Martin Mörike

Expression of osteoblastic markers in cultured human bone and fracture callus cells

We compared the expression of osteoblastic markers in cultured human cells isolated from fracture calluses of various histological states of development with that in cells from adult and fetal bone. Adult osteoblasts and all callus cells produced almost exclusively type I collagen, whereas fetal osteoblasts produced also considerable amounts of type III collagen in vitro. 1,25-Dihydroxyvitamin D3 induced the synthesis of osteocalcin in all bone and callus cells but to varying extents. Fetal bone cells and early-stage callus cells synthesized less than 10% the amount of osteocalcin produced by adult bone cells. Late-stage callus cells produced intermediate levels of osteocalcin. Fetal bone cells and early-stage callus cells responded to parathyroid hormone with a less pronounced increase in intracellular cAMP than did adult bone cells. Late-stage callus cells showed the best response to parathyroid hormone. The activity of alkaline phosphatase was highest in fetal bone cells. These observations show that cells isolated from fetal bone and from fracture callus tissues express a pattern of markers clearly relating them to the osteoblastic lineage. On the basis of the different patterns of osteoblastic markers expressed in vitro we conclude that functionally distinct subtypes of osteoblasts do exist in different mineralized tissues and at different developmental stages.

Relationship of phosphorylation to the oligomerization of SV40 T antigen and its association with p53

The potential significance of the phosphorylation of SV40 large T antigen for oligomers and complexes with the cellular protein p53 was investigated. We observed that T antigen oligomers remain stable after enzymatic dephosphorylation by alkaline phosphatase up to 80%. Separate analysis of free and p53‐bound T antigen revealed a considerably lower phosphorylation of the p53‐bound subclass. Therefore, a simple correlation between the overall phosphorylation of T antigen and the formation of oligomers and T‐p53 complexes is highly unlikely.

146 PROLIFERATION AND COLLAGEN BIOSYNTHESIS OF CHONDROCYTES AND OSTEOBLASTS IN LETHAL SKELETAL DYSPLASIAS

We studied in vitro proliferation and collagen biosynthesis of chondrocytes and osteoblasts in thanatophoric dysplasia (TD), chondroectodermal dysplasia (CED), short rib polydaktyly-syndrome type III (SRP-III), short rib syndrome type Beemer (SR-Beemer) and osteogenesis imperfecta type II (OI-II). In TD morphologically proliferation zone is markedly reduced and in 2 out of 3 cases studied clonal growth of articular chondrocytes in methylcellulose was not or only slightly stimulated by IGF-I (1,25 ng/ml:.0%; 31% of control) and IGF-II (1,25 ng/ml:10%; 22% of control) but almost normally by TGF-β (1,25 ng/ml: 153%; 63% of control). In CED and SR-Beemer we found persistent hypertrophic cartilage islands in metaphyseal bone and in vitro elevated sensitivity of chondrocyte proliferation to TGF-β (1,25 ng/ml:376%; 213% of control). In OI-II osteoblasts synthesised electro-phoretically slower migrating collagen α1(I)-chains indicating posttranslational overmodification.We conclude that both, defects in matrix synthesis and regulation of chondrocyte proliferation play an important role in the pathogenesis of lethal skeletal dysplasias.

144 OSTEOGENESIS IMPERFECTA (OI): COLLAGEN METABOLISH OF OSTEOBLASTS IN VITRO

OI is characterized by disorders in collagen I metabolism. Biochemical aspects of OI have been studied mostly with skin fibroblasts although bone is the most affected tissue in OI. We studied collagen metabolism of osteoblasts in culture (OBC) derived from 30 OI patients (types I-IV). All OI OBC produced low lewels of collagen. Qualitative abnormal collagen I as determined by PAGE was found among all OI types. In single cases procollagen processing was slow. Many OI OBC showed a decreased pericellular accumulation of collagen I (coll.I).Conclusion: Decreased synthesis and pericellular accumulation of collagen was found associated with, but also irrespective of, obvious structural defects in collagen I and may play a crucial role in the phenotype of OI osteoblasts.