocytes. Heparin alone was not sufficient to induce adipogenesis, but enhanced the induction under a variety of adipogenic cocktails. This effect was both dose- and time-dependent. Heparin showed a positive effect at concentrations > 0.1 microg/ml when applied before day 3 during the induction course. Heparin's effect on adipogenesis was independent of cell proliferation, cell density, and extracellular lipid. This effect is likely related to the unique structure of heparin because another polyanionic glycosaminoglycan, dextran sulfate, did not promote adipogenic differentiation. Heparin treatment altered morphology and adhesion characteristics of progenitor cells, resulting in cell rounding and aggregation. As well, heparin counteracted the known inhibitory effect of fibronectin on adipogenesis and decreased basal focal adhesion kinase and paxillin phosphorylation. We conclude that heparin-mediated disruption of cell-matrix adhesion enhances adipogenic potential.

Mesenchymal precursor cells have the potential to differentiate into several cell types, including adipocytes and osteoblasts. Activation of Wnt/beta-catenin signaling shifts mesenchymal cell fate toward osteoblastogenesis at the expense of adipogenesis; however, molecular mechanisms by which Wnt signaling alters mesenchymal cell fate have not been fully investigated. Our prior work indicates that multipotent precursors express adipogenic and osteoblastogenic transcription factors at physiological levels and that ectopic expression of Wnt10b in bipotential ST2 cells suppresses expression of CCAAT/enhancer-binding protein alpha (C/EBPalpha) and peroxisome proliferator-activated receptor gamma (PPARgamma) and increases expression of Runx2, Dlx5, and osterix. Here, we demonstrate that transient activation of Wnt/beta-catenin signaling rapidly suppresses C/EBPalpha and PPARgamma, followed by activation of osteoblastogenic transcription factors. Enforced expression of C/EBPalpha or PPARgamma partially rescues lipid accumulation and decreases mineralization in ST2 cells expressing Wnt10b, suggesting that suppression of C/EBPalpha and PPARgamma is required for Wnt/beta-catenin to alter cell fate. Furthermore, knocking down expression of C/EBPalpha, PPARgamma, or both greatly reduces adipogenic potential and causes spontaneous osteoblastogenesis in ST2 cells and mouse embryonic fibroblasts, suggesting that Wnt signaling alters the fate of mesenchymal precursor cells primarily by suppressing C/EBPalpha and PPARgamma.

Bone marrow-derived mesenchymal stem cells (MSC) are multipotent, self-renewing, mesodermal-origin stem cells that are sequestered in the endosteal compartment. MSC are maintained in a relative state of quiescence in vivo but in response to a variety of physiological and pathological stimuli, proliferate and differentiate into osteoblasts, chondrocytes, adipocytes, or hematopoiesis-supporting stromal cells. Little is understood regarding the cellular or molecular events underlying MSC fate decisions. We report that human MSC (hMSC) cultured in defined, serum-free conditions respond to a narrow spectrum of growth factors with osteogenic commitment, differentiation, and hydroxyapatite deposition. Of the osteogenic factors we examined, only treatment with bone morphogenetic protein (BMP) results in osteoinduction under defined serum-free conditions. Among BMP-2, 4, 6, and 7, BMP-6 is the most consistent and potent regulator of osteoblast differentiation and, of these BMPs, only BMP-6 gene expression is detected prior to hMSC osteoblast differentiation. Addition of exogenous BMP-6 to hMSC induces the expression or upregulation of a repertoire of osteoblast-related genes including type I collagen, osteocalcin, bone sialoprotein, and their regulatory transcription factors Cbfa1/Runx2, and Osterix. This translates into increased production of osteogenic extracellular matrix (ECM) with subsequent hydroxyapatite deposition., ((c) 2005 Wiley-Liss, Inc.)

Hexadecyl-4-[(18)F]fluorobenzoate ([(18)F]HFB), a long chain fluorinated benzoic acid ester, was prepared in a one-step synthesis by aromatic nucleophilic substitution of [(18)F]fluoride ion on hexadecyl-4-(N,N,N-trimethylammonio)benzoate. The radiolabeled ester was obtained in good yields (52% decay corrected) and high purity (97%). [(18)F]HFB was used to radiolabel rat mesenchymal stem cells (MSCs) by absorption into cell membranes. MicroPET imaging of [(18)F]HFB-labeled MSCs following intravenous injection into the rat showed the expected high and persistent accumulation of radioactivity in the lungs. [(18)F]HFB is thus simple to prepare and uses labeling agent for short-term distribution studies of injected stem cells.

Adult stem cells offer the potential to treat many diseases through a combination of ex vivo genetic manipulation and autologous transplantation. Mesenchymal stem cells (MSCs, also referred to as marrow stromal cells) are adult stem cells that can be isolated as proliferating, adherent cells from bones. MSCs can differentiate into multiple cell types present in several tissues, including bone, fat, cartilage, and muscle, making them ideal candidates for a variety of cell-based therapies. Here, we have used adeno-associated virus vectors to disrupt dominant-negative mutant COL1A1 collagen genes in MSCs from individuals with the brittle bone disorder osteogenesis imperfecta, demonstrating successful gene targeting in adult human stem cells.

The biology of bone healing is a rapidly developing science. Advances in transgenic and gene-targeted mice have enabled tissue and cell-specific investigations of skeletal regeneration. As an example, only recently has it been recognized that chondrocytes convert to osteoblasts during healing bone, and only several years prior, seminal publications reported definitively that the primary tissues contributing bone forming cells during regeneration were the periosteum and endosteum. While genetically modified animals offer incredible insights into the temporal and spatial importance of various gene products, the complexity and rapidity of healing-coupled with the heterogeneity of animal models-renders studies of regenerative biology challenging. Herein, cells that play a key role in bone healing will be reviewed and extracellular mediators regulating their behavior discussed. We will focus on recent studies that explore novel roles of inflammation in bone healing, and the origins and fates of various cells in the fracture environment. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Thrombospondins (TSPs) belong to a functional class of ECM proteins called matricellular proteins that are not primarily structural, but instead influence cellular interactions within the local extracellular environment. The 3D arrangement of TSPs allow interactions with other ECM proteins, sequestered growth factors, and cell surface receptors. They are expressed in mesenchymal condensations and limb buds during skeletal development, but they are not required for patterning. Instead, when absent, there are alterations in musculoskeletal connective tissue ECM structure, organization, and function, as well as altered skeletal cell phenotypes. Both functional redundancies and unique contributions to musculoskeletal tissue structure and physiology are revealed in mouse models with compound TSP deletions. Crucial roles of individual TSPs are revealed during musculoskeletal injury and regeneration. The interaction of TSPs with mesenchymal stem cells (MSC), and their influence on cell fate, function, and ultimately, musculoskeletal phenotype, suggest that TSPs play integral, but as yet poorly understood roles in musculoskeletal health. Here, unique and overlapping contributions of trimeric TSP1/2 and pentameric TSP3/4/5 to musculoskeletal cell and matrix physiology are reviewed. Opportunities for new research are also noted. [ABSTRACT FROM AUTHOR]

Age-related osteoporosis is characterized by a decrease in bone-forming capacity mediated by defects in the number and function of osteoblasts. An important cellular mechanism that may in part explain osteoblast dysfunction that occurs with aging is senescence of mesenchymal progenitor cells (MPCs). In the telomere-based Wrn−/−Terc−/− model of accelerated aging, the osteoporotic phenotype of these mice is also associated with a major decline in MPC differentiation into osteoblasts. To investigate the role of MPC aging as a cell-autonomous mechanism in senile bone loss, transplantation of young wild-type whole bone marrow into Wrn−/−Terc−/− mutants was performed and the ability of engrafted cells to differentiate into cells of the osteoblast lineage was assessed. We found that whole bone marrow transplantation in Wrn−/−Terc−/− mice resulted in functional engraftment of MPCs up to 42 weeks, which was accompanied by a survival advantage as well as delays in microarchitectural features of skeletal aging. S TEM C ELLS 2013;31:607-611 [ABSTRACT FROM AUTHOR]

The collagen 2.3 and 3.6 promoters have been used to drive Cre expression for generation of conditional transgenic mutant mice. Within the bone, Col3.6 is expressed by mesenchymal precursor cells and their downstream progeny, while Col2.3 is more osteoblast specific. Our generation of transgenic mice with Col2.3-Cre- and Col3.6-Cre-driven deletion of the long-form leptin receptor (ObRb) necessitated a thorough analysis of the nonspecific expression of these promoters in the central nervous system. Both Col2.3 and Col3.6 were capable of forcing loxP recombination in the brain as demonstrated by EGFP expression in ROSA reporter mice. Expression of Col2.3 was limited to the central base of the brain near the third ventricle. In contrast, robust expression of Col3.6 was noted throughout the brain, centering near the distal third ventricle, third ventricle, and aqueduct. We subsequently analyzed the colocalization of leptin-responsive P-Stat3 neurons with Col3.6-expressing neurons. Approximately 5-10% colocalization was noted in leptin-responsive brain areas such as the arcuate nucleus, dorsal medial hypothalamus, ventral premammillary nucleus, and lateral hypothalamus. Injection of 3.6Cre+F/F ObRb knockout mice with leptin confirmed the presence of an intact P-Stat3 response that was dampened in the lateral hypothalamus (p < 0.050). This test was done to explore the contribution of neural leptin signaling to the bone phenotype of the 3.6Cre+F/F mice. Our analysis indicates that neural ObRb deletion, while present, is likely not the sole driver of femoral changes through traditional sympathetic circuits. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Bones are constantly remodeled throughout life to maintain robust structure and function. Dysfunctional remodeling can result in pathological conditions such as osteoporosis (bone loss) or osteosclerosis (bone gain). Bone contains 100s of extracellular matrix (ECM) proteins and the ECM of the various bone tissue compartments plays essential roles directing the remodeling of bone through the coupled activity of osteoclasts (which resorb bone) and osteoblasts (which produce new bone). One important role for the ECM is to serve as a scaffold upon which mineral is deposited. This scaffold is primarily type I collagen, but other ECM components are involved in binding of mineral components. In addition to providing a mineral scaffolding role, the ECM components provide structural flexibility for a tissue that would otherwise be overly rigid. Although primarily secreted by osteoblast-lineage cells, the ECM regulates cells of both the osteoblast-lineage (such as progenitors, mature osteoblasts, and osteocytes) and osteoclast-lineage (including precursors and mature osteoclasts), and it also influences the cross-talk that occurs between these two oppositional cells. ECM influences the differentiation process of mesenchymal stem cells to become osteoblasts by both direct cell-ECM interactions as well as by modulating growth factor activity. Similarly, the ECM can influence the development of osteoclasts from undifferentiated macrophage precursor cells, and influence osteoclast function through direct osteoclast cell binding to matrix components. This comprehensive review will focus on how networks of ECM proteins function to regulate osteoclast- and osteoblast-mediated bone remodeling. The clinical significance of these networks on normal bone and as they relate to pathologies of bone mass and geometry will be considered. A better understanding of the dynamic role of ECM networks in regulating tissue function and cell behavior is essential for the development of new treatment approaches for bone loss. [ABSTRACT FROM AUTHOR]

Abstract: The matricellular protein thrombospondin-2 (TSP2) inhibits proliferation and enhances osteoblastogenesis of multipotent mesenchymal progenitor cells (MPC). Osteoblastogenesis and adipogenesis are reciprocally regulated, thus we hypothesized that TSP2 could be an inhibitor of adipogenesis. TSP2 gene expression is up-regulated during MPC osteoblast differentiation and down-regulated during adipogenic differentiation through a cAMP–PKA pathway, relative to control cells. Next, the importance of TSP2 in adipogenesis was studied by comparing gene-targeted knockout mice that lack TSP2 protein (TSP2-null) and control wild-type mice. TSP2-null marrow-derived MPC show 25% increased adipocytes. Similarly, TSP2-null adipose-derived MPC show increased adipocytes (25–50%) and proliferation (2-fold) relative to wild-type cells. However, the increase in TSP2-null adipocytes is not due to an increase in MPC number, because the percentage of adipocytes relative to total MPC is still significantly increased. Surprisingly, there are only minor alterations in the adipogenic transcriptional program (PPARγ and C/EBPα expression). Weight gain over time was evaluated in TSP2-null and control wild-type mice fed normal and high-fat diets. Female TSP2-null mice are 30% heavier than wild-type controls due to an increase in non-visceral adipose tissue, measured by dual-energy X-ray absorptiometry (DEXA) and direct weighing of fat pads. These results show that TSP2 is an endogenous matricellular protein produced by MPC that in addition to enhancing osteoblastogenesis, inhibits adipocytes and decreases subcutaneous adiposity. [Copyright &y& Elsevier]