Post-Traumatic Caspase-3 Expression in the Adjacent Areas of Growth Plate Injury Site: A Morphological Study

The epiphyseal plate is a hyaline cartilage plate that sits between the diaphysis and the epiphysis. The objective of this study was to determine the impact of an injury in the growth plate chondrocytes through the study of histological morphology, immunohistochemistry, histomorphometry and Western Blot analyses of the caspase-3 and cleaved PARP-1, and levels of the inflammatory cytokines, Interleukin-6 (IL-6) and Tumor Necrosis Factor alpha (TNF-α), in order to acquire more information about post-injury reactions of physeal cell turnover. In our results, morphological analysis showed that in experimental bones, neo-formed bone trabeculae—resulting from bone formation repair—invaded the growth plate and reached the metaphyseal bone tissue (bone bridge), and this could result in some growth arrest. We demonstrated, by ELISA, increased expression levels of the inflammatory cytokines IL-6 and TNF-α. Immunohistochemistry, histomorphometry and Western Blot analyses of the caspase-3 and cleaved PARP-1 showed that the physeal apoptosis rate of the experimental bones was significantly higher than that of the control ones. In conclusion, we could assume that the inflammation process causes stress to chondrocytes that will die as a biological defense mechanism, and will also increase the survival of new chondrocytes for maintaining cell homeostasis. Nevertheless, the exact stimulus leading to the increased apoptosis rate, observed after injury, needs additional research to understand the possible contribution of chondrocyte apoptosis to growth disturbance.     

BMP3 Affects Cortical and Trabecular Long Bone Development in Mice

Bone morphogenetic proteins (BMPs) have a major role in tissue development. BMP3 is synthesized in osteocytes and mature osteoblasts and has an antagonistic effect on other BMPs in bone tissue. The main aim of this study was to fully characterize cortical bone and trabecular bone of long bones in both male and female Bmp3^−/− mice. To investigate the effect of Bmp3 from birth to maturity, we compared Bmp3^−/− mice with wild-type littermates at the following stages of postnatal development: 1 day (P0), 2 weeks (P14), 8 weeks and 16 weeks of age. Bmp3 deletion was confirmed using X-gal staining in P0 animals. Cartilage and bone tissue were examined in P14 animals using Alcian Blue/Alizarin Red staining. Detailed long bone analysis was performed in 8-week-old and 16-week-old animals using micro-CT. The Bmp3 reporter signal was localized in bone tissue, hair follicles, and lungs. Bone mineralization at 2 weeks of age was increased in long bones of Bmp3^−/− mice. Bmp3 deletion was shown to affect the skeleton until adulthood, where increased cortical and trabecular bone parameters were found in young and adult mice of both sexes, while delayed mineralization of the epiphyseal growth plate was found in adult Bmp3^−/− mice.     

The Potential Role of Hepatocyte Growth Factor in Degenerative Disorders of the Synovial Joint and Spine

This paper aims to provide a comprehensive review of the changing role of hepatocyte growth factor (HGF) signaling in the healthy and diseased synovial joint and spine. HGF is a multifunctional growth factor that, like its specific receptor c-Met, is widely expressed in several bone and joint tissues. HGF has profound effects on cell survival and proliferation, matrix metabolism, inflammatory response, and neurotrophic action. HGF plays an important role in normal bone and cartilage turnover. Changes in HGF/c-Met have also been linked to pathophysiological changes in degenerative joint diseases, such as osteoarthritis (OA) and intervertebral disc degeneration (IDD). A therapeutic role of HGF has been proposed in the regeneration of osteoarticular tissues. HGF also influences bone remodeling and peripheral nerve activity. Studies aimed at elucidating the changing role of HGF/c-Met signaling in OA and IDD at different pathophysiological stages, and their specific molecular mechanisms are needed. Such studies will contribute to safe and effective HGF/c-Met signaling-based treatments for OA and IDD.     

Fibroblast Growth Factor 23 and Osteoporosis: Evidence from Bench to Bedside

Osteoporosis is a chronic debilitating disease caused by imbalanced bone remodeling processes that impair the structural integrity of bone. Over the last ten years, the association between fibroblast growth factor 23 (FGF23) and osteoporosis has been studied in both pre-clinical and clinical investigations. FGF23 is a bone-derived endocrine factor that regulates mineral homeostasis via the fibroblast growth factor receptors (FGFRs)/αKlotho complex. These receptors are expressed in kidney and the parathyroid gland. Preclinical studies have supported the link between the local actions of FGF23 on the bone remodeling processes. In addition, clinical evidence regarding the effects of FGF23 on bone mass and fragility fractures suggest potential diagnostic and prognostic applications of FGF23 in clinical contexts, particularly in elderly and patients with chronic kidney disease. However, inconsistent findings exist and there are areas of uncertainty requiring exploration. This review comprehensively summarizes and discusses preclinical and clinical reports on the roles of FGF23 on osteoporosis, with an emphasis on the local action, as opposed to the systemic action, of FGF23 on the bone. Current gaps in knowledge and future research directions are also suggested to encourage further rigorous research in this important field.     

Abnormal Response of the Proliferation and Differentiation of Growth Plate Chondrocytes to Melatonin in Adolescent Idiopathic Scoliosis

Abnormalities in the melatonin signaling pathway and the involvement of melatonin receptor MT2 have been reported in patients with adolescent idiopathic scoliosis (AIS). Whether these abnormalities were involved in the systemic abnormal skeletal growth in AIS during the peripubertal period remain unknown. In this cross-sectional case-control study, growth plate chondrocytes (GPCs) were cultured from twenty AIS and ten normal control subjects. Although the MT2 receptor was identified in GPCs from both AIS and controls, its mRNA expression was significantly lower in AIS patients than the controls. GPCs were cultured in the presence of either the vehicle or various concentrations of melatonin, with or without the selective MT2 melatonin receptor antagonist 4-P-PDOT (10 µM). Then the cell viability and the mRNA expression of collagen type X (COLX) and alkaline phosphatase (ALP) were assessed by MTT and qPCR, respectively. In the control GPCs, melatonin at the concentrations of 1, 100 nM and 10 µM significantly reduced the population of viable cells, and the mRNA level of COLX and ALP compared to the vehicle. Similar changes were not observed in the presence of 4-P-PDOT. Further, neither proliferation nor differentiation of GPCs from AIS patients was affected by the melatonin treatment. These findings support the presence of a functional abnormality of the melatonin signaling pathway in AIS GPCs, which might be associated with the abnormal endochondral ossification in AIS patients.     

Lamin-Related Congenital Muscular Dystrophy Alters Mechanical Signaling and Skeletal Muscle Growth

Laminopathies are a clinically heterogeneous group of disorders caused by mutations in the LMNA gene, which encodes the nuclear envelope proteins lamins A and C. The most frequent diseases associated with LMNA mutations are characterized by skeletal and cardiac involvement, and include autosomal dominant Emery–Dreifuss muscular dystrophy (EDMD), limb-girdle muscular dystrophy type 1B, and LMNA-related congenital muscular dystrophy (LMNA-CMD). Although the exact pathophysiological mechanisms responsible for LMNA-CMD are not yet understood, severe contracture and muscle atrophy suggest that mutations may impair skeletal muscle growth. Using human muscle stem cells (MuSCs) carrying LMNA-CMD mutations, we observe impaired myogenic fusion with disorganized cadherin/β catenin adhesion complexes. We show that skeletal muscle from Lmna-CMD mice is unable to hypertrophy in response to functional overload, due to defective fusion of activated MuSCs, defective protein synthesis and defective remodeling of the neuromuscular junction. Moreover, stretched myotubes and overloaded muscle fibers with LMNA-CMD mutations display aberrant mechanical regulation of the yes-associated protein (YAP). We also observe defects in MuSC activation and YAP signaling in muscle biopsies from LMNA-CMD patients. These phenotypes are not recapitulated in closely related but less severe EDMD models. In conclusion, combining studies in vitro, in vivo, and patient samples, we find that LMNA-CMD mutations interfere with mechanosignaling pathways in skeletal muscle, implicating A-type lamins in the regulation of skeletal muscle growth.     

Factors Involved in Morphogenesis in the Muscle–Tendon–Bone Complex

A decline in the body’s motor functions has been linked to decreased muscle mass and function in the oral cavity and throat; however, aging of the junctions of the muscles and bones has also been identified as an associated factor. Basic and clinical studies on the muscles, tendons and bones, each considered independently, have been published. In recent years, however, research has focused on muscle attachment as the muscle–tendon–bone complex from various perspectives, and there is a growing body of knowledge on SRY-box9 (Sox9) and Mohawk(Mkx), which has been identified as a common controlling factor and a key element. Myostatin, a factor that inhibits muscle growth, has been identified as a potential key element in the mechanisms of lifetime structural maintenance of the muscle–tendon–bone complex. Findings in recent studies have also uncovered aspects of the mechanisms of motor organ complex morphostasis in the superaged society of today and will lay the groundwork for treatments to prevent motor function decline in older adults.     

The Clinical Significance of the Insulin-Like Growth Factor-1 Receptor Polymorphism in Non-Small-Cell Lung Cancer with Epidermal Growth Factor Receptor Mutation

The insulin-like growth factor 1 (IGF1) signaling pathway mediates multiple cancer cell biological processes. IGF1 receptor (IGF1R) expression has been used as a reporter of the clinical significance of non-small-cell lung carcinoma (NSCLC). However, the association between IGF1R genetic variants and the clinical utility of NSCLC positive for epidermal growth factor receptor (EGFR) mutation is not clear. The current study investigated the association between the IGF1R genetic variants, the occurrence of EGFR mutations, and clinicopathological characteristics in NSCLC patients. A total of 452 participants, including 362 adenocarcinoma lung cancer and 90 squamous cell carcinoma lung cancer patients, were selected for analysis of IGF1R genetic variants (rs7166348, rs2229765, and rs8038415) using real-time polymerase chain reaction (PCR)genotyping. The results indicated that GA + AA genotypes of IGF1R rs2229765 were significantly associated with EGFR mutation in female lung adenocarcinoma patients (odds ratio (OR) = 0.39, 95% confidence interval (CI) = 0.17–0.87). Moreover, The GA + AA genotype IGF1R rs2229765 was significantly associated with EGFR L858R mutation (p = 0.02) but not with the exon 19 in-frame deletion. Furthermore, among patients without EGFR mutation, those who have at least one polymorphic A allele of IGF1R rs7166348 have an increased incidence of lymph node metastasis when compared with those patients homozygous for GG (OR, 2.75; 95% CI, 1.20–2.31). Our results showed that IGF1R genetic variants are related to EGFR mutation in female lung adenocarcinoma patients and may be a predictive factor for tumor lymph node metastasis in Taiwanese patients with NSCLC.     

Mechanical Properties of Human Concentrated Growth Factor (CGF) Membrane and the CGF Graft with Bone Morphogenetic Protein-2 (BMP-2) onto Periosteum of the Skull of Nude Mice

Concentrated growth factor (CGF) is 100% blood-derived, cross-linked fibrin glue with platelets and growth factors. Human CGF clot is transformed into membrane by a compression device, which has been widely used clinically. However, the mechanical properties of the CGF membranes have not been well characterized. The aims of this study were to measure the tensile strength of human CGF membrane and observe its behavior as a scaffold of BMP-2 in ectopic site over the skull. The tensile test of the full length was performed at the speed of 2mm/min. The CGF membrane (5 × 5 × 2 mm³) or the CGF/BMP-2 (1.0 μg) membrane was grafted onto the skull periosteum of nude mice (5-week-old, male), and harvested at 14 days after the graft. The appearance and size of the CGF membranes were almost same for 7 days by soaking at 4 °C in saline. The average values of the tensile strength at 0 day and 7 days were 0.24 MPa and 0.26 MPa, respectively. No significant differences of both the tensile strength and the elastic modulus were found among 0, 1, 3, and 7 days. Supra-periosteal bone induction was found at 14 days in the CGF/BMP-2, while the CGF alone did not induce bone. These results demonstrated that human CGF membrane could become a short-term, sticky fibrin scaffold for BMP-2, and might be preserved as auto-membranes for wound protection after the surgery.     

Deep Sequencing Analysis of miRNA Expression in Breast Muscle of Fast-Growing and Slow-Growing Broilers

Growth performance is an important economic trait in chicken. MicroRNAs (miRNAs) have been shown to play important roles in various biological processes, but their functions in chicken growth are not yet clear. To investigate the function of miRNAs in chicken growth, breast muscle tissues of the two-tail samples (highest and lowest body weight) from Recessive White Rock (WRR) and Xinghua Chickens (XH) were performed on high throughput small RNA deep sequencing. In this study, a total of 921 miRNAs were identified, including 733 known mature miRNAs and 188 novel miRNAs. There were 200, 279, 257 and 297 differentially expressed miRNAs in the comparisons of WRRh vs. WRRl, WRRh vs. XHh, WRRl vs. XHl, and XHh vs. XHl group, respectively. A total of 22 highly differentially expressed miRNAs (fold change > 2 or < 0.5; p-value < 0.05; q-value < 0.01), which also have abundant expression (read counts > 1000) were found in our comparisons. As far as two analyses (WRRh vs. WRRl, and XHh vs. XHl) are concerned, we found 80 common differentially expressed miRNAs, while 110 miRNAs were found in WRRh vs. XHh and WRRl vs. XHl. Furthermore, 26 common miRNAs were identified among all four comparisons. Four differentially expressed miRNAs (miR-223, miR-16, miR-205a and miR-222b-5p) were validated by quantitative real-time RT-PCR (qRT-PCR). Regulatory networks of interactions among miRNAs and their targets were constructed using integrative miRNA target-prediction and network-analysis. Growth hormone receptor (GHR) was confirmed as a target of miR-146b-3p by dual-luciferase assay and qPCR, indicating that miR-34c, miR-223, miR-146b-3p, miR-21 and miR-205a are key growth-related target genes in the network. These miRNAs are proposed as candidate miRNAs for future studies concerning miRNA-target function on regulation of chicken growth.     

Enhanced Nitrite-Mediated Relaxation of Placental Blood Vessels Exposed to Hypoxia Is Preserved in Pregnancies Complicated by Fetal Growth Restriction

Nitric oxide (NO) is essential in the control of fetoplacental vascular tone, maintaining a high flow−low resistance circulation that favors oxygen and nutrient delivery to the fetus. Reduced fetoplacental blood flow is associated with pregnancy complications and is one of the major causes of fetal growth restriction (FGR). The reduction of dietary nitrate to nitrite and subsequently NO may provide an alternative source of NO in vivo. We have previously shown that nitrite induces vasorelaxation in placental blood vessels from normal pregnancies, and that this effect is enhanced under conditions of hypoxia. Herein, we aimed to determine whether nitrite could also act as a vasodilator in FGR. Using wire myography, vasorelaxant effects of nitrite were assessed on pre-constricted chorionic plate arteries (CPAs) and veins (CPVs) from normal and FGR pregnancies under normoxic and hypoxic conditions. Responses to the NO donor, sodium nitroprusside (SNP), were assessed in parallel. Nitrate and nitrite concentrations were measured in fetal plasma. Hypoxia significantly enhanced vasorelaxation to nitrite in FGR CPAs (p < 0.001), and in both normal (p < 0.001) and FGR (p < 0.01) CPVs. Vasorelaxation to SNP was also potentiated by hypoxia in both normal (p < 0.0001) and FGR (p < 0.01) CPVs. However, compared to vessels from normal pregnancies, CPVs from FGR pregnancies showed significantly lower reactivity to SNP (p < 0.01). Fetal plasma concentrations of nitrate and nitrite were not different between normal and FGR pregnancies. Together, these data show that nitrite-mediated vasorelaxation is preserved in FGR, suggesting that interventions targeting this pathway have the potential to improve fetoplacental blood flow in FGR pregnancies.     

Novel Single Nucleotide Polymorphisms of the Insulin-Like Growth Factor-I Gene and Their Associations with Growth Traits in Common Carp (Cyprinus carpio L.)

Insulin-like growth factor-I (IGF-I) plays an important role in the growth and development of vertebrates. To study polymorphisms of IGF-I, we screened a total of 4555 bp of genomic sequences in four exons and partial introns for the discovery of single nucleotide polymorphism (SNP) in common carp (Cyprinus carpio). Three SNPs (g.3759T>G, g.7627T>A and g.7722T>C) in intron 2 and a nonsynonymous SNP (g.7892C>T) in exon 3 were identified in a pilot population including random parents and their progenies. 289 progenies were further genotyped for studying possible associations between genotypes or combined genotypes and growth traits. The results showed that the locus g.7627T>A was significantly associated with body weight and body length, and fish with genotype AA had a mean body weight 5.9% higher than those with genotype TT. No significant associations were observed between genotypes of other loci and growth traits. However, when both g.7627T>A and g.7722T>C were considered, the combined genotype TT/TT was extremely associated with the lowest values of body length and body weight and the highest K value in comparison with other diplotypes (p < 0.01). These results suggest that genotype AA at g.7627T>A and its combined genotypes with alleles from another locus have positive effects on growth traits, which would be a candidate molecular marker for further studies in marker-assisted selection in common carp.     

Cellular and Molecular Alterations Underlying Abnormal Bone Growth in X-Linked Hypophosphatemia

X-linked hypophosphatemia (XLH), the most common form of hereditary hypophosphatemic rickets, is caused by inactivating mutations of the phosphate-regulating endopeptidase gene (PHEX). XLH is mainly characterized by short stature, bone deformities and rickets, while in hypophosphatemia, normal or low vitamin D levels and low renal phosphate reabsorption are the principal biochemical aspects. The cause of growth impairment in patients with XLH is not completely understood yet, thus making the study of the growth plate (GP) alterations necessary. New treatment strategies targeting FGF23 have shown promising results in normalizing the growth velocity and improving the skeletal effects of XLH patients. However, further studies are necessary to evaluate how this treatment affects the GP as well as its long-term effects and the impact on adult height.     

Identification of MicroRNA 395a in 24-Epibrassinolide-Regulated Root Growth of Arabidopsis thaliana Using MicroRNA Arrays

Brassinosteroids (BRs) are endogenous plant hormones and are essential for normal plant growth and development. MicroRNAs (miRNAs) of Arabidopsis thaliana are involved in mediating cell proliferation in leaves, stress tolerance, and root development. The specifics of BR mechanisms involving miRNAs are unknown. Using customized miRNA array analysis, we identified miRNAs from A. thaliana ecotype Columbia (Col-0) regulated by 24-epibrassinolide (EBR, a highly active BR). We found that miR395a was significantly up-regulated by EBR treatment and validated its expression under these conditions. miR395a was over expressed in leaf veins and root tissues in EBR-treated miR395a promoter::GUS plants. We integrated bioinformatics methods and publicly available DNA microarray data to predict potential targets of miR395a. GUN5—a multifunctional protein involved in plant metabolic functions such as chlorophyll synthesis and the abscisic acid (ABA) pathway—was identified as a possible target. ABI4 and ABI5, both genes positively regulated by ABA, were down-regulated by EBR treatment. In summary, our results suggest that EBR regulates seedling development and root growth of A. thaliana through miR395a by suppressing GUN5 expression and its downstream signal transduction.     

Transforming Growth Factor-Beta in Skeletal Muscle Wasting

Transforming growth factor-beta (TGF-β) is part of a family of molecules that is present in many body tissues and performs many different functions. Evidence has been obtained from mice and human cancer patients with bony metastases and non-metastatic disease, as well as pediatric burn patients, that inflammation leads to bone resorption and release of TGF-β from the bone matrix with paracrine effects on muscle protein balance, possibly mediated by the generation of reactive oxygen species. Whether immobilization, which confounds the etiology of bone resorption in burn injury, also leads to the release of TGF-β from bone contributing to muscle wasting in other conditions is unclear. The use of anti-resorptive therapy in both metastatic cancer patients and pediatric burn patients has been successful in the prevention of muscle wasting, thereby creating an additional therapeutic niche for this class of drugs. The liberation of TGF-β may be one way in which bone helps to control muscle mass, but further investigation will be necessary to assess whether the rate of bone resorption is the determining factor for the release of TGF-β. Moreover, whether different resorptive conditions, such as immobilization and hyperparathyroidism, also involve TGF-β release in the pathogenesis of muscle wasting needs to be investigated.     

The Effect of Orally Supplemented Melatonin on Larval Performance and Skeletal Deformities in Farmed Gilthead Seabream (Sparus aurata)

The gilthead seabream larval rearing in continuous light is common in most Mediterranean hatcheries to stimulate larval length growth and increase food consumption. Several studies have shown that continuous light affects larval development and increases the prevalence of skeletal deformities. Melatonin is a crucial pineal neurohormone that displays daily secretion patterns, stimulates cell proliferation and embryonic development in Atlantic salmon and zebrafish, and improves osseointegration in mice and humans. However, no studies have examined the effects of orally supplemented melatonin on skeletal deformities in Sparus aurata larvae. We administered exogenous melatonin to gilthead seabream larvae via enriched rotifers and nauplii of Artemia. Exogenous melatonin induced bone deformities and stimulated parathyroid hormone-related protein-coding gene (PTHrP) mRNA expression. In addition to the melatonin-induced PTHrP high expression level, the recorded non coordinated function of skeletal muscle and bone during growth can be the fountainhead of bone deformities. Both myosin light chain 2 (mlc2) and bone gamma-carboxyglutamate protein-coding gene (bglap) expression levels were significantly affected by melatonin administration in an inverse dose–response manner during the exogenous melatonin administration. This is the first study to report the effect of inducing melatonin bone deformities on Sparus aurata larvae reared under ordinary hatchery conditions.     

The mTORC2 Regulator Homer1 Modulates Protein Levels and Sub-Cellular Localization of the CaSR in Osteoblast-Lineage Cells

We recently found that, in human osteoblasts, Homer1 complexes to Calcium-sensing receptor (CaSR) and mediates AKT initiation via mechanistic target of rapamycin complex (mTOR) complex 2 (mTORC2) leading to beneficial effects in osteoblasts including β-catenin stabilization and mTOR complex 1 (mTORC1) activation. Herein we further investigated the relationship between Homer1 and CaSR and demonstrate a link between the protein levels of CaSR and Homer1 in human osteoblasts in primary culture. Thus, when siRNA was used to suppress the CaSR, we observed upregulated Homer1 levels, and when siRNA was used to suppress Homer1 we observed downregulated CaSR protein levels using immunofluorescence staining of cultured osteoblasts as well as Western blot analyses of cell protein extracts. This finding was confirmed in vivo as the bone cells from osteoblast specific CaSR−/− mice showed increased Homer1 expression compared to wild-type (wt). CaSR and Homer1 protein were both expressed in osteocytes embedded in the long bones of wt mice, and immunofluorescent studies of these cells revealed that Homer1 protein sub-cellular localization was markedly altered in the osteocytes of CaSR−/− mice compared to wt. The study identifies additional roles for Homer1 in the control of the protein level and subcellular localization of CaSR in cells of the osteoblast lineage, in addition to its established role of mTORC2 activation downstream of the receptor.