Application of 3D MAPs pipeline identifies the morphological sequence chondrocytes undergo and the regulatory role of GDF5 in this process.

The activity of epiphyseal growth plates, which drives long bone elongation, depends on extensive changes in chondrocyte size and shape during differentiation. Here, we develop a pipeline called 3D Morphometric Analysis for Phenotypic significance (3D MAPs), which combines light-sheet microscopy, segmentation algorithms and 3D morphometric analysis to characterize morphogenetic cellular behaviors while maintaining the spatial context of the growth plate. Using 3D MAPs, we create a 3D image database of hundreds of thousands of chondrocytes. Analysis reveals broad repertoire of morphological changes, growth strategies and cell organizations during differentiation. Moreover, identifying a reduction in Smad 1/5/9 activity together with multiple abnormalities in cell growth, shape and organization provides an explanation for the shortening of Gdf5 KO tibias. Overall, our findings provide insight into the morphological sequence that chondrocytes undergo during differentiation and highlight the ability of 3D MAPs to uncover cellular mechanisms that may regulate this process.

Characterization of the growth plate-bone interphase region using cryo-FIB SEM 3D volume imaging.

The interphase region at the base of the growth plate includes blood vessels, cells and mineralized tissues. In this region, cartilage is mineralized and replaced with bone. Blood vessel extremities permeate this space providing nutrients, oxygen and signaling factors. All these different components form a complex intertwined 3D structure. Here we use cryo-FIB SEM to elaborate this 3D structure without removing the water. As it is challenging to image mineralized and unmineralized tissues in a hydrated state, we provide technical details of the parameters used. We obtained two FIB SEM image stacks that show that the blood vessels are in intimate contact not only with cells, but in some locations also with mineralized tissues. There are abundant red blood cells at the extremities of the vessels. We also documented large multinucleated cells in contact with mineralized cartilage and possibly also with bone. We observed membrane bound mineralized particles in these cells, as well as in blood serum, but not in the hypertrophic chondrocytes. We confirm that there is an open pathway from the blood vessel extremities to the mineralizing cartilage. Based on the sparsity of the mineralized particles, we conclude that mainly ions in solution are used for mineralizing cartilage and bone, but these are augmented by the supply of mineralized particles.

Chondrocytes in the resting zone of the growth plate are maintained in a Wnt-inhibitory environment.

Chondrocytes in the resting zone of the postnatal growth plate are characterized by slow cell cycle progression, and encompass a population of parathyroid hormone-related protein (PTHrP)-expressing skeletal stem cells that contribute to the formation of columnar chondrocytes. However, how these chondrocytes are maintained in the resting zone remains undefined. We undertook a genetic pulse-chase approach to isolate slow cycling, label-retaining chondrocytes (LRCs) using a chondrocyte-specific doxycycline-controllable Tet-Off system regulating expression of histone 2B-linked GFP. Comparative RNA-seq analysis identified significant enrichment of inhibitors and activators for Wnt signaling in LRCs and non-LRCs, respectively. Activation of Wnt/ß-catenin signaling in PTHrP+ resting chondrocytes using Pthlh-creER and Apc-floxed allele impaired their ability to form columnar chondrocytes. Therefore, slow-cycling chondrocytes are maintained in a Wnt-inhibitory environment within the resting zone, unraveling a novel mechanism regulating maintenance and differentiation of PTHrP+ skeletal stem cells of the postnatal growth plate.

Loss of Foxc1 and Foxc2 function in chondroprogenitor cells disrupts endochondral ossification.

Endochondral ossification initiates the growth of the majority of the mammalian skeleton and is tightly controlled through gene regulatory networks. The forkhead box transcription factors Foxc1 and Foxc2 regulate aspects of osteoblast function in the formation of the skeleton, but their roles in chondrocytes to control endochondral ossification are less clear. Here, we demonstrate that Foxc1 expression is directly regulated by the activity of SRY (sex-determining region Y)-box 9, one of the earliest transcription factors to specify the chondrocyte lineage. Moreover, we demonstrate that elevated expression of Foxc1 promotes chondrocyte differentiation in mouse embryonic stem cells and loss of Foxc1 function inhibits chondrogenesis in vitro. Using chondrocyte-targeted deletion of Foxc1 and Foxc2 in mice, we reveal a role for these factors in chondrocyte differentiation in vivo. Loss of both Foxc1 and Foxc2 caused a general skeletal dysplasia predominantly affecting the vertebral column. The long bones of the limbs were smaller, mineralization was reduced, and organization of the growth plate was disrupted; in particular, the stacked columnar organization of the proliferative chondrocyte layer was reduced in size and cell proliferation was decreased. Differential gene expression analysis indicated disrupted expression patterns of chondrogenesis and ossification genes throughout the entire process of endochondral ossification in chondrocyte-specific Foxc1/Foxc2 KO embryos. Our results suggest that Foxc1 and Foxc2 are required for normal chondrocyte differentiation and function, as loss of both genes results in disorganization of the growth plate, reduced chondrocyte proliferation, and delays in chondrocyte hypertrophy that prevents ossification of the skeleton.

The hypertrophic chondrocyte: To be or not to be.

Hypertrophic chondrocytes are the master regulators of endochondral ossification; however, their ultimate cell fates cells remain largely elusive due to their transient nature. Historically, hypertrophic chondrocytes have been considered as the terminal state of growth plate chondrocytes, which are destined to meet their inevitable demise at the primary spongiosa. Chondrocyte hypertrophy is accompanied by increased organelle synthesis and rapid intracellular water uptake, which serve as the major drivers of longitudinal bone growth. This process is delicately regulated by major signaling pathways and their target genes, including growth hormone (GH), insulin growth factor-1 (IGF-1), indian hedgehog (Ihh), parathyroid hormone-related protein (PTHrP), bone morphogenetic proteins (BMPs), sex determining region Y-box 9 (Sox9), runt-related transcription factors (Runx) and fibroblast growth factor receptors (FGFRs). Hypertrophic chondrocytes orchestrate endochondral ossification by regulating osteogenic-angiogenic and osteogenic-osteoclastic coupling through the production of vascular endothelial growth factor (VEGF), receptor activator of nuclear factor kappa-B ligand (RANKL) and matrix metallopeptidases-9/13 (MMP-9/13). Hypertrophic chondrocytes also indirectly regulate resorption of the cartilaginous extracellular matrix, by controlling formation of a special subtype of osteoclasts termed "chondroclasts". Notably, hypertrophic chondrocytes may possess innate potential for plasticity, reentering the cell cycle and differentiating into osteoblasts and other types of mesenchymal cells in the marrow space. We may be able to harness this unique plasticity for therapeutic purposes, for a variety of skeletal abnormalities and injuries. In this review, we discuss the morphological and molecular properties of hypertrophic chondrocytes, which carry out important functions during skeletal growth and regeneration.

miR-143 is implicated in growth plate injury by targeting IHH in precartilaginous stem cells.

Precartilaginous stem cells (PCSCs) are able to initiate chondrocyte and bone development. The present study aimed to investigate the role of miR-143 and the underlying mechanisms involved in PCSC proliferation. In a rat growth plate injury model, tissue from the injury site was collected and the expression of miR-143 and its potential targets was determined. PCSCs were isolated from the rabbits' distal epiphyseal growth plate. Cell viability, DNA synthesis, and apoptosis were determined with MTT, BrdU, and flow cytometric analysis, respectively. Real time PCR and western blot were performed to detect the mRNA and protein expression of the indicated genes. Indian hedgehog (IHH) was identified as a target gene for miR-143 with luciferase reporter assay. Decreased expression of miR-143 and increased expression of IHH gene were observed in the growth plate after injury. miR-143 mimics decreased cell viability and DNA synthesis and promoted apoptosis of PCSCs. Conversely, siRNA-mediated inhibition of miR-143 led to increased growth and suppressed apoptosis of PCSCs. Transfection of miR-143 decreased luciferase activity of wild-type IHH but had no effect when the 3'-UTR of IHH was mutated. Furthermore, the effect of miR-143 overexpression was neutralized by overexpression of IHH. Our study showed that miR-143 is involved in growth plate behavior and regulates PCSC growth by targeting IHH, suggesting that miR-143 may serve as a novel target for PCSC-related diseases.

SOX9 keeps growth plates and articular cartilage healthy by inhibiting chondrocyte dedifferentiation/osteoblastic redifferentiation.

Cartilage is essential throughout vertebrate life. It starts developing in embryos when osteochondroprogenitor cells commit to chondrogenesis, activate a pancartilaginous program to form cartilaginous skeletal primordia, and also embrace a growth-plate program to drive skeletal growth or an articular program to build permanent joint cartilage. Various forms of cartilage malformation and degeneration diseases afflict humans, but underlying mechanisms are still incompletely understood and treatment options suboptimal. The transcription factor SOX9 is required for embryonic chondrogenesis, but its postnatal roles remain unclear, despite evidence that it is down-regulated in osteoarthritis and heterozygously inactivated in campomelic dysplasia, a severe skeletal dysplasia characterized postnatally by small stature and kyphoscoliosis. Using conditional knockout mice and high-throughput sequencing assays, we show here that SOX9 is required postnatally to prevent growth-plate closure and preosteoarthritic deterioration of articular cartilage. Its deficiency prompts growth-plate chondrocytes at all stages to swiftly reach a terminal/dedifferentiated stage marked by expression of chondrocyte-specific (Mgp) and progenitor-specific (Nt5e and Sox4) genes. Up-regulation of osteogenic genes (Runx2, Sp7, and Postn) and overt osteoblastogenesis quickly ensue. SOX9 deficiency does not perturb the articular program, except in load-bearing regions, where it also provokes chondrocyte-to-osteoblast conversion via a progenitor stage. Pathway analyses support roles for SOX9 in controlling TGFß and BMP signaling activities during this cell lineage transition. Altogether, these findings deepen our current understanding of the cellular and molecular mechanisms that specifically ensure lifelong growth-plate and articular cartilage vigor by identifying osteogenic plasticity of growth-plate and articular chondrocytes and a SOX9-countered chondrocyte dedifferentiation/osteoblast redifferentiation process.

Isolation of Mouse Growth Plate and Articular Chondrocytes for Primary Cultures.

Cartilage is a connective tissue presenting in several forms that are all essential components of the vertebrate skeleton. Complementing in vivo models, cultures of its resident cells-chondrocytes-are important experimental models in mechanistic and preclinical studies relevant to skeletal development and adult homeostasis and to such human pathologies as chondrodysplasias and osteoarthritis. Both growth plate and articular chondrocytes produce pancartilaginous extracellular matrix components, but the two cell subtypes also have distinct phenotypic properties that account for different structural features, functions, and fates of their tissues. Based on study goals, primary chondrocyte cultures should therefore be established from either growth plate or articular cartilage. Here, we describe the methods used in our laboratory to isolate and culture growth plate and articular chondrocytes from neonatal and adult mice, respectively. Both methods involve manual and enzymatic procedures to clean cartilage samples from contaminating tissues and to release chondrocytes as single-cell suspensions from their cartilage matrix.

Fetal Growth Plate Cartilage : Histological and Immunohistochemical Techniques.

Skeletal development is a tightly regulated process that primarily occurs through two distinct mechanisms. In intramembranous ossification, mesenchymal progenitors condense and transdifferentiate directly into osteoblasts, giving rise to the flat bones of the skull. The majority of the skeleton develops through endochondral ossification, in which mesenchymal progenitors give rise to a cartilaginous template that is gradually replaced by bone. The study of these processes necessitates a suitable animal model, a requirement to which the mouse is admirably suited. Their rapid reproductive ability, developmental and physiologic similarity to humans, and easily manipulated genetics all contribute to their widespread use. Outlined here are the most common histological and immunohistochemical techniques utilized in our laboratory for the isolation and analysis of specimens from the developing murine skeleton.

Laser Capture Microdissection of Mouse Growth Plate Cartilage.

The ability to identify, isolate, and study pure populations of cells is critical for understanding normal physiology in organs and tissues, which involves spatial regulation of signaling pathways and interactions between cells with different functions, expression profiles, and lineages. Here, we focus on assessing the growth plate cartilage, composed of multiple functionally and histologically distinct zones, to investigate temporally and spatially dependent gene expression differences. In this chapter, we describe the method of laser capture microdissection to isolate chondrocytes from different zones of differentiation in the mouse growth plate cartilage for RNA isolation, and subsequent downstream applications, such as RNA-sequencing and quantitative real-time PCR. We also provide an assessment of different factors contributing to the integrity of the isolated RNA, such as staining methods and procedures in RNA isolation.

Is C-type natriuretic peptide regulated by a feedback loop? A study on systemic and local autoregulatory effect.

C-type natriuretic peptide (CNP) is a pivotal enhancer of endochondral bone growth and is expected to be a therapeutic reagent for impaired skeletal growth. Although we showed that CNP stimulates bone growth as a local regulator in the growth plate via the autocrine/paracrine system, CNP is abundantly produced in other various tissues and its blood concentration is reported to correlate positively with growth velocity. Therefore we investigated the systemic regulation of CNP levels using rodent models. In order to examine whether CNP undergoes systemic feedback regulation, we investigated blood CNP levels and local CNP expression in various tissues, including cartilage, of 4-week-old rats after systemic administration of sufficient amounts of exogenous CNP (0.5 mg/kg/day) for 3 days. This CNP administration did not alter blood NT-proCNP levels in male rats but decreased mRNA expression only in tissue that included cartilage. Decrease in expression and blood NT-proCNP were greater in female rats. To analyze the existence of direct autoregulation of CNP in the periphery as an autocrine/paracrine system, we estimated the effect of exogenous supplementation of CNP on the expression of endogenous CNP itself in the growth plate cartilage of extracted fetal murine tibias and in ATDC5, a chondrogenic cell line. We found no alteration of endogenous CNP expression after incubation with adequate concentrations of exogenous CNP for 4 and 24 hours, which were chosen to observe primary and later transcriptional effects, respectively. These results indicate that CNP is not directly autoregulated but indirectly autoregulated in cartilage tissue. A feedback system is crucial for homeostatic regulation and further studies are needed to elucidate the regulatory system of CNP production and function.

Effect of total flavonoids of Rhizoma Drynariae in thiram induced cytotoxicity of chondrocyte via BMP-2/Runx2 and IHH/PTHrP expressions.

Tibial Dyschondroplasia (TD) is a prevailing skeletal disorder that mainly affects rapidly growing avian species. It results in reduced bone strength, lameness and an increase risk of fragility fractures. Total flavonoids of Rhizoma drynariae (TFRD) have been used as an effective treatment of different bone diseases in humans. The current in vitro study was conducted to explore the therapeutic effect of TFRD on thiram-induced cytotoxicity in avian growth plate cells via bone morphogenetic protein-2/runt related transcription factor-2 (BMP-2/Runx2) and Indian hedgehog/Parathyroid hormone-related peptide (IHH/PTHrP) expressions. Chondrocytes were isolated, cultured and refined from chicken's tibial growth plates in a special medium. Then chondrocytes were treated with sublethal thiram having less concentration (2.5 µg/mL) to induce cytotoxicity of chondrocyte, and then treated with providential doses (100 µg/mL) of TFRD. Thiram caused distorted morphology of chondrocytes, nuclei appeared disintegration or lysed along with decreased expressions of BMP-2/Runx2 and IHH/PTHrP. TFRD administration not only enhanced the viability of chondrocytes by itself, but also well restored the damage caused by thiram on growth plate chondrocytes by significantly up-regulating the expressions of BMP-2/Runx2 and IHH/PTHrP. Therefore, this study provides a novel insight into the further treatment of TD and other skeletal ailments and lays the foundation for prevention and treatment.

NFI-C Is Required for Epiphyseal Chondrocyte Proliferation during Postnatal Cartilage Development.

Stringent regulation of the chondrocyte cell cycle is required for endochondral bone formation. During the longitudinal growth of long bones, mesenchymal stem cells condense and differentiate into chondrocytes. Epiphyseal chondrocytes sequentially differentiate to form growth- plate cartilage, which is subsequently replaced with bone. Although the importance of nuclear factor 1C (Nfic) in hard tissue formation has been extensively studied, knowledge regarding its biological roles and molecular mechanisms in this process remains insufficient. Herein, we demonstrated that Nfic deficiency affects femoral growth-plate formation. Chondrocyte proliferation was downregulated and the number of apoptotic cell was increased in the growth plates of Nfic-/- mice. Further, the expression of the cell cycle inhibitor p21 was upregulated in the primary chondrocytes of Nfic-/- mice, whereas that of cyclin D1 was downregulated. Our findings suggest that Nfic may contribute to postnatal chondrocyte proliferation by inhibiting p21 expression and by increasing the stability of cyclin D1 protein.

Disorganization of chondrocyte columns in the growth plate does not aggravate experimental osteoarthritis in mice.

Osteoarthritis (OA) is a multifactorial joint disease mainly affecting articular cartilage (AC) with a relevant biomechanical component. During endochondral ossification growth plate (GP) chondrocytes arrange in columns. GPs do not ossify in skeletally mature rodents. In neonatal mice, an altered joint loading induces GP chondrocyte disorganization. We aimed to study whether experimental OA involves GP disorganization in adult mice and to assess if it may have additional detrimental effects on AC damage. Knee OA was induced by destabilization of the medial meniscus (DMM) in wild-type (WT) adult mice, and in Tamoxifen-inducible Ellis-van-Creveld syndrome protein (Evc) knockouts (EvccKO), used as a model of GP disorganization due to Hedgehog signalling disruption. Chondrocyte column arrangement was assessed in the tibial GP and expressed as Column Index (CI). Both DMM-operated WT mice and non-operated-EvccKO showed a decreased CI, indicating GP chondrocyte column disarrangement, although in the latter, it was not associated to AC damage. The most severe GP chondrocyte disorganization occurred in DMM-EvccKO mice, in comparison to the other groups. However, this altered GP structure in DMM-EvccKO mice did not exacerbate AC damage. Further studies are needed to confirm the lack of interference of GP alterations on the analysis of AC employing OA mice.

CANT1 deficiency in a mouse model of Desbuquois dysplasia impairs glycosaminoglycan synthesis and chondrocyte differentiation in growth plate cartilage.

Desbuquois dysplasia (DD) type 1 is a rare skeletal dysplasia characterized by a short stature, round face, progressive scoliosis, and joint laxity. The causative gene has been identified as calcium-activated nucleotidase 1 (CANT1), which encodes a nucleotidase that preferentially hydrolyzes UDP to UMP and phosphate. In this study, we generated Cant1 KO mice using CRISPR/Cas9-mediated genome editing. All F0 mice possessing frameshift deletions at both Cant1 alleles exhibited a dwarf phenotype. Germline transmission of the edited allele was confirmed in an F0 heterozygous mouse, and KO mice were generated by crossing of the heterozygous breeding pairs. Cant1 KO mice exhibited skeletal defects, including short stature, thoracic kyphosis, and delta phalanx, all of which are observed in DD type 1 patients. The glycosaminoglycan (GAG) content and extracellular matrix space were reduced in the growth plate cartilage of mutants, and proliferating chondrocytes lost their typical flat shape and became round. Chondrocyte differentiation, especially terminal differentiation to hypertrophic chondrocytes, was impaired in Cant1 KO mice. These findings indicate that CANT1 is involved in the synthesis of GAG and regulation of chondrocyte differentiation in the cartilage and contribute to a better understanding of the pathogenesis of DD type 1.

A simple method based on confocal microscopy and thick sections recognizes seven subphases in growth plate chondrocytes.

This manuscript reports a novel procedure to imaging growth plate chondrocytes by using confocal microscopy. The method is based on fixed undecalcified bone samples, in-block staining with eosin, epoxy resin embedding and grinding to obtain thick sections. It is simple, inexpensive and provides three-dimensional images of entire chondrocytes inside their native lacunae. Quantitative analysis of volume, shape and cytoplasm density of chondrocytes at different strata of the growth plate allowed to objectively grade chondrocytes of the growth plate in seven different clusters. These seven categories of chondrocytes were subsequently evaluated by immunohistochemistry of some well-defined molecular landmarks of chondrocyte differentiation. Furthermore, immunohistochemical analysis of proteins responsible for ionic changes and water transport allowing chondrocyte swelling during hypertrophy was also performed. Results obtained indicate that four subphases can be defined in the pre-hypertrophic zone and three subphases in the hypertrophic zone, a fact that raises that chondrocytes of the growth plate are less homogeneous than usually considered when different zones are defined according to subjective cell morphological criteria. Results in the present study provide a technological innovation and gives new insights into the complexity of the process of chondrocyte differentiation in the growth plate.

Home for a rest: stem cell niche of the postnatal growth plate.

The resting zone houses a group of slowly proliferating 'reserve' chondrocytes and has long been speculated to serve as the stem cell niche of the postnatal growth plate. But are these resting chondrocytes bona fide stem cells? Recent technological advances in lineage tracing and next-generation sequencing have finally allowed researchers to answer this question. Several recent studies have also shed light into the signaling pathways and molecular mechanisms involved in the maintenance of resting chondrocytes, thus providing us with important new insights into the role of the resting zone in the paracrine and endocrine regulation of childhood bone growth.

Skeletal Stem Cells for Bone Development and Repair: Diversity Matters.

PURPOSE OF REVIEW: Skeletal stem cells (SSCs) are considered to play important roles in bone development and repair. These cells have been historically defined by their in vitro potential for self-renewal and differentiation into "trilineage" cells; however, little is known about their in vivo identity. Here, we discuss recent progress on SSCs and how they potentially contribute to bone development and repair. RECENT FINDINGS: Bone is composed of diverse tissues, which include cartilage and its perichondrium, cortical bone and its periosteum, and bone marrow and its trabecular bone and stromal compartment. We are now at the initial stage of understanding the precise identity of SSCs in each bone tissue. The emerging concept is that functionally dedicated SSCs are encased by their own unique cellular and extracellular matrix microenvironment, and locally support its own compartment. Diverse groups of SSCs are likely to work in concert to achieve development and repair of the highly functional skeletal organ.

Skeletal stem cells: insights into maintaining and regenerating the skeleton.

Skeletal stem cells (SSCs) generate the progenitors needed for growth, maintenance and repair of the skeleton. Historically, SSCs have been defined as bone marrow-derived cells with inconsistent characteristics. However, recent in vivo tracking experiments have revealed the presence of SSCs not only within the bone marrow but also within the periosteum and growth plate reserve zone. These studies show that SSCs are highly heterogeneous with regard to lineage potential. It has also been revealed that, during digit tip regeneration and in some non-mammalian vertebrates, the dedifferentiation of osteoblasts may contribute to skeletal regeneration. Here, we examine how these research findings have furthered our understanding of the diversity and plasticity of SSCs that mediate skeletal maintenance and repair.

Endochondral ossification and the evolution of limb proportions.

Mammals have remarkably diverse limb proportions hypothesized to have evolved adaptively in the context of locomotion and other behaviors. Mechanistically, evolutionary diversity in limb proportions is the result of differential limb bone growth. Longitudinal limb bone growth is driven by the process of endochondral ossification, under the control of the growth plates. In growth plates, chondrocytes undergo a tightly orchestrated life cycle of proliferation, matrix production, hypertrophy, and cell death/transdifferentiation. This life cycle is highly conserved, both among the long bones of an individual, and among homologous bones of distantly related taxa, leading to a finite number of complementary cell mechanisms that can generate heritable phenotype variation in limb bone size and shape. The most important of these mechanisms are chondrocyte population size in chondrogenesis and in individual growth plates, proliferation rates, and hypertrophic chondrocyte size. Comparative evidence in mammals and birds suggests the existence of developmental biases that favor evolutionary changes in some of these cellular mechanisms over others in driving limb allometry. Specifically, chondrocyte population size may evolve more readily in response to selection than hypertrophic chondrocyte size, and extreme hypertrophy may be a rarer evolutionary phenomenon associated with highly specialized modes of locomotion in mammals (e.g., powered flight, ricochetal bipedal hopping). Physical and physiological constraints at multiple levels of biological organization may also have influenced the cell developmental mechanisms that have evolved to produce the highly diverse limb proportions in extant mammals. This article is categorized under: Establishment of Spatial and Temporal Patterns > Regulation of Size, Proportion, and Timing Comparative Development and Evolution > Regulation of Organ Diversity Comparative Development and Evolution > Organ System Comparisons Between Species.