Adaptation of the protein translational apparatus during ATDC5 chondrogenic differentiation.

Introduction: Ribosome biogenesis is integrated with many cellular processes including proliferation, differentiation and oncogenic events. Chondrogenic proliferation and differentiation require a high cellular translational capacity to facilitate cartilaginous extracellular matrix production. We here investigated the expression dynamics of factors involved in ribosome biogenesis during in vitro chondrogenic differentiation and determined whether protein translation capacity adapts to different phases of chondrogenic differentiation. Materials: SnoRNA expression during ATDC5 differentiation was analyzed by RNA sequencing of samples acquired from day 0 (progenitor stage), 7 (chondrogenic stage) and day 14 (hypertrophic stage). RT-qPCR was used to determine expression of fibrillarin, dyskerin, UBF-1, Sox9, Col2a1, Runx2, Col10a1 mRNAs and 18S, 5.8S and 28S rRNAs. Protein expression of fibrillarin, dyskerin and UBF-1 was determined by immunoblotting. Ribosomal RNA content per cell was determined by calculating rRNA RT-qPCR signals relative to DNA content (SYBR Green assay). Total protein translational activity was evaluated with a puromycilation assay and polysome profiling. Results: As a result of initiation of chondrogenic differentiation (Δt0-t7), 21 snoRNAs were differentially expressed (DE). Hypertrophic differentiation caused DE of 23 snoRNAs (Δt7-t14) and 43 when t0 was compared to t14. DE snoRNAs, amongst others, target nucleotide modifications in the 28S rRNA peptidyl transferase center and the 18S rRNA decoding center. UBF-1, fibrillarin and dyskerin expression increased as function of differentiation and displayed highest fold induction at day 5-6 in differentiation. Ribosomal RNA content per cell was significantly increased at day 7, but not at day 14 in differentiation. Similar dynamics in translational capacity and monosomal ribosome fraction were observed during differentiation. Conclusion: The expression of a great number of ribosome biogenesis factors is altered during chondrogenic differentiation of ATDC5 cells, which is accompanied by significant changes in cellular translational activity. This elucidation of ribosome biogenesis dynamics in chondrogenic differentiation models enables the further understanding of the role of ribosome biogenesis and activity during chondrocyte cell commitment and their roles in human skeletal development diseases.

Timing of Repeat Ultrasound Examination in Treatment of Stable Developmental Dysplasia of the Hip.

BACKGROUND: Worldwide a wide variation exists in duration of Pavlik harness treatment for infants up to 6 months with stable developmental dysplasia of the hip (DDH). The purpose of this study was to evaluate whether shortening the time to first routine follow-up ultrasound after initiation of Pavlik harness treatment would reduce treatment duration and whether this influenced radiologic outcome at 1 year of age. Furthermore, predictors of higher acetabular index (AI) at 1 year of age were investigated. METHODS: A retrospective study was conducted in infants with stable DDH (Graf IIb and IIc) diagnosed and treated between 2015 and 2017. Two groups were identified: first routine follow-up ultrasound at 12 weeks after Pavlik harness initiation (group I) and first routine follow-up ultrasound at 6 weeks after Pavlik harness initiation (group II). In both groups, treatment was continued until repeat ultrasound measurements (every 6 wk) showed a normalized hip. Radiologic outcome at 1 year of age was defined as residual dysplasia measured on an anteroposterior hip radiograph according to the Tönnis table. RESULTS: A total of 222 infants were included. The median time of Pavlik harness treatment was 12 weeks (interquartile range, 11.9 to 12.3) in group I compared with 6.1 weeks (interquartile range, 6.0 to 7.5) in group II (P<0.001). Residual dysplasia at 1 year of age was detected in 20 infants (16.8%) in group I compared with 11 infants (10.7%) in group II (P=0.189). The multivariable prediction model showed that positive family history and lower baseline alpha angle correlate with a higher AI at 1 year of age. CONCLUSIONS: First routine follow-up ultrasound can be safely brought forward from 12 to 6 weeks after Pavlik harness initiation. Furthermore, infants with a positive family history for DDH and an initial low alpha angle are at higher risk to have a higher AI at 1 year of age. LEVEL OF EVIDENCE: Level III-retrospective study.

Impairment of Cyclo-oxygenase-2 Function Results in Abnormal Growth Plate Development and Bone Microarchitecture but Does Not Affect Longitudinal Growth of the Long Bones in Skeletally Immature Mice.

OBJECTIVE: Despite the general awareness that cyclo-oxygenase-2 (COX-2) is crucial for endochondral ossification, the role of COX-2 in skeletal development is largely unknown. We hypothesized that inhibition or genetic loss of COX-2 leads to impaired growth plate development and consequently impaired postnatal development of the long bones. DESIGN: Skeletally immature (5 weeks old) B6;129S-Ptgs2tm1Jed/J wildtype mice were treated for 10 weeks with celecoxib (daily oral administration 10 mg/kg) or placebo and compared with B6;129S-Ptgs2tm1Jed/J homozygous knockout mice (n = 12 per group). RESULTS: Fifteen weeks postnatally, no significant difference in growth plate (zone) thickness was found between groups. However, significantly higher proteoglycan content and lower expression levels of collagen type II and X staining in the growth plates of celecoxib-treated mice, and to a lesser extent in COX-2 knockout mice. In addition, a significantly decreased cell number and cell size were observed in the hypertrophic zone of the growth plates of both experimental groups. Micro-computed tomography analysis of the subchondral bone region directly beneath the growth plate showed significantly higher bone density and trabecular thickness, following celecoxib treatment. Despite the detected differences in growth plate extracellular matrix composition and subchondral bone morphology, no difference was found in the length of the tibia in celecoxib-treated mice or COX-2 knockout mice. CONCLUSIONS: Genetic loss of COX-2 or treatment with celecoxib did not result in detectable differences in gross murine formation of the tibia or femur. However, there were notable phenotypic features detected in the maturation of the growth plate (hypertrophic zone and subchondral bone) as a result of the celecoxib treatment.

Evaluation of impaired growth plate development of long bones in skeletally immature mice by antirheumatic agents.

Restriction of physical growth and development is a known problem in patients with juvenile idiopathic arthritis (JIA). However, the effect of medical treatment for JIA on skeletal growth in affected children has not been properly investigated. We, therefore, hypothesize that naproxen and methotrexate (MTX) affect endochondral ossification and will lead to reduced skeletal development. Treatment of ATDC5 cells, an in vitro model for endochondral ossification, with naproxen or MTX resulted in increased chondrogenic but decreased hypertrophic differentiation. In vivo, healthy growing C57BL/6 mice were treated with naproxen, MTX, or placebo for 10 weeks. At 15 weeks postnatal, both the length of the tibia and the length of the femur were significantly reduced in the naproxen- and MTX-treated mice compared to their controls. Growth plate analysis revealed a significantly thicker proliferative zone, while the hypertrophic zone was significantly thinner in both experimental groups compared to their controls, comparable to the in vitro results. Micro-computed tomography analysis of the subchondral bone region directly below the growth disc showed significantly altered bone microarchitecture in naproxen and MTX groups. In addition, the involvement of the PTHrP-Ihh loop in naproxen- and MTX-treated cells was shown. Overall, these results demonstrate that naproxen and MTX have a profound effect on endochondral ossification during growth plate development, abnormal subchondral bone morphology, and reduced bone length. A better understanding of how medication influences the development of the growth plate will improve understanding of endochondral ossification and reveal possibilities to improve the treatment of pediatric patients.

Functional electrical stimulation of the ankle dorsiflexors during walking in spastic cerebral palsy: a systematic review.

AIM: To assess the effect of functional electrical stimulation (FES) of ankle dorsiflexors in children and adolescents with spastic cerebral palsy (CP) during walking. METHOD: A systematic review was performed using the American Academy of Cerebral Palsy and Developmental Medicine methodology and the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Six databases were searched for studies applying interventions to patients aged younger than 20 years. Outcomes were classified according to the International Classification of Functioning, Disability and Health (ICF). RESULTS: Seven hundred and eighty abstracts were found, 35 articles were fully screened, and 14 articles were used for analysis. Only five articles (three studies) were of level I to III evidence. At ICF participation and activity level, there is limited evidence for a decrease in self-reported frequency of toe-drag and falls. At ICF body structure and function level, there is clear evidence (I-III) that FES increased (active) ankle dorsiflexion angle, strength, and improved selective motor control, balance, and gait kinematics, but decreased walking speed. Adverse events include skin irritation, toleration, and acceptation issues. INTERPRETATION: There are insufficient data supporting functional gain by FES on activity and participation level. However, evidence points towards a role for FES as an alternative to orthoses in children with spastic CP. WHAT THIS PAPER ADDS: Effects of functional electrical stimulation (FES) point towards a potential role as an alternative to orthoses for patients with spastic cerebral palsy (CP). Some evidence for a decrease in self-reported frequency of toe-drag and falls with the use of FES in spastic CP. Limited evidence for improvements in activity and participation in patients with spastic CP using FES.