Ahnders Franzén

Ultrastructural distribution of osteoadherin in rat bone shows a pattern similar to that of bone sialoprotein

Osteoadherin (OSAD) is a keratan sulfate proteoglycan recently isolated from bovine and rat bone. Based on results obtained from in vitro experiments, the protein was shown to bind osteoblasts via the integrin receptor avb3. Due to OSADs capacity to bind hydroxyapatite crystals, a role for the protein in the mineralization process has also been suggested. To test these hypotheses in an in vivo model, the ultrastructural localization of OSAD in bone, tibial (metaphyses and diaphyses), and calvarial samples from normal 10 to 12-day-old rats were examined by immunohistochemical techniques at the ultrastructural level. In addition to the qualitative studies, quantitative measurements of OSAD marker density were performed in relevant compartments. Immunolabeling for OSAD was located to the mineralized bone matrix, with highest concentration of marker at the border between bone and cartilage remnants in the metaphyseal trabeculi. Intracellular labeling was low and no systemic accumulation of OSAD markers was observed at the cell-matrix interface. The observed distribution pattern of OSAD is strikingly similar to that of bone sialoprotein (BSP), confirmed by double labeling. The results of the current study support a role for OSAD in the mineralization process. In this process BSP is assumed to be a nucleator of hydroxyapatite crystals, and OSAD could work in concert with BSP to regulate nucleation. However, the mechanisms involved remain to be elucidated.

Altered vascular remodeling in osteopontin-deficient atherosclerotic mice.

Background: Osteopontin (OPN) is a cell-binding phosphoprotein with proposed functions in atherosclerosis. The aim of this study was to examine how OPN deficiency affects the atherosclerotic process. Methods: ApoE/LDL receptor/OPN triple knockout (ALO) mice were generated by crossing OPN null mice with ApoE/LDL receptor-deficient (AL) mice. Analysis were made on tissue sections from the aortic arch of 8-, 20- and 34-week female AL and ALO mice and included morphometric measurements, collagen staining, TUNEL staining and immunohistochemistry with antibodies to OPN, macrophages and proliferating cellular nuclear antigen (PCNA). Results: Lesion and media areas were significantly smaller and collagen accumulation in lesions was significantly reduced in 34-week-old ALO mice compared with AL mice. The numbers of proliferating and apoptotic cells were increased in lesions of 34 weeks old ALO mice. Furthermore, the plasma levels of SAA and total cholesterol were significantly decreased in 34 weeks old ALO mice. Conclusions: The present study shows that OPN deficiency reduces atherogenesis in atherosclerotic mice. The results corroborate and extend recently published findings and also include novel data on the role of OPN in the process of remodeling, inflammation and lipid metabolism.

Acceleration of Cartilage and Bone Differentiation on Collagenous Substrata

Chondroprogenitor cells of newborn murine mandibular condyles were cultured on top of collagen sponges for up to 18 days. After 24 h in culture, new chondroblasts developed which subsequently matured showing signs of hypertrophy, while the extracellular matrix revealed positive reactivity for type II collagen, cartilage proteoglycans and mineralization. Light and electron microscopy examinations showed signs of new osteoid formation, a feature that was preceded by positive immunohistochemical reaction for type I collagen, fibronectin and bone specific sialoprotein. A close temporal and spatial association was noted between the development of mature, mineralized cartilage and new osteoid. The differentiation of new cartilage and bone cells was linked to an increased activity of DNA synthesis and cellular proliferation. The de novo bone formation was accompanied by increasing rates of alkaline phosphatase activity and uptake of [45Ca] features that were found to be tightly correlated to each other. The collagen substrata appeared also to facilitate the migration of cells, their replication and their subsequent differentiation to their respective cellular lineage. Hence, collagen sponges in vitro appear to serve as a promising substrata for culture systems involved with the growth and differentiation of mineralizing tissues such as cartilage and bone.

Transformation of fetal secondary cartilage into embryonic bone in organ cultures of human mandibular condyles

Mandibular condyles of human fetuses, 14–21 weeks in utero, were kept in an organ culture system for up to 60 days. After 6 days in culture, the cartilage of the mandibular condyle appeared to have maintained its inherent structural characteristics, including all its various layers: chondroprogenitor, chondroblastic, and hypertrophic. After 12 days in culture, no chondroblasts could be seen; instead, the entire cartilage was occupied by hypertrophic chondrocytes. At the same time, the mesenchymal cells in the vicinity of the chondroprogenitor zone differentiated into osteoblast-like cells that produced type I collagen. The progenitor cells were still actively incorporating 3H-thymidine. The newly formed osteoid-like tissue lacked both metachromatic reactivity and a response to antibodies against chondroitin sulfate. Instead, the tissue reacted positively for osteocalcin (bone gla-protein). The process of new bone formation further progressed and, by the 20th day in culture, the new bone reacted positively for type I collagen, osteonectin, and to a lesser extent for chondroitin sulfate. The osteoid also underwent mineralization as revealed by both the von Kossa stain and vital staining with tetracycline. The above feature appeared even more intense in 40-day-old cultures. After 60 days, the newly formed bone contained osteoblasts and osteocytes, whereas the extracellular matrix revealed a high degree of matrix polarization. The results of the present study recapitulate findings reported for organ cultures of mice mandibular condyles. However, the in vitro process of de novo bone formation in human specimens requires a 6-fold longer culture time than that needed for mice condyles.

Possible recruitment of osteoblastic precursor cells from hypertrophic chondrocytes during initial osteogenesis in cartilaginous limbs of young rats

The appearance of the bone phenotype during rat embryogenesis was studied by in situ hybridization using a cDNA clone to osteopontin. Radiolabeled sense and antisense RNA probes were prepared from the osteopontin cDNA by in vitro transcription. The probes were used to hybridize paraffin sections of the cartilaginous diaphysis from embryonic rats at day 17 of gestation. The hybridization pattern was analyzed by autoradiography. Hybridization with the antisense probe gave patterns of silver grain labeling, indicating the presence of osteopontin mRNA among the hypertrophic chondrocytes. No silver grains could be detected in the corresponding region following hybridization of consecutive sections with the sense probe, showing the specificity of the technique being used. Whether these results indicate that the osteopontin gene is transiently expressed by hypertrophic chondrocytes or that osteopontin is an early marker for osteoblastic precursor cells will have to be explored further.

Zonal rate centrifugation of proteoglycans in sucrose gradients

Sensitive detection of proteoglycan and protein in concentrated sucrose solutions is obtained by measuring the absorbance at 206 nm. This method for detection has been used to study conditions affecting zonal rate sedimentation of proteoglycans and proteins in sucrose gradients. The ionic environment in the gradients markedly affects the sedimentation rate of the polyanionic proteoglycans and fragments thereof, while proteins sediment at an approximately constant rate irrespective of the ionic strength. The procedure can be used to separate proteoglycan aggregates from monomers and to determine size distributions of the compounds. In separate experiments, intact and fragmented proteoglycans are separated from proteins.

ALTERED VASCULAR REMODELING IN OSTEOPONTIN DEFICIENT ATHEROSCLEROTIC MICE

BACKGROUND Osteopontin (OPN) is a cell-binding phosphoprotein with proposed functions in atherosclerosis. The aim of this study was to examine how OPN deficiency affects the atherosclerotic process. METHODS ApoE/LDL receptor/OPN triple knockout (ALO) mice were generated by crossing OPN null mice with ApoE/LDL receptor-deficient (AL) mice. Analysis were made on tissue sections from the aortic arch of 8-, 20- and 34-week female AL and ALO mice and included morphometric measurements, collagen staining, TUNEL staining and immunohistochemistry with antibodies to OPN, macrophages and proliferating cellular nuclear antigen (PCNA). RESULTS Lesion and media areas were significantly smaller and collagen accumulation in lesions was significantly reduced in 34-week-old ALO mice compared with AL mice. The numbers of proliferating and apoptotic cells were increased in lesions of 34 weeks old ALO mice. Furthermore, the plasma levels of SAA and total cholesterol were significantly decreased in 34 weeks old ALO mice. CONCLUSIONS The present study shows that OPN deficiency reduces atherogenesis in atherosclerotic mice. The results corroborate and extend recently published findings and also include novel data on the role of OPN in the process of remodeling, inflammation and lipid metabolism.