The role of biomineralization in disorders of skeletal development and tooth formation.

The major mineralized tissues are bone and teeth, which share several mechanisms governing their development and mineralization. This crossover includes the hormones that regulate circulating calcium and phosphate concentrations, and the genes that regulate the differentiation and transdifferentiation of cells. In developing endochondral bone and in developing teeth, parathyroid hormone-related protein (PTHrP) acts in chondrocytes to delay terminal differentiation, thereby increasing the pool of precursor cells.

RANKL-mediated osteoclast formation from murine RAW 264.7 cells.

Extensive research efforts over the years have provided us with great insights into how bone-resorbing osteoclasts (OCs) develop and function and, based on such work, valuable antiresorptive therapies have been developed to help combat the excessive bone loss that occurs in numerous skeletal disorders. The RAW 264.7 murine cell line has proven to be an important tool for in vitro studies of OC formation and function, having particular advantages over the use of OCs generated from primary bone marrow cell populations or directly isolated from murine bones.

Isolation and culture of primary chicken osteoclasts.

Osteoclasts originate from hematopoietic myeloid progenitors that differentiate into specialized multinucleated cells uniquely capable of resorbing bone in both physiological and pathological conditions. Osteoclast numbers and degradative activities increase in various inflammatory disorders of bone and certain bone oncologies, thereby causing bone loss that may weaken the skeleton, increase fracture incidence, and disturb marrow function. Many valuable insights have been obtained through the use of osteoclasts directly isolated from the bones of chickens fed a low calcium diet to enhance osteoclastogenesis and bone resorption.

Cellular mechanism of decreased bone in Brtl mouse model of OI: imbalance of decreased osteoblast function and increased osteoclasts and their precursors.

The Brtl mouse, a knock-in model for moderately severe osteogenesis imperfecta (OI), has a G349C substitution in half of type I collagen alpha1(I) chains. We studied the cellular contribution to Brtl bone properties. Brtl cortical and trabecular bone are reduced before and after puberty, with BV/TV decreased 40-45%.

RANKL stimulates inducible nitric-oxide synthase expression and nitric oxide production in developing osteoclasts. An autocrine negative feedback mechanism triggered by RANKL-induced interferon-beta via NF-kappaB that restrains osteoclastogenesis and bone resorption.

Nitric oxide (NO) is a multifunctional signaling molecule and a key vasculoprotective and potential osteoprotective factor. NO regulates normal bone remodeling and pathological bone loss in part through affecting the recruitment, formation, and activity of bone-resorbing osteoclasts. Using murine RAW 264.

Human microvascular endothelial cell activation by IL-1 and TNF-alpha stimulates the adhesion and transendothelial migration of circulating human CD14+ monocytes that develop with RANKL into functional osteoclasts.

Unlabelled: Circulating pre-OCs may be recruited to locally inflamed sites through specific interactions with activated microvasculature. We found that HMVECs stimulated the adhesion and TEM of circulating pre-OCs, in an ICAM-1- and CD44-dependent manner, leading to greater RANKL-induced OC formation and bone pit resorption.

Introduction: Inflammation is critical for healing processes but causes severe tissue destruction when chronic.

Organization of transcriptional regulatory machinery in osteoclast nuclei: compartmentalization of Runx1.

The osteoclast is a highly polarized multinucleated cell that resorbs bone. Using high resolution immunofluorescence microscopy, we demonstrated that all nuclei of an osteoclast are transcriptionally active. Each nucleus within the osteoclast contains punctately organized microenvironments where regulatory complexes that support transcriptional and post-transcriptional control reside.

Stromal cell-derived factor-1 binding to its chemokine receptor CXCR4 on precursor cells promotes the chemotactic recruitment, development and survival of human osteoclasts.

Osteoclasts (Oc) derive from hematopoietic precursors present in the circulation and bone marrow, and they differentiate into multinucleated bone-resorbing cells in response to the dual essential signals receptor activator of NF-kappaB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF) primarily provided by bone marrow stromal cells (BMSC) and osteoblasts (Ob). However, little is known about signals that direct Oc precursors from the circulation into bone or control their migration within the marrow. Stromal cell-derived factor-1 (SDF-1 or CXCL12) is a chemokine highly expressed by bone endothelium, BMSC, and immature Ob that is essential for the normal homing, early development, and survival of various hematopoietic progenitor cells.

CCR1 chemokines promote the chemotactic recruitment, RANKL development, and motility of osteoclasts and are induced by inflammatory cytokines in osteoblasts.

Unlabelled: Chemoattractants that recruit OC precursors to locally inflamed sites of resorption are not well known. A chemokine receptor, CCR1, was expressed in OC precursors and elevated in mature OCs, and its ligands promoted OC precursor recruitment, RANKL development, and OC motility. Cytokines induced OB release of such chemokines, which may therefore significantly contribute to inflammatory bone loss.

SDF-1 increases recruitment of osteoclast precursors by upregulation of matrix metalloproteinase-9 activity.

Although chemokines play essential roles in the trafficking and homing of many circulating hematopoietic cell types, their potential influences on osteoclast (OC) recruitment or bone remodeling are not well known. Therefore, chemokine receptor expression was analyzed by RNase protection assay during OC formation induced by RANKL in a murine mononuclear cell line (RAW 264.7).

Stromal cell-derived factor-1 (SDF-1) recruits osteoclast precursors by inducing chemotaxis, matrix metalloproteinase-9 (MMP-9) activity, and collagen transmigration.

Unlabelled: Signals targeting OCs to bone and resorption sites are not well characterized. A chemoattractant receptor (CXCR4), highly expressed in murine OC precursors, mediated their chemokine (SDF-1)-induced chemoattraction, collagen transmigration, and MMP-9 expression. Thus, bone vascular and stromal SDF-1 may direct OC precursors into bone and marrow sites for development and bone resorption.

Basic fibroblast growth factor stimulates osteoclast recruitment, development, and bone pit resorption in association with angiogenesis in vivo on the chick chorioallantoic membrane and activates isolated avian osteoclast resorption in vitro.

Increased local osteoclast (OC)-mediated bone resorption coincides with angiogenesis in normal bone development and fracture repair, as well as in pathological disorders such as tumor-associated osteolysis and inflammatory-related rheumatoid arthritis or periodontal disease. Angiogenic stimulation causes recruitment, activation, adhesion, transmigration, and differentiation of hematopoietic cells which may therefore enable greater numbers of pre-OC to emigrate from the circulation and develop into bone-resorptive OCs. A chick chorioallantoic membrane (CAM) model, involving coimplantation of a stimulus in an agarose plug directly adjacent to a bone chip was used to investigate if a potent angiogenic stimulator, basic fibroblast growth factor (bFGF), could promote OC recruitment, differentiation, and resorption in vivo.

A new cytometric method for the immunophenotypic characterization of bone-derived human osteoclasts.

Background: Osteoclast cell function relates to bone resorption. Isolation and characterization of these cells from in vivo sources remain difficult. The aim of this study was to show the feasibility of using flow cytometry to identify and characterize human mature osteoclasts obtained from bone tissues.

New alternatively spliced form of galectin-3, a member of the beta-galactoside-binding animal lectin family, contains a predicted transmembrane-spanning domain and a leucine zipper motif.

Osteoclasts or their precursors interact with the glycoprotein-enriched matrix of bone during extravasation from the vasculature, and upon attachment prior to resorption. Reverse transcriptase-PCR studies showed that two new alternatively spliced forms of chicken galectin-3, termed Gal-3TM1 and Gal-3TR1, were enriched and preferentially expressed in highly purified chicken osteoclast-like cells. Gal-3TM1 and Gal-3TR1 mRNA were also detected in chicken intestinal tissue, but not in kidney, liver, or lung.