Bone morphogenetic protein rescues the lack of secondary cartilage in Runx2-deficient mice

Secondary cartilages including mandibular condylar cartilage have unique characteristics. They originate from alkaline phosphatase (ALP)-positive progenitor cells of the periosteum, and exhibit characteristic modes of differentiation. They also have a unique extracellular matrix, and coexpress type I, II and X collagens. We have previously shown that there is a total absence of secondary cartilages in Runx2-deficient (Runx2-/-) mice. To clarify whether Runx2 is essential for chondrocytic differentiation of secondary cartilages, we performed an organ culture system using mandibular explants derived from Runx2-/- mice at embryonic day 18.0. Since mRNA for bone morphogenetic protein 2 (BMP2) was strongly expressed in osteoblasts of condylar anlagen in wild-type mice, and was down-regulated in those of Runx2-/- mice, we chose to investigate BMP2 effects on secondary cartilage formation. Condensed mesenchymal cells of mandibular condylar anlagen in precultured explants were ALP-positive and expressed type I collagen and Sox9. After culture with recombinant human (rh) BMP2, chondrocytic cells showing ALP activity and expressing Sox5, Sox9, and type I and II collagens, appeared from mesenchymal condensation. This expression profile was comparable with the reported pattern of chondrocytes in mouse secondary cartilages. However, chondrocyte hypertrophy was not observed in the explants. These findings indicate that BMP2 partially rescued chondrocyte differentiation but not chondrocyte hypertrophy in secondary cartilage formation in Runx2-/- mice. Runx2 is required for chondrocyte hypertrophy in secondary cartilage formation, and it is likely that BMP2, which is abundantly secreted by osteoblasts in condylar anlagen, contributes to the early process of secondary cartilage formation.     

In situ hybridization and immunohistochemistry of bone sialoprotein and secreted phosphoprotein 1 (osteopontin) in the developing mouse mandibular condylar cartilage compared with limb bud cartilage

Mandibular condylar cartilage is often classified as a secondary cartilage, differing from the primary cartilaginous skeleton in its rapid progress from progenitor cells to hypertrophic chondrocytes. In this study we used in situ hybridization and immunohistochemistry to investigate whether the formation of primary (tibial) and secondary (condylar) cartilage also differs with respect to the expression of two major non‐collagenous glycoproteins of bone matrix, bone sialoprotein (BSP) and secreted phosphoprotein 1 (Spp1, osteopontin). The mRNAs for both molecules were never expressed until hypertrophic chondrocytes appeared. In the tibial cartilage, hypertrophic chondrocytes first appeared at E14 and the expression of BSP and Spp1 mRNAs was detected in the lower hypertrophic cell zone, but the expression of BSP mRNA was very weak. In the condylar cartilage, hypertrophic chondrocytes appeared at E15 as soon as cartilage tissue appeared. The mRNAs for both molecules were expressed in the newly formed condylar cartilage, although the proteins were not detected by immunostaining; BSP mRNA in the condylar cartilage was more extensively expressed than that in the tibial cartilage at the corresponding stage (first appearance of hypertrophic cell zone). Endochondral bone formation started at E15 in the tibial cartilage and at E16 in the condylar cartilage. At this stage (first appearance of endochondral bone formation), BSP mRNA was also more extensively expressed in the condylar cartilage than in the tibial cartilage. The hypertrophic cell zone in the condylar cartilage rapidly extended during E15–16. These results indicate that the formation process of the mandibular condylar cartilage differs from that of limb bud cartilage with respect to the extensive expression of BSP mRNA and the rapid extension of the hypertrophic cell zone at early stages of cartilage formation. Furthermore, these results support the hypothesis that, in vivo, BSP promotes the initiation of mineralization.     

Functional analysis of CTRP3/cartducin in Meckel's cartilage and developing condylar cartilage in the fetal mouse mandible

CTRP3/cartducin, a novel C1q family protein, is expressed in proliferating chondrocytes in the growth plate and has an important role in regulating the growth of both chondrogenic precursors and chondrocytes in vitro. We examined the expression of CTRP3/cartducin mRNA in Meckel's cartilage and in condylar cartilage of the fetal mouse mandible. Based on in situ hybridization studies, CTRP3/cartducin mRNA was not expressed in the anlagen of Meckel's cartilage at embryonic day (E)11.5, but it was strongly expressed in Meckel's cartilage at E14.0, and then reduced in the hypertrophic chondrocytes at E16.0. CTRP3/cartducin mRNA was not expressed in the condylar anlagen at E14.0, but was expressed in the upper part of newly formed condylar cartilage at E15.0. At E16.0, CTRP3/cartducin mRNA was expressed from the polymorphic cell zone to the upper part of the hypertrophic cell zone, but was reduced in the lower part of the hypertrophic cell zone. CTRP3/cartducin-antisense oligodeoxynucleotide (AS-ODN) treatment of Meckel's cartilage and condylar anlagen from E14.0 using an organ culture system indicated that, after 4-day culture, CTRP3/cartducin abrogation induced curvature deformation of Meckel's cartilage with loss of the perichondrium and new cartilage formation. Aggrecan, type I collagen, and tenascin-C were simultaneously immunostained in this newly formed cartilage, indicating possible transformation from the perichondrium into cartilage. Further, addition of recombinant mouse CTRP3/cartducin protein to the organ culture medium with AS-ODN tended to reverse the deformation. These results suggest a novel function for CTRP3/cartducin in maintaining the perichondrium. Moreover, AS-ODN induced a deformation of the shape, loss of the perichondrium/fibrous cell zone, and disorder of the distinct architecture of zones in the mandibular condylar cartilage. Additionally, AS-ODN-treated condylar cartilage showed reduced levels of mRNA expression of aggrecan, collagen types I and X, and reduced BrdU-incorporation. These results suggest that CTRP3/cartducin is not only involved in the proliferation and differentiation of chondrocytes, but also contributes to the regulation of mandibular condylar cartilage.     

An in situ hybridization study of the Syndecan family in the developing condylar cartilage of fetal mouse mandible

Mandibular condylar cartilage is a representative secondary cartilage, differing from primary cartilage in various ways. Syndecan is a cell-surface heparan sulfate proteoglycan and speculated to be involved in chondrogenesis and osteogenesis. This study aimed to investigate the expression patterns of the syndecan family in the developing mouse mandibular condylar cartilage. At embryonic day (E)13.0 and E14.0, syndecan-1 and -2 mRNAs were expressed in the mesenchymal cell condensation of the condylar anlage. When condylar cartilage was formed at E15.0, syndecan-1 mRNA was expressed in the embryonic zone, wherein the mesenchymal cell condensation is located. Syndecan-2 mRNA was mainly expressed in the perichondrium. At E16.0, syndecan-1 was expressed from fibrous to flattened cell zones and syndecans-2 was expressed in the lower hypertrophic cell zone. Syndecan-3 mRNA was expressed in the condylar anlage at E13.0 and E13.5 but was not expressed in the condylar cartilage at E15.0. It was later expressed in the lower hypertrophic cell zone at E16.0. Syndecan-4 mRNA was expressed in the condylar anlage at E14.0 and the condylar cartilage at E15.0 and E16.0. These findings indicated that syndecans-1 and -2 could be involved in the formation from mesenchymal cell condensation to condylar cartilage. The different expression patterns of the syndecan family in the condylar and limb bud cartilage suggest the functional heterogeneity of chondrocytes in the primary and secondary cartilage.     

In situ hybridisation study of type I, II, X collagens and aggrecan mRNAs in the developing condylar cartilage of fetal mouse mandible

The aim of this study was to investigate the developmental characteristics of the mandibular condyle in sequential phases at the gene level using in situ hybridisation. At d 14.5 of gestation, although no expression of type II collagen mRNA was observed, aggrecan mRNA was detected with type I collagen mRNA in the posterior region of the mesenchymal cell aggregation continuous with the ossifying mandibular bone anlage prior to chondrogenesis. At d 15.0 of gestation, the first cartilaginous tissue appeared at the posterior edge of the ossifying mandibular bone anlage. The primarily formed chondrocytes in the cartilage matrix had already shown the appearance of hypertrophy and expressed types I, II and X collagens and aggrecan mRNAs simultaneously. At d 16.0 of gestation, the condylar cartilage increased in size due to accumulation of hypertrophic chondrocytes characterised by the expression of type X collagen mRNA, whereas the expression of type I collagen mRNA had been reduced in the hypertrophic chondrocytes and was confined to the periosteal osteogenic cells surrounding the cartilaginous tissue. At d 18.0 of gestation before birth, cartilage-characteristic gene expression had been reduced in the chondrocytes of the lower half of the hypertrophic cell layer. The present findings demonstrate that the initial chondrogenesis for the mandibular condyle starts continuous with the posterior edge of the mandibular periosteum and that chondroprogenitor cells for the condylar cartilage rapidly differentiate into hypertrophic chondrocytes. Further, it is indicated that sequential rapid changes and reductions of each mRNA might be closely related to the construction of the temporal mandibular ramus in the fetal stage.     

Identification and location of bone-forming cells within cartilage canals on their course into the secondary ossification centre

Osteoblasts and osteocytes derive from the same precursors, and osteocytes are terminally differentiated osteoblasts. These two cell types are distinguishable by their morphology, localization and levels of expression of various bone cell-specific markers. In the present study on the chicken femur we investigated the properties of the mesenchymal cells within cartilage canals on their course into the secondary ossification centre (SOC). We examined several developmental stages after hatching by means of light microscopy, electron microscopy, immunohistochemistry and in situ hybridization. Cartilage canals appeared as extensions of the perichondrium into the developing distal epiphysis and they were arranged in a complex network. Within the epiphysis an SOC was formed and cartilage canals penetrated into it. In addition, they were successively incorporated into the SOC during its growth in the radial direction. Thus, the canals provided this centre with mesenchymal cells and vessels. It should be emphasized that regression of cartilage canals could never be observed in the growing bone. Outside the SOC the mesenchymal cells of the canals expressed type I collagen and periostin and thus these cells had the characteristics of preosteoblasts. Periostin was also expressed by numerous chondrocytes. Within the SOC the synthesis of periostin was down-regulated and the majority of osteoblasts were periostin negative. Furthermore, osteocytes did not secret this protein. Tissue-non-specific alkaline phosphatase (TNAP) staining was only detectable where matrix vesicles were present. These vesicles were found around the blind end of cartilage canals within the SOC where newly formed osteoid started to mineralize. The vesicles originated from osteoblasts as well as from late osteoblasts/preosteocytes and thus TNAP was only expressed by these cells. Our results provide evidence that the mesenchymal cells of cartilage canals express various bone cell-specific markers depending on their position. We suggest that these cells differentiate from preosteoblasts into osteocytes on their course into the SOC and consider that cartilage canals are essential for normal bone development within the epiphysis. Furthermore, we propose that the expression of periostin by preosteoblasts and several chondrocytes is required for adhesion of these cells to the extracellular matrix.     

Electroporation-mediated transfer of Runx2 and Osterix genes to enhance osteogenesis of adipose stem cells

In the present study, we tested the hypothesis that electroporation-mediated transfer of Runx2, Osterix, or both genes enhances the in vitro and in vivo osteogenesis from adipose stem cells (ASCs). ASCs were transfected with Runx2, Osterix, or both genes using electroporation, and further cultured in monolayer or in PLGA scaffold under osteogenic medium for 14 days, then analyzed for in vitro osteogenic differentiation. Transfected ASC-PLGA scaffold hybrids were also implanted on nude mice to test for in vivo ectopic bone formation. Runx2 and Osterix genes were strongly expressed in ASCs transfected with each gene on day 7, decreasing rapidly on day 14. Runx2 protein was strongly expressed in ASCs transfected with the Runx2 gene, while Osterix protein was strongly expressed in ASCs transfected with either or both Runx2 and Osterix genes. Overexpression of Runx2 and Osterix significantly increased the gene expression of osteogenic differentiation markers (alkaline phosphatase [ALP], osteocalcin [OCN], type I collagen [COL1A1], and bone sialoprotein [BSP]) in ASCs. Transfection of Runx2 and Osterix genes enhanced the protein expression of OCN, type I collagen, and BSP, as demonstrated by Western blot analysis, and ALP activity as well as enhancing mineralization in the monolayer culture and ASC-PLGA scaffold hybrids. Runx2- or Osterix-transfected ASC-PLGA scaffold hybrids promoted bone formation in nude mice after 6 weeks of in vivo implantation.     

Morphological influence of ascorbic acid deficiency on endochondral ossification in osteogenic disorder Shionogi rat

The influences of chronic deficiency of L-ascorbic acid (AsA) on the differentiation of osteo-chondrogenic cells and the process of endochondral ossification were examined in the mandibular condyle and the tibial epiphysis and metaphysis by using Osteogenic Disorder Shionogi (ODS) rats that bear an inborn deficiency of L-gulonolactone oxidase. Weanling male rats were kept on an AsA-free diet for up to 4 weeks, until the symptoms of scurvy became evident. The tibiae and condylar processes of scorbutic rats displayed undersized and distorted profiles with thin cortical and scanty cancellous bones. In these scorbutic bones, the osteoblasts showed characteristic expanded round profiles of rough endoplasmic reticulum, and lay on the bone surface where the osteoid layer was missing. Trabeculae formation was deadlocked, although calcification of the cartilage matrix proceeded in both types of bone. Scorbutic condylar cartilage showed severe disorganization of cell zones, such as unusual thickening of the calcification zone, whereas the tibial cartilage showed no particular alterations (except for a moderately decreased population of chondrocytes). In condylar cartilage, hypertrophic chondrocytes were encased in a thickened calcification zone, and groups of nonhypertrophic chondrocytes occasionally formed cell nests surrounded by a metachromatic matrix in the hypertrophic cell zone. These results indicate that during endochondral ossification, chronic AsA deficiency depresses osteoblast function and disturbs the differentiation pathway of chondrocytes. The influence of scurvy on mandibular condyle cartilage is different from that on articular and epiphyseal cartilage of the tibia, suggesting that AsA plays different roles in endochondral ossification in the mandibular condyle and long bones.     

miR-155有助于骨髓间充质干细胞成软骨分化

背景：近年发现,miRNA是能够对基因表达产生影响的一种新型调控子,miRNA有助于多能干细胞分化增殖以及自我更新。 目的：探讨miR-155对大鼠骨髓间充质干细胞成软骨分化的调控机制。 方法：12周龄健康SD大鼠60只,随机分为研究组与对照组,每组30只。于麻醉状态下将大鼠处死,获取下肢骨髓,进行骨髓间充质干细胞分离、培养,研究组给予miR-155基因模拟物转染,对照组给予阴性对照序列转染,经成软骨诱导分化后进行RT-PCR检测Sox9、CollagenⅡ、Aggrecan、Collagen X基因的表达,Western blot检测Sox9、Runx2蛋白的表达。 结果与结论：研究组Sox9、CollagenⅡ、Aggrecan基因表达高于对照组,Collagen X基因表达低于对照组,差异均有显著性意义(P < 0.05)。研究组Sox9蛋白表达高于对照组,Runx2蛋白表达低于对照组,差异均有显著性意义(P < 0.05)。结果表明miR-155不仅有助于骨髓间充质干细胞成软骨分化,而且可以抑制骨髓间充质干细胞成软骨分化呈肥大趋势发展。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

Association of the C-Propeptide of Type II Collagen with Mineralization of Embryonic Chick Long Bone and Sternal Development

Several proteins may play a role in bone formation. The C-propeptide of type II collagen is intimately associated with endochondral bone formation in bovine growth plate. We have used an antibody against this peptide to determine its immunofluorescent distribution in early stages of embryonic chick limb development with emphasis on first bone formation which occurs in the mid-diaphyseal region. The C-propeptide II is first evident by immunofluorescent localization at stage 27 (day 5-6) of embryonic tibia development with chondrocytes in the central mid-diaphysis. In subsequent stages, there is an increase in the number of chondrocytes in which it is localized in discrete vacuoles. Up to stage 30, immunofluorescence is observed intracellularly, after which it appears in the matrix. The released C-propeptide II appears to remain only transiently associated with the cartilage matrix and becomes concentrated in the calcifying periosteum, the region outside of the cartilage core where bone formation first occurs in a sequence of events comparable to intramembranous bone formation.

These observations can be reproduced in cultures of stage 35 hypertrophic chondrocytes (core cells) and periosteum cells (collar cells). Core cells contain intensely stained intracellular vacuoles while collar cells are negative, although the collar cell osteogenic matrix concentrates exogenously added C-propeptide II. Double label immuno-staining shows that the C-propeptide II, unlike type II collagen and proteoglycan, which are secreted and incorporated into extracellular sites, is initially stored in intracellular vacuoles. The matrix localization of the C-propeptide II during the transition from cartilage to bone indicates a close association with the initiation of mineralization events of cartilage and bone and its specific origin in chondrocytes and not osteoblasts. These observations suggest that the C-propeptide II made by chondrocytes is associated with the formation of bone.