Growth Plate Chondrocytes Research Articles

Runx2 controls the osteogenic fate of growth plate chondrocytes

Runx2 controls the osteogenic fate of growth plate chondrocytes

Vitamin C Alleviates Heat-Stress-Induced Damages in Pig Thoracic Vertebral Chondrocytes via the Ubiquitin-Mediated Proteolysis Pathway

Heat stress can impair organismal growth by inducing ubiquitination, proteasome-mediated degradation, and subsequent cellular damage. Vitamin C (VC) has been shown to potentially mitigate the detrimental effects of abiotic stresses on cells. Nevertheless, the impact of heat stress on growth plate chondrocytes remains unclear, and the underlying protective mechanisms of VC in these cells warrant further investigation. In this study, we focused on pig thoracic vertebral chondrocytes (PTVCs) that are crucial for promoting the body’s longitudinal elongation and treated them with 41 °C heat stress for 24 h, under varying concentrations of VC. Our findings reveal that, while oxidative stress induced by heat triggers apoptosis and inhibits the ubiquitin-mediated proteolysis pathway, the addition of VC alleviates heat-stress-induced oxidative stress and apoptosis, mitigates cell cycle arrest, and promotes cellular viability. Furthermore, we demonstrate that VC enhances the ubiquitin-proteasome proteolysis pathway by promoting the expression of ubiquitin protein ligase E3A, which thereby stabilizes the ubiquitin-mediated degradation machinery, alleviates the apoptosis, and enhances cell proliferation. Our results suggest the involvement of the ubiquitin-mediated proteolysis pathway in the effects of VC on PTVCs under heat stress, and offer a potential strategy to make use of VC to ensure the skeletal growth of animals under high temperature pressures in summer or in tropical regions.

Single cell analysis of Idh mutant growth plates identifies cell populations responsible for longitudinal bone growth and enchondroma formation

Enchondromas are a common tumor in bone that can occur as multiple lesions in enchondromatosis, which is associated with deformity of the affected bone. These lesions harbor somatic mutations in IDH and driving expression of a mutant Idh1 in Col2 expressing cells in mice causes an enchondromatosis phenotype. Here we compared growth plates from E18.5 mice expressing a mutant Idh1 with control littermates using single cell RNA sequencing. Data from Col2 expressing cells were analysed using UMAP and RNA pseudo-time analyses. A unique cluster of cells was identified in the mutant growth plates that expressed genes known to be upregulated in enchondromas. There was also a cluster of cells that was underrepresented in the mutant growth plates that expressed genes known to be important in longitudinal bone growth. Immunofluorescence showed that the genes from the unique cluster identified in the mutant growth plates were expressed in multiple growth plate anatomic zones, and pseudo-time analysis also suggested these cells could arise from multiple growth plate chondrocyte subpopulations. This data supports the notion that a subpopulation of chondrocytes become enchondromas at the expense of contributing to longitudinal growth.

8519 Investigating the Role of Protein Inhibitor of Activated STAT1 (Pias1) in Growth Plate Chondrocyte Maturation

Abstract Disclosure: J. Baronas: None. U. Ahmed: None. J.N. Hirschhorn: None. N.E. Renthal: None. Development of growth plate chondrocytes is a complex process that involves a wide array of regulatory elements. Recently our laboratory conducted a genome wide CRISPR knockout (KO) screen of growth plate chondrocyte maturation to discover novel genes and gene networks in the proliferation and maturation of chondrocytes. Among targets uncovered is Protein Inhibitor of Activated STAT 1 (Pias1), an E3 SUMO ligase that plays a multi-faceted role in genetic regulation, driving post-translational modifications, directly binding to transcription factors, and functioning as a DNA-binding protein. While Pias1 has been shown to be involved in the regulation of many cellular processes across multiple tissues, its function in growth plate development and chondrocyte maturation has been previously unstudied. In our genome-wide screen, our laboratory identified that loss of Pias1 led to early maturation of chondrocytes. This project seeks to investigate the role of Pias1 in chondrocyte maturation and differentiation. We first developed a Pias1-KO chondrocyte cell line, observing upregulation of hypertrophic markers Cd200 and Col10a1 in KOs relative to control via qPCR, in addition to proliferative regulators IHH and Pth1R. Through Bulk-RNAseq of the same cell line, we identified additional chondrocyte markers as upregulated in KO, as well as osteoblast marker Sp7. Members of the HoxD family, regulators of limb development, were downregulated. Cell culture data suggests that loss of Pias1 leads to increased cell proliferation and increased matrix production, indicating dysregulation of multiple chondrocyte processes. Immunohistochemistry of micromass culture sections also suggests morphological differences between KO and control, with KO cells significantly larger in diameter. Early SUMO-1 profiling yields limited evidence of decreased global SUMOylation in KO cells, one possible mechanistic explanation for the observed dysregulation. Ongoing studies are focused on identifying specific SUMOylation targets of Pias1 in chondrocytes, analyzing proteomic differences in KO and control, seeking putative DNA-binding sites of Pias1, and observing in vivo murine Pias1-KO growth plates. Our findings suggest that PIAS1 plays a crucial role in the growth plate by prolonging growth and delaying terminal hypertrophy. Our study aims to provide a better understanding of the regulatory pathways involved in growth plate development and related phenotypes such as height, and contribute to the development of novel therapies for growth-related disorders, such as achondroplasia and hypochondroplasia. Presentation: 6/1/2024

7598 A Humanized PTH Receptor Mouse Model Of Eiken Syndrome: Delayed Bone Mineralization Caused By A Receptor C-tail Truncation

Abstract Disclosure: J. Höppner: None. P. Hanna: None. M.N. Wein: None. H. Jueppner: None. T.J. Gardella: None. R. Civitelli: None. I.A. Portales-Castillo: None. Abstract The parathyroid hormone receptor-1 (PTH1R) plays a key role in bone development and acts in the growth plates in response to PTHrP ligand. Eiken syndrome is characterized by a delay in bone mineralization and is caused by homozygous PTH1R mutations. One such mutation, R485X, truncates the receptor's C-tail and removes serine phosphorylation sites involved in βarrestin binding. In HEK293 cells, R485X-hPTH1R exhibits deficient interaction with βarrestin and increased cAMP signaling both basally and in response to PTHrP (Portales-Castillo et al., Comms. Biology 2023). To understand how the R485X mutation causes delayed bone mineralization, we generated humanized R485X-hPTH1R knock-in mice. Homozygous hPTH1RR485X/R485X mice closely recapitulate the delayed bone mineralization seen in Eiken patients, as skeletal whole mount preparations from neonatal mutant mice showed reduced Alizarin red staining compared to WT controls, and metatarsal explants from the mutant mice showed a pronounced absence of mineralized bone. Tails of hPTH1RR485X/R485X mice were ∼50% shorter than those of WT littermates, and H&E-stained sections revealed only proliferative chondrocytes in the growth plates of mutant mice. This delay in growth plate chondrocyte maturation in hPTH1RR485X/R485X mice resolved with age, although older long bones and tails remained smaller in size than WT controls (Höppner et al. Presented at ASBMR 2023).Based on our in vitro findings and initial mouse characterization, we reasoned that the phenotype of Eiken mice may be explained by increased PTHrP/PTH1R signaling in growth plate chondrocyte. The class IIa histone deacetylase HDAC4 suppresses chondrocyte maturation downstream of PTH1R signaling. Therefore, we generated compound mutant mice bearing both Hdac4 deletion and the mutant R485X PTH1R allele. Indeed, Hdac4 deletion largely rescued the mineralization defect apparent in metatarsals of P1 hPTH1RR485X/R485X mice. We then assessed the contribution of endogenous PTHrP to the Eiken-like phenotype by generating hPTH1RR485X/R485X/PTHrPflox/+/Col2-Cre(tg) mice. Remarkably, these mice exhibit approximately the same tail lengths as hPTH1R-WT littermate controls, indicating that reduced PTHrP production can rescue the effects of homozygosity for R485X-PTH1R. In line, vertebral growth plates of the rescued mice exhibited normal zones of chondrocytes, including hypertrophic cells. The overall results support a disease mechanism for Eiken syndrome by which excess cAMP signaling by PTH1R-R485X, as induced in part by endogenous PTHrP, results in delayed chondrocyte differentiation and bone mineralization. Further studies employing βarr1/2-KO mice may help shed light on the specific roles of βarrestins in growth plate development by PTH1R in Eiken syndrome. Presentation: 6/3/2024

7096 Variants in WASHC3, a component of the WASH complex, cause distinctive facial dysmorphism, short stature, and variable neurodevelopmental abnormalities

Abstract Disclosure: Y. Jee: None. J. Lui: None. D. Marafi: None. Z. Xia: None. R. Bhatia: None. E. Zhou: None. I. Herman: None. A.G. Temnycky: None. P. Whalen: None. G. Elliot: None. E. Leschek: None. R. Wijngaard: None. R. Beek: None. A. Vreugd: None. M. De Vries: None. C. Karnebeek: None. M. Oud: None. T. Markello: None. K. Barnes: None. H. Alrohaif: None. H.H. Freeze: None. W. Gahl: None. M. Malicdan: None. J. Posey: None. J. Lupski: None. J. Baron: None. Genetic defects that impair growth plate chondrogenesis can result in a spectrum of phenotypes, ranging from skeletal dysplasia to mild short stature, sometimes accompanied by syndromic features. However, the genetic underpinnings of impaired skeletal growth often remain elusive. In our study, we investigated three unrelated families with short stature (height standard deviation score z < -2), distinctive facies (down-slanting eyes, low set ears, high arched palate, and short neck), and various neurodevelopmental abnormalities (from leaning difficulties to low intelligence quotient). In these families, exome sequencing (ES) identified rare, predicted-pathogenic variant alleles in WASHC3 that segregated with the phenotype. WASHC3 is a component of the WASH complex, which is involved in endosome receptor recycling. In the first family, a de novo-dominant missense variant (p.L69F) impaired WASHC3 participation in the WASH complex. This variant led to altered PTH1R endosomal trafficking, resulting in diminished PTH1R signaling and impairing growth plate chondrocyte hypertrophic differentiation, providing a likely explanation for the short stature. Knockdown of other WASH complex components (WASH1, SWIP, Strumpellin) also diminished PTH1R signaling, confirming the importance of an intact WASH complex. In the second and third families, a homozygous variant in the start codon (p.M1?) markedly reduced WASHC3 protein expression by approximately 75%. In conclusion, our findings strongly suggest that the WASH complex is required for normal skeletal growth and that, consequently, genetic abnormalities impairing the function of the WASH complex (WASHopathy) cause short stature as well as distinctive facies and variable neurodevelopmental abnormalities. These effects are likely mediated through disruptions in endosomal receptor trafficking. This new pathological information sheds light on the molecular basis of impaired longitudinal bone growth. Presentation: 6/3/2024

Caspase-12 affects chondrogenesis in mice

Caspase-12 is a molecule whose functions are still not well understood. Although its expression has been found in various tissues, specific roles have been described in only a few cases. These include the effect of caspase-12 on murine bone cell differentiation during craniofacial development. This work focused on the development of the limbs taking place through endochondral ossification, which precedes the formation of the cartilaginous growth plate. Caspase-12 was described here for the first time in growth plate chondrocytes during physiological development. Using pharmacological inhibition, caspase-12 was found to affect chondrogenesis. Limb-derived micromass cultures showed a significantly increased area of chondrogenic nodules after caspase-12 inhibition and there were changes in gene expression, the most significant of which was the reduction of Mmp9. These data point to potential new functions of caspase-12 in chondrogenesis.

Phlpp1 alters the murine chondrocyte phospho-proteome during endochondral bone formation

Appendicular skeletal growth and bone mass acquisition are controlled by a variety of growth factors, hormones, and mechanical forces in a dynamic process called endochondral ossification. In long bones, chondrocytes in the growth plate proliferate and undergo hypertrophy to drive bone lengthening and mineralization. Pleckstrin homology (PH) domain and leucine rich repeat phosphatase 1 and 2 (Phlpp1 and Phlpp2) are serine/threonine protein phosphatases that regulate cell proliferation, survival, and maturation via Akt, PKC, Raf1, S6k, and other intracellular signaling cascades. Germline deletion of Phlpp1 suppresses bone lengthening in growth plate chondrocytes. Here, we demonstrate that Phlpp2 does not regulate endochondral ossification, and we define the molecular differences between Phlpp1 and Phlpp2 in chondrocytes. Phlpp2−/− mice were phenotypically indistinguishable from their wildtype (WT) littermates, with similar bone length, bone mass, and growth plate dynamics. Deletion of Phlpp2 had moderate effects on the chondrocyte transcriptome and proteome compared to WT cells. By contrast, Phlpp1/2−/− (double knockout) mice resembled Phlpp1−/− mice phenotypically and molecularly, as the chondrocyte phospho-proteomes of Phlpp1−/− and Phlpp1/2−/− chondrocytes had similarities and were significantly different from WT and Phlpp2−/− chondrocyte phospho-proteomes. Data integration via multiparametric analysis showed that the transcriptome explained less variation in the data as a result of Phlpp1 or Phlpp2 deletion than proteome or phospho-proteome. Alterations in cell proliferation, collagen fibril organization, and Pdpk1 and Pak1/2 signaling pathways were identified in chondrocytes lacking Phlpp1, while cell cycle processes and Akt1 and Aurka signaling pathways were altered in chondrocytes lacking Phlpp2. These data demonstrate that Phlpp1, and to a lesser extent Phlpp2, regulate multiple and complex signaling cascades across the chondrocyte transcriptome, proteome, and phospho-proteome and that multi-omic data integration can reveal novel putative kinase targets that regulate endochondral ossification.

The metalloproteinase PAPP-A is required for IGF-dependent chondrocyte differentiation and organization

Insulin-like growth factor (IGF) signaling is required for proper growth and skeletal development in vertebrates. Consequently, its dysregulation may lead to abnormalities of growth or skeletal structures. IGF is involved in the regulation of cell proliferation and differentiation of chondrocytes. However, the availability of bioactive IGF may be controlled by antagonizing IGF binding proteins (IGFBPs) in the circulation and tissues. As the metalloproteinase PAPP-A specifically cleaves members of the IGFBP family, we hypothesized that PAPP-A activity liberates bioactive IGF in cartilage. In PAPP-A knockout mice, the femur length was reduced and the mice showed a disorganized columnar organization of growth plate chondrocytes. Similarly, zebrafish lacking pappaa showed reduced length of Meckel’s cartilage and disorganized chondrocytes, reminiscent of the mouse knockout phenotype. Expression of chondrocyte differentiation markers (sox9a, ihha, and col10a1) was markedly affected in Meckel’s cartilage of pappaa knockout zebrafish, indicating that differentiation of chondrocytes was compromised. Additionally, the zebrafish pappaa knockout phenotype was mimicked by pharmacological inhibition of IGF signaling, and it could be rescued by treatment with exogenous recombinant IGF-I. In conclusion, our data suggests that IGF activity in the growing cartilage, and hence IGF signaling in chondrocytes, requires the presence of PAPP-A. The absence of PAPP-A causes aberrant chondrocyte organization and compromised growth in both mice and zebrafish.

Apolipoprotein E is a novel marker for chondrocytes in the growth plate resting zone.

The resting zone (RZ) in mammalian growth plates is critical for maintaining and regulating chondrocyte turnover during longitudinal bone growth as a control tower and stem cell reservoir. Although recent lineage tracing studies have identified several markers for stem cells in the RZ, these markers only partially label chondrocytes in the RZ, suggesting that the resting chondrocytes (RCs) are a heterogeneous population with different types of stem cells. Since a comprehensive marker for RCs is still lacking, the RZ is generally determined based on ambiguous histological criteria, such as small and round chondrocytes without columnar formation, which may lead to inconsistencies among researchers. Therefore, in this study, we used single-cell RNA sequencing (scRNAseq) of growth plate chondrocytes followed by validation by fluorescence in situ hybridization (FISH) to precisely annotate cell clusters in scRNAseq and search for a marker of RCs. The scRNAseq analysis revealed that apolipoprotein E (Apoe) was the top-hit gene, which was ubiquitously expressed in the RC cluster. FISH confirmed that Apoe was exclusively localized to the histologically defined RZ. In newly generated Apoe-mCherry knock-in mice, we further confirmed that mCherry expression mirrored the distribution of Apoe-expressing chondrocytes in the RZ particularly after the formation of the secondary ossification center. These mCherry+ RCs were slow cycling in vivo and exhibited stem cell properties both in vitro and in vivo. Moreover, APOE was detected in human growth plate RCs. These findings suggest that Apoe is a novel pan-RC marker in both mouse and human growth plates.

Effect of manganese amino acid complexes on growth performance, meat quality, breast muscle and bone development in broilers

ABSTRACT 1. This study was conducted to investigate the effects of dietary supplementation of manganese (Mn) amino acid complexes on growth performance, Mn deposition, meat quality, breast muscle and bone development of broilers. 2. A total of 504, one-day-old male Arbor Acres broilers were randomly divided into seven treatments; control diet (CON; basal diet, no extra Mn addition), manganese diet (MnN as Numine®-Mn; CON + 40, 80, 120 or 160 mg Mn/kg), manganese-S group (MnS; CON + 120 mg Mn/kg as MnSO4·H2O), manganese-A diet (MnA as Mn from hydrolysed feather meal; CON + 40 mg Mn/kg as MnA). 3. There were no significant differences for average daily gain (ADG) or feed intake (ADFI) among diets during the feed phases (p > 0.05). The FCR in the starter and over the whole period were quadratically affected by dietary MnN dosage and gave the lowest FCR at 80 mg/kg (p < 0.05). The Mn content of thigh muscle, jejunum, heart, pancreas, liver and tibia increased linearly with MnN addition (p < 0.05). 4. For meat quality, MnN significantly increased colour (a*), pH45 min and pH24 h, reduced shear force, drip loss and pressure loss of breast muscle (p < 0.05). 5. Moreover, MnN significantly upregulated MYOD expression at d 21 and SOD expression at d 42, decreased MuRF1 and Atrogin-1 mRNA level at d 42 in breast muscle. Transcriptome analysis revealed that the regulating effect of MnN on muscle development significantly enriched signalling pathways such as adhesion, ECM-receptor, MAPK, mTOR and AMPK. Furthermore, dietary MnN significantly affected tibia length and growth plate development (p < 0.05) and promoted growth plate chondrocytes by increasing SOX-9, Runx-2, Mef2c, TGF-β, Ihh, Bcl-2 and Beclin1 and decreasing Bax and Caspase-3 (p < 0.05) expression which affect longitudinal tibial development. 6. In conclusion, Mn amino acid complexes could improve growth performance, tissue Mn deposition, breast muscle development, meat quality and bone development.

FOXC1 and FOXC2 regulate growth plate chondrocyte maturation towards hypertrophy in the embryonic mouse limb skeleton.

The Forkhead box transcription factors FOXC1 and FOXC2 are expressed in condensing mesenchyme cells at the onset of endochondral ossification. We used the Prx1-cre mouse to ablate Foxc1 and Foxc2 in limb skeletal progenitor cells. Prx1-cre;Foxc1Δ/Δ;Foxc2Δ/Δ limbs were shorter than controls, with worsening phenotypes in distal structures. Cartilage formation and mineralization was severely disrupted in the paws. The radius and tibia were malformed, whereas the fibula and ulna remained unmineralized. Chondrocyte maturation was delayed, with fewer Indian hedgehog-expressing, prehypertrophic chondrocytes forming and a smaller hypertrophic chondrocyte zone. Later, progression out of chondrocyte hypertrophy was slowed, leading to an accumulation of COLX-expressing hypertrophic chondrocytes and formation of a smaller primary ossification center with fewer osteoblast progenitor cells populating this region. Targeting Foxc1 and Foxc2 in hypertrophic chondrocytes with Col10a1-cre also resulted in an expanded hypertrophic chondrocyte zone and smaller primary ossification center. Our findings suggest that FOXC1 and FOXC2 direct chondrocyte maturation towards hypertrophic chondrocyte formation. At later stages, FOXC1 and FOXC2 regulate function in hypertrophic chondrocyte remodeling to allow primary ossification center formation and osteoblast recruitment.

Protein phosphatase SCP4 regulates cartilage development and endochondral osteogenesis via FoxO3a dephosphorylation.

The regulatory mechanisms involved in embryonic development are complex and yet remain unclear. SCP4 represents a novel nucleus-resident phosphatase identified in our previous study. The primary aim of this study was to elucidate the function of SCP4 in the progress of cartilage development and endochondral osteogenesis. SCP4-/- and SCP4Col2ER mice were constructed to assess differences in bone formation using whole skeleton staining. ABH/OG staining was used to compare chondrocyte differentiation and cartilage development. Relevant biological functions were analysed using RNA-sequencing and GO enrichment, further validated by immunohistochemical staining, Co-IP and Western Blot. Global SCP4 knockout led to abnormal embryonic development in SCP4-/- mice, along with delayed endochondral osteogenesis. In parallel, chondrocyte-specific removal of SCP4 yielded more severe embryonic deformities in SCP4Col2ER mice, including limb shortening, reduced chondrocyte number in the growth plate, disorganisation and cell enlargement. Moreover, RNA-sequencing analysis showed an association between SCP4 and chondrocyte apoptosis. Notably, Tunnel-positive cells were indeed increased in the growth plates of SCP4Col2ER mice. The deficiency of SCP4 up-regulated the expression levels of pro-apoptotic proteins both in vivo and in vitro. Additionally, phosphorylation of FoxO3a (pFoxO3a), a substrate of SCP4, was heightened in chondrocytes of SCP4Col2ER mice growth plate, and the direct interaction between SCP4 and pFoxO3a was further validated in chondrocytes. Our findings underscore the critical role of SCP4 in regulating cartilage development and endochondral osteogenesis during embryonic development partially via inhibition of chondrocytes apoptosis regulated by FoxO3a dephosphorylation.

Single-cell transcriptomic analyses of mouse idh1 mutant growth plate chondrocytes reveal distinct cell populations responsible for longitudinal growth and enchondroma formation.

Enchondromas are a common tumor in bone that can occur as multiple lesions in enchondromatosis, which is associated with deformity of the effected bone. These lesions harbor mutations in IDH and driving expression of a mutant Idh1 in Col2 expressing cells in mice causes an enchondromatosis phenotype. In this study we compared growth plates from E18.5 mice expressing a mutant Idh1 with control littermates using single cell RNA sequencing. Data from Col2 expressing cells were analyzed using UMAP and RNA pseudo-time analyses. A unique cluster of cells was identified in the mutant growth plates that expressed genes known to be upregulated in enchondromas. There was also a cluster of cells that was underrepresented in the mutant growth plates that expressed genes known to be important in longitudinal bone growth. Immunofluorescence showed that the genes from the unique cluster identified in the mutant growth plates were expressed in multiple growth plate anatomic zones, and pseudo-time analysis also suggested these cells could arise from multiple growth plate chondrocyte subpopulations. This data identifies subpopulations of cells in control and mutant growth plates, and supports the notion that a mutant Idh1 alters the subpopulations of growth plate chondrocytes, resulting a subpopulation of cells that become enchondromas at the expense of other populations that contribute to longitudinal growth.

Melatonin ameliorates Slc26a2-associated chondrodysplasias by attenuating endoplasmic reticulum stress and apoptosis of chondrocytes

Although the pathogenesis and mechanism of congenital skeletal dysplasia are better understood, progress in drug development and intervention research remains limited. Here we report that melatonin treatment elicits a mitigating effect on skeletal abnormalities caused by SLC26A2 deficiency. In addition to our previous finding of endoplasmic reticulum stress upon SLC26A2 deficiency, we found calcium (Ca2+) overload jointly contributed to SLC26A2-associated chondrodysplasias. Continuous endoplasmic reticulum stress and cytosolic Ca2+ overload in turn triggered apoptosis of growth plate chondrocytes. Melatonin, known for its anti-oxidant and anti-inflammatory properties, emerged as a promising therapeutic approach in our study, which enhanced survival, proliferation, and maturation of chondrocytes by attenuating endoplasmic reticulum stress and Ca2+ overload. Our findings not only demonstrated the efficacy of melatonin in ameliorating abnormal function and cell fate of SLC26A2-deficient chondrocytes in vitro but also underscored its role in partially alleviating the skeletal dysplasia seen in Col2a1-CreERT2; Slc26a2fl/fl mice. As revealed by histology and micro-CT analyses, melatonin significantly improved retarded cartilage growth, defective trabecular bone formation, and tibial genu varum in vivo. Collectively, these data shed translational insights for drug development and support melatonin as a potential treatment for SLC26A2-related chondrodysplasias.

Alcohol intake during pregnancy reduces offspring bone epiphyseal growth plate chondrocyte proliferation through transforming growth factor β-1 inhibition in the Sprague Dawley rat humerus.

Intrauterine alcohol exposure delays bone maturation and intensifies osteoporosis and fracture risk. As most studies emphasize the neurological aspects of intrauterine alcohol exposure, there is a lack of research on the implications pertaining to osseous tissue. Previous studies investigated these effects in fetuses, with limited studies on postnatal life. Postnatal studies are crucial since peak bone growth occurs during adolescence. This study aimed at assessing the effects of prenatal alcohol exposure on the humerus proximal and distal growth plate chondrocytes in 3-week-old rats. Sprague Dawley rats (n=9) were assigned to either the ethanol group (n=3), saline (n=3), and untreated (n=3) group and time-mated. Once pregnant, as confirmed by the presence of a copulation plug, the former 2 groups were treated with 0.015 ml/g of 25.2% ethanol and 0.9% saline. The untreated group received no treatment. The left humeri belonging to 6 pups per group were used. Serial sections were cut with a microtome at 5 μm thickness. These sections were stained with haematoxylin and eosin for assessment of normal morphology or immunolabeled with anti-Ki-67 and transforming growth factor β-1 (TGFβ-1) antibody. Prenatal alcohol exposure adversely effected the growth plate sizes and the number of cells in the proliferative zone. Fewer TGFβ-1 immunopositive and proliferative chondrocytes were found using the anti-Ki-67 antibody. This may explain the growth retardation in offspring exposed to gestational alcohol, showing that gestational alcohol exposure inhibits cell proliferation, aiding the diminished stature.

Molecular mechanisms of environmental pollutant-induced cartilage damage: from developmental disorders to osteoarthritis.

The objective of the present study was to review the molecular mechanisms of the adverse effects of environmental pollutants on chondrocytes and extracellular matrix (ECM). Existing data demonstrate that both heavy metals, including cadmium (Cd), lead (Pb), and arsenic (As), as well as organic pollutants, including polychlorinated dioxins and furans (PCDD/Fs) and polychlorinated biphenyls (PCB), bisphenol A, phthalates, polycyclic aromatic hydrocarbons (PAH), pesticides, and certain other organic pollutants that target cartilage ontogeny and functioning. Overall, environmental pollutants reduce chondrocyte viability through the induction apoptosis, senescence, and inflammatory response, resulting in cell death and impaired ECM production. The effects of organic pollutants on chondrocyte development and viability were shown to be mediated by binding to the aryl hydrocarbon receptor (AhR) signaling and modulation of non-coding RNA expression. Adverse effects of pollutant exposures were observed in articular and growth plate chondrocytes. These mechanisms also damage chondrocyte precursors and subsequently hinder cartilage development. In addition, pollutant exposure was shown to impair chondrogenesis by inhibiting the expression of Sox9 and other regulators. Along with altered Runx2 signaling, these effects also contribute to impaired chondrocyte hypertrophy and chondrocyte-to-osteoblast trans-differentiation, resulting in altered endochondral ossification. Several organic pollutants including PCDD/Fs, PCBs and PAHs, were shown to induce transgenerational adverse effects on cartilage development and the resulting skeletal deformities. Despite of epidemiological evidence linking human environmental pollutant exposure to osteoarthritis or other cartilage pathologies, the data on the molecular mechanisms of adverse effects of environmental pollutant exposure on cartilage tissue were obtained from studies in laboratory rodents, fish, or cell cultures and should be carefully extrapolated to humans, although they clearly demonstrate that cartilage should be considered a putative target for environmental pollutant toxicity.

Periodic static compression of micro-strain pattern regulates endochondral bone formation.

Introduction: Developmental engineering based on endochondral ossification has been proposed as a potential strategy for repairing of critical bone defects. Bone development is driven by growth plate-mediated endochondral ossification. Under physiological conditions, growth plate chondrocytes undergo compressive forces characterized by micro-mechanics, but the regulatory effect of micro-mechanical loading on endochondral bone formation has not been investigated. Methods: In this study, a periodic static compression (PSC) model characterized by micro-strain (with 0.5% strain) was designed to clarify the effects of biochemical/mechanical cues on endochondral bone formation. Hydrogel scaffolds loaded with bone marrow mesenchymal stem cells (BMSCs) were incubated in proliferation medium or chondrogenic medium, and PSC was performed continuously for 14 or 28days. Subsequently, the scaffold pretreated for 28days was implanted into rat femoral muscle pouches and femoral condylar defect sites. The chondrogenesis and bone defect repair were evaluated 4 or 10weeks post-operation. Results: The results showed that PSC stimulation for 14days significantly increased the number of COL II positive cells in proliferation medium. However, the chondrogenic efficiency of BMSCs was significantly improved in chondrogenic medium, with or without PSC application. The induced chondrocytes (ichondrocytes) spontaneously underwent hypertrophy and maturation, but long-term mechanical stimulation (loading for 28days) significantly inhibited hypertrophy and mineralization in ichondrocytes. In the heterotopic ossification model, no chondrocytes were found and no significant difference in terms of mineral deposition in each group; However, 4weeks after implantation into the femoral defect site, all scaffolds that were subjected to biochemical/mechanical cues, either solely or synergistically, showed typical chondrocytes and endochondral bone formation. In addition, simultaneous biochemical induction/mechanical loading significantly accelerated the bone regeneration. Discussion: Our findings suggest that microstrain mechanics, biochemical cues, and in vivo microenvironment synergistically regulate the differentiation fate of BMSCs. Meanwhile, this study shows the potential of micro-strain mechanics in the treatment of critical bone defects.

Skeletal abnormalities in mice with Dnmt3a missense mutations

Overgrowth and intellectual disability disorders in humans are typified by length/height and/or head circumference ≥ 2 standard deviations above the mean as well as intellectual disability and behavioral comorbidities, including autism and anxiety. Tatton-Brown-Rahman Syndrome is one type of overgrowth and intellectual disability disorder caused by heterozygous missense mutations in the DNA methyltransferase 3A (DNMT3A) gene. Numerous DNMT3A mutations have been identified in Tatton-Brown-Rahman Syndrome patients and may be associated with varying phenotype severities of clinical presentation. Two such mutations are the R882H and P904L mutations which result in severe and mild phenotypes, respectively. Mice with paralogous mutations (Dnmt3aP900L/+ and Dnmt3aR878H/+) exhibit overgrowth in their long bones (e.g., femur, humerus), but the mechanisms responsible for their skeletal overgrowth remain unknown. The goal of this study is to characterize skeletal phenotypes in mouse models of Tatton-Brown-Rahman Syndrome and identify potential cellular mechanisms involved in the skeletal overgrowth phenotype. We report that mature mice with the Dnmt3aP900L/+ or Dnmt3aR878H/+ mutation exhibit tibial overgrowth, cortical bone thinning, and weakened bone mechanical properties. Dnmt3aR878H/+ mutants also contain larger bone marrow adipocytes while Dnmt3aP900L/+ mutants show no adipocyte phenotype compared to control animals. To understand the potential cellular mechanisms regulating these phenotypes, growth plate chondrocytes, osteoblasts, and osteoclasts were assessed in juvenile mutant mice using quantitative static histomorphometry and dynamic histomorphometry. Tibial growth plates appeared thicker in mutant juvenile mice, but no changes were observed in osteoblast activity or osteoclast number in the femoral mid-diaphysis. These studies reveal new skeletal phenotypes associated with Tatton-Brown-Rahman Syndrome in mice and provide a rationale to extend clinical assessments of patients with this condition to include bone density and quality testing. These findings may be also informative for skeletal characterization of other mouse models presenting with overgrowth and intellectual disability phenotypes.

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

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. 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. 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. 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.