VWC2 Increases Bone Formation Through Inhibiting Activin Signaling.

By a bioinformatics approach, we have identified a novel cysteine knot protein member, VWC2 (von Willebrand factor C domain containing 2) previously known as Brorin. Since Brorin has been proposed to function as a bone morphogenetic protein (BMP) antagonist, we investigated the binding of Brorin/VWC2 to several BMPs; however, none of the BMPs tested were bound to VWC2. Instead, the ßA subunit of activin was found as a binding partner among transforming growth factor (TGF)-ß superfamily members. Here, we show that Vwc2 gene expression is temporally upregulated early in osteoblast differentiation, VWC2 protein is present in bone matrix, and localized at osteoblasts/osteocytes. Activin A-induced Smad2 phosphorylation was inhibited in the presence of exogenous VWC2 in MC3T3-E1 osteoblast cell line and primary osteoblasts. The effect of VWC2 on ex vivo cranial bone organ cultures treated with activin A was investigated, and bone morphometric parameters decreased by activin A were restored with VWC2. When we further investigated the biological mechanism how VWC2 inhibited the effects of activin A on bone formation, we found that the effects of activin A on osteoblast cell growth, differentiation, and mineralization were reversed by VWC2. Taken together, a novel secretory protein, VWC2 promotes bone formation by inhibiting Activin-Smad2 signaling pathway.

Restoration contour is a risk indicator for peri-implantitis: A cross-sectional radiographic analysis.

AIM: The purpose of this study was to determine whether restoration emergence angle was associated with peri-implantitis. MATERIALS AND METHODS: A data set consisting of 96 patients with 225 implants (mean follow-up: 10.9 years) was utilized. Implants were divided into bone-level and tissue-level groups, and radiographs were analysed to determine the restoration emergence angles, as well as restoration profiles (convex or concave). Peri-implantitis was diagnosed based on probing depth and radiographic bone loss. Associations between peri-implantitis and emergence angles/profiles were assessed using generalized estimating equations. RESULTS: Eighty-three patients with 168 implants met inclusion criteria. The prevalence of peri-implantitis was significantly greater in the bone-level group when the emergence angle was >30 degrees compared to an angle ≤30 degrees (31.3% versus 15.1%, p = .04). In the tissue-level group, no such correlation was found. For bone-level implants, when a convex profile was combined with an angle of >30 degrees, the prevalence of peri-implantitis was 37.8% with a statistically significant interaction between emergence angle and profile (p = .003). CONCLUSIONS: Emergence angle of >30 degrees is a significant risk indicator for peri-implantitis and convex profile creates an additional risk for bone-level implants, but not for tissue-level implants.

Characterization of the bone matrix and its contribution to tooth loss in human cadaveric mandibles.

OBJECTIVE: It is uncertain as to what extent the major bone matrix constituents, mineral and collagen, show inter-individual variation and dependence on age and sex in jawbones. The purpose of this study was to clarify this uncertainty using cadaveric mandibles and investigate the association of bone matrix with the number of existing teeth. MATERIALS AND METHODS: Cortical bone samples (1 × 1 cm) collected from the mental of 48 cadaveric mandibles (27 men and 21 women; age range = 56-93 years and 63-103 years, respectively) were used to quantify three bone matrix indices: mineral content, collagen content and extent of lysine hydroxylation of collagen. Associations with age and comparisons by sex were evaluated based on bone matrix indices and the numbers of existing teeth. The numbers of existing teeth were compared between the groups showing low and high bone matrix index values. RESULTS: A great amount of inter-individual variation was seen in all bone matrix indices. No bone matrix indices were associated with age, while the number of existing teeth was negatively associated with age. The bone matrix indices and number of existing teeth did not differ by sex. The number of existing teeth was nearly twice as high in the group showing high collagen content as in the low collagen group; however, an analysis of covariance showed a significant inter-group difference not from bone matrix indices, but rather from age. Interestingly, in comparison to femoral collagen, mandibular collagen showed lower lysine hydroxylation, which can represent an aspect of bone quality. CONCLUSIONS: Mandibular bone matrix shows great inter-individual variation and is independent of age and sex, but did not show as strong a relationship with tooth loss as age. Even so, mandibular collagen may represent a unique characteristic of bone matrix and deserves to be further investigated.

Modulation of matrix mineralization by Vwc2-like protein and its novel splicing isoforms.

In search of new cysteine knot protein (CKP) family members, we found a novel gene called von Willebrand factor C domain-containing protein 2-like (Vwc2l, also known as Brorin-like) and its transcript variants (Vwc2l-1, Vwc2l-2 and Vwc2l-3). Based on the deduced amino acid sequence, Vwc2l-1 has a signal peptide and 2 cysteine-rich (CR) domains, while Vwc2l-2 lacks a part of 2nd CR domain and Vwc2l-3 both CR domains. Although it has been reported that the expression of Brorin-like was predominantly observed in brain, we found that Vwc2l transcript variants were detected in more ubiquitous tissues. In osteoblasts, the induction of Vwc2l expression was observed at matrix mineralization stage. When Vwc2l was stably transfected into osteoblasts, the matrix mineralization was markedly accelerated in Vwc2l-expressing clones compared to that in the control, indicating the modulatory effect of Vwc2l protein on osteoblastic cell function. The mechanistic insight of Vwc2l-modulation was further investigated and we found that the expression of Osterix, one of the key osteogenic markers, was significantly increased by addition of all Vwc2l isoform proteins. Taken together, Vwc2l is a novel secreted protein that promotes matrix mineralization by modulating Osterix expression likely through TGF-ß superfamily growth factor signaling pathway. Our data may provide mechanistic insights into the biological functions of this novel CKP member in bone and further suggest a novel approach to enhance osteoblast function, which enables to accerelate bone formation, regeneration and healing.

Podocan-like protein: a novel small leucine-rich repeat matrix protein in bone.

Recently, significant attention has been drawn to the biology of small leucine-rich repeat proteoglycans (SLRPs) due to their multiple functionalities in various cell types and tissues. Here, we characterize a novel SLRP member, "Podocan-like (Podnl) protein" identified by a bioinformatics approach. The Podnl protein has a signal peptide, a unique cysteine-rich N-terminal cluster, 21 leucine-rich repeat (LRR) motifs, and one putative N-glycosylation site. This protein is structurally similar to podocan in SLRPs. The gene was highly expressed in mineralized tissues and in osteoblastic cells and the high expression level was observed at and after matrix mineralization in vitro. Podnl was enriched in newly formed bones based on immunohistochemical analysis. When Podnl was transfected into osteoblastic cells, the protein with N-glycosylation was detected mainly in the cultured medium, indicating that Podnl is a secreted N-glycosylated protein. The endogenous Podnl protein was also present in bone matrix. These data provide a new insight into our understanding of the emerging SLRP functions in bone formation.

A novel proteolytic processing of prolysyl oxidase.

Lysyl oxidase (LOX) is an amine oxidase that is critical for the stability of connective tissues. The secreted proLOX is enzymatically quiescent and is activated through proteolytic cleavage between residues Gly(162) and Asp(163) (residue numbers according to the mouse LOX) by bone morphogenetic protein (BMP)-1 gene products. Here we report a novel processing of proLOX identified in vitro and in vivo. Two forms of mature LOX were identified and characterized by their immunoreactivity to specific antibodies, amine oxidase activity, and mass spectrometry. One form was identified as a well-characterized BMP-1 processed LOX protein. Another was found to be a truncated form of LOX resulting from the cleavage at the carboxy terminus of Arg(192). The truncated form of LOX still appeared to retain amine oxidase activity. The results from the proLOX gene deletion and mutation experiments indicated that the processing occurs independent of the cleavage of proLOX by BMP-1 gene products and likely requires the presence of LOX propeptide. These results indicate that proLOX could be processed by two different mechanisms producing two forms of active LOX.

Anatomy of the mandibular canal and surrounding structures: Part I: Morphology of the superior wall of the mandibular canal.

INTRODUCTION: Previous studies of the mandibular canal (MC) have raised questions about the structure of its superior wall that have not been answered. The goal of this anatomical and radiological study was to investigate how CBCT imaging could predict the structure of the superior wall of the MC. METHODS: Twenty sides from ten dry mandibles derived from six females and four males were used for this study. The mandibles were examined with CBCT. The specimens were then prepared by the methods of our previous study and observed inferiorly. The inferior views were classified into four groups by gross observation of the surface of the superior wall of the MC: class I (trabecular pattern), class II (osteoporotic pattern), class III (dense/irregular pattern), and class IV (smooth pattern). Coronal section CBCT images were classed according to whether the superior wall of the MC was visible. RESULTS: Class I was most common in dentulous sections in both genders, and class IV was most common class in edentulous sections in both genders. The superior wall was visible in 59.1% in dentulous and 84.9% in edentulous sections, and non-visible in the remainder. CONCLUSION: Tooth presence and sex are important factors influencing the superior wall of the MC. When the superior wall cannot be seen on CBCT, it is more likely to belong to class II (osteoporotic) than other classes.

Distinct characteristics of mandibular bone collagen relative to long bone collagen: relevance to clinical dentistry.

Bone undergoes constant remodeling throughout life. The cellular and biochemical mechanisms of bone remodeling vary in a region-specific manner. There are a number of notable differences between the mandible and long bones, including developmental origin, osteogenic potential of mesenchymal stem cells, and the rate of bone turnover. Collagen, the most abundant matrix protein in bone, is responsible for determining the relative strength of particular bones. Posttranslational modifications of collagen, such as intermolecular crosslinking and lysine hydroxylation, are the most essential determinants of bone strength, although the amount of collagen is also important. In comparison to long bones, the mandible has greater collagen content, a lower amount of mature crosslinks, and a lower extent of lysine hydroxylation. The great abundance of immature crosslinks in mandibular collagen suggests that there is a lower rate of cross-link maturation. This means that mandibular collagen is relatively immature and thus more readily undergoes degradation and turnover. The greater rate of remodeling in mandibular collagen likely renders more flexibility to the bone and leaves it more suited to constant exercise. As reviewed here, it is important in clinical dentistry to understand the distinctive features of the bones of the jaw.

Lysyl oxidase binds transforming growth factor-beta and regulates its signaling via amine oxidase activity.

Lysyl oxidase (LOX), an amine oxidase critical for the initiation of collagen and elastin cross-linking, has recently been shown to regulate cellular activities possibly by modulating the functions of growth factors. In this study, we investigated the interaction between LOX and transforming growth factor-beta1 (TGF-beta1), a potent growth factor abundant in bone, the effect of LOX on TGF-beta1 signaling, and its potential mechanism. The specific binding between mature LOX and mature TGF-beta1 was demonstrated by immunoprecipitation and glutathione S-transferase pulldown assay in vitro. Both proteins were colocalized in the extracellular matrix in an osteoblastic cell culture system, and the binding complex was identified in the mineral-associated fraction of bone matrix. Furthermore, LOX suppressed TGF-beta1-induced Smad3 phosphorylation likely through its amine oxidase activity. The data indicate that LOX binds to mature TGF-beta1 and enzymatically regulates its signaling in bone and thus may play an important role in bone maintenance and remodeling.

Biochemical characterization of collagen in alveolar mucosa and attached gingiva of pig.

Alveolar mucosa and attached gingiva are two continuous but functionally distinct connective tissues covering alveolar bone of the jaw. In this study, the major matrix component of these tissues, collagen, was biochemically characterized and compared. The tissues were obtained from mature pigs and analyzed for collagen content, amino acid composition, collagen types, collagen cross-linking, and gene expression. We found that alveolar mucosa is primarily composed of fibrillar collagens and the collagen content is higher than attached gingiva. The content of type III relative to type I collagen was higher in alveolar mucosa when compared with attached gingiva. The collagen cross-linking pattern also was distinct between the two tissues demonstrating that alveolar mucosa contained fewer reducible cross-links but more non-reducible cross-links in comparison to attached gingiva. The mRNA expression level of type I collagen in alveolar mucosa was significantly lower than that of attached gingiva. These results indicate that alveolar mucosa is a fibrillar collagen-rich tissue and, in comparison to gingival tissue, re-models slowly.