Cartilage matrix protein (CMP) is expressed specifically in mature cartilage and consists of two von Willebrand factor A domains (CMP-A1 and CMP-A2) that are separated by an epidermal growth factor-like domain, and a coiled-coil tail domain at the carboxyl terminal end. We have shown previously that CMP interacts with type II collagencontaining fibrils in cartilage. In this study, we describe a type II collagen-independent CMP filament and we analyze the structural requirement for the formation of this type of filament. Recombinant wild-type CMP and two mutant forms were expressed in chick primary cell cultures using a retrovirus expression system. In chondrocytes, the wildtype virally encoded CMP is able to form disulfide bonded trimers and to assemble into filaments. Filaments also form with CMP whose Cys455 and Cys457 in the tail domain were mutagenized to prevent interchain disulfide bond formation. Therefore, intermolecular disulfide bonds are not necessary for the assembly of CMP into filaments. Both the wild-type and the double cysteine mutant also form filaments in fibroblasts, indicating that chondrocyte-specific factors are not required for filament formation. A truncated form of CMP that consists only of the CMP-A2 domain and the tail domain can form trimers but fails to form filaments, indicating that the deleted CMP-A1 domain and/or the epidermal growth factor domain are necessary for filament assembly but not for trimer formation. Furthermore, the expression of the virally encoded truncated CMP in chondrocyte culture disrupts endogenous CMP filament formation. Together these data suggest a role for CMP in cartilage matrix assembly by forming filamentous networks that require participation and coordination of individual domains of CMP.

Cartilage matrix protein (CMP) exists as a disulfidebonded homotrimer in the matrix of cartilage. Each monomer consists of two CMP-A domains that are separated by an epidermal growth factor-like domain. A heptad repeat-containing tail makes up the carboxyl-terminal domain of the protein. The secreted form of CMP contains 12 cysteine residues numbered C1 through C12. Two of these are in each of the CMP-A domains, six are in the epidermal growth factor-like domain, and two are in the heptad repeat-containing tail. Two major categories of mutant CMPs were generated to analyze the oligomerization process of CMP: a mini-CMP and a heptadless full-length CMP. The mini-CMP consists of the CMP-A2 domain and the heptad repeat-containing tail. In addition, a number of mutations affecting C9 through C12 were generated within the full-length, the mini-, and the heptad-less CMPs. The mutational analysis indicates that the heptad repeats are necessary for the initiation of CMP trimerization and that the two cysteines in the heptad repeat-containing tail are both necessary and sufficient to form intermolecular disulfide bonds in either full-length or mini-CMP. The two cysteines within a CMP-A domain form an intradomain disulfide bond.The macromolecular composition of the matrix of cartilage results from the expression of a unique repertoire of genes by chondrocytes. The matrix macromolecules synthesized by the chondrocytes have multiple domains that permit interactions with other matrix molecules or with cell surface components. These complex interactions determine the structure and the integrity of cartilage. The major components of the cartilage extracellular matrix are collagens, proteoglycans, and noncollagenous proteins. Cartilage matrix protein (CMP) 1 is one of the most abundant noncollagenous extracellular proteins in cartilage (1, 2) and has been shown to associate with the cartilage collagen fibril that consists of collagen types II, IX, and XI (3) as well as with proteoglycans (1).The deduced amino acid sequence of CMP reveals that a CMP monomer is made up of a unique combination of structural domains (4 -6). Two highly homologous domains, CMP-A1 and CMP-A2, are separated from each other by a domain with homology to epidermal growth factor (EGF). The last domain is the carboxyl-terminal tail, which contains a series of heptad repeats (7). Each domain has significant sequence or structural homology to portions of other proteins. Homology to the CMP-A domains is found in soluble proteins including von Willebrand factor, the complement components C2 and B, matrix proteins such as collagen types VI, VII, XII, and XIV, undulin, transmembrane proteins such as the ␣-chains of the integrins VLA-1, VLA-2, LFA-1, Mac-1, p150,95, and a Caenorhabditis elegans protein involved in muscle attachment as well as the dihydropyridine-sensitive calcium channel and the inter-␣-trypsin inhibitor (reviewed in Refs. 8 and 9). The A domains of several proteins have been shown to bind extracellular matrix molecules such as collagen (10 -15)...

Study Design:Review of literature.Objectives:This review of literature investigates the application of mesenchymal stem cells (MSCs) in spinal fusion, highlights potential uses in the development of bone grafts, and discusses limitations based on both preclinical and clinical models.Methods:A review of literature was conducted looking at current studies using stem cells for augmentation of spinal fusion in both animal and human models.Results:Eleven preclinical studies were found that used various animal models. Average fusion rates across studies were 59.8% for autograft and 73.7% for stem cell–based grafts. Outcomes included manual palpation and stressing of the fusion, radiography, micro–computed tomography (μCT), and histological analysis. Fifteen clinical studies, 7 prospective and 8 retrospective, were found. Fusion rates ranged from 60% to 100%, averaging 87.1% in experimental groups and 87.2% in autograft control groups.Conclusions:It appears that there is minimal clinical difference between commercially available stem cells and bone marrow aspirates indicating that MSCs may be a good choice in a patient with poor marrow quality. Overcoming morbidity and limitations of autograft for spinal fusion, remains a significant problem for spinal surgeons and further studies are needed to determine the efficacy of stem cells in augmenting spinal fusion.

This work presents the first direct evidence of multivalent binding between bone morphogenetic protein-2 (BMP-2) and cartilage oligomeric matrix protein (COMP) using highresolution atomic force microscopy (AFM) imaging. AFM topographic images reveal the molecular morphology of COMP, a pentameric protein whose five identical monomer units bundle together at N-termini, extending out with flexible chains to C-termini. Upon addition of BMP-2, COMP molecules undergo conformational changes at the C-termini to enable binding with BMP-2 molecules. AFM enables local structural changes of COMP to be revealed upon binding various numbers, 1-5, of BMP-2 molecules. These BMP-2/COMP complexes exhibit very different morphologies from those of COMP: much more compact and thus less flexible. These molecular level insights deepen current understanding of the mechanism of how BMP-2/COMP complex enhances osteogenesis among osteoprogenitor cells: i.e., multivalent presentation of BMP-2 via the stable and relatively rigid BMP-2/COMP complex could form a lattice of interaction between multiple BMP-2 and BMP-2 receptors. These ligand-receptor clusters lead to fast initiation and sustained activation of the Smad signaling pathway, resulting in enhanced osteogenesis. This work is also of translational importance, as the outcome may enable usage of lower BMP-2 dosage for bone repair and regeneration.