NKX3.2 plays a key role in regulating HIF1α-directed angiogenesis in chondrocytes

The cellular fraction of cartilage is mainly composed of one single cell type, the chondrocyte, which secretes, shapes and maintains the cartilaginous matrix that functions, in the case of epiphyseal cartilage, as the template for bone elongation. The biomechanical properties of cartilage are dependent mainly on the composition, as well as the macromolecular integrity of its matrix contributing to the functional and phenotypic differences between cartilage subtypes (1). For example, articular cartilage (AC) is a highly resilient tissue that allows low-friction joint articulation. In contrast, growth plate (GP) cartilage is populated by highly proliferative chondrocytes that undergo hypertrophic differentiation in an angiogenesis-promoting spatiotemporal manner, serving as a mold for longitudinal bone growth. As such, an in sharp contrast to AC, GP cartilage is a transient tissue and is replaced by bone in a process known as endochondral ossification (2)

Differential association of protein subunits with the human RNase MRP and RNase P complexes

RNase MRP is a eukaryotic endoribonuclease involved in nucleolar and mitochondrial RNA processing events. RNase MRP is a ribonucleoprotein particle, which is structurally related to RNase P, an endoribonuclease involved in pre-tRNA processing. Most of the protein components of RNase MRP have been reported to be associated with RNase P as well. In this study we determined the association of these protein subunits with the human RNase MRP and RNase P particles by glycerol gradient sedimentation and coimmunoprecipitation. In agreement with previous studies, RNase MRP sedimented at 12S and 60–80S. In contrast, only a single major peak was observed for RNase P at 12S. The analysis of individual protein subunits revealed that hPop4 (also known as Rpp29), Rpp21, Rpp20, and Rpp25 only sedimented in 12S fractions, whereas hPop1, Rpp40, Rpp38, and Rpp30 were also found in 60–80S fractions. In agreement with their cosedimentation with RNase P RNA in the 12S peak, coimmunoprecipitation with VSV-epitope-tagged protein subunits revealed that hPop4, Rpp21, and in addition Rpp14 preferentially associate with RNase P. These data show that hPop4, Rpp21, and Rpp14 may not be associated with RNase MRP. Furthermore, Rpp20 and Rpp25 appear to be associated with only a subset of RNase MRP particles, in contrast to hPop1, Rpp40, Rpp38, and Rpp30 (and possibly also hPop5), which are probably associated with all RNase MRP complexes. Our data are consistent with a transient association of Rpp20 and Rpp25 with RNase MRP, which may be inversely correlated to its involvement in pre-rRNA processing

ELISA-based detection of gentamicin and vancomycin in protein-containing samples

Background: Orthopaedic implant infections are treated by surgical debridement, systematic antibiotic treatment or local antibiotic treatment with antibiotic-loaded beads. Currently antibiotic concentrations in wound exudate, serum, urine or tissue samples are determined with HPLC or fluorescent spectrometric assays. Both methods are heavily influenced due to proteins in the samples. Questions/purposes: Is ELISA capable to detect gentamicin and vancomycin in protein-containing samples like serum and wound exudate. Methods: Two specific competitive ELISA-assays were set-up to detect either gentamicin or vancomycin in protein-rich samples. An antibiotic-BSA hapten was generated as a coatable antigen and commercially available antibodies were applied for downstream immunodetection. Results: The developed ELISAs perform at a detection range of 2–500 ng/ml gentamycin and 20–5000 ng/ml vancomycin. Both ELISAs were capable of detecting these antibiotics in human serum and wound exudate without being compromised by the presence of proteins. We did not detect cross-reactivity for gentamicin in the vancomycin ELISA or vice versa. Conclusions: The antibiotic ELISAs detect gentamicin and vancomycin at low concentrations in protein-rich samples and they can be used as a high throughput and cost-effective alternative for chromatographic or fluorescent methods. Clinical relevance: These ELISAs can be used to detect very low gentamicin or vancomycin concentrations in clinical samples or assess novel orthopaedic antibiotic release systems in in vitro and in vivo studies

Development of a Synovium-on-Chip Model with a Porous Membrane to Study Inflammatory Arthritis

Laurens Spoelstra*1,2,3, Nuno Araújo-Gomes1, Mariia Zakharova2,Tim Welting4, Marcel Karperien1, Loes Segerink2, Séverine Le Gac31Developmental BioEngineering, TechMed Centre, University of Twente, Enschede, The Netherlands2BIOS Lab on a Chip group, MESA+ Institute, TechMed Centre, University of Twente, Enschede, The Netherlands3Applied Microfluidics for BioEngineering Research, MESA+ Institute, University of Twente, Enschede, The Netherlands4Laboratory of Experimental Orthopedics, Maastricht UMC, Maastricht, The Netherlands*Corresponding author: [email protected] affects millions of people globally and commonly involves synovial inflammation.Unfortunately, for many arthritic diseases, such as Rheumatoid Arthritis (RA) and Osteoarthritis (OA) inparticular, there is a limited number of disease-modifying treatments available. This is in part due to alack of physiologically relevant preclinical models to assess these inflammatory forms of arthritis.Recently, we have developed a Synovium-on-Chip (SoC) platform that modeled the synovial lining,consisting (mainly) of synovial fibroblasts and macrophages, and the synovial vasculature in the subintimawith endothelial cells (Figure 1A) [1,2]. However, this model had the limitation of possessing a ~20µmthick nonporous PDMS membrane acting as physical barrier separating the endothelial cells from thehuman synovial fibroblasts (hSFBs) and macrophages, limiting intercellular communication and thepossibility to mimic key pathogenic events such as monocyte extravasation [1].Here, we address these limitations and integrate a previously developed [3] porous 2µm thick PDMSmembrane (pore diameter of 5µm and a 30µm pitch). First, SoC devices were fabricated at a wafer scalewith up to 20 devices in a single process to enable higher throughput experiments (Figure 1B). Next, weco-cultured hSFBs from donors with THP-1-derived macrophages (top chamber) and Human UmbilicalVein Endothelial Cells (HUVECs, bottom chamber) for up to 10 days. All three cell types integrated wellin the devices (Figure 1C) and confluent cell layers were observed on both sides of the membrane (Figure1D-E). After verification of their integration and viability, the cells were challenged using 1 ng/mL TNF-α as a proof-of-concept for studying inflammatory arthritis (Figure 1F). After 4 days of stimulation (T10),RT-qPCR was performed on the hSFB and THP-1 cells (top chamber) for IL6, CCL2, MMP1, andTNFAIP6, which were all found to be upregulated by ~2-5 fold (Figure 1G).Surprisingly, microscopic analysis of the devices showed that HUVECs had started to delaminate fromthe walls of the channels while fibroblast-like cells were visible in the bottom channel (not shown). Thisprompted the hypothesis that the hSFBs could migrate through the porous membrane, which was testedin devices with nonporous membranes and in devices with only fibroblasts seeded in the top chamber.Interestingly, fibroblast migration was observed through the 5µm pores (Figure 2A), and 3D confocalmicroscopy revealed that the HUVECs formed a lumen-like structure (Figure 2B). Strikingly, hSFBs(labeled with green CellTracker) were found next to the HUVECs at the bottom of the chip and colocalized with the HUVECs in the lumen-like structure’s lining (Figure 2C). Future research will focuson how the interactions between HUVECs and hSFBs occur, leading to this lumen formation, assessingstability in long-term culture through microscopy analysis.In short, we have successfully developed a novel SoC model with an integrated porous membrane and infuture experiments, we will investigate different inflammatory stimuli, drug efficacy, monocyteextravasation, and the apparent self-organization of HUVECs and hSFBs.References1. Paggi, C. A., et al. Nat. Rev. Rheumatol. 18, 217-231 (2022).2. Paggi, C. A., et al. MicroTAS Conference (2021).3. Zakharova, M. et al. Adv. Mater. Technol. 6, 2100138 (2021

Heterodimerization regulates RNase MRP/RNase P association, localization, and expression of Rpp20 and Rpp25

Rpp20 and Rpp25 are subunits of the human RNase MRP and RNase P endoribonucleases belonging to the Alba superfamily of nucleic acid binding proteins. These proteins, which bind very strongly to each other, transiently associate with RNase MRP. Here, we show that the Rpp20-Rpp25 heterodimer is resistant to both high concentrations of salt and a nonionic detergent. The interaction of Rpp20 and Rpp25 with the P3 domain of the RNase MRP RNA appeared to be strongly enhanced by their heterodimerization. Coimmunoprecipitation experiments demonstrated that only a single copy of each of these proteins is associated with the RNase MRP and RNase P particles in HEp-2 cells. Both proteins accumulate in the nucleoli, which in case of Rpp20 is strongly dependent on its interaction with Rpp25. Finally, the results of overexpression and knock-down experiments indicate that their expression levels are codependent. Taken together, these data indicate that the Rpp20-Rpp25 heterodimerization regulates their RNA-binding activity, subcellular localization, and expression, which suggests that their interaction is also crucial for their role in RNase MRP/P function

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-Ptgs2 tm1Jed /J wildtype mice were treated for 10 weeks with celecoxib (daily oral administration 10 mg/kg) or placebo and compared with B6;129S-Ptgs2 tm1Jed /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

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

ObjectiveDespite 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.DesignSkeletally 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).ResultsFifteen 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.ConclusionsGenetic 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