Stages Of Ossification Research Articles

Osteopetrosis in a six-month-old infant

Osteopetrosis is a severe and frequently fatal condition characterized by the formation of thick, dense bone, with a concomitant loss of bone marrow spaces secondary to defective osteoclast function or development. Clinical symptoms include pancytopenia, hepatosplenomegaly, fractures, cranial nerve impingement, and increased cranial pressure, among others. The condition requires rapid diagnosis and clinical intervention, as hematopoietic stem cell transplantation performed early in life can be curative in the majority of cases and can prevent the irreversible neurological impairment associated with the disease. We report a six-month-old male with osteopetrosis where bone marrow examination and molecular studies allowed a definitive diagnosis. Microscopic examination of a bone marrow biopsy demonstrated increased bone formation at various stages of ossification, decreased marrow spaces, and increased osteoclasts rimming the bone. Next-generation sequencing (NGS) demonstrated that the patient was homozygous for mutations in the TCIRG1 gene, which encodes an osteoclast vacuolar proton pump and is the most common mutation associated with the autosomal recessive or infantile malignant form of osteopetrosis. The patient underwent a cord blood stem cell transplant, but unfortunately expired four months after diagnosis. This case highlights the importance of bone marrow biopsy evaluation as part of the integrated approach to diagnosing osteopetrosis.

Reliability of Mandibular Canine Maturation Stages in Predicting Pubertal Growth Spurt in a Sample of Egyptian Orthodontic Patients

Objective: to investigate the reliability of mandibular canine maturation stages in predicting pubertal growth spurt in a sample of Egyptian orthodontic patients. Materials and Methods: A retrospective, cross-sectional study was carried out on the radiographic records (left lateral cephalometric, panoramic and left hand and wrist x-rays) obtained from 250 patients who were attending private clinics on Delta area, Egypt. The age was selected between 7 and 18 years. Mandibular canine calcification stages (MCC) were assessed according to Demirjian’s method for dental maturity assessment. Skeletal maturity was assessed by the ossification stages of the middle phalanx of the third finger (MP3) using Hagg and Taranger method and the cervical vertebrae maturation index (CVMI) was evaluated using Hassel and Farman method. MCC stages were statistically correlated to both MP3 stages and CVMI stages. The significance level was set at P ≤ 0.05. Statistical analysis was performed with IBM® SPSS® Statistics Version 20 for Windows. Results: Chi-square tests have shown highly significant associations between the mandibular canine calcification stages (MCC) and both CVMI and MP3 maturation stages (p < 0.001). MCC stage F had shown a moderate correlation with MP3 FG and CVMI 2 that corresponded to the onset of pubertal growth. Conclusions: MCC stage F can be used for prediction of the onset of pubertal growth, while prediction of the peak or the post peak periods using MCC method may be unreliable. Key words: Pubertal growth, MP3 stages, CVMI stages, MCC stages.

Evaluation of distal femoral epiphysis and proximal tibial epiphysis ossification using the Vieth method in living individuals: applicability in the estimation of forensic age

ABSTRACT In this study, ossification stages of distal femoral epiphysis and proximal tibial epiphysis were retrospectively evaluated with a 1.5-T magnetic resonance imaging (MRI) scanner according to the newly developed five-stage Vieth method. A total of 367 male and 231 female patients aged 12–30 years were scanned between January 2012 and July 2019. A 1.5-T MRI scanner was used to obtain a T1-weighted turbo spin-echo (TSE) sequence and a fat-suppressed proton density-weighted TSE image. The kappa statistics were good level of agreement for inter- and intra-observer evaluations of the ossification stages for knee. Comparison of male and female data revealed significant between-sex differences in the ages when the stages 3, 4 and 5 ossifications of the distal femoral epiphysis and the stages 4 and 5 ossifications of the proximal tibial epiphysis were first detected. In the proximal tibial epiphysis, stage 6 was observed at age 18 years for males and at age 17 years for females. In the distal femoral epiphysis, stage 6 was initially observed at age 20 years for both sexes. Thus, stage 6 ossification, which is determined through 1.5-T MRI of the knee joint, allows for the determination of the completion of the 18th year of life in either sex.

Mesenchymal Stem Cell-Specific and Preosteoblast-Specific Ablation of TSC1 in Mice Lead to Severe and Slight Spinal Dysplasia, Respectively.

Background TSC1-related signaling plays a pivotal role in intramembranous and endochondral ossification processes during skeletogenesis. This study was aimed at determining the significance of the TSC1 gene at different stages of spinal development. Materials and Methods. TSC1-floxed mice (TSC1flox/flox) were crossed with Prrx1-Cre or BGLAP-Cre transgenic mice or mesenchymal stem cell- and osteoblast-specific TSC1-deficient mice, respectively. Somatic and vertebral differences between WT and Prrx1-TSC1 null mice were examined at 4 weeks after birth. Results No apparent body size abnormalities were apparent in newborn and 4-week- to 2-month-old mice with BGLAP-Cre driver-depleted TSC1. Vertebral and intervertebral discs displayed strong dysplasia in Prrx1-TSC1 null mice. In contrast, vertebrae were only slightly affected, and intervertebral discs from skeletal preparations displayed no apparent changes in BGLAP-TSC1 null mice. Conclusion Our data suggest that the TSC1 gene is crucial for endochondral ossification during postnatal spine development but plays discriminative roles at different stages. Mesenchymal stem cell-specific ablation of TSC1 led to severe spinal dysplasia at early stages of endochondral ossification while osteoblast-specific deletion of TSC1 affected vertebrae slightly and had no detectable effects on intervertebral discs.

Pointing on the early stages of maxillary bone and tooth development - histological findings.

Although the morphological stages of tooth development, in parallel with maxillary bone construction, are known for decades, the intimate mechanisms of early development of the oral cavity structures and tooth’s proper and associated tissues are still incompletely elucidated. Nowadays, the research in embryology was shifted from the morphological to the molecular and genetic approach. This new approach is accomplished by using in vivo and in vitro experimental studies performed on animal models and cell lines. The interest in the knowledge of these events at gene and molecular level is still current, aiming to sustain the progress in the endorsement of novel regenerative and restorative therapies. However, the morphological standpoint maintains its interest, because the extrapolation of the results of experimental studies in humans requires a strong confirmation. Within this context, our work aims to analyze the histological characteristics of the maxillary bone and integrated tooth germs during the early stages of embryonic development. The study group consisted in mandible fragments obtained by dissection of the cephalic extremities collected from fetuses aged from 10 to 24 weeks, after medical or spontaneous abortions. The tissue specimens were processed for the histological exam. The histoarchitectonic traits of the initial stages of mandibular bone tissue and tooth development were assessed. The results revealed the dynamics of the ossification stages, from stages of early-dispersed intramembranous ossification to the organization of the dental alveoli, incorporated step-by-step in the maxillary body, and the simultaneous presence of tooth germs with different sizes and shapes, in accordance with the development stage. Our study complements the existing data regarding the embryonic period, bringing an important contribution for the enlargement of existing morphological, visual information for maxillary bone and tooth development.

Study of nutrient foramina in fibula: its applied importance

The external opening of the nutrient canal is usually referred to as the nutrient foramen. The term 'Nutrient' itself describes the role of nutrient foramina in the nutrition and overall growth of the bones. The diaphyseal nutrient artery is the primary source of blood to entire osteal tissue and bone marrow of a long bone, especially during its active growth period and the early stages of ossification. Therefore, they play a very significant role in the healing of fractures and prevention of avascular bone necrosis. Every nutrient foramen has a particular position in each bone and fibula is of no exception. However, the number and location of nutrient foramina may very sometimes. In bone graft and vascularised surgery, the fibula is commonly used. Therefore, an understanding of the position and number of nutrient foramina in fibula is essential. Moreover, fibula flap is the most widely accepted flaps used in the mandibular reconstruction surgery, especially in the malignancy of oral and oropharyngeal regions. This review paper outlines the anatomical variation of the nutrient foramen of the fibula and its applied importance in clinical practice.

Histopathological features of condylar hyperplasia and condylar Osteochondroma: a comparison study

BackgroundBoth mandibular condylar hyperplasia and condylar osteochondroma can lead to maxillofacial skeletal asymmetry and malocclusion, although they exhibit different biological behavior. This study attempted to compare the histological features of mandibular condylar hyperplasia and condylar osteochondroma using hematoxylin-and-eosin (H&E) staining, and immunohistochemistry staining of PCNA and EXT1 with quantitative analysis method.ResultsThe H&E staining showed that condylar hyperplasia and condylar osteochondroma could be divided into four histological types and exhibited features of different endochondral ossification stages. There was evidence of a thicker cartilage cap in condylar osteochondroma as compared condylar hyperplasia (P = 0.018). The percentage of bone formation in condylar osteochondroma was larger than was found in condylar hyperplasia (P = 0.04). Immunohistochemical staining showed that PCNA was mainly located in the undifferentiated mesenchymal layer and the hypertrophic cartilage layer, and there were more PCNA positive cells in the condylar osteochondroma (P = 0.007). EXT1 was mainly expressed in the cartilage layer, and there was also a higher positive rate of EXT1 in condylar osteochondroma (P = 0.0366). The thicker cartilage cap, higher bone formation rate and higher PCNA positive rate indicated a higher rate of proliferative activity in condylar osteochondroma. The more significant positive rate of EXT1 in condylar osteochondroma implied differential biological characteristic as compared to condylar hyperplasia.ConclusionsThese features might be useful in histopathologically distinguishing condylar hyperplasia and osteochondroma.

Comparison of different iterative CT reconstruction techniques and filtered back projection for assessment of the medial clavicular epiphysis in forensic age estimation

To assess the impact of iterative reconstruction and filtered back projection (FBP) on image quality in computed tomography (CT)-based forensic age estimation of the medial clavicular epiphysis. CT of the clavicle was performed in 19 patients due to forensic reasons (70 mAs/140 kVp). Raw data were reconstructed with FBP and with an iterative algorithm at level 4 and 6. Clavicular ossification stage was determined by two radiologists in consensus, firstly on FBP reconstructed images and secondly after reviewing all reconstructions including iDose 4 and 6. In addition, the 3 reconstructions were compared regarding artefacts and delineation of the meta-/epiphyseal interface. Quantitative image noise was measured. Quantitative noise was lower in iDose 6 reconstructed images than in FBP (P < 0.042), but not significantly lower between iDose 4 and FBP (P = 0.127). Side by side comparison revealed lesser qualitative image noise on both iDose reconstructed images than for FBP. The meta-/epiphyseal interface delineation was rated better on both iDose levels than with FBP. In 3 of 19 patients, the clavicular ossification stage was reclassified after iterative reconstructions had been additionally reviewed. Using iterative CT reconstruction algorithms, a reduction of image noise and an enhancement of image quality regarding the meta-/epiphyseal clavicular interface can be achieved. The study highlights the importance of image standardization as variation of reconstruction technique has impact on forensic age estimation.

FRACTURE HEALING SIMULATION REGULATED BY FORCE AND OXYGEN

According to the mechanical conditions of fracture fixation and the oxygen levels in the tissues, a simulation model of fracture healing process was built to describe the relationship among mechanical stability, oxygen levels in tissues and tissue differentiation during the second fracture healing. Different from the previous simulation model, in this paper, we took the three-dimensional model as the research object, solved the mechanical stimulation by finite element method, established the partial differential equation to solve the spatial and temporal variation of the oxygen in tissues. The process of tissue differentiation was described by fuzzy control method. The initial stage of fracture healing, intramembranous ossification, chondrogenesis, cartilage calcification and endochondral ossification during the fracture healing process were simulated, and the properties of tissue materials were continuously updated to complete the iterative process. The simulation program of fracture healing process was independently developed in Eclipse environment, and the simulation results were compared with experimental data and those of other fracture healing simulation models to verify the simulation program in this paper. Finally, the processes of transverse fracture healing in rats with different axial stability under normoxic, hypoxic and hyperoxic conditions was simulated, and the effects of different tissue oxygen levels and interosseous stabilities on fracture healing were analyzed. It is concluded by simulation that the delayed healing or non-union of bone will occur when in state of tissue hypoxia or interosseous instability, normal healing will occur when in state of tissue normoxia, and the healing will be accelerated when in state of tissue hyperoxia.

Imaging of osteochondrosis.

Osteochondrosis is an abnormality of the epiphyses or epiphyseal equivalents (round bones and apophyses) during later stages of endochondral ossification. This process of abnormal endochondral ossification can occur at various locations throughout the body. The pathogenesis of osteochondrosis is under active investigation. In humans, the process of abnormal endochondral ossification has been attributed to a combination of vascular insult and trauma. Although the proposed etiology of osteochondrosis varies based on body part affected, the overall process is defined by necrosis, revascularization and repair. As such, common radiologic findings include those of osseous destruction and associated inflammation. The purpose of this review is to discuss the current understanding of osteochondroses as a disease entity and explore imaging features of osteochondroses throughout the body.

Early growth dynamics of titanosaur sauropods inferred from bone histology

Histological studies have proven to be a powerful tool for addressing sauropod paleobiology, with most focus on adult and subadult individuals. The aim of this study is to describe long bone histology (femur) in early juvenile titanosaur sauropods, to expand our knowledge about early stages of development. The material consists of two small femora of indeterminate titanosaurs from the Upper Cretaceous Bajo de la Carpa and Allen formations of Argentina. Both femora from juvenile specimens show predominant fibrolamellar bone tissue with plexiform vascularization pattern, suggesting a fast growth rate. Despite the early stage of development of the studied specimens, secondary remodelling is evident in one of them. In the other sample, a growth mark is observed, which suggests unexpected cyclical growth and a possible departure from the typical growth strategy of sauropod dinosaurs. In addition, we examine indeterminate long bones of titanosaur embryos from the Anacleto Formation of Argentina. The embryos shows poor development of cortical bone, high density of large simple canals and presence of Kastschenko's line suggest that these individuals died during an early stage of ossification. This study reveals that the early growth of at least some titanosaurs departs from the reported in neosauropod dinosaurs.

Radiographic assessment of pectoral flipper bone maturation in bottlenose dolphins (Tursiops truncatus), as a novel technique to accurately estimate chronological age.

Accurate age estimation in wildlife conservation is an important diagnostic tool in the interpretation of biological data, necropsy examination, reproductive status and population demographics. The most frequently utilized methods to age bottlenose dolphins (Tursiops truncatus) include tooth extraction; counting dental growth layer groups and dental radiography. These methods are inaccurate in dolphins > 13 years old, due to overlapping of the growth layer groups in dolphins and worn teeth. Establishing a non-invasive method of accurately aging bottlenose dolphins across the entire age range is important to long term conservation efforts to understand health status, lifespan, reproduction and survivability. A database of 126 radiographs from 94 dolphins of known chronological age was utilized to establish the stages of skeletal ossification over time. A numerical score from -1 to 8 was assigned to 16 anatomic locations on the pectoral radiograph, to create a formula to estimate age. The most informative areas to evaluate morphologically were the metaphyseal regions of the radius and ulna, and the proximal and distal epiphysis of metacarpals II and III. Third order polynomial regression calculated separate age predictor formulas for male and female dolphins, with females reaching sexual maturity earlier than males. Completion of epiphyseal closure of the long bones correlated with average sexual maturity. Managed care dolphin ages could be properly estimated with decreasing precision from within 3 months in animals < 5 years old, to within 5 years in animals > 30 years old. This diagnostic tool could also be applied to diagnose atypical ossification patterns consistent with nutritional, developmental or growth abnormalities, and identifying subclinical health issues. In conclusion, knowledge of the lifespan and the onset of sexual maturity for each species will allow this model to be applied to other cetaceans, facilitating age estimation via pectoral radiography in future research.

Age estimation from anterior cervical vertebral ring apophysis ossification in South Africans.

Age estimation in living individuals around the age of 18years remains a difficult challenge. In this study, the anterior inferior vertebral ring apophysis development of cervical vertebrae C2, C3, and C4 of 496 white and 478 black South African individuals aged between 15 and 22years was assessed from cephalometric radiographs. Apophysis development was scored according to a four-stage scoring system. Ancestry and sex differences in apophysis maturation were assessed and likelihood values determined for individuals in each population group being 18years, based on developmental stages. Regression equations were developed for each ancestry and sex group. The results indicated that the median ages for attainment of stages 0, 1, and 2 were below the 18-year threshold for all ancestry and sex groups. Additionally, WSA males and BSA females attained stage 3 for C2, and WSA females attained stage 3 for C2, C3, and C4, below the 18-year threshold. The presence of stage 0 for black and white males in all three observed vertebrae and stage 1 for black males for C2, C3, and C4, white females for C2 and C3, and white males for C4 indicates an age below 18years (with a 95% or higher probability). The results indicate that anterior inferior apophysis ossification stages of C2, C3, and C4 can be used as a reliable indicator to determine the likelihood of being 18years of age at a 95% confidence index level. Apophysis development provides a valuable addition to the methods that can be used to assess age in the adolescent years.

Developmental tuning of mineralization drives morphological diversity of gill cover bones in sculpins and their relatives

The role of osteoblast placement in skeletal morphological variation is relatively well understood, but alternative developmental mechanisms affecting bone shape remain largely unknown. Specifically, very little attention has been paid to variation in later mineralization stages of intramembranous ossification as a driver of morphological diversity. We discover the occurrence of specific, sometimes large, regions of nonmineralized osteoid within bones that also contain mineralized tissue. We show through a variety of histological, molecular, and tomographic tests that this “extended” osteoid material is most likely nonmineralized bone matrix. This tissue type is a significant determinant of gill cover bone shape in the teleostean suborder Cottoidei. We demonstrate repeated evolution of extended osteoid in Cottoidei through ancestral state reconstruction and test for an association between extended osteoid variation and habitat differences among species. Through measurement of extended osteoid at various stages of gill cover development in species across the phylogeny, we gain insight into possible evolutionary developmental origins of the trait. We conclude that this fine‐tuned developmental regulation of bone matrix mineralization reflects heterochrony at multiple biological levels and is a novel mechanism for the evolution of diversity in skeletal morphology. This research lays the groundwork for a new model in which to study bone mineralization and evolutionary developmental processes, particularly as they may relate to adaptation during a prominent evolutionary radiation of fishes.

On the traces of tcf12: Investigation of the gene expression pattern during development and cranial suture patterning in zebrafish (Danio rerio).

The transcription factor 12 (tcf12) is a basic Helix-Loop-Helix protein (bHLH) of the E-protein family, proven to play an important role in developmental processes like neurogenesis, mesoderm formation, and cranial vault development. In humans, mutations in TCF12 lead to craniosynostosis, a congenital birth disorder characterized by the premature fusion of one or several of the cranial sutures. Current research has been primarily focused on functional studies of TCF12, hence the cellular expression profile of this gene during embryonic development and early stages of ossification remains poorly understood. Here we present the establishment and detailed analysis of two transgenic tcf12:EGFP fluorescent zebrafish (Danio rerio) reporter lines. Using these transgenic lines, we analyzed the general spatiotemporal expression pattern of tcf12 during different developmental stages and put emphasis on skeletal development and cranial suture patterning. We identified robust tcf12 promoter-driven EGFP expression in the central nervous system (CNS), the heart, the pronephros, and the somites of zebrafish embryos. Additionally, expression was observed inside the muscles and bones of the viscerocranium in juvenile and adult fish. During cranial vault development, the transgenic fish show a high amount of tcf12 expressing cells at the growth fronts of the ossifying frontal and parietal bones and inside the emerging cranial sutures. Subsequently, we tested the transcriptional activity of three evolutionary conserved non-coding elements (CNEs) located in the tcf12 locus by transient transgenic assays and compared their in vivo activity to the expression pattern determined in the transgenic tcf12:EGFP lines. We could validate two of them as tcf12 enhancer elements driving specific gene expression in the CNS during embryogenesis. Our newly established transgenic lines enhance the understanding of tcf12 gene regulation and open up the possibilities for further functional investigation of these novel tcf12 enhancer elements in zebrafish.

The Murine Limb Bud in Culture as an In Vitro Teratogenicity Test System.

There is widespread interest today in the use of in vitro methods to study normal and abnormal development. The limb is attractive in this context since much is known about pattern formation during limb development. The murine limb bud culture technique described in this chapter was developed and refined in the 1970s. In this culture system, limb development mimics the in vivo process, although at a slower rate, where growth and cartilage differentiation lead to the formation of proximal and distal structures with an "in vivo-like" 3D shape. Uniform developmental stages are selected for assessment, exposures are controlled precisely, and the confounding influences of maternal metabolism and transport are avoided. The existence of transgenic mice with fluorescent markers for the different stages of endochondral ossification adds a further dimension to the technique by allowing striking time course observations of the developing limb. Today, limb bud cultures are used to study the roles of genes during embryogenesis and the mechanisms by which chemicals interfere with critical signalling pathways.

A novel approach to estimate age and sex from mri measurement of liver dimensions in an Indian (Bengali) Population – A pilot study

Radiological methods have been used to assess the ossification stages of bones to estimate age and sex, but studies using the growth stages of organs such as the liver for identification purposes have not yet been performed. Liver weight increases with age, reaching a maximum between 41 and 50 years in men and between 51 and 60 years in women. Thereafter, above the age of 50 years, the liver weight decreases again, and the differences in liver weight between men and women are lost. For this reason, we have conducted this study in a population aged between 10 and 40 years. In this study, we attempted to find correlations between liver dimensions (from magnetic resonance imaging [MRI] images) and age and sex in an eastern Indian (Bengali) population. MRI images showing the liver were acquired from people aged between 10 and 40 years visiting the MRI Centre of Calcutta National Medical College. Liver MRI was acquired from 104 people. Chi-square tests showed a significant correlation between age and the mid hepatic point anteroposterior (MHP AP) dimension. However, there was no such significant correlation between age and maximum craniocaudal (Max CC) dimension or between age and maximum transverse dimension. The following discriminant function equation was derived (Df) = 0.04 MHP AP + 0.006 Max CC + 0.031 Max transverse −11.873 (Constant). Using this formula, 60.6% of the original grouped cases from the Eastern Indian Bengali population would be correctly classified. From the forensic point of view, the dimensions of the liver may be of the value of corroborating age and sex in doubtful cases in living individuals, aged between 10 and 40 years.

Analysis of the development of human foetal nasal turbinates using CBCT imaging.

The morphological structure of the nasal cavity (NC) is important for endoscopic surgical treatment. The location of nasal turbinates, including the superior turbinate (ST), middle turbinate (MT) and inferior turbinate (IT), are well presented during the formation of the human NC in cone beam CT (CBCT) images. There is a complex relationship between the nasal sinuses, the maxillary sinus (MS), ethmoidal sinus and sphenoid sinus, during formation of the NC structure at the morphological level. There is a need to clearly define the relationships of these nasal elements at the ossification level, during development. We investigated the three-dimensional construction of human foetal NC elements, including ST, MT, IT and vomer, using CBCT images from 16weeks gestation (E16) to E31 (25 foetuses) and compared it to histochemical observations (E25). At the stage of ossification, the studied elements are elongated in the posterior region near the sphenoidal bone, showing that the locations of the ST, MT, and IT are important during formation of the NC. CBCT analysis revealed that the horizontal and vertical directions of nasal turbinates affect the formation of the human NC. The location and elongated development of the MT is one of the most important elements for NC formation. The relationship between the nasal sinus and nasal turbine at the level of ossification may provide useful information in clinical treatment of children.

Mkx-Deficient Mice Exhibit Hedgehog Signaling-Dependent Ectopic Ossification in the Achilles Tendons.

Heterotopic ossification is the abnormal formation of mineralized bone in skin, muscle, tendon, or other soft tissues. Tendon ossification often occurs from acute tendon injury or chronic tendon degeneration, for which current treatment relies heavily on surgical removal of the ectopic bony tissues. Unfortunately, surgery creates additional trauma, which often causes recurrence of heterotopic ossification. The molecular mechanisms of heterotopic ossification are not well understood. Previous studies demonstrate that Mkx is a transcription factor crucial for postnatal tendon fibril growth. Here we report that Mkx-/- mutant mice exhibit ectopic ossification in the Achilles tendon within 1 month after birth and the tendon ossification deteriorates with age. Genetic lineage labeling revealed that the tendon ossification in Mkx-/- mice resulted from aberrant differentiation of tendon progenitor cells. Furthermore, tissue-specific inactivation of Mkx in tendon cells postnatally resulted in a similar ossification phenotype, indicating that Mkx plays a key role in tendon tissue homeostasis. Moreover, we show that Hedgehog signaling is ectopically activated at early stages of tendon ossification and that tissue-specific inactivation of Smoothened, which encodes the obligatory transducer of Hedgehog signaling, in the tendon cell lineage prevented or dramatically reduced tendon ossification in Mkx-/- mice. Together, these studies establish a new genetic mouse model of tendon ossification and provide new insight into its pathogenic mechanisms. © 2018 American Society for Bone and Mineral Research.

Effects of Organophosphate Ester Flame Retardants on Endochondral Ossification in Ex Vivo Murine Limb Bud Cultures.

Phasing out the usage of polybrominated diphenyl ether (PBDE) flame retardants (FRs) in consumer products led to their widespread replacement with organophosphate ester (OPE) FRs, despite scarce safety data. PBDE exposures were associated with the suppression of endochondral ossification but little is known about the effects of OPEs on bones. Here, we used a novel ex vivo murine limb bud culture system to compare the effects of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) with those of several OPEs. Gestation day 13 embryos were collected from transgenic CD1 mice expressing fluorescent markers for the major stages of endochondral ossification: COL2A1-ECFP (chondrogenesis), COL10A1-mCherry (early osteogenesis), and COL1A1-YFP (late osteogenesis). Limbs were excised and cultured for 6 days in the presence of vehicle, BDE-47, or an OPE FR: triphenyl phosphate (TPHP), tert-butylphenyl diphenyl phosphate (BPDP), tris(methylphenyl) phosphate (TMPP), or isopropylated triphenyl phosphate (IPPP). BDE-47 (50 μM) decreased the extent of chondrogenesis in the digits and COL1A1-YFP expression in the radius and ulna relative to control. In comparison, concentrations of ≥1 μM of all 4 OPEs limited chondrogenesis; osteogenesis (both COL10A1-mCherry and COL1A1-YFP fluorescence) was markedly inhibited at concentrations ≥3 μM. The expression of Sox9, the master regulator of chondrogenesis, was altered by BDE-47, TPHP, and BPDP. BDE-47 exposure had minimal impact on the expression of Runx2 and Sp7, which drive osteogenesis, whereas TPHP and BPDP both suppressed the expression of these transcription factors. These data suggest that OPE FRs may be more detrimental to bone formation than their brominated predecessors.