Thomas Szuwart

Magnesium in newly formed dentin mineral of rat incisor.

Small amounts of magnesium are always detectable in addition to calcium and phosphorus in mineralized tissues such as dentin or bone. Magnesium has been considered to influence the mineralization process, especially crystal growth. The present study reports on the location and enrichment of magnesium in the newly mineralized dentin by using the high lateral resolution of energy dispersive X‐ray microanalysis combined with scanning transmission electron microscopy. To this end, we have used the continuously growing rat incisor as a model for a collagenous mineralizing system. Dental tissue was dissected free and cryofixed in liquid nitrogen–cooled propane. The distribution of elements was measured in freeze‐dried ultrathin cryosections. The magnesium distribution of the newly formed dentin area near the predentin area was found to be inhomogeneous. In certain small dentin areas, characteristical magnesium enrichments were observed. Further, high magnesium‐to‐phosphate molar ratios were found in these areas, and these were correlated with low calcium‐to‐phosphate molar ratios. Our results support the theory that magnesium is involved in the process of biological apatite crystal formation.

Periosteally derived osteoblast-like cells differentiate into chondrocytes in suspension culture in agarose

Pluripotent cells from the periosteal layer adjacent to cortical bone attain an osteoblast‐like phenotype in culture when reaching confluence in monolayer. It is unknown whether such newly differentiated osteoblast‐like cells preserve the chondrogenic potential characteristics for stem cells derived from the periosteum. Primary osteoprogenitor cells derived from bovine metacarpal periosteum were differentiated into alkaline phosphatase‐positive osteoblast‐like cells by an established monolayer culture protocol. After transfer into suspension culture in agarose gels, the cells differentiated into chondrocytes demonstrated by the production of collagen II, but not of collagen I, as well as alkaline phosphatase activity was abated. Contrarily, with continuation of monolayer culture, the cells maintained their osteoblast‐like phenotype and secreted large amounts of collagen I and a minor quantity of collagen III and V. The alkaline phosphatase activity steadily increased during the entire culture period of 2 weeks. Thus, our culture techniques can serve as useful tools to study mechanisms of differentiation by modulating the phenotypic potential of osteogenic cells. The results presented here support the notion that the extracellular environment strongly influences the cell type and its metabolism. Anat Rec 259:124–130, 2000.

The mineralization of mantle dentine and of circumpulpal dentine in the rat: an ultrastructural and element-analytical study

Abstract The purpose of this study was to compare the biomineralization of circumpulpal dentine with that of mantle dentine by ultrastructural and element-analytical techniques. Forty upper second molar germs of 10-day-old albino rats were cryofixed in liquid nitrogen-cooled propane and embedded in resin after freeze drying. Semithin dry sections were cut for analyzing the calcium and phosphorus concentration in initial mantle dentine, at the mineralization front of circumpulpal dentine, in the middle region of circumpulpal dentine and in mantle dentine peripheral to circumpulpal dentine. For the morphological evaluation of mineral deposits we compared ultrathin and unstained sections of cryofixed molars with chemically fixed molars. For both dentine types it was found that they develop via identical steps of mineral formation at collagen fibrils and non-collagenous matrix molecules. In circumpulpal dentine no globular mineral protrusions along the mineralization front (i.e. calcospherites) and no indications of interglobular dentine at the transition from circumpulpal dentine to mantle dentine were present. The von Korff fibres were not only visible in mantle dentine but also in circumpulpal dentine. Matrix vesicles were present only during the formation of an initial coherent layer of mantle dentine and could not be observed during successive formation of mantle dentine and circumpulpal dentine. The element-analytical data did not demonstrate any difference in the mineral content between the two dentine types. Therefore, we conclude that mantle dentine and circumpulpal dentine in the rat molar possess a high degree of structural and chemical similarity and that only the extent of terminal branching of the odontoblast processes gives an approximate estimation of the thickness of mantle dentine.

An in vitro study of osteoblast vitality influenced by the vitamins C and E

Vitamin C and vitamin E are known as important cellular antioxidants and are involved in several other non-antioxidant processes. Generally vitamin C and vitamin E are not synthesized by humans and therefore have to be applied by nutrition. The absence or deficiency of the vitamins can lead to several dysfunctions and even diseases (e.g. scurvy). The main interest in this study is that vitamin C and E are known to influence bone formation, e.g. vitamin C plays the key role in the synthesis of collagen, the major component of the extracellular bone matrix.In the present study we evaluate the effect of ascorbic acid (vitamin C) and α-tocopherol (vitamin E) on the proliferation and differentiation of primary bovine osteoblasts in vitro. Starting from standard growth medium we minimized the foetal calf serum to reduce their stimulatory effect on proliferation.An improved growth and an increased synthesis of the extracellular matrix proteins collagen type I, osteonectin and osteocalcin was observed while increasing the ascorbic acid concentration up to 200 μg/ml. Furthermore the effects of α-tocopherol on cell growth and cell differentiation were examined, whereby neither improved growth nor increased synthesis of the extracellular matrix proteins collagen type I, osteonectin and osteocalcin were detected.Further investigations are necessary to target at better supportive effect of vitamins on bone regeneration, and healing.

Light microscopic observations on the ossification process in the early developing pedicle of fallow deer (Dama dama)

The ossification process of the early developing pedicle was studied in five male fallow deer fawns, aged about seven months. The incipient pedicle was covered by a periosteum, the cambial layer of which was significantly thicker at the apex of the outgrowth than in the more peripheral areas of the pedicle. As was demonstrated histologically, in the central part of the pedicle elongation occurred by a process corresponding to endochondral ossification, whereas in the more peripheral areas the pedicle became enlarged by typical intramembranous ossification. Thus, cartilage formation must be regarded as a normal feature in pedicle growth of fallow deer. The assumption that the transition from pedicle to first antler growth in cervids is reflected by a switch from intramembranous ossification to chondrogenesis at the apex of the growing primary cranial appendage, based mainly on observations in roe deer, does, therefore, not hold for fallow deer. Furthermore, histogenesis of the central part of the fallow deer pedicle closely resembles the developmental events leading to formation of subsequent antlers.

A light microscopic study of primary antler development in fallow deer (Dama dama)

Summary The ossification process of primary antlers was studied in five fallow bucks aged about ten months. In the growing primary antler the following tissue zones could be distinguished histologically in a disto-proximal direction: a proliferative zone, a zone of cartilage maturation and hypertrophy, a zone of cartilage mineralization and degeneration, a zone of primary spongiosa and a zone of secondary spongiosa. Although the strict disto-proximal zonation of tissue changes typical for endochondral ossification of somatic cartilage was not seen in primary antler formation, the histological and histochemical findings clearly demonstrated that this process can be best described as a modified form of endochondral ossification. Our study produced no evidence for a direct (metaplastic) conversion af cartilage into bone during primary antler development. Generally, the histogenesis of primary antlers closely resembles the process of secondary antler growth as described by others. Minor differences between the two processes can be ascribed to the fact that during secondary antler formation a much bigger structure has to be built up in an even shorter time span than in the case of primary antler development and, therefore, the processes of cartilage formation, cartilage destruction and bone remodelling interdigitate even more closely during subsequent antler growth, compared to that of primary antlers. Our study clearly revealed that the transition from pedicle to first antler growth in fallow deer cannot be defined as a change in the ossification pattern, as was previously assumed for cervids in general.

Histochemical and ultrastructural studies of cartilage resorption and acid phosphatase activity during antler growth in fallow deer (Dama dama)

Cartilage resorption in forming primary fallow deer antlers was studied by histochemistry and electron microscopy. A high activity of tartrate‐resistant acid phosphatase (TRAP), a histochemical marker of skeletal resorbing cells, was first detected in cells located in the mesenchymal tissue separating the columns of hypertrophic cartilage. No cartilage resorption was observed in this region. Intense TRAP staining occurred in large multinucleated cells (identified as inactive osteoclasts) as well as in smaller cells (regarded as mononuclear osteoclast progenitors). On the basis of these findings it was concluded that this was the region where osteoclasts differentiated from progenitor cells. Further proximally, the mineralized cartilage was eroded by active osteoclasts that were located in Howships lacunae and exhibited an intense TRAP staining. Electron microscopy showed that the cells identified as inactive osteoclasts lacked a polarized organization. In contrast, the active osteoclasts in the zone of cartilage resorption exhibited a typical polarized organization: the nuclei congregated near the basolateral cell surface, and there was a zone of deep membrane infoldings (ruffled border) surrounded by a clear zone at the apical cell pole adjacent to the resorption surface of the mineralized cartilage. The multinucleated cartilage‐resorbing cells of the forming antler thus exhibited the typical histochemical and morphological features of active mammalian osteoclasts. Low levels of TRAP activity were also observed in hypertrophic chondrocytes; however, the specificity and potential significance of this staining remain to be elucidated. Anat Rec 268:66–72, 2002.

Ultrastructural aspects of cartilage formation, mineralization, and degeneration during primary antler growth in fallow deer (Dama dama)

Due to their rapid growth, regular replacement and easy accessibility, deer antlers are considered a useful model for the study of cartilage and bone differentiation and mineralization in mammals. The present study describes, for the first time, the cellular and extracellular matrix changes associated with cartilage formation, mineralization and degeneration in primary antlers on the ultrastructural level. Growing primary antlers of 3 to 4 cm length were obtained from six fallow bucks, aged about 10 months. It was shown that the chondroblasts were derived from progenitor cells of the antler perichondrium and differentiated into mature chondrocytes that subsequently underwent hypertrophic changes. Concomitant with cell hypertrophy, formation of a lacunar and a perilacunar extracellular matrix was observed, the latter containing numerous collagenous fibers. Mineralization of the extracellular matrix occurred via matrix vesicles and the formation of apatite crystals at distinct sites of the collagenous fibers. The hypertrophic chondrocytes of the mineralized cartilage then degenerated, a process that was also occasionally observed in more distally located cells surrounded by still unmineralized matrix. No morphological indications of a transdifferentiation of hypertrophic chondrocytes into bone forming cells, i.e., co-occurrence of a degenerating chondrocyte and a viable osteogenic cell in intact lacunae, were found. The cellular and extracellular matrix changes seen in primary antlers resemble those described for secondary antlers. Our results further indicate that the hypertrophic chondrocytes of primary antlers eventually undergo apoptosis, thereby providing further evidence that metaplastic conversion of cartilage into bone does not play a role in antler growth.

Impact of testosterone on the expression of organic anion transporting polypeptides (OATP-1A2, OATP-2B1, OATP-3A1) in malignant and non-malignant human breast cells in vitro

OBJECTIVES Postmenopausal hormone therapy (HT) increases local estrogen formation in breast tissue. The enzymatic substrates depend on transmembrane anion transporting polypeptides (OATPs) to reach intracellular enzymes. The aim of this study was to investigate the effect of testosterone (T) on the expression of OATP-1A2, OATP-2B1, and OATP-3A1 in malignant (MCF-7, BT-474) and non-malignant (HBL-100) breast cells in vitro. STUDY DESIGN Cells were incubated in RPMI 1640 medium containing 5% steroid-depleted fetal calf serum for 3d, and subsequently incubated in the absence or presence of T, anastrozole (A), and T+A (10(-6)M) for 24h at 37°C. MAIN OUTCOME MEASURES OATP expression was determined by immunocytochemical staining. Expression intensity was graded as low, moderate, or strong. Hormone receptor (AR, PR, ESR1, ESR2) expression was investigated by qPCR and Western blotting. Rank variance analysis was performed for statistical analysis (p≤0.05). RESULTS OATP-1A2, OATP-2B1, and OATP-3A1 expression was present in all untreated breast cell lines examined, with OATP-1A2 and OATP-3A1 being the predominant ones. There was a trend for a higher baseline expression in untreated HBL-100 and BT-474 in comparison to MCF-7 cells, which was significant for OATP-2B1. T treatment led to decreased OATP-1A2, -2B1, and -3A1 expression in BT-474 and HBL-100 cells, respectively. In contrast, in MCF-7 cells, OATP-2B1 expression was significantly increased. T-induced upregulation of AR and PR protein expression in BT-474 and MCF-7 cells was reduced by A treatment. CONCLUSIONS T may constitute a signal for differential regulation of mammary OATP expression. In non-malignant breast cells T seems to have a beneficial effect by reducing the availability of substrates for the intracellular formation of potent estrogens.

Eggshells as natural calcium carbonate source in combination with hyaluronan as beneficial additives for bone graft materials, an in vitro study

IntroductionIn bone metabolism and the formation especially in bone substitution, calcium as basic module is of high importance. Different studies have shown that the use of eggshells as a bone substitute material is a promising and inexpensive alternative. In this in vitro study, the effects of eggshell granulate and calcium carbonate towards primary bovine osteoblasts were investigated. Hyaluronan (HA) was used as artificial extracellular matrix (ECM) for the used cells to facilitate proliferation and differentiation and to mimic the physiological requirements given by the egg in vivo.MethodsHyaluronan, eggshells, a combination of hyaluronan and eggshells and CaCO3 were applied to the cells as additive to the used standard medium (modified High Growth Enhancement Medium) in a concentration of 0,1 g/l. The effect of the additives in the culture medium was examined by proliferation tests, immunohistochemical staining (anti-collagen type I, anti-osteopontin, anti-osteonectin and anti-osteocalcin) and kinetic oxygen measurements.ResultsOur investigations revealed that all investigated additives show beneficial effect on osteoblast activity. Cell proliferation, differentiation and the metabolic activity of the differentiated cells could be influenced positively. Especially in the case cell cultures treated with eggshells the strongest effects were detected, while for the hyaluronan compared with eggshells, a weaker increase in cell activity was observed.ConclusionIn summary, it can be stated that the investigated components come into consideration as beneficial supplements for bone graft materials especially for maxillo facial surgery application.