Osteoadherin Research Articles

Glycoprotein Injectable Hydrogels Promote Accelerated Bone Regeneration through Angiogenesis and Innervation.

Glycoproteins are gaining prominence as multifunctional biomaterials. The study reports development of glycoprotein mucin as biomaterial promoting bone regeneration. Mucin 1 deletion has resulted in stiffer femoral bones with scarce presence of osteoblasts in trabecular linings and its role has been established in determining bone mass and mineralization. Limited information about its structure limits its processability, exploration as biomaterial, which is discussed in this study. The role of mucin in ECM (extracellular cellular matrix) formation validated by RNA sequencing analysis of human bone marrow derived mesenchymal stem cells is reported. The structure and stability of mucins is dependent on the presence of glycans in its structure. A thermosensitive hydrogel acquired from thermosensitive Poly (N-isopropyl acrylamide)-(PNIPAM) modified mucin and collagen is developed. The hydrogel demonstrates porous structure and mechanical strength. Newly formed bone tissue is observed at 8 weeks post-implantation in the hydrogel treated groups. The formation of blood vessels, nerves, and bone is observed with upregulation of angiopoietin (ANG), neurofilament heavy chain (NF-H), and osteoadherin (OSAD) or osteocalcin (OCN) respectively in rat calvarial defects. The outcome demonstrates that the thermosensitive injectable hydrogel accelerates repair and healing in calvarial bone defects making it a promising biodegradable biomaterial capable of regenerating bone by promoting angiogenesis and innervation.

Short Leucine-Rich Proteoglycans Modulate Complement Activity and Increase Killing of the Respiratory Pathogen Moraxella catarrhalis.

The respiratory pathogen Moraxella catarrhalis is a human-specific commensal that frequently causes acute otitis media in children and stimulates acute exacerbations in chronic obstructive pulmonary disease patients. The exact molecular mechanisms defining host-pathogen interactions promoting pathogenesis are not clearly understood. Limited knowledge hampers vaccine and immunotherapeutic development required to treat this emerging pathogen. In this study, we reveal in detail a novel antibacterial role displayed by short leucine-rich proteoglycans (SLRPs) in concert with complement. We show that fibromodulin (FMOD), osteoadherin (OSAD), and biglycan (BGN) but not decorin (DCN) enhance serum killing of M. catarrhalis. Our results suggest that M. catarrhalis binding to SLRPs is a conserved feature, as the overwhelming majority of clinical and laboratory strains bound all four SLRPs. Furthermore, we resolve the binding mechanism responsible for this interaction and highlight the role of the ubiquitous surface protein (Usp) A2/A2H in mediating binding to host SLRPs. A conserved immune evasive strategy used by M. catarrhalis and other pathogens is the surface acquisition of host complement inhibitors such as C4b-binding protein (C4BP). We observed that FMOD, OSAD, and BGN competitively inhibit binding of C4BP to the surface of M. catarrhalis, resulting in increased C3b/iC3b deposition, membrane attack complex (MAC) formation, and subsequently decreased bacterial survival. Furthermore, both OSAD and BGN promote enhanced neutrophil killing in vitro, both in a complement-dependent and independent fashion. In summary, our results illustrate that SLRPs, FMOD, OSAD, and BGN portray complement-modulating activity enhancing M. catarrhalis killing, defining a new antibacterial role supplied by SLRPs.

Evaluation of bone repair of critical size defects treated with simvastatin-loaded poly(lactic-co-glycolic acid) microspheres in rat calvaria.

Statins are hypolipemiant drugs with osteoinductive effect. We evaluated the potential of simvastatin loaded into poly(lactic-co-glycolic acid) (PLGA) microspheres to heal critical size defects in rat calvaria. PLGA scaffolds (50:50 ratio) were synthesized as pure membranes or as microspheres loaded with 2.5% simvastatin. Critical size defects (5-mm diameter) were created in the parietal bone of 3-month-old male Wistar rats; they were either left filled with blood clot (C group), covered with a PLGA membrane (M group) or with PLGA microspheres loaded with simvastatin (MSI group) or not (MM group), and then covered with the PLGA membrane. The defects were evaluated after 30 or 60 days by light and electron microscopy, immunohistochemistry for osteopontin (OPN), bone sialoprotein (BSP) and osteoadherin (OSAD), and immunocytochemistry for OPN. Scanning electron microscopy showed that the calvarial defects treated with MSI were almost completely healed after 60 days, while groups M and C presented less bone formation, whereas the bone matrix formed into the defects of MSI group was more organized and mature. The immunolabeling for OPN and BSP on the matrix in groups C and M showed typical areas of primary bone unlike the MSI that presented weak labeling at the formed area. In the MSI group, there was an intense immunostaining for OSAD in osteoid, as well as in osteocyte cytoplasm. The immunocytochemistry showed intense labeling for OPN with homogeneous distribution in the interfibrillar spaces in all groups after 30 days and after 60 days; however, while C and M groups exhibited similar aspect, the MSI specimens showed weak labeling. The ultrastructural evaluation showed the interaction between the biomaterial and the surrounding tissue where some cells established intimate contact with microspheres. The repair of critical size bone defects was accelerated and enhanced by the implantation of simvastatin-loaded PLGA microspheres.

The glycosylation profile of osteoadherin alters during endochondral bone formation

Endochondral bone formation involves the dynamic interplay between the cells and their extracellular environment to facilitate the deposition of a calcified matrix. Numerous molecules are involved within this process, including collagens and non-collagenous proteins, and their post-translational modifications have been shown to effect their biomolecular interactions. Osteoadherin (OSAD), a keratin sulfate (KS)-substituted small leucine-rich proteoglycan has been isolated from mineralized tissues and is considered to be a mineralized tissue-specific protein. However, to date, information is limited concerning the dynamic expression and role of this proteoglycan during bone formation and the biomineralization process. The current study aimed to examine the dynamic expression of this protein throughout mouse metatarsal long bone development, from the cartilage anlagen (E15) to the fully formed bone (Adult). Using quantitative gene expression analysis we observed that OSAD was produced with the onset of mineralization and the formation of the ossification center. This finding was reflected in the localization studies, using both light and electron microscopy, and showed that initial OSAD localization was restricted to the endosteal surfaces of the diaphysis and forming metaphysis. Furthermore, we analyzed protein extracts, both mineral and non-mineral associated fractions, and showed that OSAD was substituted with varying patterns of glycosylation during bone development. Sequential enzymatic digestions of the non-mineral bound protein extracts demonstrated that OSAD lacked any KS chains throughout all development stages. Whereas, in the mineral bound fractions, with long bone maturation the substitution with KS became more apparent with development. Therefore, it can be concluded that different pools of OSAD are produced during endochondral bone formation and these may have specific roles in directing the mineralization process.

In vitro effects of ascorbic acid and β-glycerophosphate on human gingival fibroblast cells

Ascorbic acid (AA) and β-glycerophosphate (βG) are considered in vitro osteogenic factors important to the differentiation of osteoblastic progenitor and dental pulp cells into mineralized tissue-forming cells. So, the present study investigated in vitro if these mineralizing inducible factors (AA and βG) could influence differentiation of human gingival fibroblasts when compared with human pulp cells and osteogenic cells derived from rat calvaria cultured. The expression of osteopontin (OPN) and osteoadherin (OSAD) was analyzed by indirect immunofluorescence, immunocytochemistry as well as Western-blotting. In addition, the main ultrastructural aspects were also investigated. No mineralized matrix formation occurred on gingival fibroblasts induced with AA+βG. On these cells, no expression of OPN and OSAD was observed when compared with pulp cells, pulp cells induced with AA+βG as well as osteogenic cells. Ultrastructure analysis additionally showed that gingival fibroblasts exhibited typical fibroblast morphology with no nodule formation. The present findings showed that AA and βG could not promote a mineralized cell differentiation of human gingival fibroblasts and confirm that human dental pulp cells, as the osteogenic cells, are capable to form a mineralized extracellular.

Effect of alendronate on endochondral ossification in mandibular condyles of growing rats

The replacement of the calcified cartilage by bone tissue during the endochondral ossification of the mandibular condyle is dependent of the resorbing activity of osteoclats. After partial resorption, calcified cartilage septa are covered by a primary bone matrix secreted by osteoblasts. Osteoadherin (OSAD) is a small proteoglycan present in bone matrix but absent in cartilage during the endochondral ossification. The aim of this study was to analyze the effect of alendronate, a drug known to inhibit bone resorption by osteoclasts, on the endochondral ossification of the mandibular condyle of young rats, by evaluating the distribution of osteoclasts and the presence of OSAD in the bone matrix deposited. Wistar newborn rats (n=45) received daily injections of alendronate (n=27) or sterile saline solution as control (n=18) from the day of birth until the ages of 4, 14 and 30 days. At the days mentioned, the mandibular condyles were collected and processed for transmission electron microscopy analysis. Specimens were also submitted to tartrate resistant acid phosphatase (TRAP) histochemistry and ultrastructural immunodetection of OSAD. Alendronate treatment did not impede the recruitment and fusion of osteoclasts at the ossification zone during condyle growth, but they presented inactivated phenotype. The trabeculae at the ossification area consisted of cartilage matrix covered by a layer of primary bone matrix that was immunopositive to OSAD at all time points studied. Apparently, alendronate impeded the removal of calcified cartilage and maturation of bone trabeculae in the mandibular ramus, while in controls they occurred normally. These findings highlight for giving attention to the potential side-effects of bisphosphonates administered to young patients once it may represent a risk of disturbing maxillofacial development.

Osteoadherin Accumulates in the Predentin towards the Mineralization Front in the Developing Tooth

BackgroundProteoglycans (PG) are known to be involved in the organization and assembly of the extracellular matrix (ECM) prior to mineral deposition. Osteoadherin (OSAD), a keratan sulphate PG is a member of the small leucine-rich (SLRP) family of PGs and unlike other SLRPs, OSAD expression is restricted to mineralized tissues. It is proposed to have a high affinity for hydroxyapatite and has been shown to be expressed by mature osteoblasts but its exact role remains to be elucidated.Methodology/Principal FindingsWe investigated the protein distribution of OSAD in the developing mouse tooth using immunohistochemistry and compared its expression with other SLRPs, biglycan (BGN), decorin (DCN) and fibromodulin (FMD). OSAD was found to be specifically localized in the predentin layer of the tooth and focused at the mineralization front. These studies were confirmed at the ultrastructural level using electron microscopy (iEM), where the distribution of immunogold labeled OSAD particles were quantified and significant amounts were found in the predentin, forming a gradient towards the mineralization front. In addition, iEM results revealed OSAD to lie in close association with collagen fibers, further suggesting an important role for OSAD in the organization of the ECM. The expression profile of mineralization-related SLRP genes by rat dental pulp cells exposed to mineralization inducing factors, showed an increase in all SLRP genes. Indeed, OSAD expression was significantly increased during the mineralization process, specifically following, matrix maturation, and finally mineral deposition. Alizarin Red S staining for calcium deposition showed clear bone-like nodules, which support matrix maturation and mineralization.ConclusionsThese studies provide new evidence for the role of OSAD in the mineralization process and its specific localization in the predentin layer accumulating at the mineralization front highlighting its role in tooth development.

Osteoadherin is Upregulated by Mature Osteoblasts and Enhances Their In Vitro Differentiation and Mineralization

During the process of differentiation, osteoblasts commit through strictly controlled checkpoints under the influence of several growth factors, cytokines, and extracellular matrix (ECM) proteins. The mineralized tissue-specific ECM component osteoadherin (OSAD) belongs to the small leucine-rich repeat protein family of proteoglycans. Proteoglycans modulate cellular behavior either through the attached glycosaminoglycan chains or by direct protein-protein interactions via the core protein sequences. Leucine-rich repeats have been shown to directly interact with cell-surface receptors such as epidermal growth factor receptor, blocking its ability to bind its ligand. In the present study, we investigated the influence of OSAD on the behavior and maturation of MC3T3E1 osteoblasts. OSAD overexpression and repression clones were created by stably transfecting with plasmids coding for either mouse OSAD cDNA or small-hairpin RNA, targeted against mouse OSAD. Overexpression of OSAD resulted in an increase of osteoblast differentiation features, such as increased alkaline phosphatase (ALP) activity and increased in vitro mineralization, as well as reduced proliferation and migration. Bone sialoprotein (BSP) levels were unchanged, while upregulation of osteocalcin (OC) and osteoglycin (OGN) was observed. Conversely, repression of OSAD expression resulted in increased cell proliferation and migration. BSP and OC were unaffected, while OGN was downregulated. ALP activity was reduced, though no change in in vitro mineralization was observed. We conclude that OSAD overexpression enhanced the differentiation and maturation of osteoblasts in vitro.

Differential regulation of osteoadherin (OSAD) by TGF-β1 and BMP-2

Osteoadherin (OSAD) is a member of the small leucine rich-repeat proteoglycan (SLRP) family. SLRPs are normally found in extracellular matrices, but OSAD is the only member restricted to mineralized tissues. We investigated the promoter region of OSAD by in silico analysis and found that the proximal promoter region contains sites for Smad-3, Smad-4, and AP-1. All are effectors of TGF-β family signalling. We tested sensitivity of the promoter to the two TGF-β family members TGF-β1 and BMP-2. We found TGF-β1 to down regulate OSAD, while BMP-2 up regulates OSAD. As a consequence of how OSAD is regulated by TGF-β1 and BMP-2 and its temporal expression pattern in osteoblasts and bone development, we can conclude OSAD as an early marker for terminally differentiated matrix producing osteoblasts.

Heat-induced retrieval of immunogold labeling for nucleobindin and osteoadherin from Lowicryl sections of bone

The main purpose of this study was to examine whether antigens can be retrieved by heating Lowicryl sections of paraformaldehyde-fixed (PFF) tissues. Thus the intensity of the immunogold signal for two bone proteins (Nucleobindin (Nuc) and osteoadherin (OSAD)) was compared in retrieved and non-retrieved sections of PFF rat bone. As an additional experiment, the effect of antigen retrieval (for Nuc) in sections of tissue primary stabilized by high pressure freezing with subsequent freeze substitution (HPF-FS) was studied. Finally, the tissue distribution patterns of Nuc labeling were compared in non-retrieved HPF-FS sections to that of retrieved and non-retrieved PFF sections. Antigen retrieval in Lowicryl sections of PFF tissues showed significantly enhanced labeling intensity for both proteins in all compartments where they are known to occur. Retrieved PFF Lowicryl sections showed only minor ultrastructural differences compared to non-retrieved ones. Retrieval of HPF-FS sections exhibited no enhancement of labeling but rather a slight reduction, which was significant in the cytoplasm and in cartilage. Furthermore, striking ultrastructural differences were observed in retrieved HPF-FS sections compared to non-retrieved ones with loss of coherence and structure in sections subjected to heating. Comparison of the distribution patterns of Nuc in the sections of PFF and HPF-FS tissues showed discrepancy in most compartments. Antigen retrieval by heating Lowicryl sections of PFF tissues significantly enhances immunogold labeling in all cell compartments where the bone proteins are known to occur. However, the procedure may distort the tissue distribution pattern of bone proteins.

Immunodetection of osteoadherin in murine tooth extracellular matrices.

An antiserum was generated from synthetic peptides highly conserved between different mammalian species to immunolocalise the small leucine-rich proteoglycan osteoadherin (OSAD) in murine teeth. In 19-day-old embryos of rats and mice, a positive staining was found in incisor predentin and alveolar bone surrounding developing incisors and molars. In newborns, OSAD was detected at the tip of the first molar cusp where it accumulated in predentin concomitantly with odontoblast differentiation. In 2-day-old rats and mice, in the first molar, immunostaining revealed positive predentin, enamel matrix close to the apical pole of ameloblasts and a strong signal in dentin. At this stage, OSAD was detected in predentin in the second molar. Ultrastructural immunocytochemistry showed gold particles associated with collagen fibres in predentin and in foci at the dentin mineralisation front. Gold particles were also detected near the secretory pole of ameloblasts where enamel crystallites elongate. No staining was detected in pulp tissue and dental follicle. Restriction of OSAD expression to the extracellular matrix of bone, dentin and enamel suggests a role of this proteoglycan in the organisation of mineralised tissues.

Identification, distribution and expression of osteoadherin during tooth formation.

Osteoadherin (OSAD) is a keratan sulfate-containing proteoglycan, belonging to the small leucine-rich proteoglycan (SLRP) family. In bone OSAD has been localized in primary spongiosa within the bovine fetal rib growth plate. Moreover, in situ hybridization has shown expression of OSAD in osteoblasts close to the cartilage and bone border in the growth plate of rat femur. mRNA expression has also detected OSAD in mature osteoblasts on the surface of bone trabeculae. We have identified OSAD in extracts of bovine dentin, and the identity was verified by N-terminal sequencing. Western blot analysis detected two bands in bovine bone and dentin at 85 kDa and 60 kDa. Northern blot analysis of RNA samples from 5-d-old-rat tooth and femur showed a 1.9-kb transcript for OSAD in both tissues. OSAD was located to the mineralized dentin matrix, cementum and surrounding alveolar bone by immunohistochemistry, and in situ hybridization showed OSAD to be highly expressed during early crown formation in the entire odontoblast cell layer, in the area of Hertwig's epithelial root sheath, in the cells of the newly formed mantle dentin, and in the odontoblasts at the fissures. Ultrastructural studies indicated that OSAD might be associated with collagen fibrils. Thus, OSAD may play an important role during tooth development and biomineralization of dentin.

Ultrastructural distribution of osteoadherin in rat bone shows a pattern similar to that of bone sialoprotein.

Osteoadherin (OSAD) is a keratan sulfate proteoglycan recently isolated from bovine and rat bone. Based on results obtained from in vitro experiments, the protein was shown to bind osteoblasts via the integrin receptor alpha v beta 3. Due to OSAD's capacity to bind hydroxyapatite crystals, a role for the protein in the mineralization process has also been suggested. To test these hypotheses in an in vivo model, the ultrastructural localization of OSAD in bone, tibial (metaphyses and diaphyses), and calvarial samples from normal 10 to 12-day-old rats were examined by immunohistochemical techniques at the ultrastructural level. In addition to the qualitative studies, quantitative measurements of OSAD marker density were performed in relevant compartments. Immunolabeling for OSAD was located to the mineralized bone matrix, with highest concentration of marker at the border between bone and cartilage remnants in the metaphyseal trabeculi. Intracellular labeling was low and no systemic accumulation of OSAD markers was observed at the cell-matrix interface. The observed distribution pattern of OSAD is strikingly similar to that of bone sialoprotein (BSP), confirmed by double labeling. The results of the current study support a role for OSAD in the mineralization process. In this process BSP is assumed to be a nucleator of hydroxyapatite crystals, and OSAD could work in concert with BSP to regulate nucleation. However, the mechanisms involved remain to be elucidated.

TGFβ1 Signaling and Stimulation of Osteoadherin in Human Odontoblasts In Vitro

Transforming growth factor beta 1 (TGF g 1) is generally considered to be a potent inducer of dentin formation. In order to further assess this role, we studied the influence of this factor in human dental pulp cells on the expression of osteoadherin (OSAD), a newly described proteoglycan found in bone and dentin and suspected to play a role in mineralization events. We performed TGF g 1 stimulation both in cultures of human tooth thick slices including mature odontoblasts and in pulp explant cultures giving rise to early secretory odontoblasts or pulpal fibroblasts. We first showed by immunohistochemistry that molecules involved in TGF g 1 signal transduction, that is, membrane receptors T g RI and T g RII and intracellular proteins SMAD-2, SMAD-3, and SMAD-4, were present in human dental cells in vivo and were all maintained after culture of thick-sliced teeth in cells undergoing TGF g 1 stimulation. In this culture system, OSAD synthesis was increased in mature odontoblasts close to the TGF g 1 delivery system. In explant cultures, semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis indicated that the growth factor stimulated OSAD gene expression in early secretory odontoblasts and in pulpal fibroblasts. Taken together, these results indicate that OSAD expression is stimulated by TGF g 1 in pulpal fibroblasts and in early secretory and mature odontoblasts. We suggest that TGF g 1 in this way could control the organization and the mineralization of the extracellular matrix deposited by these cells during dentin formation.

Expression of the small leucine-rich proteoglycan osteoadherin/osteomodulin in human dental pulp and developing rat teeth

Because the extracellular matrices of dentin and bone are composed mainly of type I collagen, their characteristics are determined by the nature of noncollagenous proteins (NCPs). Among these NCPs, some proteoglycans (PGs) belong to the small leucine-rich proteoglycans (SLRPs). Recently, osteoadherin (OSAD) has been described as a new member of this family, that is expressed by mature bovine osteoblasts. Here, we report the expression of OSAD messenger RNA (mRNA) in human dental tissues and during the development of rat molars, using in situ hybridization. For this purpose, we constructed a probe for OSAD mRNA transcripts from human odontoblast cells cultured in vitro. Our results indicate that the mature human odontoblasts overexpress the OSAD gene as compared with cells present in the pulp core. In rat developing molars, mRNA transcripts were first detected in alveolar bone in 19-day-old embryos. At the same age, no signal was detected in any cell of the first molar. In more mature teeth (newborn and 2-day-old rats), OSAD expression starts in the polarized odontoblasts and increases in the secretory and mature odontoblasts, respectively. Interestingly, a similar pattern of expression was observed in the ameloblast layer responsible for the deposition of enamel mineralized matrix. Together, these results lead us to speculate that OSAD may be implicated in biomineralization processes.