The multifaceted role of mast cells in joint inflammation and arthritis.

OBJECTIVE: To review current knowledge surrounding the role of mast cells in joint inflammation and arthritis. METHOD: Narrative review. RESULTS: Mast cells (MCs) are commonly observed in the synovium of the joint, particularly surrounding blood vessels and nerve endings. Some studies have reported increased MC number and degranulation in patients with osteoarthritis (OA). In two studies, MCs were the only immune cell type found in higher concentrations in synovium of OA patients compared to rheumatoid arthritis patients. Activation of MCs in OA includes signaling pathways such as immunoglobulin E/Fc epsilon Receptor 1 (IgE/FcεR1), immunoglobulin G/Fc gamma receptor (IgG/FcγR), complement, and toll-like cell surface receptor-mediated signaling, resulting in context-dependent release of either pro-inflammatory and/or anti-inflammatory mediators within the joint. Activation of MCs results in the release of pro-inflammatory mediators that ultimately contribute to inflammation of the synovium, bone remodeling, and cartilage damage. However, some studies have proposed that MCs can also exhibit anti-inflammatory effects by secreting mediators that inactivate pro-inflammatory cytokines such as interleukin 6 (IL-6). CONCLUSIONS: MCs may play a role in mediating synovial inflammation and OA progression. However, the mechanisms governing MC activation, the downstream pro- and/or anti-inflammatory effects, and their impact on osteoarthritis pathogenesis remains to be elucidated and requires extensive further study. Furthermore, it is important to establish the pathways of MC activation in OA to determine whether MCs exhibit varying phenotypes as a function of disease stage. Ultimately, such research is needed before understanding whether MCs could be targeted in OA treatments.

Calvaria critical-size defects in rats using piezoelectric equipment: a comparison with the classic trephine.

Calvarial critical-size defects in rats are used to study regeneration of both craniofacial bone and long-bones. For decades, the trephine technique has been used with no notable refinements in the procedure. The use of piezoelectric surgical equipment has increased in human clinical oral and maxillofacial surgery, neurosurgery, traumatology, and orthopedics, because the devices are easy to handle, and can cut bone without damaging sensitive soft tissues such as blood vessels, nerves, and membranes. This study evaluated and compared the surgical technique and bone regeneration process between a traditional hand-drill trephine and piezoelectric equipment in a critical-size calvaria defect in a rat model. Thirty SD male rats were randomly divided into two groups and had either a 7.9mm diameter circular defect created with trephine or a 7.0mm square defect using the piezoelectric device, both creating 49 mm2 defect areas. MicroCT and histology were performed at 45 and 75d after surgery. While trephine surgeries were performed faster than piezoelectric (25.5 minutes vs 38.5 minutes), the rate of complications was much higher, with 36% of trephine rats taking 20 minutes to achieve hemostasis. Although the extent of new bone formation was similar between the two surgical groups, the piezoelectric technique resulted in 50% less variability. No additional new bone formation was observed from 45 to 75d in both techniques. Piezoelectric technique represents a refined and more reproducible technique for calvarial defect generation in comparison to classic trephine methods.

R-spondin 2 mediates neutrophil egress into the alveolar space through increased lung permeability.

OBJECTIVE: R-spondin 2 (RSPO2) is required for lung morphogenesis, activates Wnt signaling, and is upregulated in idiopathic lung fibrosis. Our objective was to investigate whether RSPO2 is similarly important in homeostasis of the adult lung. While investigating the characteristics of bronchoalveolar lavage in RSPO2-deficient (RSPO2-/-) mice, we observed unexpected changes in neutrophil homeostasis and vascular permeability when compared to control (RSPO2+/+) mice at baseline. Here we quantify these observations to explore how tonic RSPO2 expression impacts lung homeostasis. RESULTS: Quantitative PCR (qPCR) analysis demonstrated significantly elevated myeloperoxidase (MPO) expression in bronchoalveolar lavage fluid (BALF) cells from RSPO2-/- mice. Likewise, immunocytochemical (ICC) analysis demonstrated significantly more MPO+ cells in BALF from RSPO2-/- mice compared to controls, confirming the increase of infiltrated neutrophils. We then assessed lung permeability/barrier disruption via Fluorescein Isothiocyanate (FITC)-dextran instillation and found a significantly higher dextran concentration in the plasma of RSPO2-/- mice compared to identically treated RSPO2+/+ mice. These data demonstrate that RSPO2 may be crucial for blood-gas barrier integrity and can limit neutrophil migration from circulation into alveolar spaces associated with increased lung permeability and/or barrier disruption. This study indicates that additional research is needed to evaluate RSPO2 in scenarios characterized by pulmonary edema or neutrophilia.

Jagged1 expression by osteoblast-lineage cells regulates trabecular bone mass and periosteal expansion in mice.

Loss-of-function mutations in the Notch ligand, Jagged1 (Jag1), result in multi-system developmental pathologies associated with Alagille syndrome (ALGS). ALGS patients present with skeletal manifestations including hemi-vertebrae, reduced bone mass, increased fracture incidence and poor bone healing. However, it is not known whether the increased fracture risk is due to altered bone homeostasis (primary) or nutritional malabsorption due to chronic liver disease (secondary). To determine the significance of Jag1 loss in bone, we characterized the skeletal phenotype of two Jag1-floxed conditional knockout mouse models: Prx1-Cre;Jag1(f/f) to target osteoprogenitor cells and their progeny, and Col2.3-Cre;Jag1(f/f) to target mid-stage osteoblasts and their progeny. Knockout phenotypes were compared to wild-type (WT) controls using quantitative micro-computed tomography, gene expression profiling and mechanical testing. Expression of Jag1 and the Notch target genes Hes1 and Hey1 was downregulated in all Jag1 knockout mice. Osteoblast differentiation genes were downregulated in whole bone of both groups, but unchanged in Prx1-Cre;Jag1(f/f) cortical bone. Both knockout lines exhibited changes in femoral trabecular morphology including decreased bone volume fraction and increased trabecular spacing, with males presenting a more severe trabecular osteopenic phenotype. Prx1-Cre;Jag1(f/f) mice showed an increase in marrow mesenchymal progenitor cell number and, counterintuitively, developed increased cortical thickness resulting from periosteal expansion, translating to greater mechanical stiffness and strength. Similar alterations in femoral morphology were observed in mice with canonical Notch signaling disrupted using Prx1-Cre-regulatable dominant-negative mastermind like-protein (dnMAML). Taken together, we report that 1) Jag1 negatively regulates the marrow osteochondral progenitor pool, 2) Jag1 is required for normal trabecular bone formation and 3) Notch signaling through homotypic Jag1 signaling in osteochondral progenitors, but not mature osteoblasts, inhibits periosteal expansion. Therefore, Jag1 signaling within the osteoblast lineage regulates bone metabolism in a compartment-dependent manner. Moreover, loss of Jag1 function in osteoblast lineage cells may contribute to the skeletal phenotype associated with ALGS.

An Injured Psittacosaurus (Dinosauria: Ceratopsia) From the Yixian Formation (Liaoning, China): Implications for Psittacosaurus Biology.

We describe a Psittacosaurus specimen from the Lujiatun beds of the Yixian Formation in Liaoning, China with an abnormality on its left fibula. Although a large number of Psittacosaurus specimens are known, only a single example of a pathologic Psittacosaurus has been previously noted. The specific pathology in the current specimen is believed to be a healed fibular fracture as assessed through a combination of gross morphology, microcomputed tomography (microCT), and histology data. The fracture can be identified using microCT, but the degree of remodeling and the stage of fracture repair are best determined histologically. The fracture callus is made up of radially oriented spokes of woven bone in a cartilage matrix and the original cortical bone prior to the fracture has been largely eroded. A transverse histologic section taken at the level of the fracture shows the displacement of the proximal and distal parts of the fibula. The Psittacosaurus appears to have survived the break considering the deposition of circumferential non-pathologic bone at the periosteal surface outside of the callus. The combination of gross morphological description, microCT data, and histologic data allowed for a full diagnosis of the abnormality. While some previous authors have preferred gross morphological description above other methods for assessing paleopathologies, it is evident based on this specimen that an amalgam of techniques provides greater clarity to paleopathology diagnoses. Although this Psittacosaurus lived in an environment with many predators, it was able to survive with a fracture on its hindlimb, which undoubtedly would have impacted its locomotion. Anat Rec, 299:897-906, 2016. © 2016 Wiley Periodicals, Inc.

Extracellular signaling molecules to promote fracture healing and bone regeneration.

To date, the delivery of signaling molecules for bone regeneration has focused primarily on factors that directly affect the bone formation pathways (osteoinduction) or that serve to increase the number of bone forming progenitor cells. The first commercialized growth factors approved for bone regeneration, Bone Morphogenetic Protein 2 and 7 (BMP2 and BMP7), are direct inducers of osteoblast differentiation. As well, newer generations of potential therapeutics that target the Wnt signaling pathway are also direct osteoinducers. On the other hand, some signaling molecules may play a role as mitogens and serve to increase the number of bone producing cells or may increase vascularization. This is true for factors such as Platelet Derived Growth Factor (PDGF) or Fibroblast Growth Factor (FGF). Vascular Endothelial Growth Factor (VEGF) likely has a special role. Not only does it induce new blood vessel formation, it also has direct effects on osteoblasts through endothelial cell-based BMP production. In addition to these pathways that classically have targeted bone production, there are also opportunities to target other aspects of the bone healing process such as inflammation, vascularization, and cell ingress to the fracture site. Bone regeneration is highly complex with defined, yet overlapping stages of healing. We will review established and novel extracellular signaling factors associated with various stages of fracture healing that could be targeted to promote enhanced bone regeneration. Importantly, multiple potential cell and tissues could be targeted to enhance healing in addition to focusing solely on osteoinductive therapeutics.

Osteopathology in the Equine Distal Phalanx Associated With the Development and Progression of Laminitis.

Although the equine distal phalanx and hoof lamellae are biomechanically and physiologically integrated, bony changes in the distal phalanx are poorly described in laminitis. The aims of this study were (1) to establish a laminitis grading scheme that can be applied to the wide spectrum of lesions seen in naturally occurring cases and (2) to measure and describe changes in the distal phalanx associated with laminitis using micro-computed tomography (micro-CT) and histology. Thirty-six laminitic and normal feet from 15 performance and nonperformance horses were evaluated. A laminitis grading scheme based on radiographic, gross, histopathologic, and temporal parameters was developed. Laminitis severity grades generated by this scheme correlated well with clinical severity and coincided with decreased distal phalanx bone volume and density as measured by micro-CT. Laminitic hoof wall changes included progressive ventral rotation and distal displacement of the distal phalanx with increased thickness of the stratum internum-corium tissues with lamellar wedge formation. Histologically, there was epidermal lamellar necrosis with basement membrane separation and dysplastic regeneration, including acanthosis and hyperkeratosis, corresponding to the lamellar wedge. The changes detected by micro-CT corresponded to microscopic findings in the bone, including osteoclastic osteolysis of trabecular and osteonal bone with medullary inflammation and fibrosis. Bone changes were identified in horses with mild/early stages of laminitis as well as severe/chronic stages. The authors conclude that distal phalangeal pathology is a quantifiable and significant component of laminitis pathology and may have important implications for early detection or therapeutic intervention of equine laminitis.

Biological perspectives of delayed fracture healing.

Fracture healing is a complex biological process that requires interaction among a series of different cell types. Maintaining the appropriate temporal progression and spatial pattern is essential to achieve robust healing. We can temporally assess the biological phases via gene expression, protein analysis, histologically, or non-invasively using biomarkers as well as imaging techniques. However, determining what leads to normal versus abnormal healing is more challenging. Since the ultimate outcome of fracture healing is to restore the original functions of bone, assessment of fracture healing should include not only monitoring the restoration of structure and mechanical function, but also an evaluation of the restoration of normal bone biology. Currently few non-invasive measures of biological factors of healing exist; however, recent studies that have correlated non-invasive measures with fracture healing outcome in humans have shown that serum TGFbeta1 levels appear to be an indicator of healing versus non-healing. In the future, developing additional measures to assess biological healing will improve the reliability and permit us to assess stages of fracture healing. Additionally, new functional imaging technologies could prove useful for better understanding both normal fracture healing and predicting dysfunctional healing in human patients.

A potential role for the myeloid lineage in leptin-regulated bone metabolism.

Leptin influences bone formation centrally through the hypothalamus and peripherally by acting on osteoblasts or their precursors. However, neither mechanism explains the divergent, gender-specific correlation between leptin and bone mineral density in humans. Although leptin is a potent regulator of pro-inflammatory immune responses, a potential role for leptin as an osteoimmunologic intermediate in bone metabolism has not been tested. Mice with myeloid-specific ablation of the long-form leptin receptor (ObRb) were generated using mice expressing cre-recombinase from the lysoszyme M promoter. At 12 weeks of age, the conditional knockout mice did not display any appreciable phenotype. However, at 52 weeks 2 changes were noted. First, there was a mild increase in liver inflammation. Second, a gender-specific, divergent bone phenotype was observed. Female mice displayed a consistent trend toward decreased trabecular bone parameters including reductions in bone volume fraction, trabecular number, and bone mineral content as well as a significant increase in marrow adipogenesis. Conversely, male mice lacked trabecular changes, but had statistically significant increases in cortical bone volume, thickness, and bone mineral density with equivalent total cortical volume. Since the year 2000, over 25 studies on more than 10,000 patients have sought to determine the correlation between leptin and bone mineral density. The results revealed a gender-specific correlation similar to that observed in our LysM transgenic animals. We hypothesize and show new evidence that regulation of myeloid lineage cells by leptin may facilitate their actions as an osteoimmunologic intermediate and contribute to leptin-regulated bone formation and metabolism in a gender-specific manner.

Increased osteoblastogenesis and decreased bone resorption protect against ovariectomy-induced bone loss in thrombospondin-2-null mice.

Although bone is composed primarily of extracellular matrix (ECM), the dynamic role that the ECM plays in regulating bone remodeling secondary to estrogen loss is relatively unexplored. Previous studies have shown that mice deficient in the matricellular protein thrombospondin-2 (TSP2-null) form excess endocortical bone; thus, we postulated that enhanced bone formation in TSP2-null mice could protect against ovariectomy (OVX)-induced bone loss. Wild-type (WT) OVX mice showed a significant loss of both midfemoral endocortical and proximal tibial trabecular bone, but OVX did not significantly alter TSP2-null bone. TSP2-null mice showed an increase in bone formation, as indicated by a 70% increase in serum osteocalcin two weeks post OVX and a two-fold increase in bone formation rate (BFR) five weeks post OVX as measured by dynamic histomorphometry. WT animals showed only a 20% increase in serum osteocalcin at two weeks and no change in BFR at five weeks. This increase in bone formation in TSP2-null OVX mice was accompanied by a three-fold increase in osteoprogenitor number. Although these results provide a partial explanation for the maintenance of bone geometry post-OVX, TSP2-null mice five weeks post-OVX also showed a significantly lower level of bone resorption than OVX WT mice, as determined by serum levels of the amino-terminal telopeptide of type I collagen (NTx). We conclude that the absence of TSP2 protects against OVX-induced bone loss by two complementary processes: increased formation and decreased resorption.

Skeletal dysplasia and male infertility locus on mouse chromosome 9.

In mice and humans, growth insufficiency and male infertility are common disorders that are genetically and phenotypically complex. We describe a spontaneously arising mouse mutant, chagun, that is affected by both dwarfism and male infertility. Dwarfism disproportionately affects long bones and is characterized by a defect in the proliferative zone of chondrocytes in the growth plate. Gonads of mutant males are small, with apparent germ cell loss and no evidence of mature sperm. The locus responsible for chagun is recessive and maps to distal chromosome 9, in a region homologous to human chromosome 3. This location is consistent with chagun defining a novel locus. Identification of the mutant gene will uncover the basis for another type of skeletal dysplasia and male infertility.

Thrombospondin 2, a matricellular protein with diverse functions.

Thrombospondin (TSP) 2 is a close relative of TSP1 but differs in its temporal and spatial distribution in the mouse. This difference in expression undoubtedly reflects the marked disparity in the DNA sequences of the promoters in the genes encoding the two proteins. The synthesis of TSP2 occurs primarily in connective tissues of the developing and growing mouse. In the adult animal the protein is again produced in response to tissue injury and in association with the growth of tumors. Despite the abnormalities in collagen fibrillogenesis, fragility of skin, and laxity of tendons and ligaments observed in the TSP2-null mouse, TSP2 does not appear to contribute directly to the structural integrity of connective tissue elements. Instead, emerging evidence supports a mode of action of TSP2 'at a distance', i.e. by modulating the activity and bioavailability of proteases and growth factors in the pericellular environment and, very likely, by interaction with cell-surface receptors. Thus, TSP2 qualifies as a matricellular protein, as defined in the introduction to this minireview series. The phenotype of TSP2-null mice has been very helpful in providing clues to the functions of TSP2. In addition to histological and functional abnormalities in connective tissues, these mice display an increased vascularity of the dermis and subdermal tissues, increased endosteal bone growth, a bleeding defect, and a marked adhesive defect of dermal fibroblasts. Our laboratory has established that TSP2 binds matrix metalloproteinase 2 (MMP2) and that the adhesive defect in TSP2-null fibroblasts results from increased MMP2 activity. The investigation of the basis for the other defects in the TSP2-null mouse is likely to yield equally interesting results.

Increased marrow-derived osteoprogenitor cells and endosteal bone formation in mice lacking thrombospondin 2.

The phenotype of thrombospondin 2 (TSP2)-null mice includes abnormalities in collagen fibrils and increases in ligamentous laxity, vascular density, and bleeding time. In this study, analyses by computerized tomography (CT) revealed that cortical density was increased in long bones of TSP2-null mice. Histomorphometric analysis showed that the mid-diaphyseal endosteal bone formation rate (BFR) of TSP2-null mice was increased in comparison with that of wild-type (WT) animals. Although microgeometric analysis showed that periosteal and endosteal radii were reduced, the mechanical properties of femurs from TSP2-null mice were not significantly different from those of controls, presumably because of the concomitant increase in endosteal bone mass. Bone loss in ovariectomized mice was equivalent for WT and mutant mice, a finding that indicates that TSP2-null animals are capable of normal bone resorption. To further explore the cellular basis for the increased endosteal BFR in TSP2-null mice, marrow stromal cells (MSCs) were isolated and examined in vitro. These cells were found to be present in increased numbers in a colony forming unit (CFU) assay and showed an increased rate of proliferation in vitro. We conclude that TSP2 regulates the proliferation of osteoblast progenitors, directly or indirectly, and that in its absence endosteal bone formation is increased.

Omega-3 fatty acids enhance ligament fibroblast collagen formation in association with changes in interleukin-6 production.

Altering dietary ratios of n-3 and n-6 polyunsaturated fatty acids (PUFA) represents an effective nonpharmaceutical means to improve systemic inflammatory conditions. An effect of PUFA on cartilage and bone formation has been demonstrated, and the purpose of this study was to determine the potential of PUFA modulation to improve ligament healing. The effects of n-3 and n-6 PUFA on the in vitro healing response of medial collateral ligament (MCL) fibroblasts were investigated by studying the cellular coverage of an in vitro wound and the production of collagen, PGE2, IL-1, IL-6, and TNF. Cells were exposed to a bovine serum albumin (BSA) control or either eicosapentaenoic acid (EPA, 20:5n-3) or arachidonic acid (AA, 20:4n-6) in the form of soaps loaded onto BSA for 4 days and wounded on Day 5. AA and EPA improved the healing of an in vitro wound over 72 hr. EPA increased collagen synthesis and the overall percentage of collagen produced, but AA reduced collagen production and total protein. PGE2 production was increased in the AA-treated group and decreased in the EPA-treated group, but was not affected by wounding. IL-1 was not produced at the time point evaluated, but TNF and IL-6 were both produced, and their levels varied relative to the PUFA or wounding treatment. There was a significant linear correlation (r2 = 0.57, P = 0.0045) between IL-6 level and collagen production. These results demonstrate that n-3 PUFA (represented by EPA in this study) positively affect the healing characteristics of MCL cells and therefore may represent a possible noninvasive treatment to improve ligament healing. Additionally, these results show that MCL fibroblasts produce PGE2, IL-6, and TNF and that IL-6 production is related to MCL collagen synthesis.

Porcine anterior cruciate ligament fibroblasts are similar to cells derived from the ligamentum teres, another non-healing intra-articular ligament.

Porcine ligament fibroblasts were cultured from the anterior cruciate (ACL), medial collateral (MCL), and ligamentum teres (LT). There were no apparent differences in confluent cellular morphology among the ligament cell types as evaluated by phase contrast microscopy. The proliferation rate of MCL cells from 24-120 h was significantly higher (p < 0.05) than that of cells from either the LT or the ACL. MCL cells produced more collagen and less non-collagenous protein than the LT and ACL as determined by [3H]proline incorporation. This resulted in MCL cells producing a higher percentage (37%, p < 0.05) of collagen relative to total protein than either the ACL (28%) or the LT (32%). The MCL cells produced a significantly higher percentage (34.7%, p < 0.05) of type-III collagen relative to total type-I and III collagen than either the ACL (29.2%) or the LT (29.5%). The LT and MCL cells had similar and significantly greater coverage of in vitro wounds than the ACL. This study provides the first in vitro study of the LT and demonstrates that fibroblasts from the LT and ACL, two ligaments that heal poorly, have similar in vitro characteristics, with the exception of wound healing.