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1.
J Periodontol ; 2024 Jun 26.
Article in English | MEDLINE | ID: mdl-38923568

ABSTRACT

BACKGROUND: The aim of this study was to evaluate the incidence of preloading crestal bone loss (PLCBL) and to identify the patient-related and implant-related factors associated with PLCBL. METHODS: This retrospective cohort examined the dental records of patients who received at least one dental implant. PLCBL was defined as a reduction ⩾0.5 mm and severe PLCBL (primary variable) as a reduction ⩾1.5 mm in mesial and/or distal bone level, measured from the day of implant placement to uncovering or abutment installation/crown delivery. The incidence of PLCBL and patient and implant variables were recorded. Bivariate analysis and binary logistic regression identified factors associated with PLCBL ⩾0.5 mm and ⩾1.5 mm. RESULTS: A total of 746 dental implants placed in 361 patients from January 2011 to July 2021 was included in the analyses. Of the implants assessed, 24.4% (n = 182) exhibited PLCBL ⩾ 0.5 mm and 10.5% (n = 78) presented severe PLCBL (i.e., ⩾1.5 mm). Males (odds ratio [OR] = 1.85, 95% confidence interval [CI] = 1.11-3.07), patients with diabetes (OR = 3.33, 95% CI = 1.73-6.42), and those allergic to penicillin (OR = 3.13, 95% CI = 1.57-6.22) were more likely to experience severe PLCBL (p < 0.05). Implants placed in the anterior area (OR = 2.08, 95% CI = 1.16-3.73), with bone-level platform-abutment connection (OR = 4.73, 95% CI = 1.94-11.49) and inserted supracrestally (OR = 3.77, 95% CI = 1.84-7.72), presented a greater risk of developing severe PLCBL (p < 0.05). Implants placed in a previously grafted area presented a lower likelihood of developing severe PLCBL (OR = 0.489, 95% CI = 0.28-0.84). CONCLUSION: The incidence of PLCBL ⩾ 0.5 mm and ⩾1.5 mm was 24.4% and 10.5%, respectively. Male sex, diabetes, allergy to penicillin, anterior location, bone-level platform-abutment connection, and supracrestal implant placement are potential risk factors for severe PLCBL. A previously grafted area is a potential protective factor.

2.
Clin Cosmet Investig Dent ; 8: 79-87, 2016.
Article in English | MEDLINE | ID: mdl-27217799

ABSTRACT

Abfraction is a type of noncarious cervical lesion (NCCL) characterized by loss of tooth tissues with different clinical appearances. Evidence supports that abfraction lesions, as any NCCLs, have a multifactorial etiology. Particularly, the cervical wear of abfraction can occur as a result of normal and abnormal tooth function and may also be accompanied by pathological wear, such as abrasion and erosion. The interaction between chemical, biological, and behavioral factors is critical and helps to explain why some individuals exhibit more than one type of cervical wear mechanism than others. In an era of personalized dentistry, patient risk factors for NCCLs must be identified and addressed before any treatment is performed. Marked variations exist in dental practice concerning the diagnosis and management of these lesions. The lack of understanding about the prognosis of these lesions with or without intervention may be a major contributor to variations in dentists' management decisions. This review focuses on the current knowledge and available treatment strategies for abfraction lesions. By recognizing that progressive changes in the cervical area of the tooth are part of a physiologically dynamic process that occurs with aging, premature and unnecessary intervention can be avoided. In cases of asymptomatic teeth, where tooth vitality and function are not compromised, abfraction lesions should be monitored for at least 6 months before any invasive procedure is planned. In cases of abfraction associated with gingival recession, a combined restorative-surgical approach may be performed. Restorative intervention and occlusal adjustment are not indicated as treatment options to prevent further tooth loss or progression of abfraction. The clinical decision to restore abfraction lesions may be based on the need to replace form and function or to relieve hypersensitivity of severely compromised teeth or for esthetic reasons.

3.
ROBRAC ; 19(50)jul.-set. 2010. graf, ilus
Article in Portuguese | LILACS | ID: lil-564350

ABSTRACT

As tendências atuais na terapia com implantes odontológicostêm incluído o uso de implantes com superfícies modificadasutilizando nanotecnologia. Ciência que permite a construçãode novos materiais e dispositivos pela manipulação de átomosindividuais e moléculas (escala menor do que 100nm). O objetivodeste trabalho foi avaliar o papel das modificações em escalananométrica de superfícies de implantes osseointegradospara melhorar o processo de osseointegração. Nanotecnologiaoferece a engenheiros e profissionais da área de biologia e saúdenovos meios para entender e otimizar funções e respostasespecíficas de células. As várias técnicas utilizadas para adicionarcaracterísticas nanométricas às superfícies de implantesosseointegrados são descritas neste trabalho. Vários trabalhostem apresentado os efeitos da nanotecnologia na modulaçãode etapas fundamentais do processo de osseointegração. Asvantagens e desvantagens da utilização da nanotecnologia nasuperfície de implantes também são discutidas nesse trabalho.Posteriormente, em uma série de experimentos in vitro e in vivo,foi possível avaliar o efeito específico destas modificações emdois diferentes modelos. Como efeitos observados da aplicaçãode nanoestruturas à superfície dos implantes osseointegradosfoi possível verificar-se uma melhor e mais rápida resposta deosseointegração destes materiais, atuando efetivamente na cascatade diferenciação de osteoblastos.


Current trends in clinical dental implant therapy include useof endosseous dental implant surfaces embellished with nanoscaletopographies. Nanotechnology deals with materials withat least one significant dimension less than 100nm. The goal ofthis study was to consider the role of nanoscale topographic modificationof titanium substrates for the purpose of improvingosseointegration. Nanotechnology offers engineers and biologistsnew ways of interacting with relevant biological processes.Moreover, nanotechnology has provided means of understandingand achieving cell specific functions. The various techniquesthat can impart nanoscale topographic features to titaniumendosseous implants are described. Existing data supportingthe role of nanotopography suggests that critical steps in osseointegrationcan be modulated by nanoscale modification ofthe implant surface. Important distinctions between nanoscaleand micron-scale modification of the implant surface are presentlyconsidered. The advantages and disadvantages of nanoscalemodification of the dental implant surface are discussed.Finally, available data concerning the current dental implantsurfaces that utilize nanotopography in clinical dentistry aredescribed. Nanoscale modification of titanium endosseous implantsurfaces can alter cellular and tissue responses that maybenefit osseointegration and dental implant therapy. In a seriesof in vitro and in vivo experiments it was possible to evaluatethe effect of this modifications in different study designs. Theadvantages of the use of nanocues added to the surface of theosseointegrated dental implants allowed to a better and fasterosseointegration response of these materials, by acting on thedifferentiation of the osteoblasts.

4.
Biomaterials ; 30(25): 4053-62, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19464052

ABSTRACT

This study investigated the influence of nanoscale implant surface features on osteoblast differentiation. Titanium disks (20.0 x 1.0 mm) with different nanoscale materials were prepared using sol-gel-derived coatings and characterized by scanning electron microscopy, atomic force microscopy and analyzed by X-ray Photoelectron Spectrometer. Human Mesenchymal Stem Cells (hMSCs) were cultured on the disks for 3-28 days. The levels of ALP, BSP, Runx2, OCN, OPG, and OSX mRNA and a panel of 76 genes related to osteogenesis were evaluated. Topographical and chemical evaluation confirmed nanoscale features present on the coated surfaces only. Bone-specific mRNAs were increased on surfaces with superimposed nanoscale features compared to Machined (M) and Acid etched (Ac). At day 14, OSX mRNA levels were increased by 2-, 3.5-, 4- and 3-fold for Anatase (An), Rutile (Ru), Alumina (Al), and Zirconia (Zr), respectively. OSX expression levels for M and Ac approximated baseline levels. At days 14 and 28 the BSP relative mRNA expression was significantly up-regulated for all surfaces with nanoscale coated features (up to 45-fold increase for Al). The PCR array showed an up-regulation on Al coated implants when compared to M. An improved response of cells adhered to nanostructured-coated implant surfaces was represented by increased OSX and BSP expressions. Furthermore, nanostructured surfaces produced using aluminum oxide significantly enhanced the hMSC gene expression representative of osteoblast differentiation. Nanoscale features on Ti implant substrates may improve the osseointegration response by altering adherent cell response.


Subject(s)
Gene Expression , Nanostructures , Osteoblasts/physiology , Prostheses and Implants , Aluminum Oxide/chemistry , Animals , Cell Differentiation/physiology , Coated Materials, Biocompatible/chemistry , Coated Materials, Biocompatible/metabolism , Humans , Materials Testing , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/physiology , Microscopy, Atomic Force , Microscopy, Electron, Scanning , Osseointegration/physiology , Osteoblasts/cytology , Surface Properties , Titanium/chemistry , Zirconium/chemistry
5.
Quintessence Int ; 39(1): 33-7, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18551214

ABSTRACT

OBJECTIVE: To evaluate the capability of different adhesive systems to inhibit in vitro caries formation. METHOD AND MATERIALS: Four materials were tested: Gluma Comfort Bond (GL; Heraeus Kulzer), Gluma Comfort Bond + Desensitizer (GL+; Heraeus Kulzer), iBond (iB; Heraeus Kulzer), and One-up Bond F (OUB; Tokuyama). Bovine roots were ground to obtain flat mesial and distal dentin surfaces. Nail varnish was applied to the entire root surface except for two 10 mm X 2 mm windows on the flattened surfaces. The adhesives were applied to the exposed areas according to the manufacturers' instructions. The roots were incubated at 37 degrees C for 1 week in a suspension of StreptococcuS mutans and Lactobacillus acidophilus. Control groups either received no root surface treatment or were etched with 37% phosphoric acid (without immersion in the bacterial suspension). Specimens were sectioned perpendicular to the long axis of the roots, stained for 24 hours with Rhodamine B, and analyzed with confocal laser microscopy. The data were analyzed by 1-way ANOVA and Fisher's PLSD test at 95% confidence level. RESULTS: Lesions in the OUB, iB, and GL+ groups were significantly shallower than those in the no-treatment group. Acid etching did not produce any measurable lesions. Mean lesion depth in the GL group was similar to that in the no-treatment group. There was no significant difference between mean lesion depths in the GL+, GL, and iB groups. CONCLUSION: Fluoride- and glutaraldehyde-containing adhesive systems might be an aid in root caries prevention.


Subject(s)
Dentin-Bonding Agents/chemistry , Resin Cements/chemistry , Root Caries/microbiology , Acid Etching, Dental , Animals , Cattle , Fluorescent Dyes , Lactobacillus acidophilus/physiology , Materials Testing , Methacrylates/chemistry , Microscopy, Confocal , Phosphoric Acids/chemistry , Rhodamines , Root Caries/pathology , Streptococcus mutans/physiology , Temperature , Time Factors
6.
J Biomed Mater Res B Appl Biomater ; 85(1): 261-6, 2008 Apr.
Article in English | MEDLINE | ID: mdl-17910048

ABSTRACT

The aim of this study was to investigate the effect of trypsin digestion on removal of chondroitin sulfate proteoglycans (CS-PGs) in demineralized dentin, and subsequent dentin bonding. Bovine dentin fragments were demineralized, treated with or without trypsin, stained with cupromeronic blue, and observed under transmission electron microscopy. Demineralized sections with or without trypsin digestion were also subjected to immunohistochemical analysis with anti-chondroitin-4-sulfate (C4S) monoclonal antibody, 2-B-6. The presence of galactosamine and glucosamine in the trypsin digest was confirmed by amino acid analysis. Bond strength testing was performed on trypsin treated and control specimens where samples were either kept moist or dried and re-wet, then bonded. Bond strength significantly decreased after trypsin treatment (p < 0.05). TEM, immunohistochemical, and amino acid analyses demonstrated that trypsin digestion efficiently removed C4S-PGs from demineralized dentin matrix. This study indicates that the detrimental effects observed on dentin bonding by trypsinization may be due in part to the removal/cleavage of the C4S-PGs, and further underscore the importance of C4S-PGs on dentin bonding.


Subject(s)
Chondroitin Sulfate Proteoglycans/metabolism , Dental Bonding , Dentin/metabolism , Trypsin/metabolism , Amino Acids/analysis , Animals , Cattle , Chondroitin Sulfate Proteoglycans/genetics , Dentin/chemistry , Dentin/ultrastructure , Materials Testing , Tensile Strength
7.
Oper Dent ; 32(4): 388-93, 2007.
Article in English | MEDLINE | ID: mdl-17695612

ABSTRACT

OBJECTIVES: This study evaluated the antibacterial potential of four different adhesive systems. METHODS & MATERIALS: Gluma Comfort Bond + Desensitizer, Gluma Comfort Bond, iBond and One-Up Bond F were tested against Streptococcus mutans, Streptococcus sobrinus, Lactobacillus acidophilus and Actinomyces viscosus. The inhibition of growth by calibrated preparations was quantified by the measurement of zones of inhibition on bacterial lawns. Bactericidal activity was determined as reductions in recoverable colony-forming units in bacterial suspensions exposed to test preparations. RESULTS: All the preparations exhibited detectable zones of inhibition for all target bacteria through six months. When the bactericidal action was evaluated, all the materials were able to kill all the tested bacteria when tested immediately after polymerization. After one week of aging, iBond was the only material that continued to kill all of the test strains.


Subject(s)
Anti-Infective Agents, Local/pharmacology , Dentin-Bonding Agents/pharmacology , Gram-Positive Bacteria/drug effects , Resin Cements/pharmacology , Actinomyces viscosus/drug effects , Colony Count, Microbial , Dental Caries/prevention & control , Glutaral/pharmacology , Lactobacillus/drug effects , Methacrylates/pharmacology , Streptococcus/drug effects , Time Factors
8.
J Biomed Mater Res B Appl Biomater ; 80(1): 268-72, 2007 Jan.
Article in English | MEDLINE | ID: mdl-16767722

ABSTRACT

The stabilization of dentin collagen with biocompatible crosslinking agents may be of clinical importance to improve dentin bond strength. The present study aimed to evaluate the effect of three collagen crosslinking agents on the ultimate tensile strength (UTS) of undemineralized and demineralized dentin. Ten freshly extracted sound molars were sectioned into 0.5 x 0.5 mm2 thick beams. The beams were either demineralized or kept undemineralized. Then, specimens were subdivided into four groups according to treatments--PBS solution (control), 5% glutaraldehyde (GD), 0.5% proanthocyanidin PBS solution (PA), and 0.625% genipin PBS solution (GE). Specimens were kept in their respective solutions for either 4 or 40 h. To assess UTS, specimens were subjected to tensile forces at a crosshead speed of 1 mm/min. Statistical analysis was performed using two-way ANOVA and Fisher's PLSD test (p < 0.05). Statistically significant increases in UTS were observed for demineralized dentin after PA and GE dentin treatment, when compared with those of the control group. Dentin treated with GD showed no statistically significant differences in UTS when compared with that the control. Undemineralized dentin revealed no significant differences as compared to that of the control, regardless of the collagen crosslinkers. The application of two naturally occurring crosslinkers, i.e., PA and GE, to dentin collagen significantly improves UTS, indicating its potential value in restorative dentistry.


Subject(s)
Collagen/chemistry , Cross-Linking Reagents/chemistry , Dentin-Bonding Agents/chemistry , Dentin/chemistry , Molar/chemistry , Dental Bonding , Humans , Tensile Strength
9.
J Bone Miner Res ; 20(10): 1878-86, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16160746

ABSTRACT

UNLABELLED: MC3T3-E1 cell-derived clones expressing higher (S) or lower (AS) levels of biglycan were generated and characterized. The processes of cell differentiation and matrix mineralization were accelerated in S but delayed in AS, indicating that BGN modulates osteoblastic cell differentiation. INTRODUCTION: Biglycan (BGN), a member of the small leucine-rich proteoglycan family, is one of the major proteoglycans found in bone and has been implicated in bone formation. In this study, the effects of over- or underexpression of BGN on osteoblastic cell phenotypes and matrix mineralization were studied. MATERIALS AND METHODS: MC3T3-E1 cells were transfected with vectors containing the BGN cDNA in a sense or antisense orientation to generate clones expressing higher (S clones) or lower (AS clones) levels of BGN. MC3T3-E1 cells and those transfected with an empty vector (EV) were used as controls. The levels of BGN synthesized by these clones were evaluated by Western blot analysis. Cell growth was analyzed by cell counting and cell differentiation by the gene expression patterns of several osteoblastic markers using quantitative real-time PCR. The abilities of these clones to form mineralized matrices were evaluated by in vitro and in vivo mineralization assays. Furthermore, the clones were treated with BMP-4 and their responsiveness was assessed. RESULTS: The cell growth in these clones was unaffected; however, osteoblast differentiation was significantly accelerated in S clones and suppressed in AS clones. The in vitro matrix mineralization in S clones was significantly enhanced but severely impaired in AS clones. When transplanted into immunodeficient mice, S clone transplants exhibited larger areas of lamellar bonelike matrices, whereas only minute amounts of woven bone-type structure was found in AS transplants. The response to BMP-4 was higher in S clones but poorer in AS clones compared with that of controls. CONCLUSIONS: BGN modulates osteoblast differentiation, possibly by regulating BMP signaling, and consequently matrix mineralization.


Subject(s)
Autocrine Communication , Calcification, Physiologic , Cell Differentiation , Extracellular Matrix Proteins/metabolism , Osteoblasts/metabolism , Proteoglycans/metabolism , Animals , Autocrine Communication/drug effects , Autocrine Communication/genetics , Biglycan , Bone Morphogenetic Protein 4 , Bone Morphogenetic Proteins/pharmacology , Calcification, Physiologic/drug effects , Calcification, Physiologic/genetics , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cell Line , Extracellular Matrix/metabolism , Extracellular Matrix Proteins/genetics , Genetic Vectors , Mice , Proteoglycans/genetics , Signal Transduction/drug effects , Signal Transduction/genetics
10.
Eur J Oral Sci ; 113(3): 225-31, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15953247

ABSTRACT

Titanium implants create a unique ultrastructure (composed of a collagenous zone with relatively disorganized fibril morphology) at the bone-implant interface. The objective of this study was to investigate the temporal mRNA expression patterns, using real-time polymerase chain reaction, of type I collagen (COLI) and regulators for collagen fibrillogenesis, collagen-binding small leucine-rich proteoglycans (SLRPs) and lysyl hydroxylases (LHs), during mineralization, by MC3T3-E1 cells cultured on titanium (Ti). Lysates of the cultures on Ti and on plastic wells (Pl) for 10-50 d were used for the quantification of calcium and mRNA. Although the onset of calcium accumulation in the cultures on Ti (30-40 d) was slower than that of cultures on Pl (20-30 d), the gene expression patterns during mineralization were similar in cells cultured on either material. COLI and fibromodulin were up-regulated just before the onset of mineralization and then down-regulated. Lumican and LH1 were up-regulated just before the onset of mineralization and then returned to the baseline level. Decorin and LH2 were up-regulated at the late mineralization stage. Biglycan was down-regulated once at the early mineralization stage and then returned to the original level. LH3 was maintained at a steady level throughout. This study suggests actual but distinct roles of SLRPs and LHs in the formation of a unique ultrastructure at the bone-implant interface.


Subject(s)
Calcification, Physiologic/physiology , Collagen Type I/analysis , Dental Materials , Leucine/analysis , Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase/analysis , Proteoglycans/analysis , Titanium , 3T3 Cells , Animals , Calcium/analysis , Chondroitin Sulfate Proteoglycans/analysis , Chondroitin Sulfate Proteoglycans/genetics , Collagen Type I/genetics , Decorin , Dental Implants , Dental Materials/chemistry , Down-Regulation , Extracellular Matrix Proteins/analysis , Extracellular Matrix Proteins/genetics , Fibromodulin , Gene Expression Regulation/genetics , Keratan Sulfate/analysis , Keratan Sulfate/genetics , Leucine/genetics , Lumican , Mice , Osteoblasts/metabolism , Plastics/chemistry , Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase/genetics , Proteoglycans/genetics , RNA, Messenger/analysis , Titanium/chemistry , Transforming Growth Factor beta/antagonists & inhibitors , Up-Regulation
11.
Biochem Biophys Res Commun ; 326(1): 147-53, 2005 Jan 07.
Article in English | MEDLINE | ID: mdl-15567164

ABSTRACT

Periodontal ligament (PDL) is a thin fibrous connective tissue located between alveolar bone and cementum that remains unmineralized physiologically. It is thus thought that PDL cells possess mechanisms to inhibit mineralization. It has been demonstrated that S100A4, a member of the S100 calcium-binding protein family, is synthesized and secreted by PDL cells, and that it may act as an inhibitor of mineralization. However, the mechanisms of action of S100A4 in mineralization have not been thoroughly clarified. In the present study we investigated the effects of S100A4 inhibition by a short interfering RNA (siRNA) on the expression of osteoblast related genes by human PDL cells. Inhibition of S100A4 by siRNA resulted in increased expression of osteoblastic markers such as osteopontin and osteocalcin, and the osteoblast-specific transcription factors, Runx2/Cbfa1 and Osterix. These results indicate that S100A4 suppresses the expression of osteoblastic genes in PDL cells and may thus inhibit mineralization in the PDL.


Subject(s)
Osteoblasts/metabolism , Osteogenesis/physiology , Periodontal Ligament/metabolism , RNA Interference , S100 Proteins/metabolism , Transcription Factors/metabolism , Cell Differentiation/physiology , Cells, Cultured , Core Binding Factor Alpha 1 Subunit , DNA-Binding Proteins/metabolism , Gene Expression Regulation/physiology , Gene Silencing/physiology , Humans , Osteoblasts/cytology , Osteocalcin/physiology , Osteopontin , Periodontal Ligament/cytology , S100 Calcium-Binding Protein A4 , S100 Proteins/genetics , Sialoglycoproteins/metabolism , Transcription Factor AP-2 , Transcription Factors/genetics , Up-Regulation/physiology
12.
J Bone Miner Res ; 20(1): 81-7, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15619673

ABSTRACT

UNLABELLED: Several MC3T3-E1 cell-derived clones expressing higher levels of LH2b were analyzed for their abilities to form collagen fibrils and mineralization. The clones all exhibited smaller collagen fibrils and defective matrix mineralization in vitro and in vivo, indicating a critical role of LH2b-catalyzed post-translational modifications of collagen in bone matrix formation and mineralization. INTRODUCTION: We have recently shown that lysyl hydroxylase (LH) 2b, through its action on the telopeptidyl lysine residues of collagen, regulates collagen cross-linking pathway in the osteoblastic cell line, MC3T3-E1. To further elucidate the roles of LH2b in bone physiology, the effects of overexpression of LH2b on collagen fibrillogenesis and matrix mineralization were investigated. MATERIALS AND METHODS: Several MC3T3-E1-derived osteoblastic cell clones expressing higher levels of LH2b (S clones) and two controls (i.e., MC3T3-E1 cells and those transfected with an empty vector) were cultured. MALDI-TOF mass spectrometry was used to identify the LH2b. The collagen fibrillogenesis in the cultures was characterized by transmission electron microscopy, and the ability of these clones and cells to form mineralized matrix was analyzed by both in vitro and in vivo mineralization assays. RESULTS: The diameter of collagen fibrils in the S clone cultures was markedly smaller than that of the controls. The onset of matrix mineralization in the S clones was significantly delayed, and considerably fewer mineralized nodules were formed in their cultures in comparison with the controls. When transplanted into immunodeficient mice, the S clones failed to form mineralized matrices in vivo, whereas a bone-like mineralized matrix was well formed by the controls. The diameter of the collagen fibrils and the timing/extent of matrix mineralization in vitro were inversely correlated with the level of LH2b. In vitro cell differentiation was unaffected by the LH2b overexpression. CONCLUSIONS: These results indicate a critical role of LH2b catalyzed post-translational modification of collagen (i.e., telopeptidyl lysine hydroxylation and subsequent cross-linking) in collagen matrix formation and mineralization in bone.


Subject(s)
Calcification, Physiologic/physiology , Fibrillar Collagens/metabolism , Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase/physiology , Protein Processing, Post-Translational/physiology , Animals , Biomarkers/metabolism , Calcification, Physiologic/genetics , Cells, Cultured , Collagen Type I/genetics , Collagen Type I/metabolism , Fibrillar Collagens/ultrastructure , Gene Expression , Integrin-Binding Sialoprotein , Mice , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Osteoblasts/metabolism , Osteocalcin/genetics , Osteocalcin/metabolism , Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase/genetics , Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase/metabolism , Protein Processing, Post-Translational/genetics , Sialoglycoproteins/genetics , Sialoglycoproteins/metabolism , Sp7 Transcription Factor , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptional Activation
13.
J Bone Miner Res ; 19(8): 1349-55, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15231023

ABSTRACT

UNLABELLED: To elucidate the roles of LH2b in collagen cross-linking, MC3T3-E1 cell clones expressing higher (S) or lower (AS) levels of LH2b were established. Compared with controls, the collagen cross-linking pattern was shifted toward hydroxylysine-aldehyde (S clones)- or lysine-aldehyde (AS clones)-derived pathways. The data indicate that LH2b directs collagen cross-linking pathways through its action on telopeptidyl lysine residues. INTRODUCTION: Lysine (Lys) hydroxylation is a post-translational modification of collagen critical for cross-linking and glycosylation. Currently, three isoforms of lysyl hydroxylase (LH) have been identified, but their specific functions are still not well defined. Recently, we proposed that LH2 might modulate collagen cross-linking pattern through its action on Lys residues located in the telopeptide domains of collagen. MATERIALS AND METHODS: To directly test this hypothesis, several MC3T3-E1 cell-derived clones expressing higher (sense [S]) or lower (antisense [AS]) levels of LH2b, the predominant form of LH2 in this cell line, were established and cultured for 2 weeks, and collagen cross-links and precursor aldehydes in the matrices were analyzed. RESULTS: In S clones tested, the ratio of dihydroxylysinonorleucine (DHLNL) to hydroxylysinonorleucine (HLNL) was significantly higher than the average of controls (76% and 140% increase, respectively), and the level of pyridinoline (Pyr) was elevated (100% and 150% increase, respectively). In contrast, when MC3T3-E1 cells were transfected with a LH2b antisense construct (AS clones), the DHLNL/HLNL ratios were significantly lower than that of controls (56% and 73% decrease, respectively), and Pyr was not detected. Furthermore, significant amounts of an aldol-derived cross-link, dehydrohistidinohydroxymerodesmosine, were produced ( approximately 0.3 mol/mol of collagen) in AS clones. CONCLUSIONS: The data clearly show a critical role of LH2b in determining collagen cross-linking pathways, most likely through its action on telopeptidyl Lys residues.


Subject(s)
Collagen/biosynthesis , Desmosine/analogs & derivatives , Histidine/analogs & derivatives , Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase/physiology , 3T3 Cells , Amino Acids/analysis , Animals , Blotting, Western , Collagen/chemistry , DNA, Antisense/genetics , DNA, Complementary/genetics , Desmosine/analysis , Dipeptides/analysis , Gene Expression/genetics , Genetic Vectors/genetics , Histidine/analysis , Immunoprecipitation , Isoenzymes/analysis , Isoenzymes/genetics , Isoenzymes/physiology , Lysine/metabolism , Mice , Osteoblasts/chemistry , Osteoblasts/metabolism , Osteoblasts/physiology , Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase/analysis , Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase/genetics , Protein Processing, Post-Translational , RNA/genetics , RNA/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction , Transfection
14.
Biochem Biophys Res Commun ; 305(1): 6-9, 2003 May 23.
Article in English | MEDLINE | ID: mdl-12732188

ABSTRACT

Decorin (DCN), a member of small leucine-rich proteoglycans, is known to modulate collagen fibrillogenesis. In order to investigate the potential roles of DCN in collagen matrix mineralization, several stable osteoblastic cell clones expressing higher (sense-DCN, S-DCN) and lower (antisense-DCN, As-DCN) levels of DCN were generated and the mineralized nodules formed by these clones were characterized. In comparison with control cells, the onset of mineralization by S-DCN clones was significantly delayed; whereas it was markedly accelerated and the number of mineralized nodules was significantly increased in As-DCN clones. The timing of mineralization was inversely correlated with the level of DCN synthesis. In these clones, the patterns of cell proliferation and differentiation appeared unaffected. These results suggest that DCN may act as an inhibitor of collagen matrix mineralization, thus modulating the timing of matrix mineralization.


Subject(s)
Calcification, Physiologic , Collagen , Neoplasm Proteins , Proteoglycans/physiology , Animals , Cell Division , Clone Cells , Decorin , Extracellular Matrix , Extracellular Matrix Proteins , Kinetics , Mice , Oligonucleotides, Antisense , Osteoblasts/physiology , Proteoglycans/genetics , Transcription Factors/metabolism
15.
J Bone Miner Res ; 18(3): 493-501, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12619934

ABSTRACT

S100A4 is an intracellular calcium-binding protein expressed by osteoblastic cells. However, its roles in bone physiology are unknown. Because before matrix mineralization, its expression is markedly diminished, we hypothesized that S100A4 negatively regulates the mineralization process. In this study, we investigated the effects of the inhibition of S100A4 synthesis on osteoblast differentiation and in vitro mineralized nodule formation. Inhibition of S100A4 synthesis was achieved by an antisense approach in the mouse osteoblastic cell line MC3T3-E1. Cell clones that synthesized low levels of S100A4 (AS clones) produced markedly increased number of mineralized nodules at much earlier stages in comparison with controls as demonstrated by Alizarin red S and von Kossa staining. The expression of type I collagen (COLI) and osteopontin (OPN) increased in AS clones compared with controls. Bone sialoprotein (BSP) and osteocalcin (OCN), molecules associated with mineralization and markers for mature osteoblastic phenotype, were expressed in AS clones before their detection in controls. Because S100A4 was not localized in the nucleus of MC3T3-E1 cells and AS clones, it is unlikely that S100A4 directly regulates the expression of these genes. Moreover, the expression of Cbfal/Osf-2 and Osx, transcription factors necessary for the expression of osteoblast-associated genes, remained unchanged in AS clones, indicating that S100A4 may be downstream to these transcription factors. These findings indicate that S100A4 is a novel negative regulator of matrix mineralization likely by modulating the process of osteoblast differentiation.


Subject(s)
Calcification, Physiologic/physiology , Cell Differentiation/physiology , Osteoblasts/cytology , S100 Proteins/physiology , 3T3 Cells , Animals , Base Sequence , Biomarkers , Blotting, Western , DNA Primers , Fluorescent Antibody Technique , Mice , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , S100 Calcium-Binding Protein A4 , S100 Proteins/genetics
16.
J Med Dent Sci ; 49(4): 109-20, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12641381

ABSTRACT

The effects of high extracellular calcium (high Ca) in the local microenvironment on osteoclasts, osteoclast progenitors and stromal cells are not fully understood. We examined high Ca effect on osteoclastogenesis in mouse bone marrow cell culture. Mouse bone marrow cells were cultured for up to 6 days in the medium supplemented with 1, 25(OH)2 vitamin D3 (D3). High Ca treatment at the early stage of culture (the initial 24 hours) reduced the number of tartrate resistant acid phosphatase-positive multinuclear cells (TRAP(+)MNCs). This treatment slightly up-regulated the mRNA expressions of receptor activator of NF-(B ligand (RANKL), RANK and osteoprotegerin (OPG). This inhibitory effect on the formation of TRAP(+)MNCs was recovered by RANKL. In contrast, high Ca treatment at the later stage of osteoclastogenesis (the last 2 days of culture) stimulated the formation of TRAP(+)MNCs, increased RANKL and RANK mRNA expressions and decreased OPG mRNA. High Ca at neither the early nor the later stage of culture affected the total number of adherent cells and the mRNA expression of alkaline phosphatase and osteopontin. In conclusion, high Ca affects osteoclastogenesis in a manner depending on the stage of osteoclastogenesis, which is partly mediated via the RANKL-RANK-OPG regulatory system.


Subject(s)
Bone Marrow Cells/drug effects , Calcium/pharmacology , Osteoclasts/drug effects , Acid Phosphatase , Analysis of Variance , Animals , Carrier Proteins/metabolism , Carrier Proteins/pharmacology , Cell Differentiation/drug effects , Cells, Cultured , Cholecalciferol/pharmacology , Glycoproteins/metabolism , Glycoproteins/pharmacology , Isoenzymes , Macrophage Colony-Stimulating Factor/metabolism , Male , Membrane Glycoproteins/metabolism , Membrane Glycoproteins/pharmacology , Mice , Mice, Inbred Strains , NF-kappa B/metabolism , Osteoprotegerin , Polymerase Chain Reaction , RANK Ligand , RNA, Messenger/analysis , Receptor Activator of Nuclear Factor-kappa B , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Tumor Necrosis Factor , Tartrate-Resistant Acid Phosphatase
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