Andrzej Wojtowicz

Alteration of Mineral Crystallinity and Collagen Cross-Linking of Bones in Osteopetrotic Toothless (tl/tl) Rats and Their Improvement After Treatment with Colony Stimulating Factor-1

A common feature of various types of mammalian osteopetroses is a marked increase in bone mass accompanied by spontaneous bone fractures. The toothless (tl/tl) rat osteopetrotic mutation is characterized by drastically reduced bone resorption due to a profound deficiency of osteoclasts and their precursors. An altered bone morphology has also been observed. The mutants cannot be cured by bone marrow transplantation, but skeletal defects are greatly reduced after treatment with colony stimulating factor 1 (CSF-1). The objectives of this study were to characterize mineral and collagen matrices in cancellous and compact bone isolated from long bones of 6-week-old normal littermates, tl/tl osteopetrotic mutants and mutants (tl/tl) treated with CSF-1. There were no differences in bone mineral content, but a significant decrease in the crystallinity of mineral evaluated by the method based on electron paramagnetic resonance spectrometry was observed in all bones of tl/tl mutants as compared to that of controls. Within the collagen matrix, slight decreases in the labile cross-links, but significant increases in the content of the stable cross-links, pyridinoline, and deoxypyridinoline, were observed in both cancellous and compact bone of osteopetrotic mutants. In tl/tl mutants treated with human recombinant CSF-1, the normalization of the crystallinity of bone mineral as well as collagen cross-links was found. Our results indicate that remodeling of bone matrix in tl/tl mutants is highly suppressed, but that after treatment with CSF-1, this activity recovers significantly. Taken together, these data provide further support for the hypothesis that CSF-1 is an essential factor for normal osteoclast differentiation and bone remodelling.

Expression of genes for bone morphogenetic proteins BMP-2, BMP-4 and BMP-6 in various parts of the human skeleton

BackgroundDifferences in duration of bone healing in various parts of the human skeleton are common experience for orthopaedic surgeons. The reason for these differences is not obvious and not clear.MethodsIn this paper we decided to measure by the use of real-time RT-PCR technique the level of expression of genes for some isoforms of bone morphogenetic proteins (BMPs), whose role is proven in bone formation, bone induction and bone turnover. Seven bone samples recovered from various parts of skeletons from six cadavers of young healthy men who died in traffic accidents were collected. Activity of genes for BMP-2, -4 and -6 was measured by the use of fluorescent SYBR Green I.ResultsIt was found that expression of m-RNA for BMP-2 and BMP-4 is higher in trabecular bone in epiphyses of long bones, cranial flat bones and corpus mandibulae then in the compact bone of diaphyses of long bones. In all samples examined the expression of m-RNA for BMP-4 was higher than for BMP-2.ConclusionIt was shown that m-RNA for BMP-6 is not expressed in the collected samples at all. It is postulated that differences in the level of activation of genes for BMPs is one of the important factors which determine the differences in duration of bone healing of various parts of the human skeleton.

BMP-4 and BMP-6 involvement in the osteogenic properties of the HeLa cell line.

The heterotopically induced ossicles are used in our research on bone tissue. The ossicles are formed in the thigh muscle of BALB/c mice under the influence of injected suspension of 3 × 106 HeLa cells. We postulate that the mechanism of bone induction is based on the secretion of bone morphogenetic proteins BMP-4 and BMP-6 by the grafted HeLa cells. This was proved by the use of specific immunohistochemical reaction and Western blots of conditioned culture medium. It seems that HeLa cells secrete BMPs continuously into the culture medium, even without contact with the mice muscle tissue, were induction of bone tissue is observed.

Persistence of birefringence and specific collagen cross-links in dentine of teeth of the ‘Nuraghi’ population living in Sardinia BC 1500–1200

Well preserved bones of the Nuraghi population, discovered in 1980, are deposited in the Department of Biomedical Sciences, Division of Human Anatomy of the University of Sassari, in Sardinia, Italy. The Giant Tomb belongs to the megalithic monuments constructed in the period BC 1500–1200. The origin, history and the fate of Nuraghi population is not well known. Many cone-shaped fortresses are found in Sicily and megalithic monuments for burial purposes were built in their proximity. Due to the good macroscopic preservation of the mineralized parts of the Nuraghi skeletons we decided to compare the histological and biochemical structure of the recovered Nuraghi teeth with contemporary ones. Crown dentine of four Nuraghi molar and four premolar teeth were analysed and compared with contemporary molars and premolars removed for orthodontic reasons from 17–40-year-old patients in the Dental School, University of North Carolina at Chapel Hill USA. Evaluation of the collagen cross-links of dentine collagen was performed using a HPLC technique to analyze for hydrolysates. Nonreducible, stable cross-links pyridinoline and its lysyl analogue were quantified by fluorescence (excitation 330 nm, emission 390 nm). The concentration of reducible, labile cross-links referred to as DHLNL and HLNL (dehydro-dihydroxylisinorleucine) were identified and calculated by chromatography after the application of NaB3H4 with integration of respective peaks and converting the numbers into a residue per mole of collagen basis. The ultrastructure of collagen was estimated by its persistent birefringence in polarized light after staining with Picrosirus Red. It was found that dentine collagen of Nuraghi teeth resisted environmental factors sufficiently well to preserve the molecular ultrastructure of collagen fibres, proved by their birefringence and by the preservation of some of the stable and reducible collagen cross-links, characteristic for skeletal tissues.