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Orthopedic surgeons sometimes combine recombinant, human BMP-2 with autograft bone when dealing with problematic osseous fractures. Although some case reports indicate success with this off-label strategy, there have been no randomized controlled trials. Moreover, a literature search revealed only one pre-clinical study and this was in a cranial defect model. The present project examined the consequences of combining BMP-2 with particles of living bone in a rat femoral defect model. Human bone particles were recovered with a reamer-irrigator-aspirator (RIA). To allow acceptance of the xenograft as surrogate autograft, rats were administered an immunosuppressive cocktail that does not interfere with bone healing. Implantation of 200 µg living bone particles generated a small amount of new bone and defects did not heal. Graded amounts of BMP-2 that alone provoked no healing (1.1 µg), borderline healing (5.5 µg), or full healing (11 µg) were added to this amount of bone particles. Addition of BMP-2 (1.1 µg) increased osteogenesis, and produced bridging in 2 of 7 defects. The combination of BMP-2 (5.5 µg) and bone particles made healing more reliable and advanced the maturation of the regenerate. Bone formation with BMP-2 (11 µg) and bone particles showed improved maturation. Thus, the combination of autograft and BMP-2 may be helpful clinically under conditions where the healing response is suboptimal. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2137-2145, 2016. Clinical significance These data support the clinical use of recombinant, human BMP-2 with autograft bone when treating large segmental osseous defects. The combination leads to greater bone formation and accelerates the maturation of the regenerate.

Purpose: We investigated the in vitro and in vivo anti-multiple myeloma activity of monoclonal antibody (mAb) 1339, a high-affinity fully humanized anti-interleukin 6 mAb (immunoglobulin G1), alone and in combination with conventional and novel anti-multiple myeloma agents, as well as its effect on bone turnover. Experimental Design: We examined the growth inhibitory effect of 1339 against multiple myeloma cell lines in the absence and in the presence of bone marrow stromal cells, alone or in combination with dexamethasone, bortezomib, perifosine, and Revlimid. Using the severe combined immunodeficient (SCID)–hu murine model of multiple myeloma, we also examined the effect of 1339 on multiple myeloma cell growth and multiple myeloma bone disease. Results: mAb 1339 significantly inhibited growth of multiple myeloma cell in the presence of bone marrow stromal cell in vitro, associated with inhibition of phosphorylation of signal transducer and activator of transcription 3, extracellular signal-regulated kinase 1/2, and Akt. In addition, mAb 1339 enhanced cytotoxicity induced by dexamethasone, as well as bortezomib, lenalidomide, and perifosine, in a synergistic fashion. Importantly mAb 1339 significantly enhanced growth inhibitory effects of dexamethasone in vivo in SCID-hu mouse model of multiple myeloma. mAb 1339 treatment also resulted in inhibition of osteoclastogenesis in vitro and bone remodeling in SCID-hu model. Conclusions: Our data confirm in vitro and in vivo anti-multiple myeloma activity of, as well as inhibition of bone turnover by, fully humanized mAb 1339, as a single agent and in combination with conventional and novel agents, providing a rationale for its clinical evaluation in multiple myeloma. (Clin Cancer Res 2009;15(23):7144–52)

Decreased activity of osteoblasts (OBs) contributes to osteolytic lesions in multiple myeloma (MM). The production of the soluble Wnt inhibitor Dickkopf-1 (DKK1) by MM cells inhibits OB activity, and its serum level correlates with focal bone lesions in MM. Therefore, we have evaluated bone anabolic effects of a DKK1 neutralizing antibody (BHQ880) in MM. In vitro BHQ880 increased OB differentiation, neutralized the negative effect of MM cells on osteoblastogenesis, and reduced IL-6 secretion. In a severe combined immunodeficiency (SCID)–hu murine model of human MM, BHQ880 treatment led to a significant increase in OB number, serum human osteocalcin level, and trabecular bone. Although BHQ880 had no direct effect on MM cell growth, it significantly inhibited growth of MM cells in the presence of bone marrow stromal cells (BMSCs) in vitro. This effect was associated with inhibition of BMSC/MM cell adhesion and production of IL-6. In addition, BHQ880 up-regulated β-catenin level while down-regulating nuclear factor-κB (NF-κB) activity in BMSC. Interestingly, we also observed in vivo inhibition of MM cell growth by BHQ880 treatment in the SCID-hu murine model. These results confirm DKK1 as an important therapeutic target in myeloma and provide the rationale for clinical evaluation of BHQ880 to improve bone disease and to inhibit MM growth.

The calcitonin receptor (CTR) is expressed in a wide variety of tissues and cell types. In bone, its expression is restricted to osteoclasts, the cells that mediate bone resorption. The human CTR (hCTR) gene has a complex structural organization that exhibits similarity to the porcine (pCTR) and mouse (mCTR) CTR genes. In these species, alternative splicing of a single gene generates multiple CTR isoforms that are distributed in both tissue-specific and species-specific patterns. However, the structural organization of the 5' putative regulatory region and transcriptional mechanisms responsible for tissue-specific expression of the different CTR isoforms are not fully defined. The present studies were undertaken to characterize the structural organization of the 5'-region of the hCTR and identify the regulatory regions involved in osteoclast-specific transcriptional activation. Analysis of mRNA prepared from human osteoclasts using reverse transcription-polymerase chain reaction (RT-PCR) and transient transfection of hCTR promoter-luciferase reporter constructs identified two regions in the 5'-flanking sequence of the hCTR gene that regulated CTR gene expression in osteoclasts. Both of these putative promoters were responsive to the osteoclast-inducing cytokine, receptor activator of NF-kappaB ligand (RANKL) and demonstrated trans-activation by the RANKL-induced transcription factor nuclear factor of activated T cells (NFATc1), consistent with a role in regulating CTR gene expression in osteoclasts.

As the only cells capable of efficiently resorbing bone, osteoclasts are central mediators of both normal bone remodeling and pathologies associates with excessive bone resorption. However, despite the clear evidence of interplay between osteoclasts and the bone surface in vivo, the role of the bone substrate in regulating osteoclast differentiation and activation at a molecular level has not been fully defined. Here, we present the first comprehensive expression profiles of osteoclasts differentiated on authentic resorbable bone substrates. This analysis has identified numerous critical pathways coordinately regulated by osteoclastogenic cytokines and bone substrate, including the transition from proliferation to differentiation, and sphingosine-1-phosphate signaling. Whilst, as expected, much of this program is dependent upon integrin beta 3, the pre-eminent mediator of osteoclast-bone interaction, a surprisingly significant portion of the bone substrate regulated expression signature is independent of this receptor. Together, these findings identify an important hitherto underappreciated role for bone substrate in osteoclastogenesis.

Analysis of tissues retrieved from the bone-pannus interface from patients with rheumatoid arthritis (RA) and studies in animal models of inflammatory arthritis provide strong evidence that osteoclasts, the cells that are essential for physiological bone resorption, are responsible for articular bone destruction in RA. However, current treatments that specifically target osteoclast-mediated bone resorption in RA have not been successful in preventing bone erosions, and new therapeutic strategies are needed. It has been noted that, although osteoclast precursors are present within the bone microenvironment at sites of pathological bone resorption, cells expressing the full morphological and functional properties of mature osteoclasts are restricted to the immediate bone surface and adjacent calcified cartilage. These findings provide evidence that, in addition to requirements for specific cytokines, interaction of osteoclast precursors with these mineralised matrices results in activation of specific signal pathways and the induction of unique gene products that are essential for terminal osteoclast differentiation and activation. These studies are designed to define the gene products and signalling pathways regulated by bone and calcified cartilage, to identify new molecular targets and novel therapeutic approaches for preventing osteoclast-mediated joint destruction in RA and related forms of pathological bone loss.

Osteoadherin is a cell binding keratan sulfate proteoglycan which was recently isolated from mineralized bovine bone and subsequently cloned and sequenced. For studies of osteoadherin expression in rat tissues we isolated and sequenced a 1.3-kbp partial cDNA covering most of the coding region using a rat calvaria cDNA library. The most 5' end of the cDNA was obtained by reverse transcription PCR from the bone total RNA preparation. The deduced, translated protein sequence containing 423 amino acid residues shows high sequence identity to mouse, bovine and human osteoadherin except in the very acidic C-terminal region. However, the rat counterpart showed a similarly high content of acidic amino acid residues. Ribonuclease protection assay showed osteoadherin mRNA to be expressed in femoral bone and calvaria tissues, while no expression was detected in cartilage, tendon or liver. Using very sensitive nested RT-PCR, however, message was detected in femoral head, rib, tendon and bone marrow total RNA preparations. An antiserum specific for the rat C-terminal region of osteoadherin was generated and used for studies of protein distribution by immunohistochemistry during femoral head development. Osteoadherin was primarily present in bone trabeculae and no staining was seen in cartilage. In situ hybridization showed the strongest expression in osteoblasts close to the cartilage/bone interface of the growth plate and lower expression in diaphyseal osteoblasts. On maturation of the femoral head on day 60 some expression was detected immediately below the forming articular cartilage. Our data indicated that osteoadherin is primarily expressed by osteoblasts and might have a role in regulation of mineralization.

While attachment to bone is required for optimal osteoclast function, the molecular events that underlie this fact are unclear, other than that the cell requires adhesion to mineralized matrix to assume a fully differentiated phenotype. To address this issue, we cultured murine bone marrow-derived osteoclasts on either cell culture plastic or devitalized mouse calvariae to identify the distinct genetic profile induced by interaction with bone. Among a number of genes previously unknown to be expressed in osteoclasts we found that Annexin A8 (AnxA8) mRNA was markedly up-regulated by bone. AnxA8 protein was present at high levels in osteoclasts present in human tissues recovered from sites of pathological bone loss. The presence of bone mineral was required for up-regulation of AnxA8 mRNA since osteoclasts plated on decalcified bone express AnxA8 at low levels as did osteoclasts plated on native or denatured type I collagen. Finally, AnxA8-regulated cytoskeletal reorganization in osteoclasts generated on a mineralized matrix. Thus, we used a novel approach to define a distinct bone-dependent genetic program associated with terminal osteoclast differentiation and identified Anxa8 as a gene strongly induced late in osteoclast differentiation and a protein that regulates formation of the cell's characteristic actin ring. J. Cell. Physiol. 226: 3413–3421, 2011. © 2011 Wiley Periodicals, Inc.

The interaction between multiple myeloma (MM) cells and the bone marrow (BM) microenvironment induces proliferation and survival of MM cells, as well as osteoclastogenesis. This study investigated the therapeutic potential of novel p38 mitogen-activated protein kinase (p38MAPK) inhibitor LY2228820 (LY) in MM. Although cytotoxicity against MM cell lines was modest, LY significantly enhanced the toxicity of bortezomib by down-regulating bortezomib-induced heat shock protein 27 phosphorylation. LY inhibited interleukin-6 secretion from long term cultured-BM stromal cells and BM mononuclear cells (BMMNCs) derived from MM patients in remission. LY also inhibited macrophage inflammatory protein-1alpha secretion from patient MM cells and BMMNCs as well as normal CD14 positive osteoclast precursor cells. Moreover, LY significantly inhibited in vitro osteoclastogenesis from CD14 positive cells induced by macrophage-colony stimulating factor and soluble receptor activator of nuclear factor-kappaB ligand. Finally, LY also inhibited in vivo osteoclatogenesis in a severe combined immunodeficiency mouse model of human MM. These results suggest that LY represents a promising novel targeted approach to improve MM patient outcome both by enhancing the effect of bortezomib and by reducing osteoskeletal events.

Osteoadherin is a recently described bone proteoglycan containing keratan sulfate. It promotes integrin (alphav beta3)-mediated cell binding (Wendel, M., Sommarin, Y., and Heinegârd, D. (1998) J. Cell Biol. 141, 839-847). The primary structure of bovine osteoadherin has now been determined by nucleotide sequencing of a cDNA clone from a primary bovine osteoblast expression library. The entire translated primary sequence corresponds to a 49,116-Da protein with a calculated isoelectric point for the mature protein of 5.2. The dominating feature is a central region consisting of 11 B-type, leucine-rich repeats ranging in length from 20 to 30 residues. The full, primary sequence contains four putative sites for tyrosine sulfation, three of which are at the N-terminal end of the molecule. There are six potential sites for N-linked glycosylation present. Osteoadherin shows highest sequence identity, 42%, to bovine keratocan and 37-38% identity to bovine fibromodulin, lumican, and human PRELP. Unique to osteoadherin is the presence of a large and very acidic C-terminal domain. The distribution of cysteine residues resembles that of other leucine-rich repeat proteins except for two centrally located cysteines. Northern blot analysis of RNA samples from various bovine tissues showed a 4.5-kilobase pair message for osteoadherin to be expressed in bone only. Osteoadherin mRNA was detected by in situ hybridization in mature osteoblasts located superficially on trabecular bone.

Chondroadherin is a cartilage protein with cell binding properties. The expression of chondroadherin was studied in rat tissues and during postnatal femoral head development. For design of oligonucleotide probes and primers a 1664 bp, full length, rat chondroadherin cDNA was isolated from a rat chondrosarcoma library and sequenced. Northern blot analysis showed chondroadherin mRNA to be present in femoral head and rib cartilage, as well as in tendon. More sensitive reverse-transcriptase PCR additionally identified the mRNA in calvaria, long bone and bone marrow. Localization of chondroadherin by immunocytochemistry in the developing femoral head from postnatal day 14 to day 60 showed presence of the protein in cartilaginous regions. With increasing age a very distinct localization of chondroadherin was seen in the territorial matrix around late proliferative cells in the growth plate as well as in the developing articular cartilage in the maturing femoral head. Localization of chondroadherin mRNA by in situ hybridization was in agreement with immunocytochemistry with strong hybridization signals in late proliferative cells in the growth plate. In the articular cartilage the expression was restricted to cells in the lower regions. A three-fold increase of cartilage chondroadherin content in the growing femoral head was demonstrated by Western blot analysis. The high expression of this cell binding protein in a dynamic region of cartilage suggests an important role for chondroadherin in the regulation of chondrocyte growth and proliferation.

Distribution and sites of synthesis of a cartilage extracellular matrix protein, cartilage oligomeric matrix protein (COMP), and of a bone extracellular matrix protein, bone sialoprotein (BSP), were studied in the femoral head of growing Wistar rats from day 14 to day 60 by immunocytochemistry and in situ hybridization. This period includes formation of the secondary ossification center and differentiation of articular cartilage. At early stages, immunoreactivity for COMP was pronounced throughout the cartilage. The localization of COMP was predominantly territorial in the center of the immature femoral head and in the growth plate at all ages studied. In the superficial parts, a shift from a uniform extracellular matrix staining at day 14 to an interterritorial localization at day 33 to day 60 was seen, apparently concurrent with formation of articular cartilage. COMP staining, representing cartilage remnants, also extended into the center of the trabecular bone in the primary spongiosa. In the secondary ossification center, the staining for COMP decreased at the onset of calcification. The protein was only synthesized by chondrocytes, as shown by in situ hybridization. The highest level of COMP mRNA was detected in chondrocytes in the central region of the growth plate. In the layer corresponding to the articular cartilage of the femoral head, mRNA levels for COMP were low from day 14 to day 33 but were increased on day 60. This shows substantial synthesis in the developing articular cartilage. Immunoreactivity for BSP was detected in bone trabeculae of primary spongiosa. In situ hybridization showed the highest levels of BSP mRNA in regions of newly formed bone. BSP mRNA was detected in hypertrophic chondrocytes in the secondary ossification center as early as day 18, well before the appearance of immunochemically detectable BSP. Interestingly, simultaneous expression of COMP and BSP mRNA was seen after day 18 in hypertrophic chondrocytes of the growth plate and later also in hypertrophic chondrocytes close to the mineralization zone of the articular cartilage.