Reduced bone loss in a murine model of postmenopausal osteoporosis lacking complement component 3.

The growing field of osteoimmunology seeks to unravel the complex interdependence of the skeletal and immune systems. Notably, we and others have demonstrated that complement signaling influences the differentiation of osteoblasts and osteoclasts, the two primary cell types responsible for maintaining bone homeostasis. However, the net effect of complement on bone homeostasis in vivo was unknown. Our published in vitro mechanistic work led us to hypothesize that absence of complement component 3 (C3), a central protein in the complement activation cascade, protects against bone loss in the ovariectomy-based model of postmenopausal osteoporosis. Indeed, we report here that, when compared to their C57BL/6J (WT) counterparts, ovariectomized C3 deficient mice experienced reduced bone loss at multiple sites and increased stiffness at the femoral neck, the latter potentially improving mechanical function. WT and B6;129S4-C3tm1Crr /J (C3-/- ) mice were either ovariectomized or sham-operated at 6 weeks of age and euthanized at 12 weeks. MicroCT on harvested bones revealed that the trabecular bone volume fraction in the metaphyses of both the proximal tibiae and distal femora of ovariectomized C3-/- mice is significantly greater than that of their WT counterparts. Lumbar vertebrae showed significantly greater osteoid content and mineral apposition rates. Mechanical testing demonstrated significantly greater stiffness in the femoral necks of ovariectomized C3-/- mice. These results demonstrate that C3 deficiency reduces bone loss at ovariectomy and may improve mechanical properties. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:118-128, 2018.

Disparate response of articular- and auricular-derived chondrocytes to oxygen tension.

PURPOSE/AIM: To determine the effect of reduced (5%) oxygen tension on chondrogenesis of auricular-derived chondrocytes. Currently, many cell and tissue culture experiments are performed at 20% oxygen with 5% carbon dioxide. Few cells in the body are subjected to this supra-physiological oxygen tension. Chondrocytes and their mesenchymal progenitors are widely reported to have greater chondrogenic expression when cultured at low, more physiological, oxygen tension (1-7%). Although generally accepted, there is still some controversy, and different culture methods, species, and outcome metrics cloud the field. These results are, however, articular chondrocyte biased and have not been reported for auricular-derived chondrocytes. MATERIALS AND METHODS: Auricular and articular chondrocytes were isolated from skeletally mature New Zealand White rabbits, expanded in culture and differentiated in high density cultures with serum-free chondrogenic media. Cartilage tissue derived from aggregate cultures or from the tissue engineered sheets were assessed for biomechanical, glycosaminoglycan, collagen, collagen cross-links, and lysyl oxidase activity and expression. RESULTS: Our studies show increased proliferation rates for both auricular and articular chondrocytes at low (5%) O2 versus standard (20%) O2. In our scaffold-free chondrogenic cultures, low O2 was found to increase articular chondrocyte accumulation of glycosaminoglycan, but not cross-linked type II collagen, or total collagen. Conversely, auricular chondrocytes accumulated less glycosaminoglycan, cross-linked type II collagen and total collagen under low oxygen tension. CONCLUSIONS: This study highlights the dramatic difference in response to low O2 of chondrocytes isolated from different anatomical sites. Low O2 is beneficial for articular-derived chondrogenesis but detrimental for auricular-derived chondrogenesis.

Gene length may contribute to graded transcriptional responses in the Drosophila embryo.

An important question in developmental biology is how relatively shallow gradients of morphogens can reliably establish a series of distinct transcriptional readouts. Current models emphasize interactions between transcription factors binding in distinct modes to cis-acting sequences of target genes. Another recent idea is that the cis-acting interactions may amplify preexisting biases or prepatterns to establish robust transcriptional responses. In this study, we examine the possible contribution of one such source of prepattern, namely gene length. We developed quantitative imaging tools to measure gene expression levels for several loci at a time on a single-cell basis and applied these quantitative imaging tools to dissect the establishment of a gene expression border separating the mesoderm and neuroectoderm in the early Drosophila embryo. We first characterized the formation of a transient ventral-to-dorsal gradient of the Snail (Sna) repressor and then examined the relationship between this gradient and repression of neural target genes in the mesoderm. We found that neural genes are repressed in a nested pattern within a zone of the mesoderm abutting the neuroectoderm, where Sna levels are graded. While several factors may contribute to the transient graded response to the Sna gradient, our analysis suggests that gene length may play an important, albeit transient, role in establishing these distinct transcriptional responses. One prediction of the gene-length-dependent transcriptional patterning model is that the co-regulated genes knirps (a short gene) and knirps-related (a long gene) should be transiently expressed in domains of differing widths, which we confirmed experimentally. These findings suggest that gene length may contribute to establishing graded responses to morphogen gradients by providing transient prepatterns that are subsequently amplified and stabilized by traditional cis-regulatory interactions.

Characterizing medullary and human mesenchymal stem cell-derived adipocytes.

Throughout postnatal years, medullary adipocytes (MAs) increase in both number and size; however, knowledge of these cells pales in comparison to that of other adipocyte depots. It is widely hypothesized that MAs derive from multipotent progenitor cells of the bone marrow, such as human mesenchymal stem cells (hMSCs). Nevertheless, there is a paucity of comparative, molecular-level studies in support of this hypothesis. In the present article, RTPCR was used to examine similarities and differences in gene expression among MAs, hMSC-derived adipocytes, and subcutaneous adipocytes. While little or no message for lineage-specific markers was detected in undifferentiated hMSCs, the data demonstrate that hMSC-derived adipocytes, MAs, and subcutaneous adipocytes commonly express mRNA encoding for adipogenic transcription factors (PPARgamma2, C/EBPalpha, and SREBP1), adipokines (adipsin, leptin, APM1, and angiotensinogen), and lipid-metabolizing agents (aP2 and LPL), among other genes. None of the cell populations examined expressed a detectable level of the brown fat marker UCP1. This suggests highly similar gene expression between human subcutaneous and MAs, not previously substantiated to this degree. Coupled with the hMSC-derived adipocyte analysis, these data provide a framework ultimately for characterizing MAs and identifying their origin and function.