Growth Plate Thickness Research Articles

The G protein-coupled receptor ADGRG6 maintains mouse growth plate homeostasis through IHH signaling.

The cartilage growth plate is essential for maintaining skeletal growth; however, the mechanisms governing postnatal growth plate homeostasis are still poorly understood. Using approaches of molecular mouse genetics and spatial transcriptomics applied to formalin-fixed, paraffin-embedded tissues, we show that ADGRG6/GPR126, a cartilage-enriched adhesion G protein-coupled receptor (GPCR), is essential for maintaining slow-cycling resting zone cells, appropriate chondrocyte proliferation and differentiation, and growth plate homeostasis in mice. Constitutive ablation of Adgrg6 in osteochondral progenitor cells with Col2a1Cre leads to a shortened resting zone, formation of cell clusters within the proliferative zone, and an elongated hypertrophic growth plate, marked by limited expression of parathyroid hormone-related protein (PTHrP) but increased Indian Hedgehog (IHH) signaling throughout the growth plate. Attenuation of smoothened-dependent hedgehog signaling restored the Adgrg6 deficiency-induced expansion of hypertrophic chondrocytes, confirming that IHH signaling can promote chondrocyte hypertrophy in a PTHrP-independent manner. In contrast, postnatal ablation of Adgrg6 in mature chondrocytes with AcanCreERT2, induced after the formation of the resting zone, does not affect PTHrP expression but causes an overall reduction of growth plate thickness marked by increased cell death specifically in the resting zone cells and a general reduction of chondrocyte proliferation and differentiation. Spatial transcriptomics reveals that ADGRG6 is essential for maintaining chondrocyte homeostasis by regulating osteogenic and catabolic genes in all the zones of the postnatal growth plates, potentially through positive regulation of SOX9 expression. Our findings elucidate the essential role of a cartilage-enriched adhesion GPCR in regulating cell proliferation and hypertrophic differentiation by regulation of PTHrP/IHH signaling, maintenance of slow-cycle resting zone chondrocytes, and safeguarding chondrocyte homeostasis in postnatal mouse growth plates.

Longitudinal skeletal growth and growth plate morphological characteristics of chondro-tissue specific CUL7 knockout mice

Background3 M syndrome is first reported in 1975，which characterized by severe pre- and postnatal growth retardation, skeletal malformation and facial dysmorphism. These three genes (CUL7, OBSL1 and CCDC8) have been identified to be respond for 3 M syndrome, of which CUL7 is accounting for approximately 70%. To date, the molecular mechanism underlying the pathogenesis of 3 M syndrome remains poorly understood. Previous studies showed that no Cul7-/- mice could survive after birth, because of growth retardation at late gestational stage and respiratory distress after birth. The establishment of the animal model of cartilage specific Cul7 knockout mice (Cul7fl/fl;Col2a1-CreERT2 mice) has confirmed that Cul7fl/fl;Col2a1-CreERT2 mice can be selective in a time- and tissue-dependent manner, which can provide an experimental basis for further research on severe genetic diseases related to growth plates. ObjectiveTo establish a model of Cul7fl/fl;Col2a1-CreERT2 mice based on Cre/LoxP system, and to further observe its phenotype and morphological changes in growth plate. MethodsThe Cul7fl/fl;Col2a1-CreERT2 mice were taken as the experimental group, while the genotype of Cul7fl/+;Col2a1-CreERT2 mice were used as the control group. The gross morphological features and X-ray films of limbs in the two groups were observed every week for 3–6 consecutive weeks, and the length of the mice from nose to the tail, the length of femur and tibia were recorded. In the meantime, The histological morphology of tibial growth plates was compared between the two groups. ResultsA preliminary model of Cul7fl/fl;Col2a1-CreERT2 mice was established. The Cul7fl/fl;Col2a1-CreERT2 mice had abnormally short and deformed limbs (P<0.05), increased thickness of growth plate, the disorderly arranged chondrocyte columns, decreased number of cells in the proliferation zone, changes in the shape from flat to round, obviously expanded extracellular matrix, and disordered arrangement, thickening and loosening of bone trabecula at the proximal metaphysis of the femur. ConclusionsThe knockout of Cul7 gene may affect both the proliferation of chondrocytes and the endochondral osteogenesis, confirming that Cul7 is essential for the normal development of bone in the body.

Chronic Iodine Intake Excess Damages the Structure of Articular Cartilage and Epiphyseal Growth Plate.

This study aimed to explore the influence of excess iodine on the articular cartilage and epiphyseal growth plate in rats. Wistar rats (n = 200) were randomly divided into five groups with 40 rats in each: normal iodine (NI), 5-fold high iodine group (5HI), 10-fold high iodine group (10HI), 50-fold high iodine group (50HI), and 100-fold high iodine group (100HI). The rats were executed in 6 and 12months. 24-h urinary iodine concentration (UIC) was monitored by arsenic-cerium catalytic spectrophotometry. The chemiluminescence method was used to determine the thyroid function. The pathological changes in the epiphyseal plate, articular cartilage, and thickness of the epiphyseal plate were observed. The mRNA expression of collagen II (ColII), collagen X, matrix metalloproteinase-13 (MMP-13), and fibroblast growth factor receptor 1 in articular chondrocytes was detected by RT-PCR. 24-h UIC increased as iodine intake increased. In the 12th month, there was a significant increase in serum sTSH and a decrease in serum FT4 in HI groups, compared to the NI group. There was a decrease in the number of proliferating cells in the epiphyseal plate and an increase in the number of mast cell layers. The chondrocytes appeared disorganized, and the tidal lines were disturbed or even broken. Growth plate thickness decreased with increasing iodine intake. Compared with the NI group, ColII and MMP-13 mRNA expression in chondrocytes in all HI groups significantly increased. Chronic iodine overdose increases the risk of hypothyroidism. Chronic iodine overdose leads to abnormal morphology of epiphyseal growth plates and articular cartilage, increasing the risk of osteoarthritis.

Fluoride-induced osteoporosis via interfering with the RANKL/RANK/OPG pathway in ovariectomized rats: Oophorectomy shifted skeletal fluorosis from osteosclerosis to osteoporosis

Osteosclerosis and osteoporosis are the two main clinical manifestations of skeletal fluorosis. However, the reasons for the different clinical manifestations are unclear. In this study, we established the fluoride (F) -exposed ovariectomized (OVX) and non-OVX rat models to assess the potential role of ovarian function loss in osteosclerosis and osteoporosis. Micro-CT scanning showed that excessive F significantly induced a high bone mass in non-OVX rats. In contrast, a low bone mass manifestation was presented in OVX F-exposed rats. Also, a prominent feature of increasing trabecular connectivity, collagen area, growth plate thickness, and reduced trabecular space was found by histopathological morphology in non-OVX F-exposed rats; an opposite result was observed in OVX F-exposed. These alterations indicated ovariectomy was a vital factor leading to osteosclerosis or osteoporosis in skeletal fluorosis. Furthermore, levels of bone alkaline phosphatase (BALP) and tartrate-resistant acid phosphatase (TRAP) increased, combined with the increasing osteoclasts number, showing a sign of high bone turnover in both OVX and non-OVX F-exposed rats. Mechanistically, oophorectomy considerably activated the RANKL/RANK/OPG signaling pathway. Meanwhile, it was discovered that upregulated NF-κB positively facilitated the accumulation of nuclear factor of activated T-cells 1 (NFATC1), significantly promoting osteoclast differentiation. To sum up, this study greatly enriched the causes of clinical skeletal fluorosis and provided a new perspective for studying the pathogenesis of skeletal fluorosis.

Impact loading intensifies cortical bone (re)modeling and alters longitudinal bone growth of pubertal rats.

Physical exercise is important for musculoskeletal development during puberty, which builds bone mass foundation for later in life. However, strenuous levels of training might bring adverse effects to bone health, reducing longitudinal bone growth. Animal models with various levels of physical exercise were largely used to provide knowledge to clinical settings. Experiments from our previous studies applied different levels of mechanical loading on rat tibia during puberty accompanied by weekly in vivo micro-CT scans. In the present article, we apply 3D image registration-based methods to retrospectively analyze part of the previously acquired micro-CT data. Longitudinal bone growth, growth plate thickness, and cortical bone (re)modeling were evaluated from rats' age of 28-77days. Our results show that impact loading inhibited proximal bone growth throughout puberty. We hypothesize that impact loading might bring different growth alterations to the distal and proximal growth plates. High impact loading might lead to pathological consequence of osteochondrosis and catch-up growth due to growth inhibition. Impact loading also increased cortical bone (re)modeling before and after the peak proximal bone growth period of young rats, of which the latter case might be caused by the shift from modeling to remodeling as the dominant activity toward the end of rat puberty. We confirm that the tibial endosteum is more mechano-sensitive than the periosteum in response to mechanical loading. To our knowledge, this is the first study to follow up bone growth and bone (re)modeling of young rats throughout the entire puberty with a weekly time interval.

Mesenchymal cell-derived Wnt1 signaling regulates subchondral bone remodeling but has no effects on the development of growth plate or articular cartilage in mice.

Chondrocyte differentiation is a principal progress in endochondral ossification and in the formation of secondary ossification center (SOC) during the long bone development. We have previously reported that targeted deletion of Wnt1 in mesenchymal progenitors (Wnt1Prrx-/-) leads to spontaneous fractures and severe osteopenia in mouse long bones, suggesting that Wnt1 is a key regulator of bone metabolism. However, the effect of Wnt1 on the regulation of cartilage development and chondrocyte differentiation remained unknown. In this study, WNT1 protein expression was observed in lateral superficial cartilage and growth plate pre-hypertrophic chondrocytes in mice. Wnt1 mRNA expression was detected in epiphyseal cartilage from E16.5 to 3month-old mice. Detailed histological analyses revealed that the average thickness and chondrocyte density of proximal tibial articular cartilage and growth plate were unchanged between Wnt1Prrx-/- and control mice. However, μCT analysis of tibial epiphyses showed that the subchondral bone mass was reduced in Wnt1Prrx-/- mice compared to control mice, as demonstrated by decreased bone volume, trabecular number, trabecular thickness, and increased trabecular separation in Wnt1Prrx-/- mice. Mechanistically, histomorphometric analyses showed that the reduced subchondral bone mass in Wnt1Prrx-/- mice was due to impaired bone formation and enhanced bone resorption. In vitro, exogenous Wnt1 inhibited chondrogenesis and chondrocyte hypertrophy in both cell autonomous and juxtacrine manners, while matrix mineralization and the expression of Mmp13, Mmp9 and Opn were induced in a juxtacrine manner. Taken together, mesenchymal cell-derived Wnt1 is an important regulator of subchondral bone remodeling, although it has no effect on the regulation of growth plate or articular cartilage.

PHEXL222P Mutation Increases Phex Expression in a New ENU Mouse Model for XLH Disease.

PhexL222P mouse is a new ENU mouse model for XLH disease due to Leu to Pro amino acid modification at position 222. PhexL222P mouse is characterized by growth retardation, hypophosphatemia, hypocalcemia, reduced body bone length, and increased epiphyseal growth plate thickness and femur diameter despite the increase in PHEXL222P expression. Actually, PhexL222P mice show an increase in Fgf23, Dmp1, and Mepe and Slc34a1 (Na-Pi IIa cotransporter) mRNA expression similar to those observed in Hyp mice. Femoral osteocalcin and sclerostin and Slc34a1 do not show any significant variation in PhexL222P mice. Molecular dynamics simulations support the experimental data. P222 might locally break the E217-Q224 β-sheet, which in turn might disrupt inter-β-sheet interactions. We can thus expect local protein misfolding, which might be responsible for the experimentally observed PHEXL222P loss of function. This model could be a valuable addition to the existing XLH model for further comprehension of the disease occurrence and testing of new therapies.

How do bones grow? A mathematical description of the mechanobiological behavior of the epiphyseal plate.

Growth modulation is an emerging method for the treatment of skeletal deformities originating in the long bones or the vertebral bodies. It requires the controlled application of mechanical loads to the affected bone, causing an alteration of the growth and ossification process occurring in a cartilaginous region called epiphyseal growth plate or physis. In order to avoid the possibility of under- or over-correction, quantification of the applied forces is necessary. Pursuing this goal, here we propose a phenomenological model of mechanobiological effects on the epiphyseal growth plate, based on the observed similarity between the mechanobiologically induced growth and viscoelastic material behavior. The model incorporates mechanical loading effects on growth direction, growth rate and ossification speed; it also allows to evaluate the occurrence of transient effects. Model consistency was tested against a rather large set of experiments existing in the literature. A generic simplified geometrical model of bones was established for this. Analytical solutions for growth and ossification evolution were obtained for different loading conditions, allowing to test the ability of the model to describe bone growth under various kinds of mechanical loading conditions. Model-predicted changes regarding epiphyseal growth plate thickness as well as longitudinal growth speed are consistent with experiments in which static tension or compression were applied to long bones. Results suggest that when the mechanical load is sinusoidally variable, conflicting data existing in the literature could be explained by a previously unconsidered effect of the the applied load initial phase. The model can accurately fit data regarding torsional loads effects on growth. Mechanobiological data for humans is very scarce. For this reason, when possible, the model parameters values were estimated, for the proposed generic geometry, after growth measurements in animal models available in the literature. Although it is not possible to assert their validity for humans, the proposed model along with the obtained parameters values give a rational foundation to be used in more advanced computational studies.

S-allylmercapto-N-acetylcysteine protects bone cells from oxidation and improves femur microarchitecture in healthy and diabetic mice.

Oxidative stress is involved in the deterioration of bone quality and mechanical strength in both diabetic and aging adults. Therefore, we studied the ability of the antioxidant compound, S-allylmercapto-N-acetylcysteine (ASSNAC) to protect bone marrow stromal cells (BMSCs) from advanced glycation end-products (AGEs) cytotoxicity and improve bone microarchitecture of adult healthy and obese/diabetic (db/db) female mice. ASSNAC effect on AGEs-treated cultured rat BMSCs was evaluated by Neutral Red and XTT cell survival and reactive oxygen species (ROS) level assays. Its effect on healthy (C57BL/6) and obese/diabetic (C57BLKS/J Leprdb+/+; db/db) female mice femur parameters, such as (1) number of adherent BMSCs, (2) percentage of CD73+/CD45- cells in bone marrow (BM), (3) glutathione level in BM cells, and (4) femur microarchitecture parameters by microcomputed tomography, was studied. ASSNAC treatment protected BMSCs by significantly decreasing AGEs-induced ROS production and increasing their cellular resistance to the cytotoxic effect of AGEs. ASSNAC treatment of healthy female mice (50 mg/kg/day; i.p.; age 12-20 weeks) significantly increased the number of BMSCs (+60%), CD73+/CD45- cells (+134%), and glutathione level (+110%) in the femur bone marrow. Furthermore, it increased the femur length (+3%), cortical diameter (+3%), and cortical areal moment of inertia (Ct.MOI; +10%) a surrogate for biomechanical strength. In db/db mice that demonstrated a compromised trabecular bone and growth plate microarchitecture, ASSNAC treatment restored the trabecular number (Tb.N, +29%), bone volume fraction (Tb.BV/TV, +130%), and growth plate primary spongiosa volumetric bone mineral density (PS-vBMD, +7%) and thickness (PS-Th, +18%). In conclusion, this study demonstrates that ASSNAC protects bone marrow cells from oxidative stress and may improve bone microarchitecture in adult healthy and diabetic female mice.

Dimensions of the anterior cruciate ligament and thickness of the distal femoral growth plate in children: a MRI-based study.

When determining the dimensions of an anterior cruciate ligament (ACL) graft in children, bone age should be considered in addition to the patient's age, gender, and body height. We aimed to determine the relationship between age, gender, and ACL dimensions as well as thickness of growth plate cartilage of the distal femur during puberty. We retrospectively analyzed MRI scans of the knee in 131 children (82 girls, 49 boys) aged 6-18years (mean age: 14.9 ± 2.6years). ACL length and width as well as thickness of the growth plate cartilage at the distal femoral epiphysis were measured. Mean ACL length increased linearly up to the age of 12years in females and 14years in males; thereafter, mean ACL length remained constant. Mean ACL length was largest at the age of 12 to < 13years (38.18mm) in females and at 15 to < 16years (39.38mm) in males. Mean ACL width increased up to the age of 12years in girls and 13years in boys. After the age of 12years, both the ACL length and width were significantly larger in boys than girls (p = 0.002 and p = 0.045, respectively). Mean thickness of the growth plate cartilage of the distal femur remained stable up to the age of 12years in girls and 14years in boys. Thickness of the growth plate cartilage changed most markedly between the age intervals of 11 to < 12years and 12 to < 13years in girls and between the age intervals of 13 to < 14years and 14 to < 15years in boys. ACL dimensions depended on both age and gender during the growth period. Measurement of cartilage thickness of the femoral distal growth plate proved to be an objective parameter to assess the maturation stage of local bone. This may be useful for the planning and timing of orthopedic ACL procedures. III-retrospective cohort study.

Dietary Complex and Slow Digestive Carbohydrates Promote Bone Mass and Improve Bone Microarchitecture during Catch-Up Growth in Rats.

Catch-up growth is a process that promotes weight and height gains to recover normal growth patterns after a transient period of growth inhibition. Accelerated infant growth is associated with reduced bone mass and quality characterized by poor bone mineral density (BMD), content (BMC), and impaired microarchitecture. The present study evaluated the effects of a diet containing slow (SDC) or rapid (RDC) digestible carbohydrates on bone quality parameters during the catch-up growth period in a model of diet-induced stunted rats. The food restriction period negatively impacted BMD, BMC, and microarchitecture of appendicular and axial bones. The SDC diet was shown to improve BMD and BMC of appendicular and axial bones after a four-week refeeding period in comparison with the RDC diet. In the same line, the micro-CT analysis revealed that the trabecular microarchitecture of tibiae and vertebrae was positively impacted by the dietary intervention with SDC compared to RDC. Furthermore, features of the cortical microstructure of vertebra bones were also improved in the SDC group animals. Similarly, animals allocated to the SDC diet displayed modest improvements in growth plate thickness, surface, and volume compared to the RDC group. Diets containing the described SDC blend might contribute to an adequate bone formation during catch-up growth thus increasing peak bone mass, which could be linked to reduced fracture risk later in life in individuals undergoing transient undernutrition during early life.

Effects of Therapeutic Ultrasound on Growth Plates: A Systematic Review.

The aim of this systematic review was to evaluate studies reporting on the effects of therapeutic ultrasound on the physis. Eight studies were included in the final analysis, all of which were animal studies. At higher doses, studies found skin and bone necrosis and inhibition of growth, while in lower doses some studies found that ultrasound had a transient stimulatory effect on growth, increased thickness of the hypertrophic zone, and increased thickness of the whole growth plate. Overall, experimental evidence in animal models suggests that therapeutic ultrasound, even at low doses, might induce microscopic changes to the histology of the growth plate. While we found no reports of growth disturbance in humans, given the histological changes found in animal studies, the current limited literature seems to support the recommendation that the application of therapeutic ultrasound around the physis should be avoided.

Targeting chondrocytes for arresting bony fusion in ankylosing spondylitis

Bony fusion caused by pathological new bone formation manifests the clinical feature of ankylosing spondylitis (AS). However, the underlying mechanism remains elusive. Here we discovered spontaneous kyphosis, arthritis and bony fusion in mature CD4-Cre;Ptpn11f/f mice, which present the pathophysiological features of AS. A population of CD4-Cre-expressing proliferating chondrocytes was SHP2 deficient, which could differentiate into pre-hypertrophic and hypertrophic chondrocytes. Functionally, SHP2 deficiency in chondrocytes impeded the fusion of epiphyseal plate and promoted chondrogenesis in joint cavity and enthesis. Mechanistically, aberrant chondrocytes promoted ectopic new bone formation through BMP6/pSmad1/5 signaling. It is worth emphasizing that such pathological thickness of growth plates was evident in adolescent humans with enthesitis-related arthritis, which could progress to AS in adulthood. Targeting dysfunctional chondrogenesis with Smo inhibitor sonidegib significantly alleviated the AS-like bone disease in mice. These findings suggest that blockade of chondrogenesis by sonidegib would be a drug repurposing strategy for AS treatment.

Effects of maternal consumption of morphine on rat skeletal system development

BackgroundOpioid abuse is among the most ubiquitous issues world-wide, and when it happens in mothers, it puts them at risk of diseases that can be transferred to the next generation. Previous studies have indicated that morphine addiction during pregnancy could inhibit development in rat embryos and infants.The present study focused on the effects of maternal consumption of morphine on rat skeletal system development and also investigate the molecular pathway of chondrogenesis and osteogenesis of infants from control and addicted rat groups.MethodsThirty-two female rats were randomly assigned to four groups. The groups consisted of one- and seven-day-old female infants which were born of morphine-dependent mothers and a control group for each of them. Experimental groups received oral morphine at the final dose of 0.4 mg/ml/day. Withdrawal signs were confirmation of morphine dependency. Female rats were crossed with male rats and coupling time was recorded. Fixed bones of all groups were processed and then stained by hematoxyline-eosin method. Thickness and cell number of proximal and distal growth plate of bones were measured. The cartilage and bone cells were stained by alcian blue/alizarin red method. Additionally, the gene expression of alkaline phosphatase, osteocalcin, and COLL2 and SOX9 gene expression were studied immuno-histochemically.ResultsUnfavorable effects of morphine on histological measurements were observed in one-day and seven-day infants, with more effects on seven-day infants. The thickness and cell number of the proximal and distal growth plate of morphine-dependent rat offsprings were reduced significantly. Furthermore, morphine reduced growth of primary and secondary ossification centers, and thus, longitudinal bone growth was reduced. Moreover, a decrease in the alkaline phosphatase, osteocalcin, COLL2 and SOX9 gene expression, and the number of stained cells was observed. More adverse effects of morphine in seven-day infants compared to one-day infants which showed the time dependent of morphine to the time length of administration.ConclusionHistochemistry and immunohistochemistry findings on cartilage and bone matrix formation, as well as protein expression of chondrogenic and osteogenic markers suggest that morphine dependence in pregnant mothers may impair intra-cartilaginous osteogenesis in post-natal rats.

Methotrexate Effects on the Lumbar Vertebrae of Young Male Albino Rat and Possible Protective Effect of Folic acid: Light Microscopic , Electron Microscopic and Morphometric Study

Background: With appearance of intensive chemotherapy regimens, methotrexate is a folic acid antagonist that is commonly used for the treatment of malignancies and rheumatic disorders. Aim of the Work: To reflect the effects of methotrexate on growing lumbar vertebrae of young male albino rat and potential protective effects of folic acid. Materials and Methods: 30 young male Albino rats were equally divided into three groups. Control group (I), methotrexate treated group (II) was subcutaneously injected once daily at 0.75 mg/kg for two separate 5-days courses and methotrexate plus folic acid group (III) was given methotrexate as group II plus folic acid at a dose of 1mg/kg. The lumbar vertebrae were extracted and processed for the light and electron microscopic examination. Morphometric measurements were also done.Results: Normal histological structures were determined in the control group whereas methotrexate treated group revealed cellular disorganization with degeneration. The proliferative zone showed decrease in chondrocyte number with failure of the secondary ossification center to appear and the centrum showed abundant fat cells and few bone marrow cells. Both reserve and proliferating cells revealed rarified vacuolated cytoplasm. The morphometric studies revealed a significant decrease in both proliferative cell count and midzonal thickness of growth plate. On the other hand, rats received folic acid with methotrexate revealed more or less apparent normal architecture.Conclusion: Folic acid proved to have remarkable protective effect against harmful effects of methotrexate. So, its supplementary should be started with methotrexate.Keywords: Methotrexate, folic acid, lumbar vertebra, centrum, epiphyseal plate, Albino rat.

Dosimetric considerations of 99mTc-MDP uptake within the epiphyseal plates of the long bones of pediatric patients

Skeletal scintigraphy is most performed in pediatric patients using the radiopharmaceutical 99mTc labelled methylene diphosphonate (99mTc-MDP). Reference biokinetic models for 99mTc-MDP indicate 50% of the administered activity is uniformly localized to the interior bone surfaces (trabecular and cortical regions), yet imaging data clearly show some preferential uptake to the epiphyseal growth plates of the long bones. To explore the dosimetric consequences of these regional activity concentrations, we have modified mesh-type computational phantoms of the International Commission on Radiological Protection (ICRP) reference pediatric series to explicitly include geometric models of the epiphyseal growth plates (2 mm in thickness) within the left/right, distal/proximal ends of the humeri, radii, ulnae, femora, tibia, and fibulae. Bone mineral activity from the ICRP Publication 128 biokinetic model for 99mTc-MDP (ICRP ) was then partitioned to the growth plates at values of 0.5%, 4.4%, 8.3%, 12.2%, 16.1%, and 20%. Radiation transport simulations were performed to compute 99mTc S-values and organ dose coefficients to the soft tissues and to bone site-specific regions of spongiosa. As the percentage of bone activity assigned to the growth plates was increased (from 0.5% to 20%), absorbed doses to the soft tissue organs, active bone marrow, bone endosteum (BE), as well as the detriment-weighted dose, were shown to decrease from their nominal values (no substantial growth plate activity), while epiphyseal plate self-doses increased. In the 15 year old male phantom, moving from 0.5% to 20% relative bone activity within the epiphyseal plates resulted in a 15% reduction in active marrow (AM) and BE dose, a 10% reduction in mean soft tissue and detriment-weighted dose, and a 6.3-fold increase in epiphyseal plate self-dose. In the newborn female phantom, we observed a 18% decrease in AM and BE dose, a 10% decrease in mean soft tissue dose, a 15% decrease in detriment-weighted dose, and 12.8-fold increase in epiphyseal plate self-dose. Increases (to 3 mm) and decreases (to 1 mm) in the assumed growth plate thickness of our models were shown to impact only the growth plate self-dose. Future work in differential quantification of 99mTc-MDP activity—growth plates versus other bone surfaces—is required to provide clinically realistic data on activity partitioning as a function of patient age, and perhaps skeletal site. The phantom series presented here may be used to develop more optimized age-related guidance on 99mTc-MDP administered activities to children.

Regulatory Effect of Kdm6b on Chondrocyte Metabolism in Mouse Cartilage Injury Repair Model

In order to study the expression of lysine-specific demethylase 6B, Kdm6b in differentiation of chondrocyte and chondrogenesis and the regulation effect of Kdm6b on chondrocyte anabolism, a mouse model of osteochondral defects was established in this study. The mice were subjected to antibiotic treatment and intestinal microbiota reconstruction by the method of specific knock out Kdm6b of embryonic cartilage. The mice were divided into Col2al-CreER Kdm6b experimental group and Kdm6b control group and then the destabilization of the medial meniscus operation was performed to model osteoarthritis in mice, which accelerated the abnormal metabolism of cartilage. In addition, transgenic mice with induced chondrocyte specific knockout of Kdm6b were constructed and embryonic samples were collected to study the effects of Kdm6b knockout on cartilage development. At the same time, the proliferation and apoptosis of chondrocytes after kdm6b gene knockout were also studied based on the obvious abnormalities in embryonic chondrogenesis. The results showed that 3 weeks after the mouse osteochondral defect modelling surgery, the mice in the Col2al-CreER; Kdm6b group still had an area without complete healing in the femoral trochle defect, while the defect areas in the Kdm6b group of mice were completely filled. After 12 weeks of mouse osteoarthritis modelling, mice in the Kdm6b group showed cartilage staining loss and cartilage defect. The mice in the Col2al-CreER Kdm6b group were more severe, and even a large area of wear and destruction of the cartilage layer appeared. However, after the growth plate thickness of mice in the Col2al-CreER Kdm6b group was observed, it was found that there was no statistical significance compared with the Kdm6b control group. qRT-PCR analysis showed that compared with the Kdm6b control group, the Col10al gene expression in the primary chondrocytes of the Col2a1-CreER Kdm6b group was significantly decreased. In addition, qRT-PCR and Western Blot sequencing verified that the expressions of Sox9, Col2al, Acan and other proteins related to anabolism were significantly reduced after Kdm6b knockout. Therefore, it can be concluded that Kdm6b directly regulated the expression of genes related to chondrocyte anabolism, and Kdm6b knockout could inhibit the anabolism of chondrocytes, impair the synthesis of cartilage matrix, and accelerate the progress of osteoarthritis.

Prolonged continuous infusion of teriparatide promotes bone metabolism in normal but not in castrated mice

To investigate the effects of continuous pumping of teriparatide (TPTD) on bone metabolism in ovariectomized and normal mice and provide experimental evidence for the selection of animal models for studying the effects of TPTD and its related peptides on osteoclasts. Twenty-four female C57BL mice (6-weeks old) were subjected to ovariectomy (OVX) or sham operation followed 7 days later by continuous pumping of TPTD or the solvent vehicle (VEH) via a micropump (SHAM-VEH, SHAM-TPTD, OVX-VEH, and OVX-TPTD groups; n=6). Two weeks later, the tibial and femoral bones were harvested for micro-CT scanning to measure the parameters of the tibia and the femoral cortical bone. Histopathological examinations of the tibial tissue were conducted using HE staining and TRAP staining and the number of osteoclasts and the growth plate thickness were determined. The serum Ca2 + levels of the mice were measured. The primary osteoblasts from the cranial bone were treated with estradiol (E2) and TPTD for 48 h, and the expressions of β-catenin and RANKL protein in the cells were analyzed. The trabecular bone mass of OVX mice was significantly lower than that of sham-operated mice (P &lt; 0.05). Continuous TPTD pumping significantly reduced tibial cancellous bone mass and femoral cortical bone area in the sham-operated mice, while in the castrated mice, TPTD pumping increased the cancellous bone mass without changing the cortical bone area. TRAP staining showed that cancellous osteoblasts in the tibia increased significantly in the castrated mice as compared with the sham-operated mice, and TPTD pumping significantly increased the number of cancellous osteoblasts in the sham-operated mice (P &lt; 0.05). In the primary cultured osteoblasts, treatment with both E2 and TPTD obviously lowered the expression of β-catenin and increased the expression of RANKL as compared with TPTD treatment alone. Continuous pumping of TPTD promotes bone resorption in normal mice but does not produce obvious bone resorption effect in the ovariectomized mice, suggesting that castrated mice are not suitable models for studying the effect of TPTD and the related peptides on the osteoclasts.

The Jejunum and Ileum Mediate Increased Calcium Absorption for Bone Mineralization During Postnatal Development via TRPV6 and Cav1.3 (OR26-07-19)

ObjectivesIntestinal Ca2+ absorption early in life is vital to achieving optimal bone mineralization. The molecular details of intestinal Ca2+ absorption have been defined in adults, after peak bone mass has been reached, but are largely unexplored during development. We sought to delineate the molecular details of transcellular Ca2+ absorption across the small intestine which facilitate a positive calcium balance during growth. MethodsWe used wildtype, Cav1.3 knockout and Trpv6 mutant mice. Expression of small intestinal and renal calcium transport genes was assessed using quantitative PCR. Net transcellular 45-calcium flux across intestinal segments was measured in Ussing chambers. Femurs we analyzed using micro-CT and histology. ResultsSignificant TRPV6 mediated Ca2+ flux across the duodenum was absent in pre-weaned (P14) mice but occurred post-weaning. In contrast, we found significant transcellular Ca2+ absorption in the jejunum and ileum at P14 but not 2 months. TRPV6 and Cav1.3 are necessary for this jejunal absorption and Cav1.3 appears to mediate absorption across the ileum although compensation is present in knockout pups. Knockout of Cav1.3 induces a compensatory increase in renal Ca2+ reabsorption in P14 mice although these pups have increased growth plate thickness suggesting delayed bone mineralization. ConclusionsThis work provides molecular details of how the small intestine facilitates increased demand for Ca2+ early in life to meet the requirements of growth and highlights the complexity of the multiple mechanisms involved in achieving a positive Ca2+ balance. Funding SourcesThis work is funded by grants from the Women and Children’s Health Research Institute, supported by the Stollery Children’s Hospital Foundation, and the National Sciences and Engineering Research Council to RTA, who is the Canada Research Chair in Renal Epithelial Transport Physiology. MRB is supported by a Vanier Canada Graduate Scholarship, Alberta Innovates Clinician Fellowship and an NSERC Michael Smith Foreign Study Supplement. H. Dimke is funded by the Danish Medical Research Council. Work at UdS was funded by Deutsche Forschungsgemeinschaft (DFG) by IRTG1830 (to JE, VF), Sonderforschungsbereich (SFB) 894 (to JE, PW) and SFB TRR152 (to VF).

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.