Intermittent mechanical loading on mouse tibia accelerates longitudinal bone growth by inducing PTHrP expression in the female tibial growth plate.

It is not clear as to whether weight bearing and ambulation may affect bone growth. Our goal was to study the role of mechanical loading (one of the components of ambulation) on endochondral ossification and longitudinal bone growth. Thus, we applied cyclical, biologically relevant strains for a prolonged time period (4 weeks) to one tibia of juvenile mice, while using the contralateral one as an internal control. By the end of the 4-week loading period, the mean tibial growth of the loaded tibiae was significantly greater than that of the unloaded tibiae. The mean height and the mean area of the loaded tibial growth plates were greater than those of the unloaded tibiae. In addition, in female mice we found a greater expression of PTHrP in the loaded tibial growth plates than in the unloaded ones. Lastly, microCT analysis revealed no difference between loaded and unloaded tibiae with respect to the fraction of bone volume relative to the total volume of the region of interest or the tibial trabecular bone volume. Thus, our findings suggest that intermittent compressive forces applied on tibiae at mild-moderate strain magnitude induce a significant and persistent longitudinal bone growth. PTHrP expressed in the growth plate appears to be one growth factor responsible for stimulating endochondral ossification and bone growth in female mice.

Radiographic evaluation of the forelimb bone development in maned wolves (Chrysocyon brachyurus).

This study aimed to assess the fusion of growth plates and the development of secondary ossification centres in the forelimb bones of maned wolves (Chrysocyon brachyurus), contrasting the findings with established data from domestic dogs. Three maned wolves, comprising one male and two females, initially aged between 3 and 4 months, were subjected to monthly radiographic evaluations until 10-11 months of age, followed by bimonthly assessments until 18-19 months of age, encompassing both forelimbs. The closure times of growth plates were observed as follows: supraglenoid tubercle (7-8 months), proximal humerus (17-19 months), distal humerus (8-9 months), medial epicondyle of the humerus (8-9 months), proximal ulna (9-10 months), proximal radius (13-15 months), distal ulna (13-15 months) and distal radius (17-19 months). Statistical analysis revealed significant differences in the areas of secondary ossification centres in the proximal epiphyses of the humerus and radius, respectively, observed from the initial evaluation at 8-9 months and 6-7 months. Conversely, the epiphyses of the supraglenoid tubercle, distal humerus, proximal ulna, distal ulna, medial epicondyle of the humerus and distal radius did not exhibit significant area differences between 3-4 months and 4-5 months, yet notable distinctions emerged at 5-6 months. In summary, while the radiographic appearance of epiphyseal growth plates and secondary ossification centres in maned wolves resembles that of domestic dogs, closure times vary. These findings contribute to understanding the dynamics of epiphyseal growth plates in this species.

Depletion of b-series ganglioside prevents limb length discrepancy after growth plate injury.

INTRODUCTION: Growth plate damage in long bones often results in progressive skeletal growth imbalance and deformity, leading to significant physical problems. Gangliosides, key glycosphingolipids in cartilage, are notably abundant in articular cartilage and regulate chondrocyte homeostasis. This suggests their significant roles in regulating growth plate cartilage repair. METHODS: Chondrocytes from 3 to 5 day-old C57BL/6 mice underwent glycoblotting and mass spectrometry. Based on the results of the glycoblotting analysis, we employed GD3 synthase knockout mice (GD3-/-), which lack b-series gangliosides. In 3-week-old mice, physeal injuries were induced in the left tibiae, with right tibiae sham operated. Tibiae were analyzed at 5 weeks postoperatively for length and micro-CT for growth plate height and bone volume at injury sites. Tibial shortening ratio and bone mineral density were measured by micro-CT. RESULTS: Glycoblotting analysis indicated that b-series gangliosides were the most prevalent in physeal chondrocytes among ganglioside series. At 3 weeks, GD3-/- exhibited reduced tibial shortening (14.7 ± 0.2 mm) compared to WT (15.0 ± 0.1 mm, P = 0.03). By 5 weeks, the tibial lengths in GD3-/- (16.0 ± 0.4 mm) closely aligned with sham-operated lengths (P = 0.70). Micro-CT showed delayed physeal bridge formation in GD3-/-, with bone volume measuring 168.9 ± 5.8 HU at 3 weeks (WT: 180.2 ± 3.2 HU, P = 0.09), but normalizing by 5 weeks. CONCLUSION: This study highlights that GD3 synthase knockout mice inhibit physeal bridge formation after growth plate injury, proposing a new non-invasive approach for treating skeletal growth disorders.

Astragalus Extract Mixture HT042 Alleviates Dexamethasone-Induced Bone Growth Retardation in Rat Metatarsal Bones.

The most widely used synthetic glucocorticoid, dexamethasone (DEX), causes stunted growth in children when used excessively or for long periods of time; however, there are still plenty of pediatric patients require long-term treatment with DEX. As an alternative, growth hormone is used in combination, but it has side effects, a high cost, and psychological factors, and it is not satisfactory in terms of effectiveness. It is necessary to develop a safe and affordable treatment that can replace it. The Korean Food and Drug Administration approved HT042, a standardized functional food ingredient, with the claim that it can help height growth of children. In this study, it was found that HT042 activated the Indian hedgehog/parathyroid hormone-related protein signaling pathway and enhanced the number of growth hormone receptors and insulin-like growth factor-1 receptors on the growth plate surface, which were reduced by DEX treatment, and restored growth retardation. In metatarsal bone and primary chondrocyte models, it was found that HT042 can promote the length of growth plate and recover DEX-induced growth retardation. It was also found that HT042 promotes cell proliferation using bromodeoxyuridine and terminal deoxynucleotidyl transferase dUTP nick end labeling assays; moreover, we verified increased expression of GHR/IGF-1R and Ihh/PTHrP pathway activity using qRT-PCR, western blotting, and siRNA analyses to verify its direct action on the growth plate. The anti-apoptotic effect of HT042 was identified by regulating the expression of apoptotic factors such as caspase-3, Bcl2, Bclx, and Bax. These results were identified using both ex vivo and in vitro models. Our study verified that co-administration of HT042 could recover the DEX induced growth retardation.

The Immature Pediatric Appendicular Skeleton.

Growth and maturation occur in a predictable pattern throughout the body and within each individual bone. In the appendicular skeleton, endochondral ossification predominates in long bones and growth plates. The ends of these long bones are sites of relative weakness in the immature skeleton and prone to injury from acute insult and overuse. We present the normal histoanatomy and physiology of the growth plate complex, highlighting the unique contribution of each component and shared similarities between primary and secondary complexes. Components of the growth plate complex include the physis proper, subjacent vascularity within the growth cartilage, and the ossification front. The second section describes imaging considerations and features of normal and abnormal growth. Finally, we review the Salter-Harris classification for acute fractures and offer examples of characteristic overuse injury patterns involving the epiphyseal (proximal humerus and distal radius), apophyseal (medial epicondyle and tibial tubercle), and secondary growth plate complexes (medial femoral condyle and capitellar osteochondritis dissecans). This article provides a foundation and basic framework to better understand and anticipate potential complications and growth disturbances and to ensure optimal follow-up and early intervention when treatment can be less invasive.

Overuse Injury of the Epiphyseal Primary Physis.

Year-round participation in youth sport that involves high levels of repetitive movement fosters an environment in which overuse injuries are likely to occur. Epiphyseal primary physeal stress injuries (PSIs), unique to skeletally immature athletes, are a particular concern, given their potential for growth disturbance. Initially observed in Little League baseball players, these injuries are now known to affect the long bones around the shoulder, elbow, wrist, hand, knee, ankle, and foot of skeletally immature athletes involved in a variety of sport activities.This article offers an epidemiological and radiologic perspective on the extent and distribution of epiphyseal PSIs in youth sport. We also review a novel framework for understanding the pathophysiologic mechanisms causing these injuries. This information is essential for the early identification of epiphyseal PSIs and devising preventive measures that can reduce a delayed diagnosis and long-term morbidity. Preventing and reducing injury to the epiphyseal growth plates is essential because impairment and dysfunction can result in lifelong morbidity and a risk of premature osteoarthritis.

Does spinopelvic alignment affect femoral head cartilage and the proximal femoral physis in slipped capital femoral epiphysis? A finite element analysis.

BACKGROUND: Slipped capital femoral epiphysis is a prevalent pediatric hip disorder. Recent studies suggest the spine's sagittal profile may influence the proximal femoral growth plate's slippage, an aspect not extensively explored. This study utilizes finite element analysis to investigate how various spinopelvic alignments affect shear stress and growth plate slip. METHODS: A finite element model was developed from CT scans of a healthy adult male lumbar spine, pelvis, and femurs. The model was subjected to various sagittal alignments through reorientation. Simulations of two-leg stance, one-leg stance, walking heel strike, ascending stairs heel strike, and descending stairs heel strike were conducted. Parameters measured included hip joint contact area, stress, and maximum growth plate Tresca (shear) stress. FINDINGS: Posterior pelvic tilt cases indicated larger shear stresses compared to the anterior pelvic tilt variants except in two leg stance. Two leg stance resulted in decreases in the posterior tilted pelvi variants hip contact and growth plate Tresca stress compared to anterior tilted pelvi, however a combination of posterior pelvic tilt and high pelvic incidence indicated larger shear stresses on the growth plate. One leg stance and heal strike resulted in higher shear stress on the growth plate in posterior pelvic tilt variants compared to anterior pelvic tilt, with a combination of posterior pelvic tilt and high pelvic incidence resulting in the largest shear. INTERPRETATION: Our findings suggest that posterior pelvic tilt and high pelvic incidence may lead to increased shear stress at the growth plate. Activities performed in patients with these alignments may predispose to biomechanical loading that shears the growth plate, potentially leading to slip.

The efficacy of physeal bar resection with guided growth in the treatment of physeal arrest with angular limb deformity.

Premature physeal arrest can cause progressive deformities and functional disabilities of the lower limbs. This study addressed the outcomes after physeal bar resection with or without guided growth (temporary hemiepiphysiodesis) for the treatment of angular limb deformities. We retrospectively analyzed 27 patients (mean 9 years; range, 3-12 years) who underwent physeal bar resection of the distal femur (15 patients), proximal tibia (3 patients), and distal tibia (9 patients) between 2002 and 2020. Fifteen patients underwent physeal bar resection only (Group A), and the other twelve underwent simultaneous guided growth (Group B). The correction angle (angle change between the preoperative and last follow-up values) was compared and analyzed. The overall mean correction angle was 2.9° (range, - 9 to 18.3°). A total of 12 (45%) patients had a > 5° angular deformity improvement (mean, 9.6°; range, 5-18.3°), 9 (33%) had a < 5° angular change; and 6 (22%) had a > 5° worsening of the angular deformity (mean, 6.7°; range, 5.2-9°). The correction angle in Group B (mean 7.6° ± 6.2) was significantly higher than that in Group A (mean - 0.77° ± 6.3) (P = 0.01). We found six (40%) and zero patients with a > 5° angular deformity increase in Groups A and B, respectively (P < 0.047). The group that underwent physeal bar resection with guided growth showed significantly higher correction angles than the group that underwent physeal bar resection alone. Additionally, none of the patients in the guided growth group experienced an increased angular deformity. Therefore, combining guided growth with physeal bar resection may lead to better outcomes in the treatment of growth arrest with angular deformities.

Neprilysin Inhibition Promotes Skeletal Growth via the CNP/NPR-B Pathway.

C-type natriuretic peptide (CNP) plays a crucial role in enhancing endochondral bone growth and holds promise as a therapeutic agent for impaired skeletal growth. To overcome CNP's short half-life, we explored the potential of dampening its clearance system. Neprilysin (NEP) is an endopeptidase responsible for catalyzing the degradation of CNP. Thus, we investigated the effects of NEP inhibition on skeletal growth by administering sacubitril, a NEP inhibitor, to C57BL/6 mice. Remarkably, we observed a dose-dependent skeletal overgrowth phenotype in mice treated with sacubitril. Histological analysis of the growth plate revealed a thickening of the hypertrophic and proliferative zones, mirroring the changes induced by CNP administration. The promotion of skeletal growth observed in wild-type mice treated with sacubitril was nullified by the knockout of cartilage-specific natriuretic peptide receptor B (NPR-B). Notably, sacubitril promoted skeletal growth in mice only at 3 to 4 weeks of age, a period when endogenous CNP and NEP expression was higher in the lumbar vertebrae. Additionally, sacubitril facilitated endochondral bone growth in organ culture experiments using tibial explants from fetal mice. These findings suggest that NEP inhibition significantly promotes skeletal growth via the CNP/NPR-B pathway, warranting further investigations for potential applications in people with short stature.

Physeal Location on the Distal Femoral Cortical Surface Is Consistently Proximal to Apparent Location on the Perfect Lateral Radiograph: A 3-Dimensional Digitally Reconstructed Radiograph Study.

INTRODUCTION: The perfect knee lateral radiograph visualizes anatomic landmarks on the distal femur for clinical and scientific purposes. However, radiographic imaging is a two-dimensional (2D) representation of a three-dimensional (3D) physis. The aim of this study was to characterize the perceived radiographic projection of the femoral physis using perfect lateral digitally reconstructed radiographs (DRRs) and to evaluate discrepancies from this projection to the physis at the lateral and medial cortices. METHODS: Pediatric patients from a cohort of CT scans were analyzed. Inclusion criteria were an open physis; exclusion criteria were any implant or pathology affecting the physis. CT scans were imported into 3D imaging software and transformed into lateral DRRs and 3D renderings of the femur. The physis was divided into four equal segments, with fiducial markers placed at the "anterior," "midpoint," and "posterior" points. Lines extended from these points in the lateral and medial direction. The vertical distance from these lines, representing the radiographic projection of the physis, was measured relative to the physis at the lateral and medial cortex of the femur on coronal CT slices. RESULTS: Thirty-one patients were included. On the perfect lateral radiograph DRR, the physis on the medial cortex was located proximal to the visualized physis by 6.64 ± 1.74 mm, 11.95 ± 1.67 mm, and 14.30 ± 1.75 mm at the anterior (25%), midpoint (50%), and posterior (75%) locations, respectively. On the lateral side, the physis on the lateral cortex was proximal to the visualized physis by 2.19 ± 1.13 mm, 3.71 ± 1.19 mm, and 6.74 ± 1.25 mm at the anterior, midpoint, and posterior locations, respectively. DISCUSSION: In this cohort of pediatric patients, the location of the cortical physis was, in all areas measured, proximal to the projection of the visualized physis as seen on the perfect knee lateral DRR. The distance from radiographic physis to cortical physis was greater at the medial cortex compared with the lateral cortex. STUDY DESIGN: Descriptive laboratory study. LEVEL OF EVIDENCE: III, observational radiographic anatomic study.

Age-related changes in densitometry and histomorphometry of long bone during the pubertal growth spurt in male rats.

Because experimental studies to determine the developmental toxicity of exposure to various substances in children are impossible, many studies use immature male rats. This study aimed to provide normative data for longitudinal bone growth with age during the puberty in male rats. In order to evaluate long bone growth and mineralization we examined bone size and bone density by dual-energy X-ray absorptiometry, analyzed histomorphometry of the growth plate, and serum hormone levels relevant to bone growth from postnatal day (PD)20 to PD60. The length and weight of long bones increased strongly by PD40, and no further increase was observed after PD50. On the other hand, tibial growth plate height decreased sharply after PD50 along with a reduction in the number of cells and columns, which was probably responsible for the absence of further lengthening of long bones. Parameters related to bone formation such as bone area ratio, and the thickness and number of trabeculae, also increased significantly between PD40 and PD50. Furthermore, serum levels of IGF-1 peaked at PD30 and testosterone increased rapidly on and after PD40, when IGF-1 levels were going down. These changes may participate in the parallel increase in mineral acquisition, as well as lengthening of long bones. Our findings provide comprehensive data for changes in bone density, histomorphometry of long bones, and hormone levels relevant to bone growth during the growth spurt. This will be useful for planning animal toxicological studies, particularly for deciding on the appropriate age of animals to use in given experiments.

Low-dose infigratinib increases bone growth and corrects growth plate abnormalities in an achondroplasia mouse model.

Achondroplasia (ACH), the most common form of disproportionate short stature, is caused by gain-of-function point mutations in fibroblast growth factor receptor 3 (FGFR3). Abnormally elevated activation of FGFR3 modulates chondrocyte proliferation and differentiation via multiple signaling pathways, such as the MAPK pathway. Using a mouse model mimicking ACH (Fgfr3Y367C/+), we have previously shown that daily treatment with infigratinib (BGJ398), a selective and orally bioavailable FGFR1-3 inhibitor, at a dose of 2 mg/kg, significantly increased bone growth. In this study, we investigated the activity of infigratinib administered at substantially lower doses (0.2 and 0.5 mg/kg, given once daily) and using an intermittent dosing regimen (1 mg/kg every 3 days). Following a 15-day treatment period, these low dosages were sufficient to observe significant improvement of clinical hallmarks of ACH such as growth of the axial and appendicular skeleton and skull development. Immunohistological labeling demonstrated the positive impact of infigratinib on chondrocyte differentiation in the cartilage growth plate and the cartilage end plate of the vertebrae. Macroscopic and microcomputed analyses showed enlargement of the foramen magnum area at the skull base, thus improving foramen magnum stenosis, a well-recognized complication in ACH. No changes in FGF23 or phosphorus levels were observed, indicating that the treatment did not modify phosphate homeostasis. This proof-of-concept study demonstrates that infigratinib administered at low doses has the potential to be a safe and effective therapeutic option for children with ACH.

Investigating Novel Therapeutic Approaches for Idiopathic Short Stature: Targeting siRNA and Growth Hormone Delivery to the Growth Plate Using Exosome Nanoparticles.

Idiopathic short stature (ISS) is a common childhood condition with largely unknown underlying causes. Recent research highlights the role of circulating exosomes in the pathogenesis of various disorders, but their connection to ISS remains unexplored. In the experiments, human chondrocytes are cocultured with plasma exosomes from ISS patients, leading to impaired chondrocyte growth and bone formation. Elevated levels of a specific long non-coding RNA (lncRNA), ISSRL, are identified as a distinguishing factor in ISS, boasting high specificity and sensitivity. Silencing ISSRL in ISS plasma exosomes reverses the inhibition of chondrocyte proliferation and bone formation. Conversely, overexpression of ISSRL in chondrocytes impedes their growth and bone formation, revealing its mechanism of action through the miR-877-3p/GZMB axis. Subsequently, exosomes (CT-Exo-siISSRL-oeGH) with precise cartilage-targeting abilities are engineered, loaded with customized siRNA for ISSRL and growth hormone. This innovative approach offers a therapeutic strategy to address ISS by rectifying abnormal non-coding RNA expression in growth plate cartilage and delivering growth hormone with precision to promote bone growth. This research provides valuable insights into ISS diagnosis and treatment, highlighting the potential of engineered exosomes.

Impact of Deep Learning Denoising Algorithm on Diffusion Tensor Imaging of the Growth Plate on Different Spatial Resolutions.

To assess the impact of a deep learning (DL) denoising reconstruction algorithm applied to identical patient scans acquired with two different voxel dimensions, representing distinct spatial resolutions, this IRB-approved prospective study was conducted at a tertiary pediatric center in compliance with the Health Insurance Portability and Accountability Act. A General Electric Signa Premier unit (GE Medical Systems, Milwaukee, WI) was employed to acquire two DTI (diffusion tensor imaging) sequences of the left knee on each child at 3T: an in-plane 2.0 × 2.0 mm2 with section thickness of 3.0 mm and a 2 mm3 isovolumetric voxel; neither had an intersection gap. For image acquisition, a multi-band DTI with a fat-suppressed single-shot spin-echo echo-planar sequence (20 non-collinear directions; b-values of 0 and 600 s/mm2) was utilized. The MR vendor-provided a commercially available DL model which was applied with 75% noise reduction settings to the same subject DTI sequences at different spatial resolutions. We compared DTI tract metrics from both DL-reconstructed scans and non-denoised scans for the femur and tibia at each spatial resolution. Differences were evaluated using Wilcoxon-signed ranked test and Bland-Altman plots. When comparing DL versus non-denoised diffusion metrics in femur and tibia using the 2 mm × 2 mm × 3 mm voxel dimension, there were no significant differences between tract count (p = 0.1, p = 0.14) tract volume (p = 0.1, p = 0.29) or tibial tract length (p = 0.16); femur tract length exhibited a significant difference (p < 0.01). All diffusion metrics (tract count, volume, length, and fractional anisotropy (FA)) derived from the DL-reconstructed scans, were significantly different from the non-denoised scan DTI metrics in both the femur and tibial physes using the 2 mm3 voxel size (p < 0.001). DL reconstruction resulted in a significant decrease in femorotibial FA for both voxel dimensions (p < 0.01). Leveraging denoising algorithms could address the drawbacks of lower signal-to-noise ratios (SNRs) associated with smaller voxel volumes and capitalize on their better spatial resolutions, allowing for more accurate quantification of diffusion metrics.

Stimulation of skeletal stem cells in the growth plate promotes linear bone growth.

Recently, skeletal stem cells were shown to be present in the epiphyseal growth plate (epiphyseal skeletal stem cells, epSSCs), but their function in connection with linear bone growth remains unknown. Here, we explore the possibility that modulating the number of epSSCs can correct differences in leg length. First, we examined regulation of the number and activity of epSSCs by Hedgehog (Hh) signaling. Both systemic activation of Hh pathway with Smoothened agonist (SAG) and genetic activation of Hh pathway by Patched1 (Ptch1) ablation in Pthrp-creER Ptch1fl/fl tdTomato mice promoted proliferation of epSSCs and clonal enlargement. Transient intra-articular administration of SAG also elevated the number of epSSCs. When SAG-containing beads were implanted into the femoral secondary ossification center of 1 leg of rats, this leg was significantly longer 1 month later than the contralateral leg implanted with vehicle-containing beads, an effect that was even more pronounced 2 and 6 months after implantation. We conclude that Hh signaling activates growth plate epSSCs, which effectively leads to increased longitudinal growth of bones. This opens therapeutic possibilities for the treatment of differences in leg length.

Polystyrene microplastic-induced endoplasmic reticulum stress contributes to growth plate endochondral ossification disorder in young rat.

BACKGROUND: Previous studies on the effects of microplastics (MPs) on bone in early development are limited. This study aimed to investigate the adverse effects of MPs on bone in young rats and the potential mechanism. METHODS: Three-week-old female rats were orally administered MPs for 28 days, and endoplasmic reticulum (ER) stress inhibitor salubrinal (SAL) and ER stress agonist tunicamycin (TM) were added to evaluate the effect of ER stress on toxicity of MPs. The indicators of growth and plasma markers of bone turnover were evaluated. Tibias were analyzed using micro-computed tomography (micro-CT). Histomorphological staining of growth plates was performed, and related gene expression of growth plate chondrocytes was tested. RESULTS: After exposure of MPs, the rats had decreased growth, shortened tibial length, and altered blood calcium and phosphorus metabolism. Trabecular bone was sparse according to micro-CT inspection. In the growth plate, the thickness of proliferative zone substantial reduced while the thickness of hypertrophic zone increased significantly, and the chondrocytes were scarce and irregularly arranged according to tibial histological staining. The transcription of the ER stress-related genes BIP, PERK, ATF4, and CHOP dramatically increased, and the transcription factors involved in chondrocyte proliferation, differentiation, apoptosis, and matrix secretion were aberrant according to RT-qPCR and western blotting. Moreover, the addition of TM showed higher percentage of chondrocyte death. Administration of SAL alleviated all of the MPs-induced symptoms. CONCLUSION: These results indicated that MPs could induce growth retardation and longitudinal bone damage in early development. The toxicity of MPs may attribute to induced ER stress and impaired essential processes of the endochondral ossification after MPs exposure.

Limb Length Discrepancy After Intraosseous Line Malpositioning: A Case Report.

CASE: We will present the case of a 6-year-old girl who presented with a 3-cm limb length discrepancy after intraosseous line placement at age 14 months without other known history of trauma or infection to account for the growth arrest. Imaging revealed a left proximal tibial physeal bar amenable to surgical resection with autologous lipotransfer. At 10 months postoperatively, physical examination and imaging demonstrated a stable 3-cm leg length discrepancy with an interval increase in the length of the left tibia in proportion to the growth of the right side with an increase in valgus alignment that will continue to be monitored and addressed as indicated. CONCLUSION: Pediatric intraosseous line placement presents unique challenges and can ultimately lead to physeal injury and growth arrest in the case of malpositioning.

Regulatory Pathways in Growth Plate Chondrocytes that Are Impacted by Matrix Vesicle microRNA Identified by Targeted RISC Pulldown and Sequencing of the Resulting Transcriptome.

During endochondral bone formation, growth plate chondrocytes are differentially regulated by various factors and hormones. As the cellular phenotype changes, the composition of the extracellular matrix is altered, including the production and composition of matrix vesicles (MV) and their cargo of microRNA. The regulatory functions of these MV microRNA in the growth plate are still largely unknown. To address this question, we undertook a targeted bioinformatics approach. A subset of five MV microRNA was selected for analysis based on their specific enrichment in these extracellular vesicles compared to the parent cells (miR-1-3p, miR-22-3p, miR-30c-5p, miR-122-5p, and miR-133a-3p). Synthetic biotinylated versions of the microRNA were produced using locked nucleic acid (LNA) and were transfected into rat growth plate chondrocytes. The resulting LNA to mRNA complexes were pulled down and sequenced, and the transcriptomic data were used to run pathway analysis pipelines. Bone and musculoskeletal pathways were discovered to be regulated by the specific microRNA, notably those associated with transforming growth factor beta (TGFß) and Wnt pathways, cell differentiation and proliferation, and regulation of vesicles and calcium transport. These results can help with understanding the maturation of the growth plate and the regulatory role of microRNA in MV.

Chondrocyte hypertrophy in the growth plate promotes stress anisotropy affecting long bone development through chondrocyte column formation.

The length of long bones is determined by column formation of proliferative chondrocytes and subsequent chondrocyte hypertrophy in the growth plate during bone development. Despite the importance of mechanical loading in long bone development, the mechanical conditions of the cells within the growth plate, such as the stress field, remain unclear owing to the difficulty in investigating spatiotemporal changes within dynamically growing tissues. In this study, the mechanisms of longitudinal bone growth were investigated from a mechanical perspective through column formation of proliferative chondrocytes within the growth plate before secondary ossification center formation using continuum-based particle models (CbPMs). A one-factor model, which simply describes essential aspects of a biological signaling cascade regulating cell activities within the growth plate, was developed and incorporated into CbPM. Subsequently, the developmental process and maintenance of the growth plate structure and resulting bone morphogenesis were simulated. Thus, stress anisotropy in the proliferative zone that affects bone elongation through chondrocyte column formation was identified and found to be promoted by chondrocyte hypertrophy. These results provide further insights into the mechanical regulation of multicellular dynamics during bone development.

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

BACKGROUND: 3 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. OBJECTIVE: To 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. METHODS: The 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. RESULTS: A 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. CONCLUSIONS: The 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.