The motor protein Kif3a and primary cilia regulate important developmental processes, but their roles in skeletogenesis remain illdefined. Here we created mice deficient in Kif3a in cartilage and focused on the cranial base and synchondroses. Kif3a deficiency caused cranial base growth retardation and dysmorphogenesis, which were evident in neonatal animals by anatomical and microcomputed tomography (CT) inspection. Kif3a deficiency also changed synchondrosis growth plate organization and function, and the severity of these changes increased over time. By postnatal day (P)7, mutant growth plates lacked typical zones of chondrocyte proliferation and hypertrophy, and were instead composed of chondrocytes with an unusual phenotype characterized by strong collagen II (Col2a1) gene expression but barely detectable expression of Indian hedgehog (Ihh), collagen X (Col10a1), Vegf (Vegfa), MMP-13 (Mmp13) and osterix (Sp7). Concurrently, unexpected developmental events occurred in perichondrial tissues, including excessive intramembranous ossification all along the perichondrial border and the formation of ectopic cartilage masses. Looking for possible culprits for these latter processes, we analyzed hedgehog signalling topography and intensity by monitoring the expression of the hedgehog effectors Patched 1 and Gli1, and of the hedgehog-binding cell-surface component syndecan 3. Compared with controls, hedgehog signaling was quite feeble within mutant growth plates as early as P0, but was actually higher and was widespread all along mutant perichondrial tissues. Lastly, we studied postnatal mice deficient in Ihh in cartilage; their cranial base defects only minimally resembled those in Kif3a-deficient mice. In summary, Kif3a and primary cilia make unique contributions to cranial base development and synchondrosis growth plate function. Their deficiency causes abnormal topography of hedgehog signaling, growth plate dysfunction, and un-physiologic responses and processes in perichondrial tissues, including ectopic cartilage formation and excessive intramembranous ossification.

No abstract

Matrix GLA protein (MGP), a γ-carboxyglutamic acid (GLA)–rich, vitamin K–dependent and apatite-binding protein, is a regulator of hypertrophic cartilage mineralization during development. However, MGP is produced by both hypertrophic and immature chondrocytes, suggesting that MGP's role in mineralization is cell stage–dependent, and that MGP may have other roles in immature cells. It is also unclear whether MGP regulates the quantity of mineral or mineral nature and quality as well. To address these issues, we determined the effects of manipulations of MGP synthesis and expression in (a) immature and hypertrophic chondrocyte cultures and (b) the chick limb bud in vivo. The two chondrocyte cultures displayed comparable levels of MGP gene expression. Yet, treatment with warfarin, a γ-carboxylase inhibitor and vitamin K antagonist, triggered mineralization in hypertrophic but not immature cultures. Warfarin effects on mineralization were highly selective, were accompanied by no appreciable changes in MGP expression, alkaline phosphatase activity, or cell number, and were counteracted by vitamin K cotreatment. Scanning electron microscopy, x-ray microanalysis, and Fourier-transform infrared spectroscopy revealed that mineral forming in control and warfarin-treated hypertrophic cell cultures was similar and represented stoichiometric apatite. Virally driven MGP overexpression in cultured chondrocytes greatly decreased mineralization. Surprisingly, MGP overexpression in the developing limb not only inhibited cartilage mineralization, but also delayed chondrocyte maturation and blocked endochondral ossification and formation of a diaphyseal intramembranous bone collar. The results show that MGP is a powerful but developmentally regulated inhibitor of cartilage mineralization, controls mineral quantity but not type, and appears to have a previously unsuspected role in regulating chondrocyte maturation and ossification processes.

Retinoids have long been known to influence skeletogenesis but the specific roles played by these effectors and their nuclear receptors remain unclear. Thus, it is not known whether endogenous retinoids are present in developing skeletal elements, whether expression of the retinoic acid receptor (RAR) genes alpha, beta, and gamma changes during chondrocyte maturation, or how interference with retinoid signaling affects skeletogenesis. We found that immature chondrocytes present in stage 27 (Day 5.5) chick embryo humerus exhibited low and diffuse expression of RARalpha and gamma, while RARbeta expression was strong in perichondrium. Emergence of hypertrophic chondrocytes in Day 8-10 embryo limbs was accompanied by a marked and selective up-regulation of RARgamma gene expression. The RARgamma-rich type X collagen-expressing hypertrophic chondrocytes lay below metaphyseal prehypertrophic chondrocytes expressing Indian hedgehog (Ihh) and were followed by mineralizing chondrocytes undergoing endochondral ossification. Bioassays revealed that cartilaginous elements in Day 5.5, 8.5, and 10 chick embryo limbs all contained endogenous retinoids; strikingly, the perichondrial tissues surrounding the cartilages contained very large amounts of retinoids. Implantation of beads filled with retinoid antagonist Ro 41-5253 or AGN 193109 near the humeral anlagens in stage 21 (Day 3.5) or stage 27 chick embryos severely affected humerus development. In comparison to their normal counterparts, antagonist-treated humeri in Day 8.5-10 chick embryos were significantly shorter and abnormally bent; their diaphyseal chondrocytes had remained prehypertrophic Ihh-expressing cells, did not express RARgamma, and were not undergoing endochondral ossification. Interestingly, formation of an intramembranous bony collar around the diaphysis was not affected by antagonist treatment. Using chondrocyte cultures, we found that the antagonists effectively interfered with the ability of all-trans-retinoic acid to induce terminal cell maturation. The results provide clear evidence that retinoid-dependent and RAR-mediated mechanisms are required for completion of the chondrocyte maturation process and endochondral ossification in the developing limb. These mechanisms may be positively influenced by cooperative interactions between the chondrocytes and their retinoid-rich perichondrial tissues.

Loss of epidermal growth factor receptor (EGFR) activity in mice alters growth plate development, impairs endochondral ossification, and retards growth. However, the detailed mechanism by which EGFR regulates endochondral bone formation is unknown. Here, we show that administration of an EGFR-specific small molecule inhibitor, gefitinib, into 1-month-old rats for 7 days produced profound defects in long bone growth plate cartilage characterized by epiphyseal growth plate thickening and massive accumulation of hypertrophic chondrocytes. Immunostaining demonstrated that growth plate chondrocytes express EGFR but endothelial cells and osteoclasts show little to no expression. Gefitinib did not alter chondrocyte proliferation or differentiation and vascular invasion into the hypertrophic cartilage. However, osteoclast recruitment and differentiation at the chondro-osseous junction was attenuated due to decreased RANKL expression in the growth plate. Moreover, gefitinib treatment inhibited the expression of matrix metalloproteinases (MMP9, 13, and 14), increased the amount of collagen fibrils, and decreased degraded extracellular matrix products in the growth plate. In vitro, the EGFR ligand TGFα strongly stimulated RANKL, MMP9 and MMP13 expression and suppressed OPG expression in primary chondrocytes. In addition, a mouse model of cartilage-specific EGFR inactivation exhibited a similar phenotype of hypertrophic cartilage enlargement. Together, our data demonstrate that EGFR signaling supports osteoclastogenesis at the chondro-osseous junction and promotes chondrogenic expression of MMPs in the growth plate. Therefore, we conclude that EGFR signaling plays an essential role in the remodeling of growth plate cartilage extracellular matrix into bone during endochondral ossification.

Heparan sulfate (HS) is an essential component of cell surface and matrix-associated proteoglycans (HSPGs). Due to their sulfation patterns, the HS chains interact with numerous signaling proteins and regulate their distribution and activity on target cells. Many of these proteins, including bone morphogenetic protein family members, are expressed in the growth plate of developing skeletal elements, and several skeletal phenotypes are caused by mutations in HS-synthesizing and modifying enzymes. The disease we discuss here is Hereditary Multiple Exostoses (HME), a disorder caused by mutations in HS synthesizing enzymes EXT1 and EXT2, leading to HS deficiency. The exostoses are benign cartilaginous-bony outgrowths, form next to growth plates, can cause growth retardation and deformities, chronic pain and impaired motion, and progress to malignancy in 2-5% of patients. We describe recent advancements on HME pathogenesis and exostosis formation deriving from studies that have determined distribution, activities and roles of signaling proteins in wild type and HS-deficient cells and tissues. Aberrant distribution of signaling factors combined with aberrant responsiveness of target cells to those same factors appear to be a major culprit in exostosis formation. Insights from these studies suggest plausible and cogent ideas about how HME could be treated in the future.

Limb synovial joints are intricate structures composed of articular cartilage, synovial membranes, ligaments and an articular capsule. Each joint has a unique shape, organization and biomechanical function, and articular cartilage itself is rather complex and organized in distinct zones, including the superficial zone that produces lubricans and contains stem/progenitor cells. There has been a great of interest for many years to decipher the mechanisms by which the joints form and come to acquire such unique structural features and diversity. Decades ago, classic embryologists discovered that the first overt sign of joint formation at each prescribed limb site is the appearance of a dense and compact population of mesenchymal cells collectively called the interzone. Work carried out since by several groups has provided evidence that the interzone cells do actively participate in joint tissue formation over developmental time. This minireview provides a succinct but comprehensive description of the many and important recent advances in this field of research. These includes: studies using various conditional reporter mice to genetically trace and track the origin, fate and possible function of joint progenitor cells; studies on the involvement and roles in signaling pathways and transcription factors in joint cell determination and functioning; and studies using advanced methods of gene expression analyses to uncover novel genetic determinants of joint formation and diversity. The overall advances are impressive, and the findings are not only of obvious interest and importance, but have major implications to conceive future translational medicine tools to repair and regenerate defective, overused or aging joints.

Introduction The purpose of this study was to compare healing after root-end surgery by using grey mineral trioxide aggregate (MTA) and EndoSequence Root Repair Material (RRM) as root-end filling material in an animal model. Methods Apical periodontitis was induced in 55 mandibular premolars of 4 healthy beagle dogs. After 6 weeks, root-end surgeries were performed by using modern microsurgical techniques. Two different root-end filling materials were used, grey MTA and RRM. Six months after surgery, healing of the periapical area was assessed by periapical radiographs, cone-beam computed tomography (CBCT), micro computed tomography (CT), and histology. Results Minimal or no inflammatory response was observed in the majority of periapical areas regardless of the material. The degree of inflammatory infiltration and cortical plate healing were not significantly different between the 2 materials. However, a significantly greater root-end surface area was covered by cementum-like, periodontal ligament–like tissue, and bone in RRM group than in MTA group. When evaluating with periapical radiographs, complete healing rate in RRM and MTA groups was 92.6% and 75%, respectively, and the difference was not statistically significant (P = .073). However, on CBCT and micro CT images, RRM group demonstrated significantly superior healing on the resected root-end surface and in the periapical area (P = .000 to .027). Conclusions Like MTA, RRM is a biocompatible material with good sealing ability. However, in this animal model RRM achieved a better tissue healing response adjacent to the resected root-end surface histologically. The superior healing tendency associated with RRM could be detected by CBCT and micro CT but not periapical radiography.