Role of the subchondral vascular system in endochondral ossification: endothelial cell-derived proteinases derepress late cartilage differentiation in vitro.

Endochondral ossification in growth plates proceeds through several consecutive steps of late cartilage differentiation leading to chondrocyte hypertrophy, vascular invasion, and, eventually, to replacement of the tissue by bone. The subchondral vascular system is essential for this process and late chondrocyte differentiation is subject to negative control at several checkpoints. Endothelial cells of subchondral blood vessels not only are the source of vascular invasion accompanying the transition of hypertrophic cartilage to bone but also produce factors overruling autocrine barriers against late chondrocyte differentiation. Here, we have determined that the action of proteases secreted by endothelial cells were sufficient to derepress the production of the hypertrophy-markers collagen X and alkaline phosphatase in arrested populations of chicken chondrocytes. Signalling by thyroid hormones was also necessary but endothelial factors other than proteinases were not. Negative signalling by PTH/PTHrP- or TGF-beta-receptors remained unaffected by the endothelial proteases whereas signalling by FGF-2 did not suppress, but rather activated late chondrocyte differentiation under these conditions. A finely tuned balance between chondrocyte-derived signals repressing cartilage maturation and endothelial signals promoting late differentiation of chondrocytes is essential for normal endochondral ossification during development, growth, and repair of bone. A dysregulation of this balance in permanent joint cartilage also may be responsible for the initiation of pathological cartilage degeneration in joint diseases.

Terminal differentiation of chondrocytes is arrested at distinct stages identified by their expression repertoire of marker genes.

During endochondral bone formation, cells in the emerging cartilaginous model transit through a cascade of several chondrocyte differentiation stages, each characterized by a specific expression repertoire of matrix macromolecules, until, as a final step, the hypertrophic cartilage is replaced by bone. In many permanent cartilage tissues, however, late differentiation of chondrocytes does not occur, due to negative regulation by the environment of the cells. Here, addressing the reason for the difference between chondrocyte fates in the chicken embryo sternum, cells from the caudal and cranial part were cultured separately in serum-free agarose gels with complements defined earlier that either permit or prevent hypertrophic development. Total RNA was extracted using a novel protocol adapted to agarose cultures, and the temporal changes in developmental stage-specific mRNA expression were monitored by Northern hybridization and phosphor image analysis. Kinetic studies of the mRNA accumulation not only showed significant differences between the expression patterns of cranial and caudal cultures after recovery, but also revealed two checkpoints of chondrocyte differentiation in keeping with cartilage development in vivo. Terminal differentiation of caudal chondrocytes is blocked at the late proliferative stage (stage Ib), while the cranial cells can undergo hypertrophic development spontaneously. The differentiation of cranial chondrocytes is reversible, since they can re-assume an early proliferative (stage Ia) phenotype under the influence of insulin, fibroblast growth factor-2 and transforming growth factor-beta in combination. Thus, the expression pattern in the latter culture resembles that of articular chondrocytes. We also provide evidence that the capacities of caudal and sternal chondrocytes to progress from the late proliferative (stage Ib) to hypertrophic stage (stage II) correlate with their differing abilities to express the Indian hedgehog gene.

Absence of the alpha1(IX) chain leads to a functional knock-out of the entire collagen IX protein in mice.

Cartilage fibrils contain collagen II as well as smaller amounts of collagens IX and XI. The three collagens are thought to co-assemble into cartilage-specific arrays. The precise role of collagen IX in cartilage has been addressed previously by generating mice harboring an inactivated Col9a1 gene encoding the alpha1(IX) chain, i.e. one of the three constituent chains of collagen IX (Fässler, R., Schnegelsberg, P. N. J., Dausman, J., Shinya, T., Muragaki, Y., McCarthy, M. T., Olsen, B. R., and Jaenisch, R. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5070-5074). The animals did not produce alpha1(IX) mRNA or polypeptides and were born with no conspicuous skeletal abnormality but post-natally developed early onset osteoarthritis. Here we show that the deficiency in alpha1(IX) chains leads to a functional knock-out of all polypeptides of collagen IX, whereas the Col9a2 and Col9a3 genes were normally transcribed. Therefore, synthesis of alpha1(IX) polypeptides is essential for the assembly of heterotrimeric collagen IX molecules. Surprisingly, cartilage fibrils of all shapes and banding patterns found in normal newborn, adolescent, or adult mice were formed in transgenic animals, although they lacked collagen IX. Therefore, collagen IX is not essential, and may be functionally redundant, in fibrillogenesis in cartilage in vivo. The protein is required, however, for long term tissue stability, presumably by mediating interactions between fibrillar and extrafibrillar macromolecules.

[Clinical importance of hepatitis B virus DNA detection in serum of children with chronic hepatitis B]. / Klinische Bedeutung des Hepatitis-B-Virus-DNS-Nachweises im Serum von Kindern mit chronischer Hepatitis B.

206 sera from 172 children with chronic hepatitis B infection were tested for HBV DNA by dot blot hybridization. 111 were positive and 95 negative for HBV DNA. 103 (78.6%) of the positive patients had HBeAg and 5 (7.7%) anti-HBe. In 60 (92.3%) of the anti-HBe positive sera no HBV DNA could be detected. Children with elevated liver enzymes had HBV DNA in 80.1%, whereas in 71.6% of the chronic HBsAg carriers with normal liver enzymes no HBV DNA was found. In 87 of the 95 dot blot negative patients polymerase chain reaction was performed. 73 (83.9%) children of this group were HBV DNA positive. All HBeAg positive patients and those with elevated aminotransferases had HBV DNA in their serum. 56 anti-HBe-positive HBsAg carriers were also positive; 14 were negative for HBV DNA. Our results demonstrate that viral sequences can be found in all HBeAg positive and in most of the anti-HBe positive children. Patients with ongoing virus replication have to be considered infectious and recommendation for vaccination of close relatives of these patients must be stressed.

Use of the polymerase chain reaction to demonstrate hepatitis B virus DNA in serum of children with chronic hepatitis B.

The polymerase chain reaction was used to investigate the presence of hepatitis B virus DNA in sera of 61 children with chronic hepatitis B and negative results on dot blot hybridization tests. Our results demonstrate that most chronic carriers of hepatitis B surface antigen in childhood have hepatitis B virus DNA detectable by polymerase chain reaction in their serum and must be considered infectious.

Detection of hepatitis B virus DNA by polymerase chain reaction in serum and liver of children with chronic hepatitis B negative for hepatitis B virus DNA by conventional hybridization tests.

Hepatitis B virus (HBV) DNA was detected by polymerase chain reaction in the serum of 87 and liver tissue of 40 children with chronic hepatitis B, negative for HBV DNA by dot blot and Southern blot hybridization, respectively. In sera HBV DNA could be detected in 73 hepatitis B surface antigen carriers; 14 were hepatitis B e antigen (HBeAg), 56 were anti-HBe-seropositive and 3 had neither HBeAg nor positive anti-HBe. In 14 anti-HBe-positive patients no HBV DNA could be found. Viral sequences in liver tissue were present in 33 specimens; 20 were HBeAg and 13 were anti-HBe-seropositive. All of the 7 negative children had anti-HBe. Our results confirm polymerase chain reaction to be a more sensitive method to detect HBV DNA in the liver compared with conventional hybridization techniques. Every HBeAg-positive carrier as well as the majority of anti-HBe-positive patients show ongoing viral replication. This is of special clinical relevance, because these children must be considered infectious.