Aspects of collagen mineralization in hard tissue formation.

Collagen is the dominant fibrous protein not only in connective tissues but also in hard tissues, bone, dentin, cementum, and even the mineralizing cartilage of the epiphyseal growth plate. It comprises about 80-90% (by weight) of the organic substance in demineralized dentin and bone. When collagen fibers are arranged in parallel to form thicker bundles, as in lamellar bone and cementum, interior regions may be less mineralized; in dentin, however, the collagen fibers form a network and collagen fibers are densely filled with a mineral substance. In the biomineralization of collagen fibers in hard tissues, matrix vesicles play a fundamental role in the induction of crystal formation. The mineralization of matrix vesicles precedes the biomineralization of the collagen fibrils and the intervening ground substance. In addition, immobilized noncollagenous fibrous macromolecules, bound in a characteristic way to the fibrous collagen surface, initiate, more intensely than collagen, mineral nucleation in the hard tissue matrix.

Quantitative analyses of the biomineralization of different hard tissues.

The primary crystallites of the different developing hard tissues have an apatite structure. However, they have crystal lattice distortions representing an intermediate state between amorphous and fully crystalline. We have applied energy-filtering transmission electron microscopy in the selected area electron diffraction mode to analyse different stages of crystal formation in dentine, bone, enamel and inorganic apatite mineral. We have obtained quantitative information on the degree of crystal lattice distortion using the paracrystal theory of Hosemann and Bagchi. We have found that the early formed crystallites of the hard tissues being analysed have a paracrystalline character comparable to biopolymers. However, with maturation, the lattice fluctuations of the crystallites of the hard tissues bone, enamel and dentine decrease to form a typical (para)crystalline character. Also the decrease of the organic proportion in the matrix corresponds to the decrease of the lattice fluctuation of the crystallites in the different hard tissues during maturation.

Endochondral ossification of costal cartilage is arrested after chondrocytes have reached hypertrophic stage of late differentiation.

Late cartilage differentiation during endochondral bone formation is a multistep process. Chondrocytes transit through a differentiation cascade under the direction of environmental signals that either stimulate or repress progression from one step to the next. In human costal cartilage, chondrocytes reach very advanced stages of late differentiation and express collagen X. However, remodeling of the tissue into bone is strongly repressed. The second hypertrophy marker, alkaline phosphatase, is not expressed before puberty. Upon sexual maturity, both alkaline phosphatase and collagen X activity levels are increased and slow ossification takes place. Thus, the expression of the two hypertrophy markers is widely separated in time in costal cartilage. Progression of endochondral ossification in this tissue beyond the stage of hypertrophic cartilage appears to be associated with the expression of alkaline phosphatase activity. Costal chondrocytes in culture are stimulated by parathyroid hormone in a PTH/PTHrP receptor-mediated manner to express the fully differentiated hypertrophic phenotype. In addition, the hormone stimulates hypertrophic development even more powerfully through its carboxyterminal domain, presumably by interaction with receptors distinct from PTH/PTHrP receptors. Therefore, PTH can support late cartilage differentiation at very advanced stages, whereas the same signal negatively controls the process at earlier stages.

Quantitative electron spectroscopic diffraction analyses of the crystal formation in dentine.

Newly formed apatitic crystallites of different hard tissues consist, according to our investigations, of chains composed of nanometre-sized particles (islands, dots) arising at nucleating sites of the collagenous and noncollagenous matrix macromolecules. In dentine these islands coalesce rapidly in longitudinal direction to form needle-like crystallites which further coalesce to ribbon-like crystallites. We have concluded that the centre-to-centre distances between these islands represent the distances between the nucleating sites of the matrix macromolecules. We have applied energy-filtering transmission electron microscopy in the selected area electron diffraction mode at different stages of crystal formation in dentine and have obtained quantitative information of the degree of crystal disorder on the basis of the paracrystal theory. The fluctuation of the lattice plane distances in c-axis direction decreases, proceeding from the region near the dentine/predentine border to the dentine/enamel border.

Sutural mineralization of rat calvaria characterized by atomic-force microscopy and transmission electron microscopy.

The application of transmission electron microscopy (TEM) and atomic-force microscopy (AFM) aid the acquisition of detailed structural information on the process of hard tissue formation. The sutural mineralization of rat calvaria is taken as a model for a collagen-related mineralization system. After cryofixation or chemical fixation an anhydrous tissue preparation technique with no staining procedures is used. The atomic-force microscope and the transmission electron microscope are used for structural analysis of the mineralizing region of the sutural tissue. With the application of AFM the collagen macroperiod is shown to be well represented in the unmineralized sutural tissue. At the mineralization front the collagen fibrils are found to be thickened and to change to a characteristic stacked platelet structure. Using TEM the macroperiod is faintly visible before mineral crystallites have formed and is more prominent after the apatite crystallization has started in the fibrils. In this step a needle-like structure of the newly formed apatitic crystals is visible.

Potassium is involved in apatite biomineralization.

The biogenetic formation of mineral crystals, one aspect of biomineralization, is a multistep process of apatite formation throughout the growth of dentin tissue. An important step is the transformation of the non-mineralized predentin matrix to mineralizing dentin matrix and its biological control. In this study, the high capacity of elemental mapping is combined with single x-ray point measurements to elucidate whether special elements are involved in initiation or regulation of mineral nucleation. Directly at the mineralization front, micro-areas with a strong co-enrichment of phosphorus (e.g., as phosphate) and potassium are found. During the beginning of the calcium enrichment and the subsequent apatite mineral formation in the characteristic micro-areas, the content of potassium decreases significantly. These findings indicate that potassium is involved in the process of dentin mineralization.

General principle of ordered apatitic crystal formation in enamel and collagen rich hard tissues.

The biomineralization processes in different hard tissues like enamel, circumpulpal dentine, epiphyseal growth plates were analyzed morphologically and ultrastructurally by an energy filtering transmission electron microscope. In the primary stage of crystal formation Ca- and phosphate-ions accumulate at charged sites, "active sites", along the fiber matrix-molecules of the extracellular matrix. After exceeding the critical radius for nucleation, crystal nuclei appear that develop to "chains" of stable nanometer-sized paracrystalline particles. In the latest studies of small area electron diffraction it was found that in the earliest stage of crystal formation these mineral chains show a parallel orientation in the direction of the c-axis of apatite. This was supported by a texture of the 002 reflection in the corresponding diffraction patterns. Since apatite is bipolar in this direction crystal growth would be in like manner in both directions. Thus the center-to-center distances between nucleating sites along the matrix macromolecules show with the chains of nanometer islands the same process of biomineralization in the different mineralizing hard tissue systems. This way of crystal formation might be a general principle of apatitic biomineralization.

Quantitative electron-spectroscopic diffraction (ESD) and electron-spectroscopic imaging (ESI) analyses of dentine mineralisation in rat incisors.

Primary crystal formations in all hard tissues are, according to our investigations, Ca-phosphate chains composed of nanometer sized particles (dots) which develop along the matrix macromolecules. In circumpulpal dentine the centre-to-centre distances between the dots inside the chains reflect the distances between the crystal nucleating sites ("active sites") along the collagen matrix macromolecule. The centre-to-centre distances at the surface of the mineralised collagen fibrils probably reflect the distances between nucleating sites of noncollagenous proteins attached to collagen. These needle-like chains of dots coalesce in lateral directions to form ribbon-like crystallites. The morphological results are supported by correlated small area diffraction studies in the same regions of dentine. We have found that the first appearing Bragg-reflection has a lattice spacing value of 0.388 nm, which corresponds to the (111) apatite value. For the earliest crystal formations the intensity of the (002) reflection is higher than that of the (300)-reflection. A maximum of the net-signal-intensity ratio of the (002) to (300) Bragg-reflection appears at the mineralisation front. This peak repeats with decreasing height 3 to 5 times with a distance range of about 8-16 microm through the whole dentine zone, which corresponds to the distances of the incremental lines, called "von Ebner lines".

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

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 phosphorous 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. Two 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.

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.

Analysis of a general principle of crystal nucleation, formation in the different hard tissues.

We have found, at high EM magnification, on ultrathin sections of shock-frozen, freeze-dried, embedded pieces of the developing hard tissues, that the primary crystallites consist of strands composed of nanometer-sized apatitic islands, which rapidly coalesce to needles and afterward to platelets. By small-area electron diffraction, with energy-filtered electrons, it was clarified that these strands are already crystallographically oriented along the bipolar c-axis so that the center-to-center distances between the islands would reflect the distances between crystal-nucleating sites along the matrix. The EM analysis of the cross-cut stained unmineralized and of the unstained mineralized collagen fibers of turkey tibia tendon shows that the staining "nuclei" and the early crystallites, appearing as dark dots, surround "light" round structures, which we interpret as the collagen microfibrils, surrounded by the apatitic crystallites.

Early mineralization of matrix vesicles in the epiphyseal growth plate.

Matrix vesicles (MVs) induce the primary mineralization in collagen-rich hard tissues such as bone, mineralizing cartilage and dentine. Calcium and phosphate ions accumulate at the inner MV membrane. This accumulation takes place in association with phospholipids alone and/or in association with Annexin V, which displays Ca ion channel activity when inserted in membranes; consequently, Annexin V may be involved in Ca uptake by matrix vesicles. The first crystal nuclei are formed at these macromolecules of the MV inner membrane. They grow to stable nanometre-sized particles, dots, which coalesce to form chains of dots along the macromolecules of the MV inner membrane. At the same time, or shortly afterwards, chains of these Ca phosphate dots also develop inside the MVs. The measured centre-to-centre distances between these dots represent approximately the distances between the nucleating sites, called active sites, along the MV matrix molecules. The mineralization does not stop at the MV membrane but expands continuously into the extravesicular region in radial directions to form nodules. These radiating Ca phosphate chains, which coalesce to form needles, are composed of such primary dots, which have developed at the nucleating sites of the corresponding macromolecules.

Mineralization during matrix-vesicle-mediated mantle dentine formation in molars of albino rats: a microanalytical and ultrastructural study.

The purpose of this study was to elucidate the mineralization process of mantle dentine by ultrastructural and element-analytical investigation of matrix vesicles and successive stages. Upper second molars of albino rats were cryofixed and embedded in resin after freeze drying. Semithin dry sections were prepared for analyzing the calcium and phosphorus concentrations in the mineralized matrix vesicles or noduli, larger mineralized islands, and the mantle dentine. For ultrastructural studies, it was necessary to reduce section contact with hydrous fluids to a minimum in order to avoid preparation artifacts. The first mineral deposits were recognized as dot-like formations both in the interior of matrix vesicles and in association with the inner vesicle membrane. This indicated the existence of mineral nucleating sites located both at the inner membrane and at calcium-phosphate-binding macromolecules in the interior of the matrix vesicles. A significantly higher mineral content was found in mineralized matrix vesicles than in the mineralized extravesicular regions of the mineralized islands, suggesting the existence of a rapidly and densely mineralized matrix in the matrix vesicles. A significant increase in mineral content per volume proceeding from the mineralized islands to mantle dentine suggested a further increase in the density of mineral.

[Neuroblastoma: diagnosis and therapy]. / Neuroblastom: Diagnostik und Therapie.

Prognosis of neuroblastoma is primarily dependent on the stage of disease. While stages 1 to 3 show a survival rate of 94 to 66%, stage 4 has a survival rate below 20%. The most important prognostic factor is the extent of disease. Age, LDH, resectability of primary tumor, cytologic and molecular parameters are defined to have a prognostic impact as well. A stage- and risk-adapted therapy of neuroblastoma needs a thorough assessment of all these factors.

Structural relationship between the primary crystal formations and the matrix macromolecules in different hard tissues. Discussion of a general principle.

For many years we have investigated the earliest crystal formations of different developing hard tissues (matrix vesicle, bone, dentine, enamel, etc.) by different electron microscopic measurements. It was observed that primarily Ca-phosphate (apatite) "chains," composed of nanometer sized particles (dots, islands), exist, which coalesce rapidly to needles. For the mineralization of collagen (e.g., bone, dentine) the center to center distances between the dots in the mineral chains represent the distances between nucleating sites, so-called "active sites" of collagen which bind primarily Ca for a subsequent nucleation. For the mineralization of noncollagen macromolecules (e.g., enamel) the same principle of mineral nucleation at such "active sites" exists being represented indirectly by corresponding center to center distances between the dots in the mineral chains.

Antidisialoganglioside ricin A-chain immunotoxins show potent antitumor effects in vitro and in a disseminated human neuroblastoma severe combined immunodeficiency mouse model.

Several monoclonal antibodies (mAbs) were screened on different neuroblastoma cell lines to evaluate ricin A-chain immunotoxins for possible use against human neuroblastoma. Four mAbs were identified that exhibited high antitumor activity against neuroblastoma cell lines as measured in an indirect cytotoxicity assay. These mAbs, including 14G2a (antidisialoganglioside), ch14.18 (a humanized switch variant), BW704 (antidisialoganglioside), and chCE7 (anti-glycoprotein of M(r) 190,000), were subsequently linked via the bivalent linker N-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-piridyldithio++ +)toluene to deglycosylated ricin A chain. The most potent immunotoxin, 14G2a.dgA, inhibited the protein synthesis of neuroblastoma cell lines IMR5 and NMB by 50% at concentrations of 6 x 10(-12) M. To test the antitumor efficacy of these immunotoxins in vivo, we developed a disseminated human neuroblastoma model in severe combined immunodeficiency mice. Treatment of tumor-bearing mice with 14G2a.dgA 12 days after tumor challenge resulted in a significant prolongation of survival as compared with phosphate-buffered saline-treated controls (16.8 versus 6.5 weeks). We conclude that ricin A-chain immunotoxins might be of potential use in the treatment of human neuroblastoma.

Investigation of the early mineralisation on collagen in dentine of rat incisors by quantitative electron spectroscopic diffraction (ESD).

The earliest crystallites in dentine appear as chains of "dots" in ultra-thin sections viewed by transmission electron microscopy. These dots rapidly coalesce along the longitudinal directions of the collagen microfibrils to form needle-like structures that coalesce preferentially in lateral directions to form ribbon-like or plate-like crystallites. This morphological interpretation is supported by line-scans of the corresponding zero-loss filtered electron spectroscopic diffraction patterns, which demonstrate the crystalline structure of the dentine mineral (apatite). The intensity ratio of the Debye-Scherrer rings of the characteristic Bragg-reflections (002 to 300, together with 1 or 2 unresolved reflections) shows a maximum in the region of early chain-like and needle-like crystallites, decreasing with maturation of the dentine mineral to the ribbon-plate-like crystallites. Detailed investigations using line-scans of the zero-loss filtered electron spectroscopic diffraction patterns through the dentine zone show that the intensity ratio found near the mineralisation front is repeated 3-5 times at distances of about 10-20 microns. This may represent a circadian pattern of mineralisation corresponding to light microscopically visible incremental lines in dentine.

Morphological and structural studies of early mineral formation in enamel of rat incisors by electron spectroscopic imaging (ESI) and electron spectroscopic diffraction (ESD).

Morphological and structural analysis of the earliest stage of crystal formation in enamel of rat incisors, by use of energy filtering transmission electron microscopy (EFTEM), has shown needlelike crystallites with a dotlike substructure. We conclude that these dots (nanometer-sized particles) have developed at nucleating, active sites along the non-collagenous matrix proteins in enamel. Calcium and phosphate groups are bound at such "active sites" and develop to nuclei, which grow to these stable dots (nanometer-sized particles). The dots coalesce rapidly in longitudinal direction, along the matrix proteins, with neighbouring dots to form parallel arranged "needlelike" crystallites. These needles grow and coalesce in lateral directions to ribbon-platelike crystallites. In enamel most of the organic substance becomes decomposed and transported to the ameloblasts. Consequently, the ribbon-platelike crystallites can coalesce to form much thicker (hydroxy)-apatite crystals than in dentine. Already in the earliest stage of crystal formation the mineral chains of dots (nanometer-sized particles) and the needlelike crystallites show a parallel orientation in the direction of the c-axis of hydroxyapatite. This is supported by the texture of the 002 reflections in the corresponding electron spectroscopic diffraction patterns (ESD), which appear as the first Bragg reflections.

Analysis of early hard tissue formation in dentine by energy dispersive X-ray microanalysis and energy-filtering transmission electron microscopy.

Thin cryosections and sections of embedded tissue were prepared from dentine of cryofixed rat incisors. Energy dispersive X-ray microanalysis (EDX) and electron energy-loss spectroscopy (EELS) have been applied to study the calcium and phosphorus distribution in predentine of these incisors. A small enrichment of calcium and phosphorus was found in the predentine zone near the dentine border. Element distributions were correlated with analyses of the early crystal formation in dentine. These investigations were carried out by parallel applications of electron spectroscopic diffraction (ESD) and electron spectroscopic imaging (ESI) using zero-loss filtering. It was found that the earliest crystal formations already showed the lattice of the hexagonal mineral apatite. They form parallelly arranged chains of dots which coalesce rapidly to form "needle-like" crystallites along the collagen microfibrils.

Analysis of the calcium distribution in predentine by EELS and of the early crystal formation in dentine by ESI and ESD.

Predentine is a collagen-rich extracellular matrix between the odontoblasts and the dentine with a width of about 15-20 microns. Electron energy-loss spectroscopy of rat incisors shows a significantly higher calcium content in the predentine at the predentine-dentine border than in the middle region of the predentine. At the predentine-dentine border in the dentine, the calcium and the phosphate groups combine to form apatite crystallites. Electron spectroscopic diffraction with zero-loss filtering revealed that the earliest crystallites contain only Debye-Scherrer rings of apatite, which are fewer in number and more diffuse than the diffraction rings from the mature crystallites. We therefore conclude that the early crystallites still contain lattice defects, which are annealed out to some degree with crystal growth. Electron spectroscopic imaging with zero-loss filtering also showed that the earliest crystallites are chains of dots (or small islands); they build up strands composed of islands, which rapidly acquire a needle-like character and coalesce laterally to form ribbon-or plate-like crystallites. The parallel strands sometimes appear to reinforce the macroperiod of the collagen microfibrils (67 nm) by tiny holes without any crystal-substance lined up perpendicular to the parallel strands of the crystallites.