Project description:The phases of fracture healing have been well characterized. However, the exact source and genetic profile of the skeletal progenitors that participate in bone repair is somewhat unclear. Sox9 expression in skeletal elements precedes bone and cartilage formation and a Sox9+ cell type is retained in the adult periosteum. We hypothesized that Sox9+ periosteal cells are multipotent skeletal progenitors normally participating in fracture repair. To test this hypothesis we used tamoxifen (TM)-mediated lineage tracing of Sox9+ cells in Sox9CreErt2:Td-Tomato mice. TM injection indelibly labels Sox9+ cells and all their descendants with the fluorescent reporter protein Td-Tomato. Intact mouse femora were harvested 2 weeks after TM injection and analyzed by histology and immunostaining and RNA sequencing, to evaluate the skeletal distribution and gene expression profile of Td-Tomato positive cells in the adult femur. To assess the role of these cells in fracture repair, mice underwent a closed mid-diaphyseal femoral fracture and their hind limbs were harvested at 3, 9 or 56 days post-fracture to assess the contribution of Sox9+ and Td-tomato+ cells in the fracture healing process. In the intact adult mouse femur, Td-Tomato-labelled cells were observed in articular and growth plate cartilage where Sox9 is known to be expressed, but also in the primary spongiosa, periosteum, and endosteum. RNA sequencing and subsequent analysis showed that Td-Tomato positive periosteal cells were enriched in Sox9 transcripts, and transcripts for various osteogenic and chondrocyte specific genes. In a femoral fracture model, we showed that pre-labeled Td-Tomato positive descendent cells were mobilized during the fracture repair process, expanding and migrating towards the fracture site 3 days post-fracture. Here, Td-Tomato positive cells differentiate into chondrocytes and osteoblasts in the soft and hard callus, respectively, 9 days post-fracture. By 2 months post-fracture, descendants of the original Sox9 labelled cell population were prevalent in the cortex and periosteum, and amongst the differentiated osteocyte population embedded within the cortical bone. Thus, a Sox9+ progenitor population resides in the adult periosteum. Fate tracing shows that these cells likely play a substantial role in repair of the fractured femur giving rise to chondrocytes, osteoblasts and mature cortical osteocytes. To our knowledge this is the first report of this Sox9+ cell population in the periosteum of the adult long bone. Taken together with developmental studies on skeletal formation, our data suggest that Sox9+ osteochondroprogenitors play a continuous role in skeletal formation throughout life.
Project description:Non-union skeletal fractures are characterized by their inability to heal six months after injury. Left untreated, the non-union fracture may cause advanced arthritis or loss of function in the affected limb. Arrays were used to identify the mechanisms that lead to lack of skeletal repair in non-unions. These profiles are the non-union callous samples pooled from five individuals Keywords: other
2003-07-16 | GSE494 | GEO
Project description:Sulfate-reducing bacteria community structure in mangrove wetlands
Project description:We reported the application of single-cell mRNA sequencing to identify a unique population of fibroblasts that exits in the fracture callus of bisphosphonate-treated rats. Such unique population of fibroblasts prevented fracture healing by secreting ECM. Further, it was found that these ECM-secreting fibroblasts were enriched for myeloid genes, suggesting a bone marrow orgin. After fractures were treated with local CGRP injection or Magnesium based biometal, such population of fibroblasts was cleared off, resulting in facilitated fracture healing of bisphosphonate-treated rats.
Project description:Structural characterization of glycosaminoglycans remains a challenge and is essential for determining not only structure-function relationships between glycosaminoglycans and the biomolecules with which they interact, but also to gain insight into the biosynthesis of glycosaminoglycans. We have recently reported cytotoxic effects of xyloside-primed chondroitin/dermatan sulfate derived from a human breast carcinoma cell line, HCC70, and shown that it differs in disaccharide composition from non-toxic chondroitin/dermatan sulfate derived from a human breast fibroblast cell line, CCD-1095Sk. To further investigate the structural requirements for the cytotoxic effect, we have here developed a novel LC-MS/MS approach based on dibutylamine ion-pairing reversed-phase chromatography and negative mode higher-energy collision dissociation (HCD), and used it in combination with cell growth studies and disaccharide fingerprinting. This allowed for detailed structural characterization of linkage regions, internal oligosaccharides, and non-reducing ends, showing not only differences between xyloside-primed chondroitin/dermatan sulfate from HCC70 cells and CCD-1095Sk cells, but also in sialylation of the linkage region as well as previously undescribed methylation and sulfation of the non-reducing ends. Although the xyloside-primed chondroitin/dermatan sulfate from HCC70 cells was less complex in terms of presence and distribution of iduronic acid than that from CCD-1095Sk cells, both glucuronic acid and iduronic acid appeared essential for the cytotoxic effect. Our data have moved us one step closer to understanding the structure of the cytotoxic chondroitin/dermatan sulfate from HCC70 cells primed on xylosides, and demonstrate the suitability of the LC-MS/MS approach for structural characterization of glycosaminoglycans.