Bone marrow adipose tissue composition and glycemic improvements after gastric bypass surgery.

Fracture risk is increased in type 2 diabetes, which may in part be due to altered bone marrow adiposity. Cross sectional studies have reported that people with type 2 diabetes have lower unsaturated BMAT lipid levels than people without diabetes, although there are limited data on longitudinal changes. We hypothesized that Roux-en-Y gastric bypass (RYGB), which dramatically improves glycemic status, would have differential effects on BMAT composition, with increases in the unsaturated lipid index in people with diabetes. Given reports that axial BMAT is responsive to metabolic stimuli while appendicular BMAT is stable, we hypothesized that BMAT changes would occur at the spine but not the tibia. We enrolled 30 obese women, stratified by diabetes status, and used magnetic resonance spectroscopy to measure BMAT at the spine in all participants, and the tibia in a subset (n = 19). At baseline, BMAT parameters were similar between those with and without diabetes, except tibial marrow fat content was lower in women with diabetes (97.4 % ± 1.0 % versus 98.2 % ± 0.4 %, p = 0.04). Six months after surgery, both groups experienced similar weight loss of 27 kg ± 7 kg. At the spine, there was a significant interaction between diabetes status and changes in both marrow fat content and the unsaturated lipid index (p = 0.02, p < 0.01 for differences, respectively). Women with diabetes had a trend towards a decline in marrow fat content (-4.3 % ± 8.2 %, p = 0.09) and increase in the unsaturated lipid index (+1.1 % ± 1.5 %, p = 0.02). In contrast, BMAT parameters did not significantly change in women without diabetes. In all women, changes in the unsaturated lipid index inversely correlated with hemoglobin A1c changes (r = -0.47, p = 0.02). At the tibia, there was little BMAT change by diabetes status. Our results suggest that vertebral BMAT composition is responsive to changes in glycemic control after RYGB.

Female-Specific Role of Progranulin to Suppress Bone Formation.

Progranulin (PGRN) is best known as a glial protein for which deficiency leads to the most common inherited form of frontotemporal dementia. Recently, PGRN has been found to be an adipokine associated with diet-induced obesity and insulin resistance. Therefore, PGRN may have homeostatic effects on bone because PGRN is reported to promote the differentiation of bone-resorbing osteoclasts. We investigated the actions of PGRN on bone using PGRN gene (Grn) knockout (KO) mice and transgenic mice with PGRN mutation and surprisingly found that loss of PGRN prevented the bone loss in female mice induced by aging and estrogen deficiency, whereas it had no effect on male bones during aging. Strikingly, bone formation was increased in female (but not male) PGRN KO mice. We also found that loss of PGRN inhibited bone resorption and osteoclastogenesis in both male and female mice and promoted the production of osteogenic factors in osteoclast lineage cells. These results indicate that PGRN serves to uncouple bone turnover in female mice by promoting bone resorption and suppressing bone formation. Furthermore, we demonstrated that microglial cells/macrophages, but not adipocytes, are an important source of PGRN in producing negative skeletal effects in females. Targeting PGRN production by microglial cells/macrophage-lineage cells may provide a therapeutic approach for the treatment of osteoporosis in females.

Estrogen signaling in arcuate Kiss1 neurons suppresses a sex-dependent female circuit promoting dense strong bones.

Central estrogen signaling coordinates energy expenditure, reproduction, and in concert with peripheral estrogen impacts skeletal homeostasis in females. Here, we ablate estrogen receptor alpha (ERα) in the medial basal hypothalamus and find a robust bone phenotype only in female mice that results in exceptionally strong trabecular and cortical bones, whose density surpasses other reported mouse models. Stereotaxic guided deletion of ERα in the arcuate nucleus increases bone mass in intact and ovariectomized females, confirming the central role of estrogen signaling in this sex-dependent bone phenotype. Loss of ERα in kisspeptin (Kiss1)-expressing cells is sufficient to recapitulate the bone phenotype, identifying Kiss1 neurons as a critical node in this powerful neuroskeletal circuit. We propose that this newly-identified female brain-to-bone pathway exists as a homeostatic regulator diverting calcium and energy stores from bone building when energetic demands are high. Our work reveals a previously unknown target for treatment of age-related bone disease.

Sirtuin-3 Promotes Adipogenesis, Osteoclastogenesis, and Bone Loss in Aging Male Mice.

Sirtuin-3 (Sirt3) is an essential metabolic regulatory enzyme that plays an important role in mitochondrial metabolism, but its role in bone marrow and skeletal homeostasis remains largely unknown. In this study, we hypothesize that increased expression of Sirt3 plays a role in skeletal aging. Using mice that overexpress Sirt3 [i.e., Sirt3 transgenic (Sirt3Tg)], we show that Sirt3 is a positive regulator of adipogenesis and osteoclastogenesis and a negative regulator of skeletal homeostasis. Sirt3Tg mice exhibited more adipocytes in the tibia compared with control mice. Bone marrow stromal cells (BMSCs) from Sirt3Tg mice displayed an enhanced ability to differentiate into adipocytes compared with control BMSCs. We found a 2.5-fold increase in the number of osteoclasts on the bone surface in Sirt3Tg mice compared with control mice (P < 0.03), and increased osteoclastogenesis in vitro. Importantly, Sirt3 activates the mechanistic target of rapamycin (mTOR) pathway to regulate osteoclastogenesis. Sirt3Tg male mice exhibited a significant reduction in cortical thickness at the tibiofibular junction (P < 0.05). In summary, Sirt3 activity in bone marrow cells is associated with increased adipogenesis, increased osteoclastogenesis through activation of mTOR signaling, and reduced bone mass. Interestingly, Sirt3 expression in bone marrow cells increases during aging, suggesting that Sirt3 promotes age-related adipogenesis and osteoclastogenesis associated with bone loss. These findings identify Sirt3 as an important regulator of adipogenesis and skeletal homeostasis in vivo and identify Sirt3 as a potential target for the treatment of osteoporosis.

Role of Osteoblast Gi Signaling in Age-Related Bone Loss in Female Mice.

Age-related bone loss is an important risk factor for fractures in the elderly; it results from an imbalance in bone remodeling mainly due to decreased bone formation. We have previously demonstrated that endogenous G protein-coupled receptor (GPCR)-driven Gi signaling in osteoblasts (Obs) restrains bone formation in mice during growth. Here, we launched a longitudinal study to test the hypothesis that Gi signaling in Obs restrains bone formation in aging mice, thereby promoting bone loss. Our approach was to block Gi signaling in maturing Obs by the induced expression of the catalytic subunit of pertussis toxin (PTX) after the achievement of peak bone mass. In contrast to the progressive cancellous bone loss seen in aging sex-matched littermate control mice, aging female Col1(2.3)+/PTX+ mice showed an age-related increase in bone volume. Increased bone volume was associated with increased bone formation at both trabecular and endocortical surfaces as well as increased bending strength of the femoral middiaphyses. In contrast, male Col1(2.3)+/PTX+ mice were not protected from age-related bone loss. Our results indicate that Gi signaling markedly restrains bone formation at cancellous and endosteal bone surfaces in female mice during aging. Blockade of the relevant Gi-coupled GPCRs represents an approach for the development of osteoporosis therapies-at least in the long bones of aging women.

Osteoblast-derived FGF9 regulates skeletal homeostasis.

FGF9 has complex and important roles in skeletal development and repair. We have previously observed that Fgf9 expression in osteoblasts (OBs) is regulated by G protein signaling and therefore the present study was done to determine whether OB-derived FGF9 was important in skeletal homeostasis. To directly test this idea, we deleted functional expression of Fgf9 gene in OBs using a 2.3kb collagen type I promoter-driven Cre transgenic mouse line (Fgf9OB-/-). Both Fgf9 knockout (Fgf9OB-/-) and the Fgf9 floxed littermates (Fgf9fl/fl) mice were fully backcrossed and maintained in an FBV/N background. Three month old Fgf9OB-/- mice displayed a significant decrease in cancellous bone and bone formation in the distal femur and a significant decrease in cortical thickness at the TFJ. Strikingly, female Fgf9OB-/- mice did not display altered bone mass. Continuous treatment of mouse BMSCs with exogenous FGF9 inhibited mouse BMSC mineralization while acute treatment increased the proliferation of progenitors, an effect requiring the activation of Akt1. Our results suggest that mature OBs are an important source of FGF9, positively regulating skeletal homeostasis in male mice. Osteoblast-derived FGF9 may serve a paracrine role to maintain the osteogenic progenitor cell population through activation of Akt signaling.

Investigating the cellular origin of rotator cuff muscle fatty infiltration and fibrosis after injury.

BACKGROUND: rotator cuff muscle atrophy, fibrosis and fatty infiltration are common complications after large and massive rotator cuff tears. Currently, there are no effective treatments for these muscle pathologies after injury. Furthermore, the cellular source for fibrotic and adipose tissues in rotator cuff muscle after injury remains unknown. In this study, we proposed that two groups of muscle resident progenitors, Tie2+ muscle mesenchymal progenitors and PDGFRα(+) fibro/adipogenic progenitor cells (FAPs), contribute significantly to rotator cuff muscle fibrosis and fatty infiltration. METHODS: we tested our hypothesis using reporter mice. Rotator cuff muscles from Tie2-GFP and PDGFRα-GFP reporter mice were harvested at 2 and 6 weeks after unilateral massive rotator cuff tear surgeries. Immunofluorescent staining for fibroblast and adipocyte markers was conducted. RESULTS: our results showed significant co-localization of Tie2+ cells with fibrotic markers vimentin and αSMA. In the PDGFRα-GFP reporter mice, GFP signal was seen in only a small fraction of cells staining positive for vimentin and αSMA. However, PDGFRα showed significant co-localization with adipocyte markers, including PPAR-γ, adiponectin, and perilipin A. Oil red O staining confirmed that the mature adipocytes appearing in rotator cuff muscles after injury are also PDGFRα(+). CONCLUSION: these data demonstrated that the Tie2(+) muscle mesenchymal progenitors are the major source of fibroblasts while PDGFRα(+) FAPs are the major source of adipocytes in rotator cuff muscle fatty infiltration. Basic Science Study.

Dietary dried plum increases bone mass, suppresses proinflammatory cytokines and promotes attainment of peak bone mass in male mice.

Nutrition is an important determinant of bone health and attainment of peak bone mass. Diets containing dried plum (DP) have been shown to increase bone volume and strength. These effects may be linked to the immune system and DP-specific polyphenols. To better understand these relationships, we studied DP in skeletally mature (6-month-old) and growing (1- and 2-month-old) C57Bl/6 male mice. In adult mice, DP rapidly (<2 weeks) increased bone volume (+32%) and trabecular thickness (+24%). These changes were associated with decreased osteoclast surface (Oc.S/BS) and decreased serum CTX, a marker of bone resorption. The reduction in Oc.S/BS was associated with a reduction in the osteoclast precursor pool. Osteoblast surface (Ob.S/BS) and bone formation rate were also decreased suggesting that the gain in bone in adult mice is a consequence of diminished bone resorption and formation, but resorption is reduced more than formation. The effects of DP on bone were accompanied by a decline in interleukins, TNF and MCP-1, suggesting that DP is acting in part through the immune system to suppress inflammatory activity and reduce the size of the osteoclast precursor pool. Feeding DP was accompanied by an increase in plasma phenolics, some of which have been shown to stimulate bone accrual. In growing and young adult mice DP at levels as low as 5% of diet (w/w) increased bone volume. At higher levels (DP 25%), bone volume was increased by as much as 94%. These data demonstrate that DP feeding dramatically increases peak bone mass during growth.

Negative Skeletal Effects of Locally Produced Adiponectin.

Epidemiological studies show that high circulating levels of adiponectin are associated with low bone mineral density. The effect of adiponectin on skeletal homeostasis, on osteoblasts in particular, remains controversial. We investigated this issue using mice with adipocyte-specific over-expression of adiponectin (AdTg). MicroCT and histomorphometric analysis revealed decreases (15%) in fractional bone volume in AdTg mice at the proximal tibia with no changes at the distal femur. Cortical bone thickness at mid-shafts of the tibia and at the tibiofibular junction was reduced (3-4%) in AdTg mice. Dynamic histomorphometry at the proximal tibia in AdTg mice revealed inhibition of bone formation. AdTg mice had increased numbers of adipocytes in close proximity to trabecular bone in the tibia, associated with increased adiponectin levels in tibial marrow. Treatment of BMSCs with adiponectin after initiation of osteoblastic differentiation resulted in reduced mineralized colony formation and reduced expression of mRNA of osteoblastic genes, osterix (70%), Runx2 (52%), alkaline phosphatase (72%), Col1 (74%), and osteocalcin (81%). Adiponectin treatment of differentiating osteoblasts increased expression of the osteoblast genes PPARγ (32%) and C/ebpα (55%) and increased adipocyte colony formation. These data suggest a model in which locally produced adiponectin plays a negative role in regulating skeletal homeostasis through inhibition of bone formation and by promoting an adipogenic phenotype.

Loss of Gi G-Protein-Coupled Receptor Signaling in Osteoblasts Accelerates Bone Fracture Healing.

G-protein-coupled receptors (GPCRs) are key regulators of skeletal homeostasis and are likely important in fracture healing. Because GPCRs can activate multiple signaling pathways simultaneously, we used targeted disruption of G(i) -GPCR or activation of G(s) -GPCR pathways to test how each pathway functions in the skeleton. We previously demonstrated that blockade of G(i) signaling by pertussis toxin (PTX) transgene expression in maturing osteoblastic cells enhanced cortical and trabecular bone formation and prevented age-related bone loss in female mice. In addition, activation of G(s) signaling by expressing the G(s) -coupled engineered receptor Rs1 in maturing osteoblastic cells induced massive trabecular bone formation but cortical bone loss. Here, we test our hypothesis that the G(i) and G(s) pathways also have distinct functions in fracture repair. We applied closed, nonstabilized tibial fractures to mice in which endogenous G(i) signaling was inhibited by PTX, or to mice with activated G(s) signaling mediated by Rs1. Blockade of endogenous G(i) resulted in a smaller callus but increased bone formation in both young and old mice. PTX treatment decreased expression of Dkk1 and increased Lef1 mRNAs during fracture healing, suggesting a role for endogenous G(i) signaling in maintaining Dkk1 expression and suppressing Wnt signaling. In contrast, adult mice with activated Gs signaling showed a slight increase in the initial callus size with increased callus bone formation. These results show that G(i) blockade and G(s) activation of the same osteoblastic lineage cell can induce different biological responses during fracture healing. Our findings also show that manipulating the GPCR/cAMP signaling pathway by selective timing of G(s) and G(i) -GPCR activation may be important for optimizing fracture repair.

Effects of blockade of endogenous Gi signaling in Tie2-expressing cells on bone formation in a mouse model of heterotopic ossification.

Available evidence indicates that some Tie2-expressing (Tie2(+) ) cells serve as multipotent progenitors that have robust BMP-dependent osteogenic activity and mediate heterotopic ossification (HO). Since signaling through the G protein Gi is required for cell motility, we hypothesized that blockade of endogenous Gi signaling in Tie2(+) cell populations would prevent HO formation. Blockade of Gi signaling in Tie2(+) cells was accomplished in transgenic mice with expression of pertussis toxin (PTX) under the control of the Tie2 promoter (Tie2(+) /PTX(+) ). Bone formation within HOs was evaluated 2 weeks after BMP injection. Expression of PTX in Tie2(+) cells significantly reduced the bone volume (BV) of HOs in male and female mice. Orthotopic bones were assessed at the distal femur and expression of PTX significantly increased trabecular bone fractional volume and bone formation rate in females only. In adult Tie2(+) /GFP(+) mice, GFP(+) cells appeared both inside and at the surfaces of bone tissue within HOs and in orthotopic bones. In summary, blockade of Gi signaling in Tie2(+) cells reduced the accrual of HOs and stimulated osteogenesis in orthotopic bones. Targeting of Gi protein coupled receptors in Tie2(+) cells may be a novel therapeutic strategy in states of abnormal bone formation such as osteoporosis and HO.

Assessing the osteoblast transcriptome in a model of enhanced bone formation due to constitutive Gs-G protein signaling in osteoblasts.

G protein-coupled receptor (GPCR) signaling in osteoblasts (OBs) is an important regulator of bone formation. We previously described a mouse model expressing Rs1, an engineered constitutively active Gs-coupled GPCR, under the control of the 2.3 kb Col I promoter. These mice showed a dramatic age-dependent increase in trabecular bone of femurs. Here, we further evaluated the effects of enhanced Gs signaling in OBs on intramembranous bone formation by examining calvariae of 1- and 9-week-old Col1(2.3)/Rs1 mice and characterized the in vivo gene expression specifically occurring in osteoblasts with activated Gs G protein-coupled receptor signaling, at the cellular level rather than in a whole bone. Rs1 calvariae displayed a dramatic increase in bone volume with partial loss of cortical structure. By immunohistochemistry, Osterix was detected in cells throughout the inter-trabecular space while Osteocalcin was expressed predominantly in cells along bone surfaces, suggesting the role of paracrine mediators secreted from OBs driven by 2.3 kb Col I promoter could influence early OB commitment, differentiation, and/or proliferation. Gene expression analysis of calvarial OBs revealed that genes affected by Rs1 signaling include those encoding proteins important for cell differentiation, cytokines and growth factors, angiogenesis, coagulation, and energy metabolism. The set of Gs-GPCRs and other GPCRs that may contribute to the observed skeletal phenotype and candidate paracrine mediators of the effect of Gs signaling in OBs were also determined. Our results identify novel detailed in vivo cellular changes of the anabolic response of the skeleton to Gs signaling in mature OBs.

Novel mouse model of spinal cord injury-induced heterotopic ossification.

Heterotopic ossification (HO) develops in about 20% to 30% of patients with spinal cord injury (SCI) and significantly impairs their rehabilitation. There is no effective prevention or treatment for this condition at this time. Our current understanding of its etiology and pathophysiology is limited partially due to the lack of clinically relevant animal models. In this study, we report a novel mouse model of SCI-induced HO by administering a subthreshold dose of bone morphogenetic protein (BMP)-2 to muscles in mice after SCI. Micro-computed tomography scanning showed that an intramuscular injection of 0.25 micrograms of BMP-2 causes significant HO in mice with SCI but not in control (sham surgery) mice. Our analysis of gene expression showed significantly increased BMP signaling in quadriceps following SCI, suggesting that BMP signaling may play a role in SCI-induced HO. Administering 0.25 micrograms of BMP-2 to the front arms of the mice with SCI also results in the development of significant HO but not in control mice. This suggests that SCI causes a systematic osteogenic effect, which is not limited to paralyzed limbs. This novel mouse model will serve as a powerful tool in exploring the molecular mechanisms of SCI-induced HO, which may lead to novel treatment for this disease.

A novel mouse model of trauma induced heterotopic ossification.

Severe soft tissue trauma is associated with heterotopic ossification (HO), the abnormal deposition of bone at extra-skeletal sites. The pathophysiology of the development of trauma-induced HO remains largely unknown due in part to the lack of appropriate animal models. In this study, we sought to develop a new trauma-induced HO mouse model using muscle impact injury combined with low dose BMP-2. BMP-2 at doses ranging from 0 to 2 µg was injected into quadriceps muscles of adult male C57/BL6 mice. Animals then received a one-time quadriceps impaction injury to mimic the trauma associated with severe injuries. HO was monitored using in vivo microCT scanning at 1, 2, 4, and 8 weeks after treatment. After trauma, the expression of BMP-2, -4, BMP receptor 1, SOX9 and RUNX2 were increased in muscle. Although little or no HO was observed in mice receiving 1 µg BMP-2, combining this dose with muscle trauma produced an abundance of HO. At higher doses of BMP-2, trauma did not augment mineral deposition. These results suggest that BMP-2 signaling can sensitize muscle to trauma-induced HO. They also provide the basis for a new model to study the pathogenesis of trauma-induced HO.

CXCL12/CXCR4 signaling in the osteoblast regulates the mesenchymal stem cell and osteoclast lineage populations.

The chemokine CXCL12 and its receptor CXCR4 play a key role in regulation of hematopoietic stem cells and cell migratory function during morphogenesis. Osteoblasts express both the ligand and the receptor, but little is known about the role of CXCL12-CXCR4 signaling in maintaining skeletal homeostasis. Using Cre-Lox technology to delete CXCR4 in mature osteoblasts in mice, we show here a significant decrease in bone mass and alterations in cancellous bone structure. CXCR4 gene ablation increased the number of colony-forming units (CFU), CFU-positive for alkaline phosphatase (CFU-AP(+)), and mineralizing nodules in bone marrow stromal cell (BMSC) cultures. The adipocyte precursor population decreased in BMSCs harvested from the KO animals. The nonadherent population of BMSCs harvested from the long bone diaphysis of KO animals formed more osteoclasts, a finding that was associated with increased circulatory levels of pyridinoline, a marker of bone resorption. Our data show that osteoblast-specific CXCR4 deletion has profound effects on the mesenchymal stem cell pool and allocation to the osteoblastic and adipocytic cell lineages. They also show that CXCL12/CXCR4 signaling in the mature osteoblast can feedback to regulate the osteoclast precursor pool size and play a multifunctional role in regulating bone formation and resorption.

Increased bone mass in mice lacking the adipokine apelin.

Adipose tissue plays an important role in skeletal homeostasis, and there is interest in identifying adipokines that influence bone mass. One such adipokine may be apelin, a ligand for the Gi-G protein-coupled receptor APJ, which has been reported to enhance mitogenesis and suppress apoptosis in MC3T3-E1 cells and primary human osteoblasts (OBs). However, it is unclear whether apelin plays a physiological role in regulating skeletal homeostasis in vivo. In this study, we compared the skeletal phenotypes of apelin knockout (APKO) and wild-type mice and investigated the direct effects of apelin on bone cells in vitro. The increased fractional cancellous bone volume at the distal femur was observed in APKO mice of both genders at 12 weeks of age and persisted until the age of 20. Cortical bone perimeter at the femoral midshaft was significantly increased in males and females at both time points. Dynamic histomorphometry revealed that APKO mice had increased rates of bone formation and mineral apposition, with evidences of accelerated OB proliferation and differentiation, without significant alteration in osteoclast activity. An in vitro study showed that apelin increased proliferation of primary mouse OBs as well as suppressed apoptosis in a dose-dependent manner with the maximum effect at 5nM. However, it had no effect on the formation of mineralized nodules. We did not observed significantly altered in osteoclast parameters in vitro. Taken together, the increased bone mass in mice lacking apelin suggested complex direct and paracrine/endocrine effects of apelin on bone, possibly via modulating insulin sensitivity. These results indicate that apelin functions as a physiologically significant antianabolic factor in bone in vivo.

Constitutive protein kinase A activity in osteocytes and late osteoblasts produces an anabolic effect on bone.

Osteocytes have been implicated in the control of bone formation. However, the signal transduction pathways that regulate the biological function of osteocytes are poorly defined. Limited evidence suggests an important role for the Gs/cAMP pathway in osteocyte function. In the present study, we explored the hypothesis that cAMP-dependent kinase A (PKA) activation in osteocytes plays a key role in controlling skeletal homeostasis. To test this hypothesis, we mated mice harboring a Cre-conditional, mutated PKA catalytic subunit allele that encodes a constitutively active form of PKA (CαR) with mice expressing Cre under the control of the osteocyte-specific promoter, DMP1. This allowed us to direct the expression of CαR to osteocytes in double transgenic progeny. Examination of Cre expression indicated that CαR was also expressed in late osteoblasts. Cortical and trabecular bone parameters from 12-week old mice were determined by µCT. Expression of CαR in osteocytes and late osteoblasts altered the shape of cortical bone proximal to the tibia-fibular junction (TFJ) and produced a significant increase in its size. In trabecular bone of the distal femur, fractional bone volume, trabecular number, and trabecular thickness were increased. These increases were partially the results of increased bone formation rates (BFRs) on the endosteal surface of the cortical bone proximal to the TFJ as well as increased BFR on the trabecular bone surface of the distal femur. Mice expressing CαR displayed a marked increase in the expression of osteoblast markers such as osterix, runx2, collagen 1α1, and alkaline phosphatase (ALP). Interestingly, expression of osteocyte marker gene, DMP1, was significantly up-regulated but the osteocyte number per bone area was not altered. Expression of SOST, a presumed target for PKA signaling in osteocytes, was significantly down-regulated in females. Importantly, no changes in bone resorption were detected. In summary, constitutive PKA signaling in osteocytes and late osteoblasts led to a small expansion of the size of the cortical bone proximal to the TFJ and an increase in trabecular bone in female mice. This was associated with down-regulation of SOST and up-regulation of several osteoblast marker genes. Activation of the PKA pathway in osteocytes and late osteoblasts is sufficient for the initiation of an anabolic skeletal response.

Cyclic AMP signaling in bone marrow stromal cells has reciprocal effects on the ability of mesenchymal stem cells to differentiate into mature osteoblasts versus mature adipocytes.

Stimulatory G protein-mediated cAMP signaling is intimately involved in skeletal homeostasis. However, limited information is available on the role of the cAMP signaling in regulating the differentiation of mesenchymal stem cells into mature osteoblasts and adipocytes. To investigate this, we treated primary mouse bone marrow stromal cells (BMSCs) with forskolin to stimulate cAMP signaling and determined the effect on osteoblast and adipocyte differentiation. Exposure of differentiating osteoblasts to forskolin markedly inhibited progression to the late stages of osteoblast differentiation, and this effect was replicated by continuous exposure to PTH. Strikingly, forskolin activation of cAMP signaling in BMSCs conditioned mesenchymal stem cells (MSCs) to undergo increased osteogenic differentiation and decreased adipogenic differentiation. PTH treatment of BMSCs also enhanced subsequent osteogenesis, but promoted an increased adipogenesis as well. Thus, activation of cAMP signaling alters the lineage commitment of MSCs, favoring osteogenesis at the expense of adipogenesis.

Lamellar spacing in cuboid hydroxyapatite scaffolds regulates bone formation by human bone marrow stromal cells.

BACKGROUND: A major goal in bone engineering is the creation of large volume constructs (scaffolds and stem cells) that bear load. The scaffolds must satisfy two competing requirements--they need be sufficiently porous to allow nutrient flow to maintain cell viability, yet sufficiently dense to bear load. We studied the effect of scaffold macroporosity on bone formation and scaffold strength, for bone formed by human bone marrow stromal cells. METHODS: Rigid cubical hydroxyapatite/tricalcium phosphate scaffolds were produced by robo-casting. The ceramic line thickness was held constant, but the distance between adjacent lines was either 50, 100, 200, 500, or 1000 µm. Cultured human bone marrow stromal cells were combined with the scaffolds in vitro; transplants were placed into the subcutis of immunodeficient mice. Transplants were harvested 9, 18, 23, 38, or 50 weeks later. Bone formation and scaffold strength were analyzed using histology and compression testing. RESULTS: Sixty transplants were evaluated. Cortical bone increased with transplant age, and was greatest among 500 µm transplants. In contrast, maximum transplant strength was greatest among 200 µm transplants. CONCLUSIONS: Lamellar spacing within scaffolds regulates the extent of bone formation; 500 µm yields the most new bone, whereas 200 µm yields the strongest transplants.

Blockade of receptor-activated G(i) signaling in osteoblasts in vivo leads to site-specific increases in cortical and cancellous bone formation.

Osteoblasts play a critical role in the maintenance of bone mass through bone formation and regulation of bone resorption. Targeted expression of a constitutively active engineered G(i)-coupled G protein-coupled receptor (GPCR) to osteoblasts in vivo leads to severe osteopenia. However, little is known about the role of endogenous receptor-mediated G(i) signaling in regulating osteoblast function. In this study, we investigated the skeletal effects of blocking G(i)-coupled signaling in osteoblasts in vivo. This was accomplished by transgenic expression of the catalytic subunit of pertussis toxin (PTX) under control of the collagen Iα 2.3-kb promoter. These mice, designated Col1(2.3)(+)/PTX(+), showed increased cortical thickness at the femoral midshaft at 12 weeks of age. This correlated with increased periosteal bone formation associated with expanded mineralizing surface observed in 8-week-old mice of both genders. The cancellous bone phenotype of the Col1(2.3)(+)/PTX(+) mice was sexually dimorphic, with increases in fractional bone volume at the distal femur seen only in females. Similarly, while cancellous bone-formation rates were unchanged in males, they could not be quantified for female Col1(2.3)(+)/PTX(+) mice owing to the disorganized nature of the labeling pattern, which was consistent with rapid formation of woven bone. Alterations in osteoclast activity did not appear to participate in the phenotype. These data demonstrate that G(i)-coupled signaling by GPCRs endogenous to osteoblasts plays a complex role in the regulation of bone formation in a manner that is dependent on both gender and the anatomic site within bone.