Runx1, the hematopoietic lineage determining transcription factor, is present in perichondrium and chondrocytes. Here we addressed Runx1 functions, by examining expression in cartilage during mouse and human osteoarthritis (OA) progression and in response to mechanical loading. Spared and diseased compartments in knees of OA patients and in mice with surgical destabilization of the medial meniscus were examined for changes in expression of Runx1 mRNA (Q‐PCR) and protein (immunoblot, immunohistochemistry). Runx1 levels were quantified in response to static mechanical compression of bovine articular cartilage. Runx1 function was assessed by cell proliferation (Ki67, PCNA) and cell type phenotypic markers. Runx1 is enriched in superficial zone (SZ) chondrocytes of normal bovine, mouse, and human tissues. Increasing loading conditions in bovine cartilage revealed a positive correlation with a significant elevation of Runx1. Runx1 becomes highly expressed at the periphery of mouse OA lesions and in human OA chondrocyte ‘clones’ where Runx1 co‐localizes with Vcam1, the mesenchymal stem cell (MSC) marker and lubricin (Prg4), a cartilage chondroprotective protein. These OA induced cells represent a proliferative cell population, Runx1 depletion in MPCs decreases cell growth, supporting Runx1 contribution to cell expansion. The highest Runx1 levels in SZC of normal cartilage suggest a function that supports the unique phenotype of articular chondrocytes, reflected by upregulation under conditions of compression. We propose Runx1 co‐expression with Vcam1 and lubricin in murine cell clusters and human ‘clones’ of OA cartilage, participate in a cooperative mechanism for a compensatory anabolic function. J. Cell. Physiol. 230: 440–448, 2015. © 2014 Wiley Periodicals, Inc.

Introduction: Epidemiological studies have shown that there are significant gender differences in both the prevalence and the clinical manifestations of osteoarthritis (OA) (1). OA appears to be more prevalent in men than women before the age of 50. With the onset of menopause in women, both the prevalence and severity of OA in women increases (1). Small animal models of OA have shown that female mice develop less OA than males, and that ovariectomized (OVX) female mice develop worse OA than female mice with intact ovaries (2). One hypothesis is that estrogen may be a chondro-protective molecule. There has been increasing evidence in recent years that suggest sex hormones and estrogen in particular may be important in maintaining homeostasis of cartilage as well as other articular tissues (3). Notably, the alphaand betareceptors for 17-β estradiol (E2) are both expressed in joint chondrocytes of both male and females, which suggest direct effects on chondrocytes (4). However, the exact cellular and molecular mechanism(s) of E2 in OA chondroprotection are unknown. It is our hypothesis that estrogen acts to inhibit the early chondrocyte apoptosis event which occurs shortly after OA-inducing soft tissue injury.

Regenerative medicine aspires to reduce reliance on or overcome limitations associated with donor tissue-mediated repair. Structural bone allografts are commonly used in orthopedic surgery, with a high percentage of graft failure due to poor tissue integration. This problem is aggravated among elderly, those suffering from metabolic conditions, or those undergoing cancer therapies that compromise graft healing. Toward this end, we developed a synthetic graft named FlexBone, in which nanocrystalline hydroxyapatite (50 wt%) was structurally integrated with crosslinked poly(hydroxyethyl methacrylate) hydrogel, which provides dimensional stability and elasticity. It recapitulates the essential role of nanocrystalline hydroxyapatite in defining the osteoconductivity and biochemical microenvironment of bone because of its affinity for biomolecules. Here, we demonstrate that FlexBone effectively absorbed endogenously secreted signaling molecules associated with the inflammation/graft healing cascade upon being press-fit into a 5-mm rat femoral segmental defect. Further, when preabsorbed with a single dose of 400 ng recombinant human (rh) bone morphogenetic protein-2/7 heterodimer, it enabled the functional repair of the critical-sized defect by 8-12 weeks. FlexBone was stably encapsulated by the bridging bony callus and the FlexBone-callus interface was continuously remodeled. In summary, FlexBone combines the dimensional stability and osteoconductivity of structural bone allografts with desirable surgical compressibility and acquired osteoinductivity in an easy-to-fabricate and scalable synthetic biomaterial.

BACKGROUND:Investigations of naturally-occurring mutations in animal models provide important insights and valuable disease models. Lamins A and C, along with lamin B, are type V intermediate filament proteins which constitute the proteinaceous boundary of the nucleus. LMNA mutations in humans cause a wide range of phenotypes, collectively termed laminopathies. To identify the mutation and investigate the phenotype of a spontaneous, semi-dominant mutation that we have named Disheveled hair and ear (Dhe), which causes a sparse coat and small external ears in heterozygotes and lethality in homozygotes by postnatal day 10.FINDINGS:Genetic mapping identified a point mutation in the Lmna gene, causing a single amino acid change, L52R, in the coiled coil rod domain of lamin A and C proteins. Cranial sutures in Dhe/+ mice failed to close. Gene expression for collagen types I and III in sutures was deficient. Skulls were small and disproportionate. Skeletons of Dhe/+ mice were hypomineralized and total body fat was deficient in males. In homozygotes, skin and oral mucosae were dysplastic and ulcerated. Nuclear morphometry of cultured cells revealed gene dose-dependent blebbing and wrinkling.CONCLUSION:Dhe mice should provide a useful new model for investigations of the pathogenesis of laminopathies.

Inflammatory mediators and drugs which affect inflammation can influence the healing of injured tissues. Leukotrienes are potent inflammatory mediators, and similar to prostaglandins, are metabolites of arachidonic acid which can have positive or negative effects on bone and cartilage tissues. Here we tested the hypothesis that blocking the negative regulation of leukotrienes, would lead to enhanced endochondral bone formation during fracture repair. A closed femoral fracture was created in mice. Animals were divided into three groups for treatment with either montelukast sodium, a cysteinyl leukotriene type 1 receptor antagonist (trade name Singulair), zileuton, a 5-lipoxygenase enzyme inhibitor (trade name Zyflo), or carrier alone. The fractures were analyzed using radiographs, quantitative gene expression, histology and histomorphometry, and immunohistochemistry. Both the montelukast sodium group and the zileuton group exhibited enhanced fracture repair when compared with controls. Both treatment groups exhibited increased callous size and earlier bone formation when compared to controls as early as day 7. Gene expression analysis of treatment groups showed increased markers of chondrocyte proliferation and differentiation, and increased early bone formation markers when compared with controls. Treatment with montelukast sodium directly targeted the cysteinyl leukotriene type 1 receptor, leading to increased chondrocyte proliferation at early time points. These novel findings suggests a potential mechanism by which the cysteinyl leukotriene type 1 receptor acts as a negative regulator of chondrocyte proliferation, with important and previously unrecognized implications for both fracture repair, and in a broader context, systemic chondrocyte growth and differentiation.

The septoclast is a specialized, cathepsin B-rich, perivascular cell type that accompanies invading capillaries on the metaphyseal side of the growth plate during endochondral bone growth. The putative role of septoclasts is to break down the terminal transverse septum of growth plate cartilage and permit capillaries to bud into the lower hypertrophic zone. This process fails in osteoclast-deficient, osteopetrotic animal models, resulting in a progressive growth plate dysplasia. The toothless rat is severely osteopetrotic because of a frameshift mutation in the colony-stimulating factor-1 (CSF-1) gene (Csf1(tl)). Whereas CSF-1 injections quickly restore endosteal osteoclast populations, they do not improve the chondrodysplasia. We therefore investigated septoclast populations in Csf1(tl)/Csf1(tl) rats and wild-type littermates, with and without CSF-1 treatment, at 2 weeks, before the dysplasia is pronounced, and at 4 weeks, by which time it is severe. Tibial sections were immunolabeled for cathepsin B and septoclasts were counted. Csf1(tl)/Csf1(tl) mutants had significant reductions in septoclasts at both times, although they were more pronounced at 4 weeks. CSF-1 injections increased counts in wild-type and mutant animals at both times, restoring mutants to normal levels at 2 weeks. In all of the mutants, septoclasts seemed misoriented and had abnormal ultrastructure. We conclude that CSF-1 promotes angiogenesis at the chondroosseous junction, but that, in Csf1(tl)/Csf1(tl) rats, septoclasts are unable to direct their degradative activity appropriately, implying a capillary guidance role for locally supplied CSF-1.

Bone morphogenetic proteins (BMPs) play pivotal roles in bone and cartilage growth and repair. Through phenotypes of short ‐ ear ( se ) mice, which have BMP‐5 mutations, a role for BMP‐5 in some specific aspects of skeletogenesis and cartilage growth is known. This report examines BMP‐5 expression in the growth plate and in differentiating cultures of primary chondrocytes, and the effects of addition of BMP‐5 or its inhibition by anti‐BMP‐5 antibody in chondrocyte cultures. By laser capture microdissection and immunohistochemistry, we found that BMP‐5 is expressed in proliferating zone (PZ) chondrocytes and that the expression increases sharply with hypertrophic differentiation. A similar pattern was observed in differentiating cultures of primary chondrocytes, with BMP‐5 expression increasing as cells differentiated, in contrast to other BMPs. BMP‐5 added to cultures increased cell proliferation early in the culture period and also stimulated cartilage matrix synthesis. Also, BMP‐5 addition to the cultures activated phosphorylation of Smad 1/5/8 and p38 MAP kinase and caused increased nuclear accumulation of phospho‐Smads. Anti‐BMP‐5 antibody inhibited the endogenous BMP‐5, reducing cell proliferation and phospho‐Smad nuclear accumulation. Together, the results demonstrate that BMP‐5 is normally an important regulator of chondrocyte proliferation and differentiation. Whether other BMPs may compensate in BMP‐5 loss‐of‐function mutations is discussed. J. Cell. Physiol. 214:56–64, 2008. © 2007 Wiley‐Liss, Inc.

Microarray and real‐time RT‐PCR were used to examine expression changes in primary bone marrow cells and RAW 264.7 cells in response to RANKL. In silico sequence analysis was performed on a novel gene which we designate OC‐STAMP. Specific siRNA and antibodies were used to inhibit OC‐STAMP RNA and protein, respectively, and tartrate‐resistant acid phosphatase (TRAP)+ multinucleated osteoclasts were counted. Antibodies were used to probe bone tissues and western blots of RAW cell extracts +/− RANKL. cDNA overexpression constructs were transfected into RAW cells and the effect on RANKL‐induced differentiation was studied. OC‐STAMP was very strongly up‐regulated during osteoclast differentiation. Northern blots and sequence analysis revealed two transcripts of 2 and 3.7 kb differing only in 3′UTR length, consistent with predictions from genome sequence. The mRNA encodes a 498 amino acid, multipass transmembrane protein that is highly conserved in mammals. It has little overall homology to other proteins. The carboxy‐terminal 193 amino acids, however, are significantly similar to the DC‐STAMP family consensus sequence. DC‐STAMP is a transmembrane protein required for osteoclast precursor fusion. Knockdown of OC‐STAMP mRNA by siRNA and protein inhibition by antibodies significantly suppressed the formation of TRAP+, multinucleated cells in differentiating osteoclast cultures, with many TRAP+ mononuclear cells present. Conversely, overexpression of OC‐STAMP increased osteoclastic differentiation of RAW 264.7 cells. We conclude that OC‐STAMP is a previously unknown, RANKL‐induced, multipass transmembrane protein that promotes the formation of multinucleated osteoclasts. J. Cell. Physiol. 215: 497–505, 2008. © 2007 Wiley‐Liss, Inc.

Osteoclasts differentiate from hematopoietic mononuclear precursor cells under the control of both colony stimulating factor-1 (CSF-1, or M-CSF) and receptor activator of NF-κB ligand (RANKL, or TRANCE, TNFSF11) to carry out bone resorption. Using high density gene microarrays, we followed gene expression changes in long bone RNA when CSF-1 injections were used to restore osteoclast populations in the CSF-1-null toothless (csf1tl/csf1tl) osteopetrotic rat. We found that ovarian cancer G-protein-coupled receptor 1 (OGR1, or GPR68) was strongly up-regulated, rising >6-fold in vivo after 2 days of CSF-1 treatments. OGR1 is a dual membrane receptor for both protons (extracellular pH) and lysolipids. Strong induction of OGR1 mRNA was also observed by microarray, real-time RT-PCR, and immunoblotting when mouse bone marrow mononuclear cells and RAW 264.7 pre-osteoclast-like cells were treated with RANKL to induce osteoclast differentiation. Anti-OGR1 immunofluorescence showed intense labeling of RANKL-treated RAW cells. The time course of OGR1 mRNA expression suggests that OGR1 induction is early but not immediate, peaking 2 days after inducing osteoclast differentiation both in vivo and in vitro. Specific inhibition of OGR1 by anti-OGR1 antibody and by small inhibitory RNA inhibited RANKL-induced differentiation of both mouse bone marrow mononuclear cells and RAW cells in vitro, as evidenced by a decrease in tartrate-resistant acid phosphatase-positive osteoclasts. Taken together, these data indicate that OGR1 is expressed early during osteoclastogenesis both in vivo and in vitro and plays a role in osteoclast differentiation.

Escherichia coli beta-galactosidase (beta-gal), encoded by the lacZ gene, has become an essential tool in studies of gene expression and function in higher eukaryotes. lac-Z is widely used as a marker gene to detect expression of transgenes or Cre recombinase driven by tissue-specific promoters. The timing and location of promoter activity is easily visualized in whole embryos or specific tissues using the cleavable, chromogenic substrate, 5-bromo-4-chloro-3-indolyl-D-galactopyranoside (X-gal). The tissue specificity of promoters in transgenic constructs is routinely tested by using a promoter of choice to drive lacZ. Alternatively, the targeted expression of Cre recombinase to perform in vivo recombination of loxP sites can be visualized by beta-gal staining in mice carrying a Cre-activated lacZ transgene, such as the ROSA26 strain. In the course of our investigations, we examined beta-gal activity in bone tissue from genetically normal mice using standard detection methodology and found very high endogenous activity in bone-resorbing osteoclasts. This was true in frozen, paraffin, and glycol methacrylate sections. X-gal staining colocalized with the osteoclast marker, tartrate-resistant acid phosphatase ( TRAP). beta-gal activity was present in osteoclasts in long bones, in the mandible, and in both neonatal and more mature animals. We present this brief article as a caution to those testing genetic models of skeletal gene expression using beta-gal as a marker gene.

The compartmentalization of myosin isoforms within a muscle cell (Gauthier: J. Cell Biol. 110:693–701, 1990) suggests that myosin might be assembled directly into thick filaments at sites where it is synthesized. We therefore examined myofibrils by immunoelectron microscopy to determine whether ribosomes are associated with thick filaments under conditions in which new myosin can be identified. We used the embryonic chick anterior latissimus dorsi (ALD), a slow muscle that is induced, by curare, to synthesize a fast myosin isoform that is not normally present. Myosin was localized in situ, using a gold‐labeled monoclonal antibody that recognizes the new isoform. The gold marker, as expected, was localized preferentially to the A band. There was an overall increase of fivefold in the number of gold particles per μm2 of A band in the curare‐treated compared to the normal ALD, indicating that the labeled isoform was largely newly formed. There was a corresponding preferential distribution of ribosomes at the A band, especially in the H‐band region, and the number of ribosomes per μm2 of A band was nearly twice as high in the curare‐treated as in the normal muscle. Ribosomes were located between thick filaments, often aligned in rows. We conclude that ribosomes are located within the filament lattice, and therefore that they are available for local myosin synthesis. © 1993 Wiley‐Liss Inc.