Dysregulated BMP signaling through ACVR1 impairs digit joint development in fibrodysplasia ossificans progressiva (FOP).

The development of joints in the mammalian skeleton depends on the precise regulation of multiple interacting signaling pathways including the bone morphogenetic protein (BMP) pathway, a key regulator of joint development, digit patterning, skeletal growth, and chondrogenesis. Mutations in the BMP receptor ACVR1 cause the rare genetic disease fibrodysplasia ossificans progressiva (FOP) in which extensive and progressive extra-skeletal bone forms in soft connective tissues after birth. These mutations, which enhance BMP-pSmad1/5 pathway activity to induce ectopic bone, also affect skeletal development. FOP can be diagnosed at birth by symmetric, characteristic malformations of the great toes (first digits) that are associated with decreased joint mobility, shortened digit length, and absent, fused, and/or malformed phalanges. To elucidate the role of ACVR1-mediated BMP signaling in digit skeletal development, we used an Acvr1R206H/+;Prrx1-Cre knock-in mouse model that mimics the first digit phenotype of human FOP. We have determined that the effects of increased Acvr1-mediated signaling by the Acvr1R206H mutation are not limited to the first digit but alter BMP signaling, Gdf5+ joint progenitor cell localization, and joint development in a manner that differently affects individual digits during embryogenesis. The Acvr1R206H mutation leads to delayed and disrupted joint specification and cleavage in the digits and alters the development of cartilage and endochondral ossification at sites of joint morphogenesis. These findings demonstrate an important role for ACVR1-mediated BMP signaling in the regulation of joint and skeletal formation, show a direct link between failure to restrict BMP signaling in the digit joint interzone and failure of joint cleavage at the presumptive interzone, and implicate impaired, digit-specific joint development as the proximal cause of digit malformation in FOP.

GR/Sp3/HDAC1/UGDH signaling participated in the maternal dexamethasone-induced dysplasia of the rat fetal growth plate.

Maternal dexamethasone decreases the body length of the newborn. However, whether dexamethasone inhibits the development of the growth plate of the fetal long bone is still unknown. Here, we found that lengths of fetal femur and growth plate were both shorter in the fetuses with maternal dexamethasone (0.2 mg/kg.d from gestation day 9 to 20), with a decreased proteoglycan content of the growth plate in the fetal rat. Notable decreases in both the gene expression and H3K9 acetylation of UDP-glucose dehydrogenase (Ugdh) gene, which codes a key enzyme in the proteoglycan biosynthesis in the chondrocyte, were also observed. Meanwhile, up-regulation of glucocorticoid receptor (GR), specific protein 3 (Sp3), and histone deacetylase 1 (Hdac1) gene expression were detected in the fetal growth plate. Similar changes were also observed in the chondrogenic rat bone marrow stromal cells (BMSCs) with excessive exogenous dexamethasone. However, antagonizing GR with RU486 and silencing Hdac1 or Sp3 with specific siRNAs could all stimulate the H3K9 acetylation and gene expression of Ugdh previously inhibited by dexamethasone. Meanwhile, dexamethasone also induced the nuclear translocation of GR, which further directly bound to the Ugdh promoter and interacted with HDAC1 and Sp3, respectively. Collectively, our study revealed that maternal dexamethasone induced the direct binding of GR to the Ugdh promoter of the chondrocyte in the rat fetal growth plate, which recruited HDAC1 and Sp3, induced deacetylation of the H3K9, and subsequently inhibited Ugdh gene expression. Such changes further led to attenuated proteoglycan synthesis in the developing chondrocyte and therefore disrupted the development of growth plate and fetal long bone.

The extended chondrocyte lineage: implications for skeletal homeostasis and disorders.

Endochondral bone formation relies on a finely controlled sequence of chondrocyte proliferation, maturation and hypertrophy that establishes the growth plate which, combined with the deposition of bone upon the cartilage template, mediates longitudinal skeletal growth. Recent lineage studies support a chondrocyte-osteoblast differentiation continuum and the presence of skeletal stem cells within cartilage. The biological significance of the lineage extension and the mechanisms controlling the process are unclear. In this review, we describe recent work on the extended chondrocyte-osteoblast lineage and its contribution to the development, growth and repair of bone and to bone disorders that provides insight into the process and the molecular controls involved. The implications for skeletal homeostasis are discussed.

Stem and progenitor cells in skeletal development.

Accumulating evidence supports the idea that stem and progenitor cells play important roles in skeletal development. Over the last decade, the definition of skeletal stem and progenitor cells has evolved from cells simply defined by their in vitro behaviors to cells fully defined by a combination of sophisticated approaches, including serial transplantation assays and in vivo lineage-tracing experiments. These approaches have led to better identification of the characteristics of skeletal stem cells residing in multiple sites, including the perichondrium of the fetal bone, the resting zone of the postnatal growth plate, the bone marrow space and the periosteum in adulthood. These diverse groups of skeletal stem cells appear to closely collaborate and achieve a number of important biological functions of bones, including not only bone development and growth, but also bone maintenance and repair. Although these are important findings, we are only beginning to understand the diversity and the nature of skeletal stem and progenitor cells, and how they actually behave in vivo.

Suppressing UPR-dependent overactivation of FGFR3 signaling ameliorates SLC26A2-deficient chondrodysplasias.

BACKGROUND: Mutations in the SLC26A2 gene cause a spectrum of currently incurable human chondrodysplasias. However, genotype-phenotype relationships of SLC26A2-deficient chondrodysplasias are still perplexing and thus stunt therapeutic development. METHODS: To investigate the causative role of SLC26A2 deficiency in chondrodysplasias and confirm its skeleton-specific pathology, we generated and analyzed slc26a2-/- and Col2a1-Cre; slc26a2fl/fl mice. The therapeutic effect of NVP-BGJ398, an FGFR inhibitor, was tested with both explant cultures and timed pregnant females. FINDINGS: Two lethal forms of human SLC26A2-related chondrodysplasias, achondrogenesis type IB (ACG1B) and atelosteogenesis type II (AO2), are phenocopied by slc26a2-/- mice. Unexpectedly, slc26a2-/- chondrocytes are defective for collagen secretion, exhibiting intracellular retention and compromised extracellular deposition of ColII and ColIX. As a consequence, the ATF6 arm of the unfolded protein response (UPR) is preferentially triggered to overactivate FGFR3 signaling by inducing excessive FGFR3 in slc26a2-/- chondrocytes. Consistently, suppressing FGFR3 signaling by blocking either FGFR3 or phosphorylation of the downstream effector favors the recovery of slc26a2-/- cartilage cultures from impaired growth and unbalanced cell proliferation and apoptosis. Moreover, administration of an FGFR inhibitor to pregnant females shows therapeutic effects on pathological features in slc26a2-/- newborns. Finally, we confirm the skeleton-specific lethality and pathology of global SLC26A2 deletion through analyzing the Col2a1-Cre; slc26a2fl/fl mouse line. INTERPRETATION: Our study unveils a previously unrecognized pathogenic mechanism underlying ACG1B and AO2, and supports suppression of FGFR3 signaling as a promising therapeutic approach for SLC26A2-related chondrodysplasias. FUND: This work was supported by National Natural Science Foundation of China (81871743, 81730065 and 81772377).

High-Phosphate Diet Improved the Skeletal Development of Fam20c-Deficient Mice.

FAM20C (family with sequence similarity 20 - member C) is a protein kinase that phosphorylates secretory proteins, including the proteins that are essential to the formation and mineralization of calcified tissues. Previously, we reported that inactivation of Fam20c in mice led to hypophosphatemic rickets/osteomalacia along with increased circulating fibroblast growth factor 23 (FGF23) levels and dental defects. In this study, we examined whether a high-phosphate (hPi) diet could rescue the skeletal defects in Fam20c-deficient mice. Fam20c conditional knockout (cKO) mice were generated by crossing female Fam20c-floxed mice (Fam20cfl/fl) with male Sox2-Cre;Fam20cfl/+ mice. The pregnant female Fam20cfi/fl mice were fed either a normal or hPi diet until the litters were weaned. The cKO and control offspring were continuously given a normal or hPi diet for 4 weeks after weaning. Plain X-ray radiography, micro-CT, histology, immunohistochemistry (FGF23, DMP1, OPN, and SOX9), and in situ hybridization (type II and type X collagen) analyses were performed to evaluate the effects of an hPi diet on the mouse skeleton. Plain X-ray radiography and micro-CT radiography analyses showed that the hPi diet improved the shape and mineral density of the Fam20c-deficient femurs/tibiae, and rescued the growth plate defects in the long bone. Histology analyses further demonstrated that an hPi diet nearly completely rescued the growth plate-widening defects in the long bone and restored the expanded hypertrophic zone to nearly normal width. These results suggested that the hPi diet significantly improved the skeletal development of the Fam20c-deficient mice, implying that hypophosphatemia partially contributed to the skeletal defects in Fam20c-deficient subjects.

Impact of prenatal hypoxia on fetal bone growth and osteoporosis in ovariectomized offspring rats.

Prenatal hypoxia causes intrauterine growth retardation. It is unclear whether/how hypoxia affects the bone in fetal and offspring life. This study showed that prenatal hypoxia retarded fetal skeletal growth in rats, inhibited extracellular matrix (ECM) synthesis and down-regulated of insulin-like growth factor 1 (IGF1) signaling in fetal growth plate chondrocytes in vivo and in vitro. In addition, ovariectomized (OVX) was used for study of postmenopausal osteoporosis. Compared with the control, OVX offspring in prenatal hypoxic group showed an enhanced osteoporosis in the femurs, associated with reduced proteoglycan and IGF1 signaling. The results indicated prenatal hypoxia not only delayed fetal skeletal growth, but also increased OVX-induced osteoporosis in the elder offspring probably through down-regulated IGF1 signaling and inhibition of ECM synthesis, providing important information of prenatal hypoxia on functional and molecular bone growth and metabolism in fetal and offspring.

Limb derived cells as a paradigm for engineering self-assembling skeletal tissues.

Mimicking developmental events has been proposed as a strategy to engineer tissue constructs for regenerative medicine. However, this approach has not yet been investigated for skeletal tissues. Here, it is demonstrated that ectopic implantation of day-14.5 mouse embryonic long bone anlagen, dissociated into single cells and randomly incorporated in a bioengineered construct, gives rise to epiphyseal growth plate-like structures, bone and marrow, which share many morphological and molecular similarities to epiphyseal units that form after transplanting intact long bone anlage, demonstrating substantial robustness and autonomy of complex tissue self-assembly and the overall organogenesis process. In vitro studies confirm the self-aggregation and patterning capacity of anlage cells and demonstrate that the model can be used to evaluate the effects of large and small molecules on biological behaviour. These results reveal the preservation of self-organizing and self-patterning capacity of anlage cells even when disconnected from their developmental niche and subjected to system perturbations such as cellular dissociation. These inherent features make long bone anlage cells attractive as a model system for tissue engineering technologies aimed at creating constructs that have the potential to self-assemble and self-pattern complex architectural structures.

Imaging of Pediatric Growth Plate Disturbances.

The growth plates, or physes, are visible on virtually all images obtained in skeletally immature children. The proper function of these growth plates depends on an intricate balance between chondrocyte proliferation, which requires nourishment from the epiphyseal vessels, and chondrocyte death, which requires the integrity of the metaphyseal vessels. Therefore, injury to the growth plate (ie, direct insult) or vascular compromise on either side of the growth plate (ie, indirect insult) can cause growth plate dysfunction. Direct growth plate insults occur most commonly with Salter-Harris fractures, and injuries that allow the transphyseal communication of vessels are at a higher risk for subsequent transphyseal bone bridge formation. Indirect insults lead to different sequelae that are based on whether the epiphyseal blood supply or metaphyseal blood supply is compromised. Epiphyseal osteonecrosis can result in slowed longitudinal bone growth, with possible growth plate closure, and is often accompanied by an abnormal secondary ossification center. In contrast, the disruption of metaphyseal blood supply alters endochondral ossification and allows the persistence of chondrocytes within the metaphysis, which appear as focal or diffuse growth plate widening. Imaging remains critical for detecting acute injuries and identifying subsequent growth disturbances. Depending on the imaging findings and patient factors, these growth disturbances may be amenable to conservative or surgical treatment. Therefore, an understanding of the anatomy and physiologic features of the normal growth plate and the associated pathophysiologic conditions can increase diagnostic accuracy, enable radiologists to anticipate future growth disturbances, and ensure optimal imaging, with the ultimate goal of timely and appropriate intervention. ©RSNA, 2017.

Cartilage-Specific Autophagy Deficiency Promotes ER Stress and Impairs Chondrogenesis in PERK-ATF4-CHOP-Dependent Manner.

Autophagy is activated during nutritionally depleted or hypoxic conditions to facilitate cell survival. Because growth plate is an avascular and hypoxic tissue, autophagy may have a crucial role during chondrogenesis; however, the functional role and underlying mechanism of autophagy in regulation of growth plate remains elusive. In this study, we generated TamCart Atg7-/- (Atg7cKO) mice to explore the role of autophagy during endochondral ossification. Atg7cKO mice exhibited growth retardation associated with reduced chondrocyte proliferation and differentiation, and increased chondrocyte apoptosis. Meanwhile, we observed that Atg7 ablation mainly induced the PERK-ATF4-CHOP axis of the endoplasmic reticulum (ER) stress response in growth plate chondrocytes. Although Atg7 ablation induced ER stress in growth plate chondrocytes, the addition of phenylbutyric acid (PBA), a chemical chaperone known to attenuate ER stress, partly neutralized such effects of Atg7 ablation on longitudinal bone growth, indicating the causative interaction between autophagy and ER stress in growth plate. Consistent with these findings in vivo, we also observed that Atg7 ablation in cultured chondrocytes resulted in defective autophagy, elevated ER stress, decreased chondrocytes proliferation, impaired expression of col10a1, MMP-13, and VEGFA for chondrocyte differentiation, and increased chondrocyte apoptosis, while such effects were partly nullified by reduction of ER stress with PBA. In addition, Atg7 ablation-mediated impaired chondrocyte function (chondrocyte proliferation, differentiation, and apoptosis) was partly reversed in CHOP-/- cells, indicating the causative role of the PERK-ATF4-CHOP axis of the ER stress response in the action of autophagy deficiency in chondrocytes. In conclusion, our findings indicate that autophagy deficiency may trigger ER stress in growth plate chondrocytes and contribute to growth retardation, thus implicating autophagy as an important regulator during chondrogenesis and providing new insights into the clinical potential of autophagy in cartilage homeostasis. © 2017 American Society for Bone and Mineral Research.

The Role of Hox in Pisiform and Calcaneus Growth Plate Formation and the Nature of the Zeugopod/Autopod Boundary.

The mesopodium forms at the boundary between the zeugopod and autopod and is composed of short nodular bones that typically lack growth plates. Hoxa11 and Hoxa13 are expressed in mutually exclusive proximal-distal domains that demarcate the zeugopod/autopod boundary. Similarly, Hoxd genes are deployed in two distinct phases during limb development. The early phase corresponds to proximal segments including the zeugopod, and a late phase occurs in the digits. This arrangement produces a gap of low Hoxd expression that is traditionally viewed to correspond to the mesopodium. In contrast to the other mesopodials, the mammalian pisiform and calcaneus form true growth plates. We show that these bones, along with other proximal mesopodials, develop within the Hoxa11 and Hoxd11 expression domains. We also observe that the pisiform growth plate becomes disorganized with Hoxa11 or Hoxd11 loss of function, indicating a direct role for Hox11 in its development. Hoxa13 loss of function has minimal effect on the pisiform and proximal calcaneus as these bones still form secondary centers and undergo longitudinal growth. Consideration of the phenotypes resulting from hypodactyly (Hd) and synpolydactyly homolog (spdh) mutations, which result from altered HOXA13 and HOXD13 proteins, respectively, confirms that Hox13 plays a limited role in the development of the pisiform and calcaneus and suggests that they lie within the early phase of Hox expression. Therefore, with respect to patterns of ossification and gene expression, these bones share much more in common with the zeugopod than the autopod. Such an interpretation fits with the timing of autopod origins during tetrapod evolution.

Systematic Reconstruction of Molecular Cascades Regulating GP Development Using Single-Cell RNA-Seq.

The growth plate (GP) comprising sequentially differentiated cell layers is a critical structure for bone elongation and regeneration. Although several key regulators in GP development have been identified using genetic perturbation, systematic understanding is still limited. Here, we used single-cell RNA-sequencing (RNA-seq) to determine the gene expression profiles of 217 single cells from GPs and developed a bioinformatics pipeline named Sinova to de novo reconstruct physiological GP development in both temporal and spatial high resolution. Our unsupervised model not only confirmed prior knowledge, but also enabled the systematic discovery of genes, potential signal pathways, and surface markers CD9/CD200 to precisely depict development. Sinova further identified the effective combination of transcriptional factors (TFs) that regulates GP maturation, and the result was validated using an in vitro EGFP-Col10a screening system. Our case systematically reconstructed molecular cascades in GP development through single-cell profiling, and the bioinformatics pipeline is applicable to other developmental processes. VIDEO ABSTRACT.

Ihha induces hybrid cartilage-bone cells during zebrafish jawbone regeneration.

The healing of bone often involves a cartilage intermediate, yet how such cartilage is induced and utilized during repair is not fully understood. By studying a model of large-scale bone regeneration in the lower jaw of adult zebrafish, we show that chondrocytes are crucial for generating thick bone during repair. During jawbone regeneration, we find that chondrocytes co-express genes associated with osteoblast differentiation and produce extensive mineralization, which is in marked contrast to the behavior of chondrocytes during facial skeletal development. We also identify the likely source of repair chondrocytes as a population of Runx2(+)/Sp7(-) cells that emanate from the periosteum, a tissue that normally contributes only osteoblasts during homeostasis. Analysis of Indian hedgehog homolog a (ihha) mutants shows that the ability of periosteal cells to generate cartilage in response to injury depends on a repair-specific role of Ihha in the induction as opposed to the proliferation of chondrocytes. The large-scale regeneration of the zebrafish jawbone thus employs a cartilage differentiation program distinct from that seen during development, with the bone-forming potential of repair chondrocytes potentially due to their derivation from osteogenic cells in the periosteum.

Analysis of Mouse Growth Plate Development.

To investigate skeletal development, pathophysiological mechanisms of cartilage and bone disease, and eventually assess innovative treatments, the mouse is a very important resource. During embryonic development, mesenchymal condensations are formed, and cells within these mesenchymal condensations either directly differentiate into osteoblasts and give origin to intramembranous bone, or differentiate into chondrocytes and form a cartilaginous anlage. The cartilaginous anlage or fetal growth plate is then replaced with bone. This process is also called endochondral bone development, and it is responsible for the generation of most of our skeleton. Here we discuss in detail the most common in vivo and in vitro techniques our laboratory is currently using for the analysis of the mouse fetal growth plate during development.

Histological and histochemical evaluations on the effects of high incubation temperature on the embryonic development of tibial growth plate in broiler chickens.

The effects of experimentally induced high incubation temperature on the embryonic development of growth plate of the chicken were investigated by means of histological and enzyme histochemical methods. In the experiments, 250 fertile eggs of Ross-308 broiler strain were divided into two groups, the control eggs were maintained under optimal conditions (37.8°C and 65% ± 2% relative humidity, rh) during the whole incubation period. Heat-stress imposed eggs were maintained under normal conditions (37.8°C and 65% ± 2% rh) until the 10th day of incubation, and then, continuously (24 h per day) exposed to high temperature (38.8°C and 65% ± 2% rh). Tissue samples were taken from 10 animals of each group at the 11th, 13th, 15th, 18th, 21st days of incubation. Tissue samples were processed by enzyme histochemical methods in addition to routine histological techniques. The relative tibia weights and tibia length were lower in the heat-stress group compared to the control group. The results of the measurements of the growth plate showed that the proliferative zone was narrowed whereas, the transitional and hypertrophic zone were thickened in the heat stress group. Alkaline phosphatase (ALP) density was significantly decreased in the heat-stress group compared to the control group. In conclusion, bone formation and growth plate formation are crucial for embryo development and 1°C higher from optimum may increase the incidence of skeletal disorders and leg problems in broiler chickens which is one of the major animal welfare concerns for the poultry industry.

A combined series of Fgf9 and Fgf18 mutant alleles identifies unique and redundant roles in skeletal development.

Fibroblast growth factor (FGF) signaling is a critical regulator of skeletal development. Fgf9 and Fgf18 are the only FGF ligands with identified functions in embryonic bone growth. Mice lacking Fgf9 or Fgf18 have distinct skeletal phenotypes; however, the extent of overlapping or redundant functions for these ligands and the stage-specific contributions of FGF signaling to chondrogenesis and osteogenesis are not known. To identify separate versus shared roles for FGF9 and FGF18, we generated a combined series of Fgf9 and Fgf18 null alleles. Analysis of embryos lacking alleles of Fgf9 and Fgf18 shows that both encoded ligands function redundantly to control all stages of skeletogenesis; however, they have variable potencies along the proximodistal limb axis, suggesting gradients of activity during formation of the appendicular skeleton. Congenital absence of both Fgf9 and Fgf18 results in a striking osteochondrodysplasia and revealed functions for FGF signaling in early proximal limb chondrogenesis. Additional defects were also noted in craniofacial bones, vertebrae, and ribs. Loss of alleles of Fgf9 and Fgf18 also affect the expression of genes encoding other key intrinsic skeletal regulators, including IHH, PTHLH (PTHrP), and RUNX2, revealing potential direct, indirect, and compensatory mechanisms to coordinate chondrogenesis and osteogenesis.

Different Roles of GRP78 on Cell Proliferation and Apoptosis in Cartilage Development.

Eukaryotic cells possess several mechanisms to adapt to endoplasmic reticulum (ER) stress and thereby survive. ER stress activates a set of signaling pathways collectively termed as the unfolded protein response (UPR). We previously reported that Bone morphogenetic protein 2 (BMP2) mediates mild ER stress and activates UPR signal molecules in chondrogenesis. The mammalian UPR protects the cell against the stress of misfolded proteins in the endoplasmic reticulum. Failure to adapt to ER stress causes the UPR to trigger apoptosis. Glucose regulated protein 78 (GRP78), as an important molecular chaperone in UPR signaling pathways, is responsible for binding to misfolded or unfolded protein during ER stress. However the influence on GRP78 in BMP2-induced chondrocyte differentiation has not yet been elucidated and the molecular mechanism underlyng these processes remain unexplored. Herein we demonstrate that overexpression of GRP78 enhanced cell proliferation in chondrocyte development with G1 phase advance, S phase increasing and G2-M phase transition. Furthermore, overexpression of GRP78 inhibited ER stress-mediated apoptosis and then reduced apoptosis in chondrogenesis induced by BMP2, as assayed by cleaved caspase3, caspase12, C/EBP homologous protein (CHOP/DDIT3/GADD153), p-JNK (phosphorylated c-Jun N-terminal kinase) expression during the course of chondrocyte differentiation by Western blot. In addition, flow cytometry (FCM) assay, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) assay and immune-histochemistry analysis also proved this result in vitro and in vivo. It was demonstrated that GRP78 knockdown via siRNA activated the ER stress-specific caspase cascade in developing chondrocyte tissue. Collectively, these findings reveal a novel critical role of GRP78 in regulating ER stress-mediated apoptosis in cartilage development and the molecular mechanisms involved.

Dynamic imaging of the growth plate cartilage reveals multiple contributors to skeletal morphogenesis.

The diverse morphology of vertebrate skeletal system is genetically controlled, yet the means by which cells shape the skeleton remains to be fully illuminated. Here we perform quantitative analyses of cell behaviours in the growth plate cartilage, the template for long bone formation, to gain insights into this process. Using a robust avian embryonic organ culture, we employ time-lapse two-photon laser scanning microscopy to observe proliferative cells' behaviours during cartilage growth, resulting in cellular trajectories with a spreading displacement mainly along the tissue elongation axis. We build a novel software toolkit of quantitative methods to segregate the contributions of various cellular processes to the cellular trajectories. We find that convergent-extension, mitotic cell division, and daughter cell rearrangement do not contribute significantly to the observed growth process; instead, extracellular matrix deposition and cell volume enlargement are the key contributors to embryonic cartilage elongation.

Human monoclonal antibody fragments targeting matrilin-3 in growth plate cartilage.

PURPOSE: Many genetic disorders, including chondrodysplasias, and acquired disorders impair growth plate function, resulting in short and sometimes malformed bones. There are multiple endocrine and paracrine factors that promote chondrogenesis at the growth plate, which could potentially be used to treat these disorders. Targeting these growth factors specifically to the growth plate might augment the therapeutic skeletal effect while diminishing undesirable effects on non-target tissues. METHODS: Using yeast display technology, we selected single-chain variable antibody fragments that bound to human and mouse matrilin-3, an extracellular matrix protein specifically expressed in cartilage tissue. The ability of the selected antibody fragments to bind matrilin-3 and to bind cartilage tissue in vitro and in vivo was assessed by ELISA and immunohistochemistry. RESULTS: We identified antibody fragments that bound matrilin-3 with high affinity and also bound with high tissue specificity to cartilage homogenates and to cartilage structures in mouse embryo sections. When injected intravenously in mice, the antibody fragments specifically homed to cartilage. CONCLUSIONS: Yeast display successfully selected antibody fragments that are able to target cartilage tissue in vivo. Coupling these antibodies to chondrogenic endocrine and paracrine signaling molecules has the potential to open up new pharmacological approaches to treat childhood skeletal growth disorders.

Fate of growth plate hypertrophic chondrocytes: death or lineage extension?

The vertebrate growth plate is an essential tissue that mediates and controls bone growth. It forms through a multistep differentiation process in which chondrocytes differentiate, proliferate, stop dividing and undergo hypertrophy, which entails a 20-fold increase in size. Hypertrophic chondrocytes are specialized cells considered to be the end state of the chondrocyte differentiation pathway, and are essential for bone growth. They are characterized by expression of type X collagen encoded by the Col10a1 gene, and synthesis of a calcified cartilage matrix. Whether hypertrophy marks a transition preceding osteogenesis, or it is the terminal differentiation stage of chondrocytes with cell death as the ultimate fate has been the subject of debate for over a century. In this review, we revisit this debate in the light of new findings arising from genetic-mediated lineage tracing studies showing that hypertrophic chondrocytes can survive at the chondro-osseous junction and further make the transition to become osteoblasts and osteocytes. The contribution of chondrocytes to the osteoblast lineage has important implications in bone development, disease and repair.