Loss of Autophagy In Tibial Growth Plate Chondrocytes Causes Increased Chondrocyte Apoptosis In Young Rats With Chronic Renal Insufficiency

Background: To observed the effect of autophagy in tibial growth plate chondrocytes on apoptosis in chronic renal insufficiency(CRI) rats.Method: Male 4-week-old Sprague Dawley(SD) rats were randomly divided into two groups (n=20/per group): (1) the normal group was intragastrically administered distilled water; and (2) the CRI group was given a 150 mg/(kg·d) adenine suspension. All rats were sacrificed after continuous gavage for 6 weeks. The tibial length and the width of the tibial growth plate were measured using micro-CT. The width of the tibial growth plate was also measured in histological sections at both 4 w and 10 w. The level of the autophagy marker Beclin-1 in chondrocytes was measured by immunofluorescence. The level of glycogenin-1, a marker of intracellular glycogen accumulation, was measured by immunohistochemistry in chondrocytes in vivo and in vitro. The apoptosis rate of chondrocytes was measured by the TUNEL method in vivo and in vitro.Results: The results showed that the length of tibia was shorter and the width of tibia growth plate was narrower in CRF young rats. Autophagy level of chondrocytes in tibial growth plate decreased, and accumulation of glycogen granules in chondrocytes increased significantly. Meanwhile, the apoptosis rate of chondrocytes in tibial growth plate increased.Conclusion: When CRF occurred in young rats, the autophagy level of chondrocytes in tibial growth plate decreased significantly.As a result, there are not enough autophagic vesicles to swallow glycogen granules in chondrocytes and degrade them into glucose for energy supply, which leads to chondrocyte apoptosis.Autophagy of chondrocytes is at least partly involved in energy metabolism of cells.

Fractions of Shen-Sui-Tong-Zhi Formula Enhance Osteogenesis Via Activation of β-Catenin Signaling in Growth Plate Chondrocytes

Background: Shen-sui-tong-zhi formula (SSTZF) has been used to treat osteoporosis for decades and shows excellent clinical efficacy. This article aims to explore the optimal anti-osteoporotic ingredient and its precise mechanisms in mice models.Methods: In this study, we first screened the optimal anti-osteoporosis fraction of SSTZF extract in vivo, and then further explored the mechanism of its effects both in vivo and in vitro. Ten-week-old female C57BL/6J mice were administrated with each fraction of SSTZF. At 10 weeks after ovariectomy (OVX), femurs were collected for tissue analyses, including histology, micro-CT, biomechanical tests, and immunohistochemistry for ALP, FABP4, and β-catenin. Additionally, we also evaluated the mRNA expression level of ALP and FABP4 and the protein expression level of β-catenin after being treated with SSTZF extract in C3H10T1/2 cells. Moreover, we investigated the anti-osteoporosis effect of SSTZF extract on mice with β-catenin conditional knockout in growth plate chondrocytes (β-cateninGli1ER mice) through μCT, histology, and immunohistochemistry analyzes.Results: At 10 weeks after treatment, osteoporosis-like phenotype were significantly ameliorated in SSTZF n-butanol extract (SSTZF-NB) group mice, as indicated by increased trabecular bone area and ALP content, and decreased lipid droplet area and FABP4 content. No such improvements were observed after being treated with other extracts, demonstrating that SSTZF-NB is the optimal anti-osteoporosis fraction. Additionally, the elevated β-catenin was revealed in both OVX mice and C3H10T1/2 cells with SSTZF-NB administered. Furthermore, a significant osteoporosis-like phenotype was observed in β-cateninGli1ER mice as expected. However, SSTZF-NB failed to rescue the deterioration in β-cateninGli1ER mice, no significant re-upregulated ALP and downregulated FABP4 were observed after being treated with SSTZF-NB, demonstrating that SSTZF-NB prevents bone loss mainly via β-catenin signaling.Conclusion: SSTZF-NB enhances osteogenesis mainly via activation of β-catenin signaling in growth plate chondrocytes. SSTZF-NB is the optimal anti-osteoporosis fraction of SSTZF and it can be considered a salutary alternative therapeutic option for osteoporosis.

C-type natriuretic peptide facilitates autonomic Ca2+ entry in growth plate chondrocytes for stimulating bone growth

The growth plates are cartilage tissues found at both ends of developing bones, and vital proliferation and differentiation of growth plate chondrocytes are primarily responsible for bone growth. C-type natriuretic peptide (CNP) stimulates bone growth by activating natriuretic peptide receptor 2 (NPR2) which is equipped with guanylate cyclase on the cytoplasmic side, but its signaling pathway is unclear in growth plate chondrocytes. We previously reported that transient receptor potential melastatin-like 7 (TRPM7) channels mediate intermissive Ca2+ influx in growth plate chondrocytes, leading to activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) for promoting bone growth. In this report, we provide experimental evidence indicating a functional link between CNP and TRPM7 channels. Our pharmacological data suggest that CNP-28 evoked NPR2 activation elevates cellular cGMP content and stimulates big-conductance Ca2+-dependent K+ (BK) channels as a substrate for cGMP-dependent protein kinase (PKG). BK channel-induced hyperpolarization likely enhances the driving force of TRPM7-mediated Ca2+ entry and seems to accordingly activate CaMKII. Indeed, ex vivo organ culture analysis indicates that CNP-facilitated bone growth is abolished by chondrocyte-specific Trpm7 gene ablation. The defined CNP signaling pathway, the NPR2-PKG-BK channel-TRPM7 channel-CaMKII axis, likely pinpoints promising target proteins for developing new therapeutic treatments for divergent growth disorders.

G-Protein-Coupled Estrogen Receptor-1 Positively Regulates the Growth Plate Chondrocyte Proliferation in Female Pubertal Mice

Estrogen enhances long bone longitudinal growth during early puberty. Growth plate chondrocytes are the main cells that contribute to long bone elongation. The role of G-protein-coupled estrogen receptor-1 (GPER-1) in regulating growth plate chondrocyte function remains unclear. In the present study, we generated chondrocyte-specific GPER-1 knockout (CKO) mice to investigate the effect of GPER-1 in growth plate chondrocytes. In control mice, GPER-1 was highly expressed in the growth plates of 4- and 8-week-old mice, with a gradual decline through 12 to 16 weeks. In CKO mice, the GPER-1 expression in growth plate chondrocytes was significantly lower than that in the control mice (80% decrease). The CKO mice also showed a decrease in body length (crown–rump length), body weight, and the length of tibias and femurs at 8 weeks. More importantly, the cell number and thickness of the proliferative zone of the growth plate, as well as the thickness of primary spongiosa and length of metaphysis plus diaphysis in tibias of CKO mice, were significantly decreased compared with those of the control mice. Furthermore, there was also a considerable reduction in the number of proliferating cell nuclear antigens and Ki67-stained proliferating chondrocytes in the tibia growth plate in the CKO mice. The chondrocyte proliferation mediated by GPER-1 was further demonstrated via treatment with a GPER-1 antagonist in cultured epiphyseal cartilage. This study demonstrates that GPER-1 positively regulates chondrocyte proliferation at the growth plate during early puberty and contributes to the longitudinal growth of long bones.

Statistical image analysis and escort histograms in characterization of articular cartilage repair in a skeleton animal model

Statistical image analysis of an ensemble of digital images of histological samples is performed as an auxiliary investigation a result of the recently proposed method of articular cartilage repair utilizing growth plate chondrocytes in a skeleton animal model. A fixed–shift model of maximal likelihood estimates of image histograms applied for monochromatic (grayscale) images or their RGB components confirms the statistically significant effect of the previously proposed medical treatment. The type of staining used to prepare images of histological samples is related to the visibility of the effectiveness of medical treatment. Hellinger distance of escort distributions for maximal likelihood estimates of image histograms of medically treated and control samples is investigated to identify grayscale (or RGB) intensities responsible for statistically significant difference of the estimates. A difference of Shannon entropy quantifying informational content of the histograms allows one to identify staining and image colors which are most suitable to visualize cluster formation typical for articular cartilage repair processes.

Hypertrophic Chondrocytes Serve as a Reservoir for Unique Marrow Associated Skeletal Stem and Progenitor Cells, Osteoblasts, and Adipocytes During Skeletal Development

Skeletal stem and progenitor cells (SSPCs) reside within niches localized to the intramedullary bone marrow and periosteal tissues surrounding bones, with most being capable of becoming osteoblasts, chondrocytes, and adipocytes during bone development and/or regeneration. SSPCs within the periosteum can give rise to intramedullary SSPCs, osteoblasts, osteocytes, and adipocytes during bone development; however, whether they are the sole source of these cells remains to be determined. Growth plate chondrocytes contribute to the osteoblast lineage and trabecular bone formation; however, the cellular process used to achieve this is unknown. We utilized hypertrophic chondrocyte genetic reporter mouse models combined with single cell RNA-sequencing, immunofluorescent staining, and bulk RNA-sequencing approaches to determine that hypertrophic chondrocytes undergo a process of dedifferentiation to generate unique bone marrow associated SSPC populations that likely serve as a primary source of osteogenic cells during skeletal development, while also contributing to the adipogenic lineage with age.

Growth Plate Genes Are Key Regulators of Growth: Lessons Learned From Children of Consanguineous Families From Kurdistan, Iraq

Introduction: The genetic basis of human growth regulation has only been partly elucidated thus far. Therefore, finding causative genes in patients with short stature help in understanding precise pathophysiological mechanisms, establishing genotype-phenotype relationships and optimizing treatment. In order to extend our knowledge about the genes involved in short stature, we studied a unique cohort of consanguineous families with children with short stature from Sulaymani in Kurdistan, Iraq. Patients and Methods: Fifty-five consanguineous families, with children shorter than -2.3SDS at the time of examination (median height -3.3SDS, range -2.3 to -15SDS), who were examined at the endocrine outpatient clinic of the Department of Pediatrics, Sulaymani University College of Medicine between January 2018 and February 2020, were included in the study. In families with more than one child with short stature, the shorter sibling was selected as the proband (median age 8 years, range 1 - 15 years). Three probands were subsequently excluded due to the diagnosis of Turner’s syndrome and Edward’s syndrome Consent was obtained from all families and probands’ DNA was analyzed by Whole Exome Sequencing (WES) methods. The data were processed by a bioinformatic pipeline and detected variants were filtered using variant analysis software. Selected potentially pathogenic variants were confirmed using Sanger sequencing methods and evaluated by the American College of Medical Genetics (ACMG) standards and guidelines. Results: A monogenic cause of short stature, which explained the patient phenotype, was elucidated in 13 of 26 families who were analyzed thus far. Seven families had multiple affected children making a total of 22 patients with a positive genetic diagnosis. Pathogenic or likely pathogenic variants (according to the ACMG standards) were found in genes involved in the GH-IGF-1 axis (GHR), in the extracellular matrix of the growth plate (COL1A2, MMP13, LTBP3, and ADAMTS17), in the regulation of chondrocytes (NPR2 and CTSK), transporter coding genes (SLC34A3), and other genes (PTCH1, GALNS, DNACJ21, ZSWIM6, GNPTG). Among them, there are 9 novel variants and 10 homozygous variants including variants in genes causing syndromic short stature. Unexpectedly, we successfully identified three cases of autosomal dominant short stature (variants in genes NPR2, COL1A2, PTCH1) as well. The remaining probands from 26 families are still being analyzed. Conclusion: With the help of NGS methods, we have successfully elucidated the genetic cause of short stature in nearly 50% of patients who were analyzed thus far. These results further strength the concept that genes affecting the growth plate (chondrocytes and the extracellular matrix) play a crucial role in growth regulation. Acknowledgements: The study was co-funded by grants AZV 18-07-00283 and GAUK 340420.