Depth distortion and angular deviation of a fully guided tooth-supported static surgical guide in a partially edentulous patient: A systematic review and meta-analysis.

PURPOSE: This systematic review and meta-analysis aimed to evaluate the depth distortion and angular deviation of fully-guided tooth-supported static surgical guides (FTSG) in partially edentulous arches compared to partially guided surgical guides or freehand. MATERIAL AND METHODS: This study followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines and was registered in the Open Science Framework (OSF). The formulated population, intervention, comparison, and outcome (PICO) question was: "In partially edentulous arches, what are the depth distortion and angular deviation of FTSG compared to partially guided surgical guides or freehand?" The search strategy involved four main electronic databases, and an additional manual search was completed in November 2023 by following an established search strategy. Initial inclusion was based on titles and abstracts, followed by a detailed review of selected studies, and clinical studies that evaluated the angular deviations or depth distortion in FTSG in partial arches, compared to partially guided surgical guides or freehand, were included. In FTSG, two surgical approaches were compared: open flap and flapless techniques, and two digital methods were assessed for surgical guide design with fiducial markers or dental surfaces. A qualitative analysis for clinical studies was used to assess the risk of bias. The certainty of the evidence was assessed according to the grading of recommendations, assessment, development, and evaluations (GRADE) system. In addition, a single-arm meta-analysis of proportion was performed to evaluate the angular deviation of freehand and FTSG. RESULTS: Ten studies, published between 2018 and 2023, met the eligibility criteria. Among them, 10 studies reported angular deviations ranging from -0.32° to 4.96° for FTSG. Regarding FTSG surgical approaches, seven studies examined the open flap technique for FTSG, reporting mean angular deviations ranging from 2.03° to 4.23°, and four studies evaluated flapless FTSG, reporting angular deviations ranging from -0.32° to 3.38°. Six studies assessed the freehand surgical approach, reporting angular deviations ranging from 1.40° to 7.36°. The mean depth distortion ranged between 0.19 mm to 2.05 mm for open flap FTSG, and between 0.15 mm to 0.45 mm for flapless FTSG. For partially guided surgical guides, two studies reported angular deviations ranging from 0.59° to 3.44°. Seven studies were eligible for meta-analysis, focusing on the FTSG in open flap technique, with high heterogeneity (I2 (95%CI) = 92.3% (88.7%-96.4%)). In contrast, heterogeneity was low in studies comparing freehand versus FTSG in open flap techniques (I2 (95%CI) = 21.3% (0.0%-67.8%)), favoring the FTSG surgical approach. CONCLUSION: In partially edentulous arches, FTSG systems exhibited less angular deviation than freehand and partially guided surgical guides. Flapless surgical approaches were associated with reduced angular deviation and depth distortion, suggesting a potential preference for the FTSG method in these procedures.

The beyond the gap filling approach: Modeling facial and interproximal tissues and improving esthetics for anterior flapless immediate implant placement.

OBJECTIVE: Maintenance of adequate interproximal tissue height between an implant and a natural tooth or between adjacent implants represents an esthetic challenge in implant dentistry. The aim of this case report is to describe a modified technique referred to as the beyond the gap filling (BGF) approach designed to improve the horizontal and vertical components of the facial aspect and particularly the height of interproximal bone peaks around immediately placed implants into fresh extraction sockets. CLINICAL CONSIDERATIONS: Four patients (five teeth) requiring anterior tooth extraction were treated with the BGF approach that included: (a) minimally traumatic tooth extraction; (b) immediate implant placement without flap elevation; (c) installation of a narrow profile healing abutment to protect the implant during grafting; (d) grafting with a construct with 90% bovine bone granules and 10% porcine collagen packed coronally to the facial and interproximal bone walls above the level of the bone crest; and (e) delivery of an immediate restoration. CONCLUSIONS: The current report suggests that the level/height of the interproximal bone crests between an implant and a natural tooth or between two adjacent implants can be improved by the BGF approach and, consequently, papilla height can be maintained in cases with a high risk of papilla height collapse and, consequently, esthetic outcomes can be maximized. CLINICAL SIGNIFICANCE: The BGF is a simple technique to be used by clinicians to prevent significant papilla collapse in anterior immediate implants and consequently achieve maximum esthetic outcomes in implant dentistry.

Preloading peri-implant crestal bone loss: A retrospective study of incidence and related factors.

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.

Linear Accuracy of Intraoral Scanners for Full-Arch Impressions of Implant-Supported Prostheses: A Systematic Review and Meta-Analysis.

This article compares the accuracy of intraoral scanners (IOSs) used in the digital impression of full arches to fabricate implant-supported complete prostheses. This study followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines and was registered in the Open Science Framework (DOI 10.17605/OSF.IO/CPM9K). Six electronic databases, gray literature databases, and a manual search were performed in April 2022. Studies that evaluated the accuracy of intraoral scan impressions compared with conventional impressions in full-arch impressions were included for complete implant-supported prostheses. In addition, an adapted checklist for reporting in vitro studies was used to assess the risk of bias. Meta-analysis was conducted using a random-effects Hunter- Schmidt model. Nine studies were included in the analysis. IOS impressions present higher accuracy (137.86 µm) than conventional impressions (182.51 µm) (p<0.001). The heterogeneity of the study's methodology was I2»18.34. However, impression accuracy varies significantly with scan body type, IOS type, scanning strategy, and modification technique. For most IOS systems, the acceptable clinical threshold of linear accuracy of 200 µm can be achieved, except for the True Definition Scanner in one of the studies. Based on the results of the included studies, digital impressions using IOS present similar or better linear accuracy than conventional impression techniques.

Dimension of the facial bone wall in the anterior maxilla: a cone-beam computed tomography study.

OBJECTIVE: To determine the thickness of the facial bone wall in the anterior dentition of the maxilla and at different locations apical to the cemento-enamel junction (CEJ). MATERIAL AND METHODS: Two-hundred and fifty subjects, aged between 17 and 66 years, with all maxillary front teeth present were included. Written informed consents were obtained. Cone-beam computed tomography scans were performed with the iCAT unit. This examination included all tooth and edentulous sites in the dentition. The images were acquired by means of the iCAT software and processed by a computer. Measurements of the (i) distance between the CEJ and the facial bone crest and (ii) the thickness of the facial bone wall were performed. The bone wall dimensions were assessed at three different positions in relation to the facial bone crest, i.e., at distances of 1, 3, and 5 mm apical to the crest. RESULTS: The measurements demonstrated that (i) the distance between the CEJ and the facial bone crest varied between 1.6 and 3 mm and (ii) the facial bone wall in most locations in all tooth sites examined was ≤1 mm thick and that close to 50% of sites had a bone wall thickness that was ≤0.5 mm. CONCLUSION: Most tooth sites in the anterior maxilla have a thin facial bone wall. Such a thin bone wall may undergo marked dimensional diminution following tooth extraction. This fact must be considered before tooth removal and the planning of rehabilitation in the anterior segment of the dentition in the maxilla.

Local effect of simvastatin combined with different osteoconductive biomaterials and collagen sponge on new bone formation in critical defects in rat calvaria.

PURPOSE: To evaluate the local effect of simvastatin (SVT) combined with deproteinized bovine bone (DBB) with hydroxyapatite/ß-tricalcium phosphate biphasic ceramics (HA/TCP) and with collagen sponge (CS) on bone repair in critical size defects (CSDs) in rat calvaria. METHODS: Forty-two 5-mm diameter CSDs were made bilaterally in the calvaria of 18 rats. The animals were allocated according to the type of biomaterial and associations used to fill the CSD. After 8 weeks, the animals were euthanized, and their calvaria were evaluated for repaired tissue composition using histologic and histometric analyses. RESULTS: In the histometric analysis, the use of SVT showed to increase bone formation in the CSDs when combined with all the bone substitutes tested in this study (p<0.05). Greater bone formation was observed in the groups with SVT compared to the groups without SVT. CONCLUSIONS: The use of SVT without the need for a vehicle and combined with a commercially available biomaterial may be a cheaper way to potentiate the formation of bone tissue without the need to produce new biomaterials. Therefore, SVT combined with DBB induced significantly greater new bone formation than did the other treatments.

A new method to assess and measure palatal masticatory mucosa by cone-beam computerized tomography.

AIM: The aim of this study was to apply a novel method to obtain high-quality images by cone-beam computerized tomography (CBCT) that consistently allowed the determination of the dimensions of the palatal mucosa. MATERIALS AND METHODS: Thirty-one patients participated in this study. At the time of the CBCT scanning, the patients wore a plastic lip retractor and wooden spatulas to retract soft tissues away from the teeth and gingiva. The thickness of the palatal mucosa was obtained at forty different locations on each patient. RESULTS: Retraction of the lips and cheek allowed a clear observation and measurements of the thickness of the palatal masticatory mucosa. The average thickness of the palatal mucosa was 2.92 mm in the canine area, 3.11 mm at the first pre-molar, 3.28 mm at the second pre-molar, 2.89 mm at the first molar and 3.15 mm at the second molar. Statistical differences were observed at different ages and heights of measurements. CONCLUSIONS: A new non-invasive method to consistently obtain high-quality images of the palatal masticatory mucosa is described. Measurements of this mucosa could be obtained at different locations on the palate.

Nanostructured alumina-coated implant surface: effect on osteoblast-related gene expression and bone-to-implant contact in vivo.

PURPOSE: The use of nanotechnology to enhance endosseous implant surfaces may improve the clinical control of interfacial osteoblast biology. This study investigated the influence of a nanostructure-coated implant surface on osteoblast differentiation and its effects on bone-to-implant contact (BIC) and removal torque values. MATERIALS AND METHODS: Titanium disks were machined (M) or machined and subsequently treated by acid etching (Ac) or by dipping in an aluminum oxide solution (Al2O3). Surfaces were characterized by scanning electron microscopy, atomic force microscopy, and x-ray microanalysis. For the in vitro experiment, rat mesenchymal stem cells (rMSCs) were grown in osteogenic supplements on the disk surfaces for 3 days. Real-time polymerase chain reaction (PCR) was used to measure mRNA levels of several gene products (bone sialoprotein, osteocalcin, osteopontin, and RUNX-2). For the in vivo experiment, titanium implants were placed in rat tibiae and harvested after 3 to 21 days for measurement of bone-specific mRNA levels by real-time PCR. Removal torque and BIC were measured 3 to 56 days after placement. RESULTS: Average height deviation (Sa, in nm) values for M, Ac, and Al2O3 implants were 86.5, 388.4, and 61.2, respectively. Nanostructured Al2O3 topographic features applied to machined implants promoted MSC commitment to the osteoblast phenotype. Greater bone-specific gene expression was observed in tissues adjacent to Al2O3 implants, and associated increases in BIC and torque removal were noted. CONCLUSION: Nanostructured alumina may directly influence cell behavior to enhance osseointegration.

Application of crosslinkers to dentin collagen enhances the ultimate tensile strength.

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.

In vitro inhibition of bacterial growth using different dental adhesive systems.

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.

Abfraction lesions: etiology, diagnosis, and treatment options.

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.

Simvastatin and biphasic calcium phosphate affects bone formation in critical-sized rat calvarial defects.

PURPOSE: To investigate the effects of locally applied simvastatin plus biphasic calcium phosphate (BoneCeramic(r)) or collagen sponge on bone formation in critical-sized bone defects. METHODS: Thirty defects of 5mm in diameter were created bilaterally with a trephine bur in the calvariae of fifteen Wistar rats. The defects were divided into five groups: group 1 - control, no treatment; group 2 (BoneCeramic(r)); group 3 (BoneCeramic(r) + 0.1mg simvastatin); group 4 (collagen sponge); and group 5 (collagen sponge + 0.1mg simvastatin). After eight weeks the animals were euthanized and their calvariae were histologically processed. Hematoxylin and eosin-stained sections were subjected to histological and histomorphometrical analyses. The area of newly formed bone was calculated and compared between groups. RESULTS: The greater amount of a bone-like tissue was formed around the carrier in group 3 (BoneCeramic(r) + 0.1mg simvastatin) followed by group 2 (BoneCeramic(r)), and almost no bone was formed in the other groups. Group 3 was significantly different compared to group 2, and both groups were significantly different compared to the other groups. CONCLUSION: Simvastatin combined with BoneCeramic(r) induced significantly greater amounts of newly formed bone and has great potential for the healing of bone defects.

Soft tissue cone beam computed tomography (ST-CBCT) for the planning of esthetic crown lengthening procedures.

OBJECTIVE: The aim of this study was to measure the clinical crown, anatomical crown, and amount of gingiva covering the enamel of anterior maxillary teeth with soft tissue cone beam computed tomography (ST-CBCT) and thus propose a protocol for the application of this noninvasive method for the planning of esthetic crown lengthening procedures to prevent undesired root exposure. MATERIALS AND METHODS: Ten young patients (mean age 22.8 years) requiring esthetic crown lengthening surgeries but not restorative procedures were included in this study. The ST-CBCT images were analyzed with an image editor and analysis software (Pixelmator 3.2), and the clinical crown, anatomical crown, and amount of gingiva covering the enamel were measured. RESULTS: The mean value of the length of the anatomical crowns of the central incisors, lateral incisors, and canines were 11.06 mm, 9.55 mm, and 9.77 mm, respectively. Thus, the canines were approximately 1.3 mm shorter than the central incisors. CONCLUSION: It was shown that ST-CBCT is a useful tool to assess the actual length of the anatomical crown and the amount of gingival tissue that needs to be surgically removed in an esthetic crown lengthening procedure to prevent undesired root exposure. Thus, ST-CBCT is a helpful noninvasive procedure for the planning of esthetic crown lengthening surgeries, making it possible to better predict their final outcome.

Overexpression of lysyl hydroxylase-2b leads to defective collagen fibrillogenesis and matrix mineralization.

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.

Biglycan modulates osteoblast differentiation and matrix mineralization.

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.

Local effect of simvastatin combined with different osteoconductive biomaterials and collagen sponge on new bone formation in critical defects in rat calvaria

Abstract Purpose To evaluate the local effect of simvastatin (SVT) combined with deproteinized bovine bone (DBB) with hydroxyapatite/β-tricalcium phosphate biphasic ceramics (HA/TCP) and with collagen sponge (CS) on bone repair in critical size defects (CSDs) in rat calvaria. Methods Forty-two 5-mm diameter CSDs were made bilaterally in the calvaria of 18 rats. The animals were allocated according to the type of biomaterial and associations used to fill the CSD. After 8 weeks, the animals were euthanized, and their calvaria were evaluated for repaired tissue composition using histologic and histometric analyses. Results In the histometric analysis, the use of SVT showed to increase bone formation in the CSDs when combined with all the bone substitutes tested in this study (p<0.05). Greater bone formation was observed in the groups with SVT compared to the groups without SVT. Conclusions The use of SVT without the need for a vehicle and combined with a commercially available biomaterial may be a cheaper way to potentiate the formation of bone tissue without the need to produce new biomaterials. Therefore, SVT combined with DBB induced significantly greater new bone formation than did the other treatments.

Lysyl hydroxylase-2b directs collagen cross-linking pathways in MC3T3-E1 cells.

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.

S100A4: a novel negative regulator of mineralization and osteoblast differentiation.

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.

High extracellular calcium affects osteoclastogenesis in mouse bone marrow cell culture.

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.

Behavioural and cognitive effects of simvastatin dose used in stimulation of bone regeneration in rats.

PURPOSE: To analyze the effects of simvastatin (SVT) in the locomotion, anxiety and memory of rats, as a reflection of the administration of a minimum dose capable of stimulating bone regeneration in defects in the calvariae. METHODS: Surgical procedures were performed in 15 female Wistar rats, 2-month old, to insert the grafting material regenerator (Bone-ceramic®) and/or SVT, followed by behavioural and cognitive assessments in the 7th, 30th and 60th days post surgery. RESULTS: The SVT locally applied with the goal of bone regeneration in defects created in rat calvariae does not interfere with locomotion, anxiety levels and/or memories of rats, except for the first week following surgery, when an anxiolytic effect was observed, as a result of a possible central action. CONCLUSION: Failure to provoke any response within 30 and 60 days post surgical procedures suggests that SVT may constitute a good choice in stimulating bone regeneration without affecting the long term neural functions.