Lipolysis products from triglyceride-rich lipoproteins induce stress protein ATF3 in osteoblasts.

High fat diets overwhelm the physiological mechanisms for absorption, storage, and utilization of triglycerides (TG); consequently TG, TG-rich lipoproteins (TGRL), and TGRL remnants accumulate, circulate systemically, producing dyslipidemia. This associates with, or is causative for increased atherosclerotic cardiovascular risk, ischemic stroke, fatty liver disease, and pancreatitis. TGRL hydrolysis by endothelial surface-bound lipoprotein lipase (LPL) generates metabolites like free fatty acids which have proinflammatory properties. While osteoblasts utilize fatty acids as an energy source, dyslipidemia is associated with negative effects on the skeleton. In this study we investigated the effects of TGRL lipolysis products (TGRL-LP) on expression of a stress responsive transcription factor, termed activating transcription factor 3 (ATF3), reactive oxygen species (ROS), ATF3 target genes, and angiopoietin-like 4 (Angptl4) in osteoblasts. As ATF3 negatively associates with osteoblast differentiation, we also investigated the skeletal effects of global ATF3 deletion in mice. TGRL-LP increased expression of Atf3, proinflammatory proteins Ptgs2 and IL-6, and induced ROS in MC3T3-E1 osteoblastic cells. Angptl4 is an endogenous inhibitor of LPL which was transcriptionally induced by TGRL-LP, while recombinant Angptl4 prevented TG-driven Atf3 induction. Atf3 global knockout male mice demonstrated increased trabecular and cortical microarchitectural parameters. In summary, we find that TGRL-LP induce osteoblastic cell stress as evidenced by expression of ATF3, which may contribute to the negative impact of dyslipidemia in the skeleton. Further, concomitant induction of Angptl4 in osteoblasts might play a protective role by reducing local lipolysis.

Circulating progenitor cells and the expression of Cxcl12, Cxcr4 and angiopoietin-like 4 during wound healing in the murine ear.

Migration of cells from both local and systemic sources is essential for the inflammatory and regenerative processes that occur during normal wound healing. CXCL12 is considered a critical regulator of CXCR4-positive cell migration during tissue regeneration. In this study, we investigated the expression of Cxcl12 and Cxcr4 during healing of a murine full thickness ear wound. We also investigated the expression of angiopoietin-like 4, which has been shown to participate in wound angiogenesis and reepithelialization. At time points up to 48hrs, complete blood counts were performed using automated hematology analysis, and the numbers of circulating stem and progenitor cells quantified using flow cytometry. Expression of both Cxcr4 and Angptl4 was significantly elevated within 3 days of wounding, and both were strongly expressed in cells of the epidermis. ANGPTL4 protein expression remained elevated in the epithelium through day 14. Cxcl12 expression was increased significantly at day 3, and remained elevated through day 21. Faint Cxcl12 staining was detectable in the epithelium at day 1, and thereafter staining was faint and more generalized. There were significantly fewer circulating total white blood cells and lymphocytes 1hr following ear punching. Similarly, there was a significant early (1hr) reduction in the number of circulating endothelial progenitor cells. Further studies are warranted to investigate whether ANGPTL4 and CXCL12/CXCR4 interact or synergize to facilitate cell recruitment and migration, and to potentiate reepithelialization and wound healing.

Age Dependence of Systemic Bone Loss and Recovery Following Femur Fracture in Mice.

The most reliable predictor of future fracture risk is a previous fracture of any kind. The etiology of this increased fracture risk is not fully known, but it is possible that fracture initiates systemic bone loss, leading to greater fracture risk at all skeletal sites. In this study, we investigated systemic bone loss and recovery after femoral fracture in young (3-month-old) and middle-aged (12-month-old) mice. Transverse femur fractures were created using a controlled impact, and whole-body bone mineral density (BMD), trabecular and cortical microstructure, bone mechanical properties, bone formation and resorption rates, mouse voluntary movement, and systemic inflammation were quantified at multiple time points post-fracture. We found that fracture led to decreased whole-body BMD in both young and middle-aged mice 2 weeks post-fracture; this bone loss was recovered by 6 weeks in young but not middle-aged mice. Similarly, trabecular bone volume fraction (BV/TV) of the L5 vertebral body was significantly reduced in fractured mice relative to control mice 2 weeks post-fracture (-11% for young mice, -18% for middle-aged mice); no significant differences were observed 6 weeks post-fracture. At 3 days post-fracture, we observed significant increases in serum levels of interleukin-6 and significant decreases in voluntary movement in fractured mice compared with control mice, with considerably greater changes in middle-aged mice than in young mice. At this time point, we also observed increased osteoclast number on L5 vertebral body trabecular bone of fractured mice compared with control mice. These data show that systemic bone loss occurs after fracture in both young and middle-aged mice, and recovery from this bone loss may vary with age. This systemic response could contribute to increased future fracture risk after fracture; these data may inform clinical treatment of fractures with respect to improving long-term skeletal health. © 2018 American Society for Bone and Mineral Research.

Gross anatomy and morphometric evaluation of the canine lacrimal and third eyelid glands.

OBJECTIVE: The lacrimal gland (LG) and the third eyelid gland (TELG) are two intraorbital glands that, in dogs, secrete the aqueous component of the tear film. Despite the central importance of these structures for maintaining ocular surface health, the gross anatomy of the glands remains understudied. We investigated the macroscopic morphometric characteristics of the LG and TELG in three different dog breeds. PROCEDURES: Twenty-six dog heads were dissected to expose the LG and TELG; the length, width, thickness, and weight of each were measured. During the dissections, the relationships between the glands and adjacent ocular structures and the blood and nerve supplies to the LG were photo-documented. RESULTS: The LG had a flat and irregular shape with morphological variations among dogs. The LG was located on the dorsolateral aspect of the globe underneath the orbital ligament. The average length, width, and thickness (SEM) of the LG (mm) were 16.5 ± 0.7, 12.5 ± 0.4, and 2.7 ± 0.1 and of the TELG 10.5 ± 0.6, 11.0 ± 0.3, and 3.3 ± 0.1, respectively. The mean weights (SD) of the LG and TELG (mg) were 315.7 ± 21.1 and 263.3 ± 13.2, respectively. Beagles were observed to have significantly smaller LGs compared to pit bull terriers and pointer mixed-breed dogs. CONCLUSIONS: The present study provides detailed normative anatomical and morphometric data for the LG and TELG. These data will aid researchers investigating alterations induced by disease states and should inform strategies for the local delivery of pharmacologic and cellular therapeutics.

Expression of angiopoietin-like protein 4 at the fracture site: Regulation by hypoxia and osteoblastic differentiation.

Vascular disruption that occurs as a consequence of bone fracture, leads to hypoxia at the site of damage. Hypoxia regulates the expression of a number of genes that can modulate energy conservation, cell survival, tissue regeneration and angiogenesis. In this study we investigated the expression of Angiopoietin-like 4, an adipocytokine that has additional roles in angiogenesis, at the fracture site. We demonstrate that Angptl4 mRNA expression increased early during fracture healing (day 3) returning close to baseline at day14. In the callus, Angptl4 mRNA was visualized in areas of condensing mesenchymal cells, callus cartilage and was especially high in mineralizing osteoblasts located in areas of new bone formation. In vitro, Angptl4 mRNA expression in osteoblasts increased under hypoxic conditions and in cells treated with the hypoxia mimetic desferrioxamine. Angptl4 levels were strongly induced at day 14 in differentiating MC3T3-E1 osteoblastic cells. Exogenous ANGPTL4 increased expression of Runx2, Spp1, vegfa, and Alp mRNA in differentiating osteoblasts. We suggest that the distribution of Angptl4 in the callus may be driven by hypoxia and that Angptl4 may play a role in osteoblastic differentiation, and possibly angiogenesis via regulation of VEGF. Further studies could reveal a dual role for Angptl4 in angiogenesis and osteogenesis.

Femoroacetabular impingement: current concepts in diagnosis and treatment.

EDUCATIONAL OBJECTIVES: As a result of reading this article, physicians should be able to: 1. Identify the etiology of femoroacetabular impingement. 2. Assess femoroacetabular impingement on physical examination. 3. Recognize femoroacetabular impingement on imaging studies. 4. Discuss modern techniques to effectively treat femoroacetabular impingement, both open and arthroscopic. Femoroacetabular impingement (FAI) is a recently proposed concept describing abnormal anatomic relationships within the hip joint that may lead to articular damage. Impingement is caused by bony deformities or spatial malorientation of the femoral head-neck junction and/or the acetabulum. These abnormalities lead to pathologic contact and shearing forces at the acetabular labrum and cartilage during physiological hip motion. There is an increasing body of evidence that these forces lead to cartilage wear and eventual osteoarthritis. Treatment options for FAI are evolving rapidly. Although the gold standard remains open hip dislocation, arthroscopic techniques have shown significant promise. It is possible that early recognition and treatment of subtle deformity about the hip may reduce the rate of hip osteoarthritis in the future.

Detection of bacterial DNA by PCR in dogs with stifle pathology.

OBJECTIVE: To determine presence of bacterial DNA in canine stifles with cranial cruciate ligament rupture (CCLR) and medial patellar luxation (MPL) compared to normal canine stifles (control). STUDY DESIGN: Prospective clinical study. ANIMALS: Dogs (n = 44). METHODS: Dogs of varying age, breed, sex, and weight residing in California were assessed for stifle pathology (CCLR, MPL, or normal control). Synovial fluid of all stifles was assessed for the presence of bacterial DNA using broad-ranging 16S rRNA primers and PCR. RESULTS: Bacterial DNA was detected in normal control stifles and those with CCLR and MPL. There were no statistical differences in the copy numbers of bacterial DNA in the stifle synovial fluid among groups (P > .05); however, synovial fluid specimens from dogs with stifle pathology (CCLR and MPL combined) tended to have higher copy numbers of bacterial DNA than those from controls (P = .06). There was no significant difference in the number of bacterial DNA between the CCLR and MPL groups (P = .57). The copy numbers of bacterial DNA had a weak positive significant correlation with the duration of lameness in CCLR group (P < .05). CONCLUSIONS: Increased detection of bacterial DNA in the stifle synovial fluid may indicate joint pathology but not be directly linked to a specific joint disease.

Mobilization of endogenous stem cell populations enhances fracture healing in a murine femoral fracture model.

BACKGROUND AIMS: Delivery of bone marrow-derived stem and progenitor cells to the site of injury is an effective strategy to enhance bone healing. An alternate approach is to mobilize endogenous, heterogeneous stem cells that will home to the site of injury. AMD3100 is an antagonist of the chemokine receptor 4 (CXCR4) that rapidly mobilizes stem cell populations into peripheral blood. Our hypothesis was that increasing circulating numbers of stem and progenitor cells using AMD3100 will improve bone fracture healing. METHODS: A transverse femoral fracture was induced in C57BL/6 mice, after which they were subcutaneously injected for 3 d with AMD3100 or saline control. Mesenchymal stromal cells, hematopoietic stem and progenitor cells and endothelial progenitor cells in the peripheral blood and bone marrow were evaluated by means of flow cytometry, automated hematology analysis and cell culture 24 h after injection and/or fracture. Healing was assessed up to 84 d after fracture by histomorphometry and micro-computed tomography. RESULTS: AMD3100 injection resulted in higher numbers of circulating mesenchymal stromal cells, hematopoietic stem cells and endothelial progenitor cells. Micro-computed tomography data demonstrated that the fracture callus was significantly larger compared with the saline controls at day 21 and significantly smaller (remodeled) at day 84. AMD3100-treated mice have a significantly higher bone mineral density than do saline-treated counterparts at day 84. CONCLUSIONS: Our data demonstrate that early cell mobilization had significant positive effects on healing throughout the regenerative process. Rapid mobilization of endogenous stem cells could provide an effective alternative strategy to cell transplantation for enhancing tissue regeneration.

Long-term administration of AMD3100, an antagonist of SDF-1/CXCR4 signaling, alters fracture repair.

Fracture healing involves rapid stem and progenitor cell migration, homing, and differentiation. SDF-1 (CXCL12) is considered a master regulator of CXCR4-positive stem and progenitor cell trafficking to sites of ischemic (hypoxic) injury and regulates their subsequent differentiation into mature reparative cells. In this study, we investigated the role of SDF-1/CXCR4 signaling in fracture healing where vascular disruption results in hypoxia and SDF-1 expression. Mice were injected with AMD3100, a CXCR4 antagonist, or vehicle twice daily until euthanasia with the intent to impair stem cell homing to the fracture site and/or their differentiation. Fracture healing was evaluated using micro-computed tomography, histology, quantitative PCR, and mechanical testing. AMD3100 administration resulted in a significantly reduced hyaline cartilage volume (day 14), callus volume (day 42) and mineralized bone volume (day 42) and reduced expression of genes associated with endochondral ossification including collagen Type 1 alpha 1, collagen Type 2 alpha 1, vascular endothelial growth factor, Annexin A5, nitric oxide synthase 2, and mechanistic target of rapamycin. Our data suggest that the SDF-1/CXCR4 signaling plays a central role in bone healing possibly by regulating the recruitment and/or differentiation of stem and progenitor cells.

Osteogenic proliferation and differentiation of canine bone marrow and adipose tissue derived mesenchymal stromal cells and the influence of hypoxia.

The aim of this study was to compare the osteogenic and proliferative potential of canine mesenchymal stromal cells (cMSCs) derived from bone marrow (BM-cMSCs) and adipose tissue (AT-cMSCs). Proliferation potential was determined under varying oxygen tensions (1%, 5%, and 21% O(2)). Effects of reduced oxygen levels on the osteogenic differentiation of AT-cMSCs were also investigated. AT-cMSCs proliferated at a significantly faster rate than BM-cMSCs, although both cell types showed robust osteogenic differentiation. Culture in 5% and 1% O(2) impaired proliferation in cMSC from both sources and osteogenic differentiation in AT-cMSCs. Our data suggests that AT-cMSCs might be more suitable for use in a clinical situation, where large cell numbers are required for bone repair, due to their rapid proliferation combined with robust osteogenic potential. Our data also suggests that the inhibitory effects of hypoxia on both cell proliferation and differentiation should be considered when using MSCs in a potentially hypoxic environment such as a fracture site.

Comparison of the osteogenic potential of equine mesenchymal stem cells from bone marrow, adipose tissue, umbilical cord blood, and umbilical cord tissue.

OBJECTIVE: To determine the optimal osteogenic source of equine mesenchymal stem cells (eMSCs) and optimize collection of and expansion conditions for those cells. ANIMALS: 10 adult Quarter Horses and 8 newborn Thoroughbred foals. PROCEDURES: eMSCs were isolated from bone marrow (BM), adipose tissue, and umbilical cord blood and tissue, and the osteogenic potential of each type was assessed. Effects of anatomic site, aspiration volume, and serum type on eMSC yield from BM were investigated. RESULTS: BM-eMSCs had the highest overall expression of the osteogenic genes Cbfa1, Osx, and Omd and staining for ALP activity and calcium deposition. There was no significant difference in BM-eMSC yield from the tuber coxae or sternum, but yield was significantly greater from the first 60-mL aspirate than from subsequent aspirates. The BM-eMSC expansion rate was significantly higher when cells were cultured in fetal bovine serum instead of autologous serum (AS). CONCLUSIONS AND CLINICAL RELEVANCE: eMSCs from BM possessed the highest in vitro osteogenic potential; eMSCs from adipose tissue also had robust osteogenic potential. The tuber coxae and the sternum were viable sources of BM-eMSCs in yearlings, and 60 mL of BM aspirate was sufficient for culture and expansion. Expanding BM-eMSCs in AS to avoid potential immunologic reactions decreased the total yield because BM-eMSCs grew significantly slower in AS than in fetal bovine serum. Additional studies are needed to determine optimal ex vivo eMSC culture and expansion conditions, including the timing and use of growth factor­supplemented AS.

Hypoxia decreases sclerostin expression and increases Wnt signaling in osteoblasts.

Mutations in sclerostin function or expression cause sclerosing bone dysplasias, involving decreased antagonism of Wnt/Lrp5 signaling. Conversely, deletion of the VHL tumor suppressor in osteoblasts, which stabilize HIF-alpha isoforms and thereby enables HIF-alpha/beta-driven gene transcription, increases bone mineral content and cross-sectional area compared to wild-type controls. We examined the influence of cellular hypoxia (1% oxygen) upon sclerostin expression and canonical Wnt signaling. Osteoblasts and osteocytes cultured under hypoxia revealed decreased sclerostin transcript and protein, and increased expression and nuclear localization of activated beta-catenin. Similarly, both hypoxia and the hypoxia mimetic DFO increased beta-catenin gene reporter activity. Hypoxia and its mimetics increased expression of the BMP antagonists gremlin and noggin and decreased Smad-1/5/8 phosphorylation. As a partial explanation for the mechanism of regulation of sclerostin by oxygen, MEF2 reporter assays revealed decreased activity. Modulation of VEGF signaling under normoxia or hypoxia revealed no influence upon Sost transcription. These data suggest that hypoxia inhibits sclerostin expression, through enhanced antagonism of BMP signaling independent of VEGF.