The Role of PTX3 in Mineralization Processes and Aging-Related Bone Diseases.

The Long Pentraxin 3 (PTX3) is a multifunctional glycoprotein released by peripheral blood leukocytes and myeloid dendritic cells in response to primary pro-inflammatory stimuli, that acts as a non-redundant component of the humoral arm of innate immunity. In addition to the primary role in the acute inflammatory response, PTX3 seems to be involved in other physiological and pathological processes. Indeed, PTX3 seems to play a pivotal role in the deposition and remodeling of bone matrix during the mineralization process, promoting osteoblasts differentiation and activity. Recently, PTX3 was seen to be involved in the ectopic calcifications' formation in breast cancer disease. In this regard, it has been observed that breast cancer tumors characterized by high expression of PTX3 and high amount of Breast Osteoblast Like Cells (BOLCs) showed several Hydroxyapatite (HA) microcalcifications, suggesting a likely role for PTX3 in differentiation and osteoblastic activity in both bone and extra-bone sites. Furthermore, given its involvement in bone metabolism, several studies agree with the definition of PTX3 as a molecule significantly involved in the pathogenesis of age-related bone diseases, such as osteoporosis, both in mice and humans. Recent results suggest that genetic and epigenetic mechanisms acting on PTX3 gene are also involved in the progression of these diseases. Based on these evidences, the aim of our systemic review was to offer an overview of the variety of biological processes in which PTX3 is involved, focusing on bone mineralization, both in a physiological and pathological context.

The Immune Response to Allogeneic Differentiated Mesenchymal Stem Cells in the Context of Bone Tissue Engineering.

The use of allogeneic differentiated mesenchymal stem cells (MSCs) to mediate bone formation may be a potential alternative to the current gold standards of bone repair. Although it is known that undifferentiated MSCs are immunomodulatory and weakly immunogenic, the host immune reaction to differentiated MSCs is less known. Implantation of allogeneic osteogenic or chondrogenically differentiated MSC pellets may be a promising route to induce bone repair via the processes of intramembranous and endochondral ossification. This review summarizes the current literature surrounding the immune response to these allogeneic differentiated stem cells in the context of bone repair and replacement. Although there have been great developments in researching the effects of allogeneic differentiated cells on the host immune system, lack of standardized preclinical assays has limited their progression to the clinics. Future investigations are required to identify the host immune cells having a positive or negative effect on bone formation mediated by these allogeneic differentiated MSCs to move the use of these cells toward future clinical bone repair therapies.

Increased interleukin (IL)-20 and IL-24 target osteoblasts and synovial monocytes in spondyloarthritis.

The pathogenesis of spondyloarthritis (SpA) involves activation of the innate immune system, inflammation and new bone formation. The two cytokines interleukin (IL)-20 and IL-24 have been shown to link innate immune activation and tissue homeostasis. We hypothesized that these two cytokines are secreted as part of activation of the innate immune system and affect bone homeostasis in SpA. IL-20 and IL-24 were measured in plasma from axial SpA patients (n = 83). Peripheral SpA patients (n = 16) were included for in-vitro cell culture studies. The plasma IL-20 and IL-24 levels were increased in SpA patients compared with healthy controls (HCs) by 57 and 83%, respectively (both P < 0·0001). The Toll-like receptor 4-induced secretion of the two cytokines was greater in SpA peripheral blood mononuclear cells (PBMCs) compared with HC PBMCs. IL-20 and IL-24 increased the production of monocyte chemoattractant protein-1 by activated SpA synovial fluid monocytes, decreased the production of Dickkopf-1 by SpA fibroblast-like synovial cells and induced mineralization in human osteoblasts. Taken together, our findings indicate disease-aggravating functions of IL-20 and IL-24 in SpA.

Characterization of a Monoclonal Antibody Directed against Mytilus spp Larvae Reveals an Antigen Involved in Shell Biomineralization.

The M22.8 monoclonal antibody (mAb) developed against an antigen expressed at the mussel larval and postlarval stages of Mytilus galloprovincialis was studied on adult samples. Antigenic characterization by Western blot showed that the antigen MSP22.8 has a restricted distribution that includes mantle edge tissue, extrapallial fluid, extrapallial fluid hemocytes, and the shell organic matrix of adult samples. Other tissues such as central mantle, gonadal tissue, digestive gland, labial palps, foot, and byssal retractor muscle did not express the antigen. Immunohistochemistry assays identified MSP22.8 in cells located in the outer fold epithelium of the mantle edge up to the pallial line. Flow cytometry analysis showed that hemocytes from the extrapallial fluid also contain the antigen intracellularly. Furthermore, hemocytes from hemolymph have the ability to internalize the antigen when exposed to a cell-free extrapallial fluid solution. Our findings indicate that hemocytes could play an important role in the biomineralization process and, as a consequence, they have been included in a model of shell formation. This is the first report concerning a protein secreted by the mantle edge into the extrapallial space and how it becomes part of the shell matrix framework in M. galloprovincialis mussels.

Interferon Gamma-treated Dental Pulp Stem Cells Promote Human Mesenchymal Stem Cell Migration In Vitro.

INTRODUCTION: Chronic inflammation disrupts dental pulp regeneration by disintegrating the recruitment process of progenitors for repair. Bone marrow-derived mesenchymal stem cells (BM-MSCs) share the common features with dental pulp stem cells (DPSCs). The aim of the study was to investigate the migration of BM-MSCs toward DPSCs in response to inflammatory chemoattractants. Additionally, our studies also delineated the signaling mechanisms from BM-MSCs in mediating the proliferation and differentiation of DPSCs in vitro. METHODS: Human DPSCs and BM-MSCs between passages 2 and 4 were used and were grown in odontogenic differentiation medium. Mineralization was determined by alizarin red staining analysis. Migration was assessed using crystal violet staining in cells grown in Boyden chamber Transwell inserts (Corning Inc Foundation, Tewksbury, MA). The mineralization potential of DPSCs was evaluated using alkaline phosphatase activity assay. Real-time polymerase chain reaction analysis was performed to assess the gene expression profile of chemokine (C-X-C motif) ligand (Cxcl) 3, 5, 6, 10, 11, 12, 14, and 16; stromal cell-derived factor (SDF) α; vascular endothelial growth factor; and fibroblast growth factor. RESULTS: Interferon gamma (FN-γ) treatment significantly abrogated the differentiation potential of DPSCs as shown by using alizarin red and alkaline phosphatase activity analysis. An increase in the migration of BM-MSCs was documented when cocultured with IFN-γ-treated DPSCs. RNA expression studies showed an increase in the levels of Cxcl6 and Cxcl12 in BM-MSCs when cocultured with IFN-γ-treated DPSCs. Additionally, an up-regulation of proangiogenic factors vascular endothelial growth factor and fibroblast growth factor were observed in DPSCs exposed to IFN-γ. CONCLUSIONS: Our findings indicate that inflamed IFN-γ-treated DPSCs release factors (presumably Cxcl6 and 12) that contribute to the homing of MSCs. This model might provide a potential research tool for studying MSC-DPSC cross talk and for future studies involving the recruitment and sustainability of progenitor stem cells sustaining the inflammatory cascade to treat pulp inflammation.

Cloning of ovocalyxin-36, a novel chicken eggshell protein related to lipopolysaccharide-binding proteins, bactericidal permeability-increasing proteins, and plunc family proteins.

The avian eggshell is a composite biomaterial composed of noncalcifying eggshell membranes and the overlying calcified shell matrix. The shell is deposited in a uterine fluid where the concentration of different protein species varies at different stages of its formation. The role of avian eggshell proteins during shell formation remains poorly understood, and we have sought to identify and characterize the individual components in order to gain insight into their function during elaboration of the eggshell. In this study, we have used direct sequencing, immunochemistry, expression screening, and EST data base mining to clone and characterize a 1995-bp full-length cDNA sequence corresponding to a novel chicken eggshell protein that we have named Ovocalyxin-36 (OCX-36). Ovocalyxin-36 protein was only detected in the regions of the oviduct where egg-shell formation takes place; uterine OCX-36 message was strongly up-regulated during eggshell calcification. OCX-36 localized to the calcified eggshell predominantly in the inner part of the shell, and to the shell membranes. BlastN data base searching indicates that there is no mammalian version of OCX-36; however, the protein sequence is 20-25% homologous to proteins associated with the innate immune response as follows: lipopolysaccharide-binding proteins, bactericidal permeability-increasing proteins, and Plunc family proteins. Moreover, the genomic organization of these proteins and OCX-36 appears to be highly conserved. These observations suggest that OCX-36 is a novel and specific chicken eggshell protein related to the superfamily of lipopolysaccharide-binding proteins/bactericidal permeability-increasing proteins and Plunc proteins. OCX-36 may therefore participate in natural defense mechanisms that keep the egg free of pathogens.

Impaired skeletal development in interleukin-6-transgenic mice: a model for the impact of chronic inflammation on the growing skeletal system.

OBJECTIVE: To identify the mediator responsible for the impact of chronic inflammation on skeletal development in children (bone loss, defective peak bone mass accrual, stunted growth), we evaluated the effects of chronic interleukin-6 (IL-6) overexpression on the skeletons of growing prepubertal mice. METHODS: We studied IL-6-transgenic mice that had high circulating IL-6 levels since birth. Trabecular and cortical bone structure were analyzed by microcomputed tomography. Epiphyseal ossification, growth plates, and calvariae were studied by histology/histomorphometry. Osteoclastogenesis, osteoblast function/differentiation, and the effects of IL-6 on bone cells were studied in vitro. Osteoblast gene expression was evaluated by reverse transcriptase-polymerase chain reaction. The mineral apposition rate was evaluated dynamically in cortical bone by in vivo double fluorescence labeling. RESULTS: In prepubertal IL-6-transgenic mice, we observed osteopenia, with severe alterations in cortical and trabecular bone microarchitecture, as well as uncoupling of bone formation from resorption, with decreased osteoblast and increased osteoclast number and activity. Increased osteoclastogenesis and reduced osteoblast activity, secondary to decreased precursor proliferation and osteoblast function, were present. IL-6-transgenic mice also showed impaired development of growth plates and epiphyseal ossification centers. Intramembranous and endochondral ossification and the mineral apposition rate were markedly affected, showing the presence of defective ossification. CONCLUSION: Chronic overexpression of IL-6 alone induces a skeletal phenotype closely resembling growth and skeletal abnormalities observed in children with chronic inflammatory diseases, pointing to IL-6 as a pivotal mediator of the impact of chronic inflammation on postnatal skeletal development. We hypothesize that IL-6-modifying drugs may reduce skeletal defects and prevent the growth retardation associated with these diseases.

Immunosuppression with FK506 increases bone induction in demineralized isogeneic and xenogeneic bone matrix in the rat.

The aim of the present study was to investigate a systemic induction of bone formation in rats by immunosuppression with FK506 (1 mg/kg body weight intraperitoneally [ip]) in a model of osteoinduction of isogeneic and xenogeneic demineralized bone matrix (DBM) for a period of 28 days. In particular, alterations of in vitro cytokine synthesis and changes of lymphocyte subsets were studied. DBM was implanted intramuscularly in the abdominal wall of Lewis rats (seven per group). Blood was sampled on days -7, 0, 7, and 28 for determination of in vitro tumor necrosis factor a (TNF-alpha) synthesis and lymphocyte subsets by flow cytometry (CD3+, CD4+, CD8+, CD45+, ED9+, and Ia+ antibodies). Ossicles of de novo formed bone and the tibias were removed on day 28 after double tetracycline labeling for histomorphometric analysis. Immunosuppression with FK506 significantly decreased lipopolysaccharide (LPS)-stimulated in vitro cytokine synthesis after 7 days and 28 days (p < 0.05). Compared with control animals FK506 treatment significantly increased the volume of induced bone in isogeneic (2.1 +/- 0.3 mm3 vs. 10.8 +/- 0.9 mm3) and xenogeneic (O mm3 vs. 4.7 +/- 0.8 mm3) DBM. Bone histomorphometry of the tibias revealed that immunosuppression increased both bone formation and bone resorption, accompanied by a significant reduction in the relative trabecular area (Tb.Ar). FK506 caused a decrease in the counts of CD8+ T cells probably because of destruction or dislocation of these cells. This suggests that the amount of CD8+ cells and the degree of T cell activation in terms of mean fluorescence intensity (MFI) may be associated with bone metabolism. In support of this, statistical analysis revealed a significant positive correlation between parameters of bone formation as well as bone resorption and the CD4+/CD8+ ratio. There was a significant negative correlation between parameters of remodeling of the metaphysis of the tibia and induced bone volume (BV), respectively, and MFI values of CD3+/Ia+ cells. These findings suggest an important role of T lymphocytes in bone formation and bone resorption in vivo. FK506 caused a marked increase of bone formation in DBM. However, the conclusion that immunosuppression increases fracture healing warrants further investigation.

Type I collagen influences cartilage calcification: an immunoblocking study in differentiating chick limb-bud mesenchymal cell cultures.

Chick limb-bud mesenchymal cells, plated in high-density micro-mass culture, differentiate and form a matrix resembling chick epiphyseal cartilage. In the presence of 4 mM inorganic phosphate or 2.5 mM beta-glycerophosphate mineral deposits upon this matrix forming a mineralized tissue that, based on electron microscopy, x-ray diffraction and Fourier Transform Infrared microspectoscopy, is like that of chick calcified cartilage. In this culture system the initial mineral deposits are found on the periphery of the chondrocyte nodules. During differentiation of the cells in the high-density micro-mass cultures there is a switch from expression of type I collagen to type II, and then to type X collagen. However, type I collagen persists in the matrix. Because there is some debate about whether type I collagen influences cartilage calcification, an immunoblocking technique was used to determine the importance of type I collagen on the mineralization process in this system. Studies using nonspecific goat anti-chick IgG demonstrated that 1-100 ng/ml antibody added with the media after the cartilage nodules had developed (day 7) had no effect on the accumulation of mineral in the cultures. Nonspecific antibody added before day 7 blocked development of the cultures. Parallel solution based cell-free studies showed that IgG did not have a strong affinity for apatite crystals, and had no significant effect on apatite crystal growth. Type I collagen antibodies (1-200 ng/ml) added to cultures one time on day 9 (before mineralization started), or on day 11 (at the start of mineralization), slightly inhibited the accumulation of mineral. There was a statistically significant decrease in mineral accretion with 100 or 200 ng/ml collagen antibody addition continuously after these times. Fab' fragments of nonspecific and type I collagen antibodies had effects parallel to those of the intact antibodies, indicating that the decreased mineralization was not attributable to the presence of the larger, bulkier antibodies. The altered accumulation of mineral was not associated with cell death in the presence of antibody (demonstrated by fluorescent labeling of DNA) or with increased apoptosis (TUNEL-stain). In the immunoblocked cultures, EM analysis demonstrated that mineral continued to deposit on collagen fibrils, but there appeared to be fewer deposits. The data demonstrate that type I collagen is important for the mineralization of these cultures.