Studies have shown that systemic PTH treatment enhanced the rate of bone repair in rodent models. However, the mechanisms through which PTH affects bone repair have not been elucidated. In these studies we show that PTH primarily enhanced the earliest stages of endochondral bone repair by increasing chondrocyte recruitment and rate of differentiation. In coordination with these cellular events, we observed an increased level of canonical Wnt-signaling in PTH-treated bones at multiple time-points across the timecourse of fracture repair, supporting the conclusion that PTH responses are at least in part mediated through Wnt signaling.Introduction: Since FDA approval of PTH ; Forteo] as a treatment for osteoporosis, there has been interest in its use in other musculoskeletal conditions. Fracture repair is one area in which PTH may have a significant clinical impact. Multiple animal studies have shown that systemic PTH treatment of healing fractures increased both callus volume and return of mechanical competence in models of fracture healing. Whereas the potential for PTH has been established, the mechanism(s) by which PTH produces these effects remain elusive. Materials and Methods: Closed femoral fractures were generated in 8-wk-old male C57Bl/6 mice followed by daily systemic injections of either saline (control) or 30 g/kg PTH(1-34) for 14 days after fracture. Bones were harvested at days 2, 3, 5, 7, 10, 14, 21, and 28 after fracture and analyzed at the tissue level by radiography and histomorphometry and at the molecular and biochemical levels level by RNase protection assay (RPA), real-time PCR, and Western blot analysis. Results: Quantitative CT analysis showed that PTH treatment induced a larger callus cross-sectional area, length, and total volume compared with controls. Molecular analysis of the expression of extracellular matrix genes associated with chondrogenesis and osteogenesis showed that PTH treated fractures displayed a 3-fold greater increase in chondrogenesis relative to osteogenesis over the course of the repair process. In addition, chondrocyte hypertrophy occurred earlier in the PTH-treated callus tissues. Analysis of the expression of potential mediators of PTH actions showed that PTH treatment significantly induced the expression of Wnts 4, 5a, 5b, and 10b and increased levels of unphosphorylated, nuclear localized ␤-catenin protein, a central feature of canonical Wnt signaling. Conclusions: These results showed that the PTH-mediated enhancement of fracture repair is primarily associated with an amplification of chondrocyte recruitment and maturation in the early fracture callus. Associated with these cellular effects, we observed an increase in canonical Wnt signaling supporting the conclusion that PTH effects on bone repair are mediated at least in part through the activation of Wnt-signaling pathways.

Background:The A2BAR signals via cAMP. Cyclic AMP signaling has been shown to regulate MSC differentiation. Results: A2BAR KO mice have reduced differentiation of osteoblasts, a mild osteopenic phenotype, and impaired fracture physiology. A2BAR activation increases the differentiation of osteoblasts. Conclusion: The A2BAR regulates bone homeostasis. Significance: A2BAR signaling is a component of bone homeostasis, particularly after injury.

Fractures are among the most common human traumas. Fracture healing represents a unique temporarily definable post-natal process in which to study the complex interactions of multiple molecular events that regulate endochondral skeletal tissue formation. Because of the regenerative nature of fracture healing, it is hypothesized that large numbers of post-natal stem cells are recruited and contribute to formation of the multiple cell lineages that contribute to this process. Bayesian modeling was used to generate the temporal profiles of the transcriptome during fracture healing. The temporal relationships between ontologies that are associated with various biologic, metabolic, and regulatory pathways were identified and related to developmental processes associated with skeletogenesis, vasculogenesis, and neurogenesis. The complement of all the expressed BMPs, Wnts, FGFs, and their receptors were related to the subsets of transcription factors that were concurrently expressed during fracture healing. We further defined during fracture healing the temporal patterns of expression for 174 of the 193 genes known to be associated with human genetic skeletal disorders. In order to identify the common regulatory features that might be present in stem cells that are recruited during fracture healing to other types of stem cells, we queried the transcriptome of fracture healing against that seen in embryonic stem cells (ESCs) and mesenchymal stem cells (MSCs). Approximately 300 known genes that are preferentially expressed in ESCs and ∼350 of the known genes that are preferentially expressed in MSCs showed induction during fracture healing. Nanog, one of the central epigenetic regulators associated with ESC stem cell maintenance, was shown to be associated in multiple forms or bone repair as well as MSC differentiation. In summary, these data present the first temporal analysis of the transcriptome of an endochondral bone formation process that takes place during fracture healing. They show that neurogenesis as well as vasculogenesis are predominant components of skeletal tissue formation and suggest common pathways are shared between post-natal stem cells and those seen in ESCs.

The effects of BMP2 on bone marrow stromal cell differentiation and bone formation after bone marrow ablation were determined using C57 BL/6J (B6) mice. Inhibition of BMP2 expression with lentiviral BMP2 shRNA prevented both mineralized nodule formation in vitro and bone formation in vivo, and blocked the expression of Runx2 and osterix, transcriptional determinants of terminal osteogenic differentiation. No effect was observed on the expression of Sox9, a transcription factor, which is the one of the first transcriptional determinant to be expressed in committed chondroprogenitor and osteoprogenitor cells. In vitro studies showed that exogenously added BMP7 rescued the expression of osterix and enhanced the expression of Sox9, but had no effect on the expression of Runx2, while it only partially recovered the development of mineral deposition in the cultures. On the other hand, the exogenous addition of BMP2 rescued both Runx2 and osterix expression, did not enhance the expression of Sox9, but fully recovered the inhibition of mineral deposition in the cultures. Using antibodies against CD146 and Sox9, immunohistological examination of the cell populations found in the medullary space three days after bone marrow ablation, showed qualitatively equal numbers of cells expressing these skeletal progenitor and stem cell markers in control and BMP2 shRNA treated animals. Fluorescence Activated Cell Sorting (FACS) analysis of the cells found with the marrow cavities at three days after marrow ablation using CD146 antibody showed near equal numbers of immunopositive cells in both control and shRNA treated animals. In summary, the differences observed in vitro for BMP2 and BMP7 on osteogenic gene expression and mineralization suggest that they have differing effects on bone cell differentiation. These results further demonstrate that in vivo BMP2 is a central morphogenetic regulator of post natal osteoprogenitor differentiation, but does not affect recruitment of progenitors to the osteoblastic lineage. AbstractThe effects of BMP2 on bone marrow stromal cell differentiation and bone formation after bone marrow ablation were determined using C57 BL/6J (B6) mice. Inhibition of BMP2 expression with lentiviral BMP2 shRNA prevented both mineralized nodule formation in vitro and bone formation in vivo, and blocked the expression of Runx2 and osterix, transcriptional determinants of terminal osteogenic differentiation. No effect was observed on the expression of Sox9, a transcription factor, which is the one of the first transcriptional determinant to be expressed in committed chondroprogenitor and osteoprogenitor cells. In vitro studies showed that exogenously added BMP7 rescued the expression of osterix and enhanced the expression of Sox9, but had no effect on the expression of Runx2, while it only partially recovered the development of mineral deposition in the cultures. On the other hand, the exogenous addition of BMP2 rescued both Runx2 and osterix expression, did not enhance the expression of Sox9, but fully re...

Pyrococcus furiosus utilizes starch and its degradation products, such as maltose, as primary carbon sources, but the pathways by which these ␣-glucans are processed have yet to be defined. For example, its genome contains genes proposed to encode five amylolytic enzymes (including a cyclodextrin glucanotransferase [CGTase] and amylopullulanase), as well as two transporters for maltose and maltodextrins (Mal-I and Mal-II), and a range of intracellular enzymes have been purified that reportedly metabolize maltodextrins and maltose. However, precisely which of these enzymes are involved in starch processing is not clear. In this study, starch metabolism in P. furiosus was examined by biochemical analyses in conjunction with global transcriptional response data for cells grown on a variety of glucans. In addition, DNA sequencing led to the correction of two key errors in the genome sequence, and these change the predicted properties of amylopullulanase (now designated PF1935*) and CGTase (PF0478*). Based on all of these data, a pathway is proposed that is specific for starch utilization that involves one transporter (Mal-II [PF1933 to PF1939]) and only three enzymes, amylopullulanase (PF1935*), 4-␣-glucanotransferase (PF0272), and maltodextrin phosphorylase (PF1535). Their expression is upregulated on starch, and together they generate glucose and glucose-1-phosphate, which then feed into the novel glycolytic pathway of this organism. In addition, the results indicate that several hypothetical proteins encoded by three gene clusters are also involved in the transport and processing of ␣-glucan substrates by P. furiosus.Pyrococcus furiosus is an obligately anaerobic, heterotrophic, hyperthermophilic archaeon that was isolated from a shallow hydrothermal vent near Vulcano Island, Italy (12). The organism grows optimally near 100°C and is able to utilize a range of sugars as a primary carbon source (4,11,28,29). These include cellobiose (28, 43), laminarin (43), chitin (16), maltose (29), barley glucan (3), and starch (29). However, the means by which these compounds are metabolized by P. furiosus are not well understood. In attempting to define the pathway by which starch, and one of its degradation products, maltose, serve as carbon sources, there are two complicating factors. First, the genome sequence of P. furiosus (36) contains an array of genes that encode putative glycosyl hydrolases and glycosyl transferases. With respect to starch, they include five enzymes that potentially hydrolyze ␣-glucans. Two of them (PF0477 and PF1935) have putative signal peptides and are presumably extracellular, while the other three (PF0272, PF0478, and PF1939) lack a signal peptide and are presumably intracellular. They are annotated as amylases (PF0272 and PF0477), amylopullulanases (PF1934 and PF1935), and cyclodextrin glucanotransferases (PF0478). The question is, are all of these enzymes involved in degrading starch? The pathway for utilizing maltose and maltodextrins, primary products of starch breakdown, is well defined in ...

Hypophosphatemia leads to rickets and osteomalacia, the latter of which results in decreased biomechanical integrity of bones, accompanied by poor fracture healing. Impaired phosphate-dependent apoptosis of hypertrophic chondrocytes is the molecular basis for rickets. However, the underlying pathophysiology of impaired fracture healing has not been characterized previously. To address the role of phosphate in fracture repair, mice were placed on a phosphate-restricted diet 2 days prior to or 3 days after induction of a mid-diaphyseal femoral fracture to assess the effects of phosphate deficiency on the initial recruitment of mesenchymal stem cells and their subsequent differentiation. Histologic and micro-computed tomographic (µCT) analyses demonstrated that both phosphate restriction models dramatically impaired fracture healing primarily owing to a defect in differentiation along the chondrogenic lineage. Based on Sox9 and Sox5 mRNA levels, neither the initial recruitment of cells to the callus nor their lineage commitment was effected by hypophosphatemia. However, differentiation of these cells was impaired in association with impaired bone morphogenetic protein (BMP) signaling. In vivo ectopic bone-formation assays and in vitro investigations in ST2 stromal cells confirmed that phosphate restriction leads to BMP-2 resistance. Marrow ablation studies demonstrate that hypophosphatemia has different effects on injury-induced intramembranous bone formation compared with endochondral bone formation. Thus phosphate plays an important role in the skeleton that extends beyond mineralized matrix formation and growth plate maturation and is critical for endochondral bone repair. © 2010 American Society for Bone and Mineral Research.

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Previous studies showed that loss of tumor necrosis factor α (TNFα) signaling delayed fracture healing by delaying chondrocyte apoptosis and cartilage resorption. Mechanistic studies showed that TNFα induced Fas expression within chondrocytes; however, the degree to which chondrocyte apoptosis is mediated by TNFα alone or dependent on the induction of Fas is unclear. This question was addressed by assessing fracture healing in Fas-deficient B6.MRL/Faslpr/J mice. Loss of Fas delayed cartilage resorption but also lowered bone fraction in the calluses. The reduced bone fraction was related to elevated rates of coupled bone turnover in the B6.MRL/Faslpr/J calluses, as evidenced by higher osteoclast numbers and increased osteogenesis. Analysis of the apoptotic marker caspase 3 showed fewer positive chondrocytes and osteoclasts in calluses of B6.MRL/Faslpr/J mice. To determine if an active autoimmune state contributed to increased bone turnover, the levels of activated T cells and Treg cells were assessed. B6.MRL/Faslpr/J mice had elevated Treg cells in both spleens and bones of B6.MRL/Faslpr/J but decreased percentage of activated T cells in bone tissues. Fracture led to ∼30% to 60% systemic increase in Treg cells in both wild-type and B6.MRL/Faslpr/J bone tissues during the period of cartilage formation and resorption but either decreased (wild type) or left unchanged (B6.MRL/Faslpr/J) the numbers of activated T cells in bone. These results show that an active autoimmune state is inhibited during the period of cartilage resorption and suggest that iTreg cells play a functional role in this process. These data show that loss of Fas activity specifically in chondrocytes prolonged the life span of chondrocytes and that Fas synergized with TNFα signaling to mediate chondrocyte apoptosis. Conversely, loss of Fas systemically led to increased osteoclast numbers during later periods of fracture healing and increased osteogenesis. These findings suggest that retention of viable chondrocytes locally inhibits osteoclast activity or matrix proteolysis during cartilage resorption. © 2014 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.