A radical switch in clonality reveals a stem cell niche in the epiphyseal growth plate.

Longitudinal bone growth in children is sustained by growth plates, narrow discs of cartilage that provide a continuous supply of chondrocytes for endochondral ossification1. However, it remains unknown how this supply is maintained throughout childhood growth. Chondroprogenitors in the resting zone are thought to be gradually consumed as they supply cells for longitudinal growth1,2, but this model has never been proved. Here, using clonal genetic tracing with multicolour reporters and functional perturbations, we demonstrate that longitudinal growth during the fetal and neonatal periods involves depletion of chondroprogenitors, whereas later in life, coinciding with the formation of the secondary ossification centre, chondroprogenitors acquire the capacity for self-renewal, resulting in the formation of large, stable monoclonal columns of chondrocytes. Simultaneously, chondroprogenitors begin to express stem cell markers and undergo symmetric cell division. Regulation of the pool of self-renewing progenitors involves the hedgehog and mammalian target of rapamycin complex 1 (mTORC1) signalling pathways. Our findings indicate that a stem cell niche develops postnatally in the epiphyseal growth plate, which provides a continuous supply of chondrocytes over a prolonged period.

The Origin and Fate of Chondrocytes: Cell Plasticity in Physiological Setting.

PURPOSE OF REVIEW: Here, we discuss the origin of chondrocytes, their destiny, and their plasticity in relationship to bone growth, articulation, and formation of the trabeculae. We also consider these processes from a biological, clinical, and evolutionary perspective. RECENT FINDINGS: Chondrocytes, which provide the template for the formation of most bones, are responsible for skeletal growth and articulation during postnatal life. In recent years our understanding of the fate of these cells has changed dramatically. Current evidence indicates a paradoxical situation during skeletogenesis, with some cells of mesenchymal condensation differentiating directly into osteoblasts, whereas others of the same kind give rise to highly similar osteoblasts via a complex process of differentiation involving several chondrocyte intermediates. The situation becomes even more paradoxical during postnatal growth when stem cells in the growth plate produce differentiated, functional progenies, which thereafter presumably dedifferentiate into another type of stem cell. Such a remarkable transition from one cell type to another under postnatal physiological conditions provides a fascinating example of cellular plasticity that may have valuable clinical implications.

Schwann cell precursors contribute to skeletal formation during embryonic development in mice and zebrafish.

Immature multipotent embryonic peripheral glial cells, the Schwann cell precursors (SCPs), differentiate into melanocytes, parasympathetic neurons, chromaffin cells, and dental mesenchymal populations. Here, genetic lineage tracing revealed that, during murine embryonic development, some SCPs detach from nerve fibers to become mesenchymal cells, which differentiate further into chondrocytes and mature osteocytes. This occurred only during embryonic development, producing numerous craniofacial and trunk skeletal elements, without contributing to development of the appendicular skeleton. Formation of chondrocytes from SCPs also occurred in zebrafish, indicating evolutionary conservation. Our findings reveal multipotency of SCPs, providing a developmental link between the nervous system and skeleton.

Implantation of Various Cell-Free Matrixes Does Not Contribute to the Restoration of Hyaline Cartilage within Full-Thickness Focal Defects.

Articular cartilage is a highly organized tissue that has a limited ability to heal. Tissue engineering is actively exploited for joint tissue reconstruction in numerous cases of articular cartilage degeneration associated with trauma, arthrosis, rheumatoid arthritis, and osteoarthritis. However, the optimal scaffolds for cartilage repair are not yet identified. Here we have directly compared five various scaffolds, namely collagen-I membrane, collagen-II membrane, decellularized cartilage, a cellulose-based implant, and commercially available Chondro-Gide® (Geistlich Pharma AG, Wolhusen, Switzerland) collagen membrane. The scaffolds were implanted in osteochondral full-thickness defects, formed on adult Wistar rats using a hand-held cutter with a diameter of 2.0 mm and a depth of up to the subchondral bone. The congruence of the articular surface was almost fully restored by decellularized cartilage and collagen type II-based scaffold. The most vivid restoration was observed 4 months after the implantation. The formation of hyaline cartilage was not detected in any of the groups. Despite cellular infiltration into scaffolds being observed in each group except cellulose, neither chondrocytes nor chondro-progenitors were detected. We concluded that for restoration of hyaline cartilage, scaffolds have to be combined either with cellular therapy or morphogens promoting chondrogenic differentiation.

Cruciate ligament, patellar tendon, and patella formation involves differential cellular sources and dynamics as joint cavitation proceeds.

BACKGROUND: Cruciate ligament (CL) and patellar tendon (PT) are important elements of the knee joint, uniting femur, patella, and tibia into a single functional unit. So far, knowledge on the developmental mechanism of CL, PT, and patella falls far behind other skeletal tissues. RESULTS: Here, employing various lineage tracing strategies we investigate the cellular sources and dynamics that drive CL, PT, and patella formation during mouse embryonic development. We show that Gdf5 and Gli1 are generally expressed in the same cell population that only contributes to CL, but not PT or patella development. In addition, Col2 is expressed in two independent cell populations before and after joint cavitation, where the former contributes to the CL and the dorsal part of the PT and the latter contributes to the patella. Moreover, Prrx1 is always expressed in CL and PT progenitors, but not patella progenitors where it is switched off after joint cavitation. Finally, we reveal that patella development employs different cellular dynamics before and after joint cavitation. CONCLUSIONS: Our findings delineate the expression changes of several skeletogenesis-related genes before and after joint cavitation, and provide an indication on the cellular dynamics underlying ligament, tendon, and sesamoid bone formation during embryogenesis.

Postnatal skeletal growth is driven by the epiphyseal stem cell niche: potential implications to pediatrics.

Children's longitudinal growth is facilitated by the activity of the growth plates, cartilage discs located near the ends of the long-bones. In order to elongate these bones, growth plates must continuously generate chondrocytes. Two recent studies have demonstrated that there are stem cells and a stem cell niche in the growth plate, which govern the generation of chondrocytes during the postnatal growth period. The niche, which allows stem cells to renew, appears at the same time as the secondary ossification center (SOC) matures into a bone epiphysis. Thus, the mechanism of chondrocyte generation differs substantially between neonatal and postnatal age, i.e., before and after the formation of the mineralized epiphyses. Hence, at the neonatal age bone growth is based on a consumption of chondro-progenitors whereas postnatally it is based on the activity of the stem cell niche. Here we discuss potential implications of these observations in relation to longitudinal growth, including the effects of estrogens, nutrition and growth hormone.

Superficial cells are self-renewing chondrocyte progenitors, which form the articular cartilage in juvenile mice.

Articular cartilage has little regenerative capacity. Recently, genetic lineage tracing experiments have revealed chondrocyte progenitors at the articular surface. We further characterized these progenitors by using in vivo genetic approaches. Histone H2B-green fluorescent protein retention revealed that superficial cells divide more slowly than underlying articular chondrocytes. Clonal genetic tracing combined with immunohistochemistry revealed that superficial cells renew their number by symmetric division, express mesenchymal stem cell markers, and generate chondrocytes via both asymmetric and symmetric differentiation. Quantitative analysis of cellular kinetics, in combination with phosphotungstic acid-enhanced micro-computed tomography, showed that superficial cells generate chondrocytes and contribute to the growth and reshaping of articular cartilage. Furthermore, we found that cartilage renewal occurs as the progeny of superficial cells fully replace fetal chondrocytes during early postnatal life. Thus, superficial cells are self-renewing progenitors that are capable of maintaining their own population and fulfilling criteria of unipotent adult stem cells. Furthermore, the progeny of these cells reconstitute adult articular cartilage de novo, entirely substituting fetal chondrocytes.-Li, L., Newton, P. T., Bouderlique, T., Sejnohova, M., Zikmund, T., Kozhemyakina, E., Xie, M., Krivanek, J., Kaiser, J., Qian, H., Dyachuk, V., Lassar, A. B., Warman, M. L., Barenius, B., Adameyko, I., Chagin, A. S. Superficial cells are self-renewing chondrocyte progenitors, which form the articular cartilage in juvenile mice.

Repair of Damaged Articular Cartilage: Current Approaches and Future Directions.

Articular hyaline cartilage is extensively hydrated, but it is neither innervated nor vascularized, and its low cell density allows only extremely limited self-renewal. Most clinical and research efforts currently focus on the restoration of cartilage damaged in connection with osteoarthritis or trauma. Here, we discuss current clinical approaches for repairing cartilage, as well as research approaches which are currently developing, and those under translation into clinical practice. We also describe potential future directions in this area, including tissue engineering based on scaffolding and/or stem cells as well as a combination of gene and cell therapy. Particular focus is placed on cell-based approaches and the potential of recently characterized chondro-progenitors; progress with induced pluripotent stem cells is also discussed. In this context, we also consider the ability of different types of stem cell to restore hyaline cartilage and the importance of mimicking the environment in vivo during cell expansion and differentiation into mature chondrocytes.

Targeted deletion of Atg5 in chondrocytes promotes age-related osteoarthritis.

OBJECTIVES: It has been suggested that the lysosomal recycling process called macro-autophagy plays a role in osteoarthritis development. We thus decided to genetically ablate the autophagy-indispensable Atg5 gene specifically in chondrocytes and analyse the development of osteoarthritis upon aging and in a post-traumatic model. METHODS: Mice lacking the Atg5 gene in their chondrocytes (Atg5cKO) were generated by crossing Atg5-floxed mice with transgenic mice that expressed cre recombinase driven by the collagen type 2 promoter. Animals were analysed at the age of 2, 6 and 12 months for age-related osteoarthritis or underwent mini-open partial medial meniscectomy at 2 months of age and were analysed 1 or 2 months after surgery. We evaluated osteoarthritis using the Osteoarthritis Research Society International (OARSI) scoring on safranin-O-stained samples. Cell death was evaluated by terminal deoxy-nucleotidyl-transferase-mediated deoxy-UTP nick end labelling (TUNEL) and by immunostaining of cleaved caspases. RESULTS: We observed the development of osteoarthritis in Atg5cKO mice with aging including fibrillation and loss of proteoglycans, which was particularly severe in males. The ablation of Atg5 was associated with an increased cell death as assessed by TUNEL, cleaved caspase 3 and cleaved caspase 9. Surprisingly, no difference in the development of post-traumatic osteoarthritis was observed between Atg5cKO and control mice. CONCLUSIONS: Autophagy protects from age-related osteoarthritis by facilitating chondrocyte survival.

Mice depleted of the coxsackievirus and adenovirus receptor display normal spermatogenesis and an intact blood-testis barrier.

The coxsackievirus and adenovirus receptor (CXADR (CAR)) is a cell adhesion molecule expressed mainly in epithelial cells. Numerous evidence indicate that CXADR has an important role in testis development and function of the blood-testis barrier (BTB) in vitro. The role of CXADR in testis physiology in vivo has, however, not been addressed. We therefore constructed a conditional CXADR knockout (cKO) mouse model in which CXADR can be depleted at any chosen timepoint by the administration of tamoxifen. We report for the first time that testicular depletion of CXADR in adult and pubertal mice does not alter BTB permeability or germ cell migration across the BTB during spermatogenesis. Adult cKO mice display normal junctional ultra-structure and localization of the junctional proteins claudin-3, occludin, junction-associated molecule-A (JAM-A), and ZO1. The BTB was intact with no leakage of biotin and lanthanum tracers into the tubular lumen. Adult CXADR cKO mice were fertile with normal sperm parameters and litter size. Breeding experiments and genotyping of the pups demonstrated that CXADR-negative sperm could fertilize WT eggs. In addition, knocking down CXADR from postnatal day 9 (P9) does not affect testicular development and BTB formation. These cKO mice were analyzed at P49 and P90 and display an intact barrier and uncompromised fertility. We conclude that CXADR possesses no direct role in testicular physiology in vivo.

Parathyroid hormone/parathyroid hormone-related protein receptor signaling is required for maintenance of the growth plate in postnatal life.

Parathyroid hormone (PTH)-related protein (PTHrP), regulated by Indian hedgehog and acting through the PTH/PTHrP receptor (PPR), is crucial for normal cartilage development. These observations suggest a possible role of PPR signaling in the postnatal growth plate; however, the role of PPR signaling in postnatal chondrocytes is unknown. In this study, we have generated tamoxifen-inducible and cartilage-specific PPR KO mice to evaluate the physiological role of PPR signaling in postnatal chondrocytes. We found that inactivation of the PPR in chondrocytes postnatally leads to accelerated differentiation of chondrocytes, followed by disappearance of the growth plate. We also observed an increase of TUNEL-positive cells and activities of caspase-3 and caspase-9 in the growth plate, along with a decrease in phosphorylation of Bad at Ser155 in postnatal PPR KO mice. Administration of a low-phosphate diet, which prevents apoptosis of chondrocytes, prevented the disappearance of the growth plate. Taken together, these observations suggest that the major consequences of PPR activation are similar in both the fetal and postnatal growth plates. Moreover, chondrocyte apoptosis through the activation of a mitochondrial pathway may be involved in the process of premature disappearance of the growth plate by postnatal inactivation of the PPR in chondrocytes.

Skeletal stem and progenitor cells in bone development and repair.

Bone development, growth, and repair are complex processes involving various cell types and interactions, with central roles played by skeletal stem and progenitor cells. Recent research brought new insights into the skeletal precursor populations that mediate intramembranous and endochondral bone development. Later in life, many of the cellular and molecular mechanisms determining development are reactivated upon fracture, with powerful trauma-induced signaling cues triggering a variety of postnatal skeletal stem/progenitor cells (SSPCs) residing near the bone defect. Interestingly, in this injury context, the current evidence suggests that the fates of both SSPCs and differentiated skeletal cells can be considerably flexible and dynamic, and that multiple cell sources can be activated to operate as functional progenitors generating chondrocytes and/or osteoblasts. The combined implementation of in vivo lineage tracing, cell surface marker-based selection, single-cell molecular analyses, and high-resolution in situ imaging has strongly improved our insights into the diversity and roles of developmental and reparative stem/progenitor subsets, while also unveiling the complexity of their dynamics, hierarchies, and relationships. Albeit incompletely understood at present, findings supporting lineage flexibility and possibly plasticity among sources of osteogenic cells challenge the classical dogma of a single primitive, self-renewing, multipotent stem cell driving bone tissue formation and regeneration from the apex of a hierarchical and strictly unidirectional differentiation tree. We here review the state of the field and the newest discoveries in the origin, identity, and fates of skeletal progenitor cells during bone development and growth, discuss the contributions of adult SSPC populations to fracture repair, and reflect on the dynamism and relationships among skeletal precursors and differentiated cell lineages. Further research directed at unraveling the heterogeneity and capacities of SSPCs, as well as the regulatory cues determining their fate and functioning, will offer vital new options for clinical translation toward compromised fracture healing and bone regenerative medicine.

Skeletal progenitor cells are crucial for bone development and growth, as they provide the cellular building blocks (chondrocytes and osteoblasts) that form the cartilage and bone tissues that the skeleton is composed of. In adult life, the occurrence of a bone fracture reactivates similar tissue-forming mechanisms, starting with the trauma triggering various postnatal skeletal stem/progenitor cells (SSPCs) residing near the bone defect to divide and migrate. These cells subsequently generate functional fracture-repairing cells by differentiating into mature chondrocytes and/or osteoblasts. In recent years, the combined use of various advanced research approaches and new techniques has strongly improved our insights into the origin, identity, fates, and roles of developmental and reparative skeletal stem cells and progenitor subsets. Concomitantly, this research also unveiled considerable complexity in their dynamics, diversity, hierarchies, and relationships, which is incompletely understood at present. In this review, we discuss the state of the field and the newest discoveries in the identity and roles of skeletal stem and progenitor cells mediating bone development, growth, and repair. Further research on these cell populations, including determining their exact nature, fate, and functioning, and how they can be harvested and regulated, is critical to develop new treatments for non-healing fractures.

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

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

The level of protein in the maternal murine diet modulates the facial appearance of the offspring via mTORC1 signaling.

The development of craniofacial skeletal structures is fascinatingly complex and elucidation of the underlying mechanisms will not only provide novel scientific insights, but also help develop more effective clinical approaches to the treatment and/or prevention of the numerous congenital craniofacial malformations. To this end, we performed a genome-wide analysis of RNA transcription from non-coding regulatory elements by CAGE-sequencing of the facial mesenchyme of human embryos and cross-checked the active enhancers thus identified against genes, identified by GWAS for the normal range human facial appearance. Among the identified active cis-enhancers, several belonged to the components of the PI3/AKT/mTORC1/autophagy pathway. To assess the functional role of this pathway, we manipulated it both genetically and pharmacologically in mice and zebrafish. These experiments revealed that mTORC1 signaling modulates craniofacial shaping at the stage of skeletal mesenchymal condensations, with subsequent fine-tuning during clonal intercalation. This ability of mTORC1 pathway to modulate facial shaping, along with its evolutionary conservation and ability to sense external stimuli, in particular dietary amino acids, indicate that the mTORC1 pathway may play a role in facial phenotypic plasticity. Indeed, the level of protein in the diet of pregnant female mice influenced the activity of mTORC1 in fetal craniofacial structures and altered the size of skeletogenic clones, thus exerting an impact on the local geometry and craniofacial shaping. Overall, our findings indicate that the mTORC1 signaling pathway is involved in the effect of environmental conditions on the shaping of craniofacial structures.

Al-Gazali Skeletal Dysplasia Constitutes the Lethal End of ADAMTSL2-Related Disorders.

Lethal short-limb skeletal dysplasia Al-Gazali type (OMIM %601356), also called dysplastic cortical hyperostosis, Al-Gazali type, is an ultra-rare disorder previously reported in only three unrelated individuals. The genetic etiology for Al-Gazali skeletal dysplasia has up until now been unknown. Through international collaborative efforts involving seven clinical centers worldwide, a cohort of nine patients with clinical and radiographic features consistent with short-limb skeletal dysplasia Al-Gazali type was collected. The affected individuals presented with moderate intrauterine growth restriction, relative macrocephaly, hypertrichosis, large anterior fontanelle, short neck, short and stiff limbs with small hands and feet, severe brachydactyly, and generalized bone sclerosis with mild platyspondyly. Biallelic disease-causing variants in ADAMTSL2 were detected using massively parallel sequencing (MPS) and Sanger sequencing techniques. Six individuals were compound heterozygous and one individual was homozygous for pathogenic variants in ADAMTSL2. In one of the families, pathogenic variants were detected in parental samples only. Overall, this study sheds light on the genetic cause of Al-Gazali skeletal dysplasia and identifies it as a semi-lethal part of the spectrum of ADAMTSL2-related disorders. Furthermore, we highlight the importance of meticulous analysis of the pseudogene region of ADAMTSL2 where disease-causing variants might be located. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Altered developmental programs and oriented cell divisions lead to bulky bones during salamander limb regeneration.

There are major differences in duration and scale at which limb development and regeneration proceed, raising the question to what extent regeneration is a recapitulation of development. We address this by analyzing skeletal elements using a combination of micro-CT imaging, molecular profiling and clonal cell tracing. We find that, in contrast to development, regenerative skeletal growth is accomplished based entirely on cartilage expansion prior to ossification, not limiting the transversal cartilage expansion and resulting in bulkier skeletal parts. The oriented extension of salamander cartilage and bone appear similar to the development of basicranial synchondroses in mammals, as we found no evidence for cartilage stem cell niches or growth plate-like structures during neither development nor regeneration. Both regenerative and developmental ossification in salamanders start from the cortical bone and proceeds inwards, showing the diversity of schemes for the synchrony of cortical and endochondral ossification among vertebrates.

The epiphyseal secondary ossification center: Evolution, development and function.

Bone age is used widely by pediatricians to assess the skeletal maturity of a child and predict growth potential. This entails measuring the size of secondary ossification centers (SOCs), which develop with age in the ends of long bones, which are initially cartilaginous. However, little is presently known about the developmental biology, evolution and functional role of these skeletal elements. Here, we summarize the knowledge currently available in this area and discuss potential primary functions of the SOC.

Niches for Skeletal Stem Cells of Mesenchymal Origin.

With very few exceptions, all adult tissues in mammals are maintained and can be renewed by stem cells that self-renew and generate the committed progeny required. These functions are regulated by a specific and in many ways unique microenvironment in stem cell niches. In most cases disruption of an adult stem cell niche leads to depletion of stem cells, followed by impairment of the ability of the tissue in question to maintain its functions. The presence of stem cells, often referred to as mesenchymal stem cells (MSCs) or multipotent bone marrow stromal cells (BMSCs), in the adult skeleton has long been realized. In recent years there has been exceptional progress in identifying and characterizing BMSCs in terms of their capacity to generate specific types of skeletal cells in vivo. Such BMSCs are often referred to as skeletal stem cells (SSCs) or skeletal stem and progenitor cells (SSPCs), with the latter term being used throughout this review. SSPCs have been detected in the bone marrow, periosteum, and growth plate and characterized in vivo on the basis of various genetic markers (i.e., Nestin, Leptin receptor, Gremlin1, Cathepsin-K, etc.). However, the niches in which these cells reside have received less attention. Here, we summarize the current scientific literature on stem cell niches for the SSPCs identified so far and discuss potential factors and environmental cues of importance in these niches in vivo. In this context we focus on (i) articular cartilage, (ii) growth plate cartilage, (iii) periosteum, (iv) the adult endosteal compartment, and (v) the developing endosteal compartment, in that order.