Cartilage Mineralization Research Articles

Salmon Nasal Cartilage-Derived Proteoglycans Alleviate Monosodium Iodoacetate-Induced Osteoarthritis in Rats

Osteoarthritis is a chronic inflammatory condition characterized by the degeneration of joint cartilage and underlying bone, resulting in pain, swelling, and reduced mobility. This study evaluates the efficacy of salmon nasal cartilage-derived proteoglycans in mitigating osteoarthritis symptoms and investigates the underlying molecular mechanisms. This study employed a rat model of osteoarthritis induced by monosodium iodoacetate (MIA) injection. The rats were orally administered salmon nasal cartilage-derived proteoglycans or ibuprofen. Key aspects of osteoarthritis pathology, including impaired exercise ability, inflammation, extracellular matrix degradation, and chondrocyte apoptosis, were assessed using histological analysis, micro-CT, treadmill testing, serum assays, and mRNA/protein expression studies. The MIA injection caused significant cartilage damage, reduced bone mineral density, and impaired exercise ability. Additionally, it elevated serum levels of prostaglandin E2 and nitric oxide, increased the mRNA and protein levels of inflammation-related factors, and activated apoptosis signaling pathways in cartilage. Treatment with salmon nasal cartilage-derived proteoglycans significantly improved cartilage morphology and mineralization, reduced inflammation, and inhibited apoptosis signaling pathways, with effects comparable to those observed with ibuprofen treatment. These findings highlight the potential of salmon nasal cartilage-derived proteoglycans as a therapeutic agent for managing osteoarthritis by effectively reducing inflammation, preventing cartilage degradation, and inhibiting chondrocyte apoptosis.

Endochondral ossification: insights into the cartilage mineralization processes achieved by an anhydrous freeze substitution protocol

Growth plate cartilage (GP) serves as a dynamic site of active mineralization and offers a unique opportunity to investigate the cell-regulated matrix mineralization process. Transmission electron microscopy (TEM) provides a means for the direct observation of these mechanisms, offering the necessary resolution and chemical analysis capabilities. However, as mineral crystallinity is prone to artifacts using aqueous fixation protocols, sample preparation techniques are critical to preserve the mineralized tissue in its native form. We optimized cryofixation by high-pressure freezing followed by freeze substitution in anhydrous acetone containing 0.5% uranyl acetate to prepare murine GP for TEM analysis. This sample preparation workflow maintains cellular and extracellular protein structural integrity with sufficient contrast for observation and without compromising mineral crystallinity. By employing appropriate sample preparation techniques, we were able to observe two parallel mineralization processes driven by chondrocytes: 1) intracellular- and 2) extracellular-originating mineralized vesicles. Both mechanisms are based on sequestering calcium phosphate (CaP) within a membrane-limited structure, albeit originating from different compartments of the chondrocytes. In the intracellular originating pathway, CaP accumulates within mitochondria as globular CaP granules, which are incorporated into intracellular vesicles (500-1000 nm) and transported as granules to the extracellular matrix (ECM). In contrast, membrane budding vesicles with a size of approximately 100-200nm, filled with needle-shaped minerals were observed only in the ECM. Both processes transport CaP to the collagenous matrix via vesicles, they can be differentiated based on the vesicle size and mineral morphologies. Their individual importance to the cartilage mineralization process is yet to be determined. Statement of SignificanceWe do not fully understand the process by which epiphyseal cartilage mineralizes - a vital step in endochondral bone formation. Previous work has proposed that mitochondria and intracellular vesicles are storage sites for the delivery of mineral to collagen fibrils. However, these concepts are founded on results from in vitro models of mineralization; no prior work has observed mineral-containing intracellular vesicles or mitochondria in developing epiphyseal cartilage. Here we developed a new cryofixation preparation route for transmission electron microscopy (TEM) imaging that has disclosed a cell-regulated process of mineralization in epiphyseal cartilage. High resolution TEM images revealed an involvement of mitochondria and intracellular and extracellular vesicles in delivering transient mineral phases to the collagen fibrils to promote cartilage mineralization.

Growth and mineralization of fetal mouse long bones under microgravity and daily 1 g gravity exposure

In a previous Space Shuttle/Spacelab experiment (STS-42), we observed direct responses of isolated fetal mouse long bones to near weightlessness. This paper aimed to verify those results and study the effects of daily 1×g exposure during microgravity on the growth and mineralization of these bones. Two experiments were conducted: one on an American Space Shuttle mission (IML-2 on STS-65) and another on a Russian Bio-Cosmos flight (Bion-10 on Cosmos-2229). Despite differences in hardware, both used 17-day-old fetal mouse metatarsals cultured for 4 days. Results showed reduced proteoglycan content under microgravity compared to 1×g conditions, with no main differences in other cellular structures. While the overall metatarsal length was unaffected, the length increase of the mineralized diaphysis was significantly reduced under microgravity. Daily 1×g exposure for at least 6 h abolished the microgravity-induced reduction in cartilage mineralization, indicating the need for long-duration exposure to 1×g as an in-flight countermeasure using artificial gravity.

Hakai, a novel Runx2 interacting protein, augments osteoblast differentiation by rescuing Runx2 from Smurf2-mediated proteasome degradation.

Runt-related transcription factor 2 (Runx2) is a key regulator of osteoblast differentiation and bone formation. In Runx2-deficient embryos, skeletal development ceases at the cartilage anlage stage. These embryos die of respiratory failure upon birth and display a complete absence of bone and cartilage mineralization. Here, we identified Hakai, a type of E3 ubiquitin ligase as a potential Runx2 interacting partner through affinity pulldown-based proteomic approach. Subsequently, we observed that similar to Runx2, Hakai was downregulated in osteopenic ovariectomized rats, suggesting its involvement in bone formation. Consistent with this observation, Hakai overexpression significantly enhanced osteoblast differentiation in mesenchyme-like C3H10T1/2 as well as primary rat calvaria osteoblast (RCO) cells in vitro. Conversely, overexpression of a catalytically inactive Hakai mutant (C109A) exhibited minimal to no effect, whereas Hakai depletion markedly reduced endogenous Runx2 levels and impaired osteogenic differentiation in both C3H10T1/2 and RCOs. Mechanistically, Hakai physically interacts with Runx2 and enhances its protein turnover by rescuing it from Smad ubiquitination regulatory factor 2 (Smurf2)-mediated proteasome degradation. Wild-type Hakai but not Hakai-C109A inhibited Smurf2 protein levels through proteasome-mediated degradation. These findings underscore Hakai's functional role in bone formation, primarily through its positive modulation of Runx2 protein turnover by protecting it from Smurf2-mediated ubiquitin-proteasomal degradation. Collectively, our results demonstrate Hakai as a promising novel therapeutic target for osteoporosis.

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

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

Internal flow field analysis of a dendritic pore scaffold for bone tissue engineering

The effective reconstruction of osteochondral biomimetic structures is a key factor in guiding the regeneration of full-thickness osteochondral defects. Due to the avascular nature of hyaline cartilage, the greatest challenge in constructing this scaffold lies in both utilizing the biomimetic structure to promote vascular differentiation for nutrient delivery to hyaline cartilage, thereby enhancing the efficiency of osteochondral reconstruction, and effectively blocking vascular ingrowth into the cartilage layer to prevent cartilage mineralization. However, the intrinsic relationship between the planning of the microporous pipe network and the flow resistance in the biomimetic structure, and the mechanism of promoting cell adhesion to achieve vascular differentiation and inhibiting cell adhesion to block the growth of blood vessels are still unclear. Inspired by the structure of tree trunks, this study designed a biomimetic tree-like tubular network structure for osteochondral scaffolds based on Murray’s law. Utilizing computational fluid dynamics, the study investigated the influence of the branching angle of micro-pores on the flow velocity, pressure distribution, and scaffold permeability within the scaffold. The results indicate that when the differentiation angle exceeds 50 degrees, the highest flow velocity occurs at the confluence of tributaries at the ninth fractal position, forming a barrier layer. This structure effectively guides vascular growth, enhances nutrient transport capacity, increases flow velocity to promote cell adhesion, and inhibits cell infiltration into the cartilage layer.

Intra-Articular Mineralization on Computerized Tomography of the Knee and Risk of Cartilage Damage: The Multicenter Osteoarthritis Study.

Intra-articular (IA) mineralization may contribute to osteoarthritis (OA) structural progression. We studied the association of IA mineralization on knee computed tomography (CT) with cartilage damage worsening on knee magnetic resonance imaging (MRI), with a focus on location- and tissue-specific effects. Participants from the Multicenter Osteoarthritis Study with knee CT and MRI scans were included. Presence of IA mineralization on CT was defined as a Boston University Calcium Knee Score >0 anywhere in the knee. Cartilage worsening on MRI was defined as any increase in the MRI OA Knee Score, including incident damage. We evaluated the association of whole-knee, compartment-specific (ie, medial or lateral), and subregion-specific (ie, location-matched) IA mineralization at baseline with cartilage worsening at two years' follow-up in the corresponding locations using binomial regression with generalized estimating equations, adjusting for age, sex, and body mass index (BMI). We included 1,673 participants (mean age 60 years, 56% female, mean BMI 29). Nine percent had any IA mineralization in the knee, and 47.4% had any cartilage worsening on follow-up. Mineralization of any tissue in the knee, regardless of location, was not associated with MRI cartilage worsening. However, cartilage mineralization was associated with 1.39 (95% confidence interval 1.04-1.88) times higher risk of cartilage worsening in the same compartment, with similar results in subregion-specific analysis. CT-detected IA mineralization in the cartilage was associated with higher risk of MRI cartilage worsening in the same compartment and subregion over two years. These findings suggest potential localized, tissue-specific effects of IA mineralization on cartilage pathology in knee OA.

Special Issue: “X-Linked Hypophosphatemia”

Rickets and osteomalacia are associated with impaired mineralization in growth plate cartilage and the bone osteoid [...]

Increased Osteoblast GαS Promotes Ossification by Suppressing Cartilage and Enhancing Callus Mineralization During Fracture Repair in Mice.

GαS, the stimulatory G protein α-subunit that raises intracellular cAMP levels by activating adenylyl cyclase, plays a vital role in bone development, maintenance, and remodeling. Previously, using transgenic mice overexpressing GαS in osteoblasts (GS-Tg), we demonstrated the influence of osteoblast GαS level on osteogenesis, bone turnover, and skeletal responses to hyperparathyroidism. To further investigate whether alterations in GαS levels affect endochondral bone repair, a postnatal bone regenerative process that recapitulates embryonic bone development, we performed stabilized tibial osteotomy in male GS-Tg mice at 8 weeks of age and examined the progression of fracture healing by micro-CT, histomorphometry, and gene expression analysis over a 4-week period. Bone fractures from GS-Tg mice exhibited diminished cartilage formation at the time of peak soft callus formation at 1 week post-fracture followed by significantly enhanced callus mineralization and new bone formation at 2 weeks post-fracture. The opposing effects on chondrogenesis and osteogenesis were validated by downregulation of chondrogenic markers and upregulation of osteogenic markers. Histomorphometric analysis at times of increased bone formation (2 and 3 weeks post-fracture) revealed excess fibroblast-like cells on newly formed woven bone surfaces and elevated osteocyte density in GS-Tg fractures. Coincident with enhanced callus mineralization and bone formation, GS-Tg mice showed elevated active β-catenin and Wntless proteins in osteoblasts at 2 weeks post-fracture, further substantiated by increased mRNA encoding various canonical Wnts and Wnt target genes, suggesting elevated osteoblastic Wnt secretion and Wnt/β-catenin signaling. The GS-Tg bony callus at 4 weeks post-fracture exhibited greater mineral density and decreased polar moment of inertia, resulting in improved material stiffness. These findings highlight that elevated GαS levels increase Wnt signaling, conferring an increased osteogenic differentiation potential at the expense of chondrogenic differentiation, resulting in improved mechanical integrity. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

A case of vitamin-D dependent type-1A rickets

Abstract Rickets is a disease of growing bones characterized by softening of bones as a result of defective mineralization of cartilage in the epiphyseal growth plate. The predominant cause is a deficiency or impaired metabolism of vitamin D. Some forms of rickets do not respond to regular doses of cholecalciferol, which leads to suspicion of vitamin D-dependent rickets (VDDR). VDDR is of two types: type I is due to defective renal tubular 25-hydroxyvitamin D 1-α hydroxylase, and type II is due to end-organ resistance to the active metabolite of vitamin D. Here, we describe a child with seizures and calcipenic rickets with biochemical profile suggestive of VDDR type 1 worsened by phenytoin which responded completely to calcium and calcitriol supplementation. Whole exome sequencing revealed a heterozygous missense mutation in exon 7 of the TRPV4 gene (chromosome 12,c.1292C>A), whereas no mutations in CYP2R1, CYP27B1, VDR, and CYP3A4 were detected.

Effect of glycosaminoglycans on the structure and composition of articular cartilage and bone of broilers

This study aimed to assess the influence of glycosaminoglycan (chondroitin and glucosamine sulfates) supplementation in the diet of broilers on the expression of matrix metallopeptidase 9 (MMP-9) and metallopeptidase inhibitor 2 (TIMP-2) genes, the synthesis of proteoglycans, collagen type II and chondrocytes, bone and cartilage macroscopy, bone mineral densitometry, bone breaking strength and mineral profile. A completely randomized design was carried out in a 3×3 factorial scheme (3 levels of chondroitin sulfate: 0.00, 0.05, and 0.10%; and 3 levels of glucosamine sulfate: 0.00, 0.15, and 0.30%), totaling 9 treatments. At 21 and 42 d of age, broilers were slaughtered, and tibias and femurs were collected for evaluation. There was an interaction (P < 0.05) of sulfates for the expression of MMP-9 and its inhibitor TIMP-2 in femur articular cartilage, as well as for the number of chondrocytes, collagen type II and proteoglycans in tibia articular cartilage, bone and cartilage macroscopy and mineral profile (P < 0.05), with better results obtained with the inclusion of chondroitin and/or glucosamine sulfates in the feed. In conclusion, chondroitin and glucosamine sulfates can be used in broiler diets in order to favor the development of the structure of the locomotor system (bones and joints), thus preventing locomotion problems.

In Vitro Mineralisation of Tissue-Engineered Cartilage Reduces Endothelial Cell Migration, Proliferation and Tube Formation.

Tissue engineering bone via endochondral ossification requires the generation of a cartilage template which undergoes vascularisation and remodelling. While this is a promising route for bone repair, achieving effective cartilage vascularisation remains a challenge. Here, we investigated how mineralisation of tissue-engineered cartilage affects its pro-angiogenic potential. To generate in vitro mineralised cartilage, human mesenchymal stromal cell (hMSC)-derived chondrogenic pellets were treated with β-glycerophosphate (BGP). After optimising this approach, we characterised the changes in matrix components and pro-angiogenic factors by gene expression analysis, histology and ELISA. Human umbilical vein endothelial cells (HUVECs) were exposed to pellet-derived conditioned media, and migration, proliferation and tube formation were assessed. We established a reliable strategy to induce in vitro cartilage mineralisation, whereby hMSC pellets are chondrogenically primed with TGF-β for 2 weeks and BGP is added from week 2 of culture. Cartilage mineralisation determines loss of glycosaminoglycans, reduced expression but not protein abundance of collagen II and X, and decreased VEGFA production. Finally, the conditioned medium from mineralised pellets showed a reduced ability to stimulate endothelial cell migration, proliferation and tube formation. The pro-angiogenic potential of transient cartilage is thus stage-dependent, and this aspect must be carefully considered in the design of bone tissue engineering strategies.

An unusual case of familial hypophosphatemic rickets

Rickets is the failure of mineralisation of osteoid and newly formed bones in a child skeleton. It is commonly associated with vitamin D deficiency; however, it can be because of a decrease in serum phosphate level leading to inadequate mineralization of cartilage and bone, consequent skeletal deformities and growth retardation. In hypophosphatemic rickets, there are both inherited and acquired forms, where X-linked hypophosphatemic rickets (XLH) is the most prevalent genetic form and caused by mutations in the phosphate-regulating endopeptidase (PHEX) gene. XLH is associated with growth retardation and bone deformities. This rare variety was diagnosed in a 2 year 4 month old child who had brought by parents with complaint of abnormal gait. In the present case, clinical and radiographic aspects of manifestation of familial hypophosphatemic rickets are discussed.

Histological Assessment of Endochondral Ossification and Bone Mineralization

Finely tuned cartilage mineralization, endochondral ossification, and normal bone formation are necessary for normal bone growth. Hypertrophic chondrocytes in the epiphyseal cartilage secrete matrix vesicles, which are small extracellular vesicles initiating mineralization, into the intercolumnar septa but not the transverse partitions of the cartilage columns. Bone-specific blood vessels invade the unmineralized transverse septum, exposing the mineralized cartilage cores. Many osteoblast precursors migrate to the cartilage cores, where they synthesize abundant bone matrices, and mineralize them in a process of matrix vesicle-mediated bone mineralization. Matrix vesicle-mediated mineralization concentrates calcium (Ca) and inorganic phosphates (Pi), which are converted into hydroxyapatite crystals. These crystals grow radially and are eventually get out of the vesicles to form spherical mineralized nodules, leading to collagen mineralization. The influx of Ca and Pi into the matrix vesicle is regulated by several enzymes and transporters such as TNAP, ENPP1, PiT1, PHOSPHO1, annexins, and others. Such matrix vesicle-mediated mineralization is regulated by osteoblastic activities, synchronizing the synthesis of organic bone material. However, osteocytes reportedly regulate peripheral mineralization, e.g., osteocytic osteolysis. The interplay between cartilage mineralization and vascular invasion during endochondral ossification, as well as that of osteoblasts and osteocytes for normal mineralization, appears to be crucial for normal bone growth.

Repairing a critical cranial defect using WISP1-pretreated chondrocyte scaffolds

In cranial flat bone fractures, spontaneous bone repair will occur only when the fracture ends are in close contact. However, in cases wherein bone discontinuity is extensive, surgical interventions are often required. To this end, autologous bone is harvested and surgically integrated into the site of fracture. Here we propose to use cartilage, as an alternative autologous source, to promote cranial fracture repair. The advantage of this approach is the potential reduction in donor site morbidity, likely due to the avascular and aneural nature of cartilage. As a first step we attempted to induce cartilage mineralization in vitro, using micromass primary chondrocyte cultures, incubated with BMP2 and/or WISP1, which were examined histologically following a 3-week culture period. Next, chondrocyte seeded collagen scaffolds were evaluated in vitro for expression profiles and ALP activity. Finally, chondrocyte-seeded collagen scaffolds were implanted in a Lewis rats 8 mm critical calvaria defect model, which was imaged via live CT for 12 weeks until sacrifice. End points were analyzed for microCT, histology, and serum levels of bone related markers. Micromass cultures exhibited an osseous inducing trend following WISP1 administration, which was maintained in chondrocyte seeded scaffolds. Accordingly, in vivo analysis was carried out to assess the impact of WISP1-pretreated chondrocytes (WCS) versus untreated chondrocytes (UCS) in calvaria defect model and compared to untreated control comprised of a defect-associated blood clot (BC) or empty collagen scaffold (CS) implant. Live CT and microCT exhibited higher mineralization volumes in critical defect implanted with UCS, with some structural improvements in WCS. Histological analysis exhibited higher anabolic bone formation in WCS and trabecular bone was detected in WCS and UCS groups. Chondrocytes implanted into critical cranial defect expedite the formation of native-like osseous tissue, especially after WISP1 priming in culture. Ultimately, these data support the use of autologous chondrocytes to repair critical maxillofacial defects.

IRE1α regulates the PTHrP-IHH feedback loop to orchestrate chondrocyte hypertrophy and cartilage mineralization

Cartilage development is controlled by the highly synergistic proliferation and differentiation of growth plate chondrocytes, in which the Indian hedgehog (IHH) and parathyroid hormone-related protein-parathyroid hormone-1 receptor (PTHrP-PTH1R) feedback loop is crucial. The inositol-requiring enzyme 1α/X-box-binding protein-1 spliced (IRE1α/XBP1s) branch of the unfolded protein response (UPR) is essential for normal cartilage development. However, the precise role of ER stress effector IRE1α, encoded by endoplasmic reticulum to nucleus signaling 1 (ERN1), in skeletal development remains unknown. Herein, we reported that loss of IRE1α accelerates chondrocyte hypertrophy and promotes endochondral bone growth. ERN1 acts as a negative regulator of chondrocyte proliferation and differentiation in postnatal growth plates. Its deficiency interrupted PTHrP/PTH1R and IHH homeostasis leading to impaired chondrocyte hypertrophy and differentiation. XBP1s, produced by p-IRE1α-mediated splicing, binds and up-regulates PTH1R and IHH, which coordinate cartilage development. Meanwhile, ER stress cannot be activated normally in ERN1-deficient chondrocytes. In conclusion, ERN1 deficiency accelerates chondrocyte hypertrophy and cartilage mineralization by impairing the homeostasis of the IHH and PTHrP/PTH1R feedback loop and ER stress. ERN1 may have a potential role as a new target for cartilage growth and maturation.

The Shape of the Jaw-Zebrafish Col11a1a Regulates Meckel's Cartilage Morphogenesis and Mineralization.

The expression of the col11a1a gene is essential for normal skeletal development, affecting both cartilage and bone. Loss of function mutations have been shown to cause abnormalities in the growth plate of long bones, as well as in craniofacial development. However, the specific effects on Meckel’s cartilage have not been well studied. To further understand the effect of col11a1a gene function, we analyzed the developing jaw in zebrafish using gene knockdown by the injection of an antisense morpholino oligonucleotide using transgenic Tg(sp7:EGFP) and Tg(Fli1a:EGFP) EGFP reporter fish, as well as wildtype AB zebrafish. Our results demonstrate that zebrafish col11a1a knockdown impairs the cellular organization of Meckel’s cartilage in the developing jaw and alters the bone formation that occurs adjacent to the Meckel’s cartilage. These results suggest roles for Col11a1a protein in cartilage intermediates of bone development, the subsequent mineralization of the bony collar of long bones, and that which occurs adjacent to Meckel’s cartilage in the developing jaw.

Biochemical characterization of a disease-causing human osteoprotegerin variant

Recently, a human mutation of OPG was identified to be associated with familial forms of osteoarthritis. This missense mutation (c.1205A = > T; p.Stop402Leu) occurs on the stop codon of OPG, which results in a 19-residue appendage to the C-terminus (OPG+19). The biochemical consequence of this unusual sequence alteration remains unknown. Here we expressed OPG+19 in 293 cells and the mutant OPG was purified to homogeneity by heparin affinity chromatography and size exclusion chromatography. We found that in sharp contrast to wildtype OPG, which mainly exists in dimeric form, OPG+19 had a strong tendency to form higher-order oligomers. To our surprise, the hyper-oligomerization of OPG+19 had no impact on how it binds cell surface heparan sulfate, how it inhibits RANKL-induced osteoclastogenesis and TRAIL-induced chondrocytes apoptosis. Our data suggest that in biological contexts where OPG is known to play a role, OPG+19 functions equivalently as wildtype OPG. The disease-causing mechanism of OPG+19 likely involves an unknown function of OPG in cartilage homeostasis and mineralization. By demonstrating the biochemical nature of this disease-causing OPG mutant, our study will likely help elucidating the biological roles of OPG in cartilage biology.

Runx2 regulates chromatin accessibility to direct the osteoblast program at neonatal stages.

SUMMARYThe transcriptional regulator Runx2 (runt-related transcription factor 2) has essential but distinct roles in osteoblasts and chondrocytes in skeletal development. However, Runx2-mediated regulatory mechanisms underlying the distinctive programming of osteoblasts and chondrocytes are not well understood. Here, we perform an integrative analysis to investigate Runx2-DNA binding and chromatin accessibility ex vivo using neonatal osteoblasts and chondrocytes. We find that Runx2 engages with cell-type-distinct chromatin-accessible regions, potentially interacting with different combinations of transcriptional regulators, forming cell-type-specific hotspots, and potentiating chromatin accessibility. Genetic analysis and direct cellular reprogramming studies suggest that Runx2 is essential for establishment of chromatin accessibility in osteoblasts. Functional enhancer studies identify an Sp7 distal enhancer driven by Runx2-dependent binding and osteoblast-specific chromatin accessibility, contributing to normal osteoblast differentiation. Our findings provide a framework for understanding the regulatory landscape encompassing Runx2-mediated and cell-type-distinct enhancer networks that underlie the specification of osteoblasts.

Mutation of foxl1 Results in Reduced Cartilage Markers in a Zebrafish Model of Otosclerosis.

Bone diseases such as otosclerosis (conductive hearing loss) and osteoporosis (low bone mineral density) can result from the abnormal expression of genes that regulate cartilage and bone development. The forkhead box transcription factor FOXL1 has been identified as the causative gene in a family with autosomal dominant otosclerosis and has been reported as a candidate gene in GWAS meta-analyses for osteoporosis. This potentially indicates a novel role for foxl1 in chondrogenesis, osteogenesis, and bone remodelling. We created a foxl1 mutant zebrafish strain as a model for otosclerosis and osteoporosis and examined jaw bones that are homologous to the mammalian middle ear bones, and mineralization of the axial skeleton. We demonstrate that foxl1 regulates the expression of collagen genes such as collagen type 1 alpha 1a and collagen type 11 alpha 2, and results in a delay in jawbone mineralization, while the axial skeleton remains unchanged. foxl1 may also act with other forkhead genes such as foxc1a, as loss of foxl1 in a foxc1a mutant background increases the severity of jaw calcification phenotypes when compared to each mutant alone. Our zebrafish model demonstrates atypical cartilage formation and mineralization in the zebrafish craniofacial skeleton in foxl1 mutants and demonstrates that aberrant collagen expression may underlie the development of otosclerosis.