Reporting stAndards for research in PedIatric Dentistry (RAPID): an expert consensus-based statement.

BACKGROUND: Reporting guidelines for different study designs are currently available to report studies with accuracy and transparency. There is a need to develop supplementary guideline items that are specific to areas within Pediatric Dentistry. This study aims to develop Reporting stAndards for research in PedIatric Dentistry (RAPID) guidelines using a pre-defined expert consensus-based Delphi process. METHODS: The development of the RAPID guidelines was based on the Guidance for Developers of Health Research Reporting Guidelines. Following a comprehensive search of the literature, the Executive Group identified ten themes in Pediatric Dentistry and compiled a draft checklist of items under each theme. The themes were categorized as: General, Oral Medicine, Pathology and Radiology, Children with Special Health Care Needs, Sedation and Hospital Dentistry, Behavior Guidance, Dental Caries, Preventive and Restorative Dentistry, Pulp Therapy, Traumatology, and Interceptive Orthodontics. A RAPID Delphi Group (RDG) was formed comprising of 69 members from 15 countries across six continents. Items were scored using a 9-point rating Likert scale. Items achieving a score of seven and above, marked by at least 70% of RDG members were accepted into the RAPID checklist items. Weighted mean scores were calculated for each item. Statistical significance was set at p < 0.05 and one-way ANOVA was used to calculate the difference in the weighted mean scores between the themes. RESULTS: The final RAPID checklist comprised of 128 items that were finalized and approved by the RDG members in the online consensus meeting. The percentage for high scores (scores 7 to 9) ranged from 69.57 to 100% for individual items. The overall weighted mean score of the final items ranged from 7.51 to 8.28 (out of 9) and the difference was statistically significant between the themes (p < 0.05). CONCLUSIONS: The RAPID statement provides guidance to researchers, authors, reviewers and editors, to ensure that all elements relevant to particular studies are adequately reported.

Prospective randomized clinical trial of primary molar crowns: 36-month results.

PURPOSE: To clinically evaluate the clinical success of a primary zirconia molar crown, compared with stainless steel crowns (SSCs). METHODS: This randomized, controlled clinical trial was designed as a split-mouth study. 50 subjects ranging in age from 3-7 years were recruited to provide a total of 50 paired teeth requiring primary molar crowns, each participant receiving a SSC and zirconia crown. Restorations were evaluated at 6-, 12-, 24-, and 36-month recall appointments examining the following criteria: gingival health, estimate of the degree crown was high in occlusion, surface roughness, staining on crown surface, wear of opposing arch tooth, color match, anatomic form, marginal integrity, marginal discoloration, proximal contact area, secondary caries at crown margin and parent/guardian satisfaction with crown appearance. RESULTS: The 36-month follow up included 23 subjects (46%). 35 crowns (35%) were evaluated; of the 18 zirconia crowns and 17 SSCs, there were no failures at the 36-month evaluation. The only significant differences in the parameters evaluated were parent satisfaction, with the zirconia crown preference (P< 0.05) and gingival health, with the zirconia crowns having healthy adjacent gingiva (P< 0.01). The 36-month results indicated that zirconia primary molar crowns performed similarly to an established SSC for restoration of primary molars. CLINICAL SIGNIFICANCE: The findings from this study indicated that at 36 months, NuSmile ZR zirconia crowns clinically performed as well as stainless steel crowns.

Reporting stAndards for research in PedIatric Dentistry (RAPID): A development protocol.

Reporting guidelines can improve the quality of reports of research findings. Some specialities in health care however require guidance on areas that are not captured within the existing guidelines, and this is the case for Paediatric Dentistry where no such standards are available to guide the reporting of different types of study designs. The 'Reporting stAndards for research in PedIatric Dentistry' (RAPID) group aims to address this need by developing guidelines on reporting elements of research of particular relevance to Paediatric Dentistry. The development of RAPID guidelines will involve a five-phase process including a Delphi study, which is an explicit consensus development method designed and implemented in accordance with the Guidance on Conducting and REporting DElphi Studies. The guideline development process will be overseen by an Executive Group. Themes specific to areas in Paediatric Dentistry will be selected, and items to be included under each theme will be identified by members of the Executive Group reviewing at least five reports of experimental and analytical study types using existing reporting guidelines. For the Delphi study, the Executive Group will identify an international multidisciplinary RAPID Delphi Group (RDG) of approximately 60 participants including academics, Paediatric Dentists, parents, and other stakeholders. Each item will be evaluated by RDG on clarity using a dichotomous scale ('well phrased' or 'needs revision') and on suitability for inclusion in the Delphi study using a 9-point Likert scale (1 = 'definitely not include' to 9 = 'definitely include'). The items will then be included in an online Delphi study of up to four rounds, with participants invited from stakeholder groups across Paediatric Dentistry. Items scored 7 or above by at least 80% of respondents will be included in the checklist and further discussed in a face-to-face Delphi consensus meeting. Following this, the Executive Group will finalize the RAPID guidelines. The guidelines will be published in peer-reviewed scientific journals and disseminated at scientific meetings and conferences. All the outputs from this project will be made freely available on the RAPID website: www.rapid-statement.org.

Early childhood caries epidemiology, aetiology, risk assessment, societal burden, management, education, and policy: Global perspective.

BACKGROUND: This paper is a summary of the proceedings of the International Association of Paediatric Dentistry Bangkok Conference on early childhood caries (ECC) held in 3-4 November 2018. AIM: The paper aims to convey a global perspective of ECC definitions, aetiology, risk factors, societal costs, management, educational curriculum, and policy. DESIGN: This global perspective on ECC is the compilation of the state of science, current concepts, and literature regarding ECC from worldwide experts on ECC. RESULTS: Early childhood caries is related to frequent sugar consumption in an environment of enamel adherent, acid-producing bacteria in a complex biofilm, as well as developmental defects of enamel. The seriousness, societal costs, and impact on quality of life of dental caries in pre-school children are enormous. Worldwide data show that ECC continues to be highly prevalent, yet infrequently treated. Approaches to reduce the prevalence include interventions that start in the first year of a child's life, evidence-based and risk-based management, and reimbursement systems that foster preventive care. CONCLUSIONS: This global perspective on ECC epidemiology, aetiology, risk assessment, global impact, and management is aimed to foster improved worldwide understanding and management of ECC.

Hidden Caries: A Case Report of a 17 Years Old Male with Two Affected Mandibular Premolars.

Hidden caries is the term used to describe carious lesions that are not visualized clinically on erupted teeth but can be detected radiographically. The exact etiology remains an area of controversy. The purpose of the current case report was to discuss the diagnosis and treatment of two mandibular premolars with hidden caries. After diagnosis was established, both premolars were treated with indirect pulp caps and resin-based composite restorations. A one year follow up appointment revealed both teeth to be free from signs and symptoms of inflammation.

Dentin and enamel demineralization inhibition at restoration margins of Vitremer, Z 100 and Cention N.

PURPOSE: To examine the in vitro caries inhibition of a resin-modified glass-ionomer cement, and a fluoride and calcium releasing resin-based composite. METHODS: Standardized Class V preparations were placed in 30 molars, the gingival margin placed below the cemento-enamel junction. Randomly, 10 Vitremer, 10 Z 100 and 10 Cention N restorations were placed according to manufacturer's instructions, in 30 teeth. The Z 100 non fluoride-releasing resin-based composite group acted as the control. All teeth had an acid-resistant varnish placed to within 1 mm of restoration margins and they were placed into artificial saliva for 2 weeks, the saliva being replenished every 48 hours. All teeth were subjected to thermocycling each day and to an artificial caries challenge (pH 4.4) for one hour twice a day. Sections of 100 µm were obtained, photographed under polarized light microscopy and then demineralized areas adjacent to restorations were quantitated. RESULTS: The mean (± S.D.) area (µm 2) demineralization 100 µm from the enamel and dentin margins were: Vitremer 1,554 ± 1,153, 4,125 ± 301; Cention N 3580 ± 1,518, 6,246 ± 630; Z 100 13,257 ± 3,794, 8,842 ± 1,799. A Mann-Whitney Rank Sum Test indicated that Vitremer had significantly less enamel demineralization then Cention N (P< 0.003) and Z 100 (P< 0.001) and Cention N had significantly less enamel demineralization than Z 100 (P< 0.001) and Z 100 (P< 0.001). Vitremer also had significantly less dentin demineralization than Cention N (P< 0.001) and Cention N had significantly less dentin demineralization than Z 100 (P< 0.001). CLINICAL SIGNIFICANCE: Recurrent caries remains a concern and this in vitro research indicates that Cention N, as well as Vitremer may clinically inhibit caries at restoration margins.

Fluoride release and re-release from a bioactive restorative material.

PURPOSE: To compare the amount of fluoride release and re-release of three different restorative materials. METHODS: The three restorative materials included a resin-based composite (Z100TM, 3M-ESPE), a resin-modified glass ionomer cement (VitremerTM, 3M-ESPE) and a bioactive material (Activa Bioactive-RestorativeTM, Pulpdent,). Ten disks were fabricated from each material. The disks were immersed in deionized water and stored. Samples were taken from each vial on Days 1, 7, 14 and 30 for fluoride ion analysis. Each disk was then exposed to 2.0% neutral sodium fluoride gel (0.9% fluoride ion, Dentsply), immersed in deionized water and stored. Samples were taken on Days 1, 7, 14 and 30 for fluoride ion analysis utilizing a fluoride-specific ion-analyzer. RESULTS: Z100 released less fluoride on Days 1 (P< 0.001), 7 (P= 0.001) and 14 (P< 0.022) for Phase I (initial release) than Phase II (re-release). Vitremer and Activa released less fluoride on Days 7, 14 and 30 (P< 0.001) for Phase II than Phase I. For all intervals of Phase I, Vitremer released the most fluoride, Activa released the second most, and Z100 released the least. These results were the same for Days 7, 14 and 30 of Phase II. The level of fluoride release from Activa was less than that of Vitremer, and greater than that of Z100 for all intervals of Phase I. The results were the same for all but one interval of Phase II. CLINICAL SIGNIFICANCE: This in vitro study evaluated the fluoride release and subsequent re-release of fluoride following a topical fluoride treatment to analyze if the materials were truly bioactive. The results indicate the bioactive material does uptake fluoride and re-release it which could offer inhibition to caries at restoration margins.

Fluoride release and re-release from various esthetic restorative materials.

PURPOSE: To measure the amount of fluoride release and re-release after re-charge from two commonly used esthetic restorative materials and compare it to a new experimental material. METHODS: 30 standardized disc-shaped specimens were fabricated using resin-based composite (Z100), resin-modified glass-ionomer cement (Vitremer) and a new experimental material which is a self-curing resin-based composite with light curing option. 10 specimens were made from each material. The specimens of each group were immersed separately in 10 ml distilled water. Fluoride release was measured after 1, 7, 14 and 30 days using a fluoride-specific ion electrode and an ion-analyzer. The specimens were then exposed to 2.0% neutral sodium fluoride foam (0.9% fluoride ion). The amount of fluoride re-released was measured at Days 1, 7, 14 and 30. RESULTS: An ANOVA indicated a statically significant variance among the groups (P< 0.001). The experimental group demonstrated significantly less fluoride release at Day 1 compared to Day 31 (first day after 2% sodium fluoride application). At Days 7, 14 and 30 there was significantly more fluoride release than Day 7, 14 and 30 after the topical fluoride application (P< 0.001). There was significantly more fluoride release from Vitremer than the experimental material at Days 1 and 7. However, similar release was observed at Days 14 and 30 for Vitremer and experimental material, but not for Z100. Both Vitremer and the experimental material showed significantly more release of fluoride compared to Z100 at all time points. CLINICAL SIGNIFICANCE: This study demonstrates that the new experimental material released fluoride, re-charged and re-released fluoride at a level comparable to Vitremer but more than Z100.

Establishment of Immortalized BMP2/4 Double Knock-Out Osteoblastic Cells Is Essential for Study of Osteoblast Growth, Differentiation, and Osteogenesis.

Bone morphogenetic proteins 2 and 4 (BMP2/4) are essential for osteoblast differentiation and osteogenesis. Generation of a BMP2/4 dual knock-out ((ko/ko)) osteoblastic cell line is a valuable asset for studying effects of BMP2/4 on skeletal development. In this study, our goal was to create immortalized mouse deleted BMP2/4 osteoblasts by infecting adenoviruses with Cre recombinase and green fluorescent protein genes into immortalized murine floxed BMP2/4 osteoblasts. Transduced BMP2/4(ko/ko) cells were verified by green immunofluorescence and PCR. BMP2/4(ko/ko) osteoblasts exhibited small size, slow cell proliferation rate and cell growth was arrested in G1 and G2 phases. Expression of bone-relate genes was reduced in the BMP2/4(ko/ko) cells, resulting in delay of cell differentiation and mineralization. Importantly, extracellular matrix remodeling was impaired in the BMP2/4(ko/ko) osteoblasts as reflected by decreased Mmp-2 and Mmp-9 expressions. Cell differentiation and mineralization were rescued by exogenous BMP2 and/or BMP4. Therefore, we for the first time described establishment of an immortalized deleted BMP2/4 osteoblast line useful for study of mechanisms in regulating osteoblast lineages.

Establishment of Immortalized Mouse Bmp2 Knock-Out Dental Papilla Mesenchymal Cells Necessary for Study of Odontoblastic Differentiation and Odontogenesis.

Bmp2 is essential for dentin formation. Bmp2 cKO mice exhibited similar phenotype to dentinogenesis imperfecta, showing dental pulp exposure, hypomineralized dentin, and delayed odontoblast differentiation. As it is relatively difficult to obtain lot of primary Bmp2 cKO dental papilla mesenchymal cells and to maintain a long-term culture of these primary cells, availability of immortalized deleted Bmp2 dental papilla mesenchymal cells is critical for studying the underlying mechanism of Bmp2 signal in odontogenesis. In this study, our goal was to generate an immortalized deleted Bmp2 dental papilla mesenchymal (iBmp2(ko/ko)dp) cell line by introducing Cre recombinase and green fluorescent protein (GFP) into the immortalized mouse floxed Bmp2 dental papilla mesenchymal (iBmp2(fx/fx)dp) cells. iBmp2(ko/ko)dp cells were confirmed by GFP and PCR. The deleted Bmp2 cells exhibited slow cell proliferation rate and cell growth was arrested in G2 phase. Expression of tooth-related marker genes and cell differentiation were decreased in the deleted cells. Importantly, extracellular matrix remodeling was impaired in the iBmp2(ko/ko)dp cells as reflected by the decreased Mmp-9 expression. In addition, with exogenous Bmp2 induction, these cell differentiation and mineralization were rescued as well as extracellular matrix remodeling was enhanced. Therefore, we for the first time described establishment of iBmp(ko/ko) cells that are useful for study of mechanisms in regulating dental papilla mesenchymal cell lineages.

Bmp2 deletion causes an amelogenesis imperfecta phenotype via regulating enamel gene expression.

Although Bmp2 is essential for tooth formation, the role of Bmp2 during enamel formation remains unknown in vivo. In this study, the role of Bmp2 in regulation of enamel formation was investigated by the Bmp2 conditional knock out (Bmp2 cKO) mice. Teeth of Bmp2 cKO mice displayed severe and profound phenotypes with asymmetric and misshaped incisors as well as abrasion of incisors and molars. Scanning electron microscopy analysis showed that the enamel layer was hypoplastic and enamel lacked a typical prismatic pattern. Teeth from null mice were much more brittle as tested by shear and compressive moduli. Expression of enamel matrix protein genes, amelogenin, enamelin, and enamel-processing proteases, Mmp-20 and Klk4 was reduced in the Bmp2 cKO teeth as reflected in a reduced enamel formation. Exogenous Bmp2 up-regulated those gene expressions in mouse enamel organ epithelial cells. This result for the first time indicates Bmp2 signaling is essential for proper enamel development and mineralization in vivo.

Space loss following premature loss of primary second molars.

This study was designed to evaluate the amount of space loss (SL) caused by premature loss of primary second molars, determine whether the eruption status of permanent first molars is an important factor in the amount of SL, and evaluate the effectiveness of space maintainers (SMs) in SL prevention. SL associated with 100 prematurely extracted primary second molars was evaluated in 87 healthy patients. Teeth were divided into groups based on the use of SMs (36 with SM and 64 without SM). Bitewing and periapical radiographs taken before extraction and 6, 12, 24, 36, and 48 months after extraction were used to determine the amount of SL. Not every patient attended every recall appointment, so the sample size varied at different evaluation times. The most significant amount of SL occurred in the first 12 months after extraction. In patients who did not use an SM, at 6 months there was a mean SL of 2.12 mm (SD, 1.65 mm) and at 12 months there was a mean of 4.02 mm (SD, 1.65), with significantly more SL in the first 6 months (P < 0.001). There was no statistically significant difference in the amount of SL found at 12 and 24 months (P > 0.05). When patients without an SM were grouped by the eruption status of the permanent first molar, there was significantly more SL in the groups with unerupted first molars than there was in the groups with erupted first molars at both 6 months (P < 0.001) and 12 months (P < 0.05). At both 6 and 12 months, the amount of SL in patients who had an SM (n = 13 and n = 14, respectively) was not significantly different from the amount of SL in those who did not have an SM (n = 33 and n = 23, respectively). SMs should be placed as soon as possible following tooth extraction to prevent undue SL. Placement of an SM a year or more after extraction has minimal benefit, since most SL takes place within the first year. SL does occur even when SMs are used.

Development and characterization of a mouse floxed Bmp2 osteoblast cell line that retains osteoblast genotype and phenotype.

Bone morphogenetic protein 2 (Bmp2) is essential for osteoblast differentiation and osteogenesis. Generation of floxed Bmp2 osteoblast cell lines is a valuable tool for studying the effects of Bmp2 on osteoblast differentiation and its signaling pathways during skeletal metabolism. Due to relatively limited sources of primary osteoblasts, we have developed cell lines that serve as good surrogate models for the study of osteoblast cell differentiation and bone mineralization. In this study, we established and characterized immortalized mouse floxed Bmp2 osteoblast cell lines. Primary mouse floxed Bmp2 osteoblasts were transfected with pSV3-neo and clonally selected. These transfected cells were verified by PCR and immunohistochemistry. To determine the genotype and phenotype of the immortalized cells, cell morphology, proliferation, differentiation and mineralization were analyzed. Also, expression of osteoblast-related gene markers including Runx2, Osx, ATF4, Dlx3, bone sialoprotein, dentin matrix protein 1, osteonectin, osteocalcin and osteopontin were examined by quantitative RT-PCR and immunohistochemistry. These results showed that immortalized floxed Bmp2 osteoblasts had a higher proliferation rate but preserved their genotypic and phenotypic characteristics similar to the primary cells. Thus, we, for the first time, describe the development of immortalized mouse floxed Bmp2 osteoblast cell lines and present a useful model to study osteoblast biology mediated by BMP2 and its downstream signaling transduction pathways.

Abnormalities in the enamel in bmp2-deficient mice.

Tooth development is regulated by epithelial-mesenchymal interactions and their reciprocal molecular signaling. Bone morphogenetic protein 2 (Bmp2) is essential for tooth formation. However, the role of Bmp2 during enamel formation remains unknown in vivo. In this study, the role of Bmp2 in the regulation of postnatal enamel formation was investigated via the conditional ablation of Bmp2 in enamel using the (Osx-Cre) mouse. Bmp2 gene ablation was confirmed by PCR analysis in Osx-Cre, Bmp2(flox/flox) mice. Bmp2-null mice displayed a severe and profound tooth phenotype with asymmetric and open forked incisors. Microradiographs revealed broken incisor tips and dental pulp chamber exposure. The enamel layer of incisors and molars was thin with hypomineralization. Scanning electron microscopy analysis showed that the enamel surface was rough with chipping and the enamel lacked a typical prismatic architecture. These results demonstrate that Bmp2 is essential for enamel formation.

Immortalized mouse floxed Bmp2 dental papilla mesenchymal cell lines preserve odontoblastic phenotype and respond to BMP2.

Bone morphogenetic protein 2 (Bmp2) is essential for odontogensis and dentin mineralization. Generation of floxed Bmp2 dental mesenchymal cell lines is a valuable application for studying the effects of Bmp2 on dental mesenchymal cell differentiation and its signaling pathways during dentinogenesis. Limitation of the primary culture of dental mesenchymal cells has led to the development of cell lines that serve as good surrogate models for the study of dental mesenchymal cell differentiation into odontoblasts and mineralization. In this study, we established and characterized immortalized mouse floxed Bmp2 dental papilla mesenchymal cell lines, which were isolated from 1st mouse mandibular molars at postnatal day 1 and immortalized with pSV40 and clonally selected. These transfected cell lines were characterized by RT-PCR, immunohistochemistry, and analyzed for alkaline phosphatase activity and mineralization nodule formation. One of these immortalized cell lines, iBmp2-dp, displayed a higher proliferation rate, but retained the genotypic and phenotypic characteristics similar to primary cells as determined by expression of tooth-specific markers as well as demonstrated the ability to differentiate and form mineralized nodules. In addition, iBmp2-dp cells were inducible and responded to BMP2 stimulation. Thus, we for the first time described the establishment of an immortalized mouse floxed Bmp2 dental papilla mesenchyma cell line that might be used for studying the mechanisms of dental cell differentiation and dentin mineralization mediated by Bmp2 and other growth factor signaling pathways.

A controlled clinical trial to evaluate the safety and whitening efficacy of a 9.5% hydrogen peroxide high-adhesion whitening strip in a teen population.

PURPOSE: To compare the efficacy and safety outcomes of two tooth-whitening systems. METHODS: 44 subjects, 12-17 years of age, participated in the study and were divided into two balanced groups. 15 subjects received 6.5% hydrogen peroxide professional whitening strip treatment and 29 subjects received 9.5% hydrogen peroxide high-adhesion whitening strips to wear for 30 minutes twice a day. Teeth were bleached for 20 days with the 9.5% hydrogen peroxide strips and 21 days with the 6.5% hydrogen peroxide strips. Digital image analysis measured color in b*, L*, and a* color spaces, where b* indicated yellowness, L* indicated lightness, and a* indicated redness at days 8, 11 and 22 for both the maxillary and mandibular arches. Oral examinations and interviews were used to ascertain any adverse events that may have occurred during treatment. RESULTS: 36 subjects completed the study. At each post-baseline visit, both of the treatment groups had statistically significant (P < 0.02) mean color improvement from baseline for b*, L* and a*. The 9.5% hydrogen peroxide strips group provided statistically greater reduction in yellowness (deltab*) relative to the 6.5% hydrogen peroxide strips group for each visit of in the maxillary arch (P < 0.02) and for Day 8 and Day 22 in the mandibular arch (P < 0.02). In addition, the 9.5% hydrogen peroxide high-adhesion strip group provided statistically greater improvement in lightness (deltaL*) relative to the 6.5% hydrogen peroxide strip group for each visit in the maxillary arch (P < or = 0.007) and for the final visit in the mandibular arch (P = 0.002). 18 subjects (62%) in the 9.5% hydrogen peroxide high-adhesion strip group reported adverse events compared to 8 subjects (53%) in the 6.5% hydrogen peroxide polyethylene strip group. Minor and transient tooth sensitivity and oral irritation were the most common adverse events.

Sealants: a review of the materials and utilization.

Dental pit and fissure sealants have been shown to be effective in the prevention of dental caries. Currently, sealants are recommended to be placed on teeth that are considered to be "at risk" to develop caries, including teeth that present with incipient enamel lesions. This paper discusses the types of sealant materials available and the placement of the sealant, including appropriate tooth preparation, acid-etching, polymerization, and use of adhesives prior to sealant placement.

Restorative dentistry for the pediatric patient.

The American Academy of Pediatric Dentistry sponsored the Pediatric Restorative Dentistry Consensus Conference in 2002. This paper will review the consensus statements that were issued as a result of the conference. Since the conference there have been advances in procedures, materials, and techniques that need to be considered in terms of some of the consensus statements. The introduction of the First Dental Home, interim therapeutic restoration and nanotechnology are examples of some of the materials and techniques that are now part of everyday pediatric dentistry. This paper will discuss the updates as it relates to each of the 2002 consensus statements.

Immortalization and characterization of mouse floxed Bmp2/4 osteoblasts.

Generation of a floxed Bmp2/4 osteoblast cell line is a valuable tool for studying the modulatory effects of Bmp2 and Bmp4 on osteoblast differentiation as well as relevant molecular events. In this study, primary floxed Bmp2/4 mouse osteoblasts were cultured and transfected with simian virus 40 large T-antigen. Transfection was verified by polymerase chain reaction (PCR) and immunohistochemistry. To examine the characteristics of the transfected cells, morphology, proliferation and mineralization were analyzed, expression of cell-specific genes including Runx2, ATF4, Dlx3, Osx, dentin matrix protein 1, bone sialoprotein, osteopontin, osteocalcin, osteonectin and collagen type I was detected. These results show that transfected floxed Bmp2/4 osteoblasts bypassed senescence with a higher proliferation rate, but retain the genotypic and phenotypic characteristics similar to the primary cells. Thus, we for the first time demonstrate the establishment of an immortalized mouse floxed Bmp2/4 osteoblast cell line.