H. J. Höhling

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.

Electron microscopy and electron microscopical measurements of collagen mineralization in hard tissues

SummaryThe best focussed members of a through focus series of electron micrographs of ultra-thin sections of freeze-dried forming rat incisor dentine and glutaraldehyde-fixed 10 months old human femoral cortex were selected for measurements. The original micrographs were prepared at magnifications of x20000 or x40000 and were photographically enlarged to a magnification from x200000 to x600000. The number of dot-like calcium phosphate nuclei per “hole zone” and per “overlapping zone” of the 64 nm collagen macro-period was determined; we found a range of 6–8 nuclei per “hole zone” and 3–5 nuclei per “overlapping zone”. Thus a total of 9–13 nuclei per 64 nm macro-period was found; this is in agreement with the total number of cross bandings per macro-period reported by previous authors. The distances between neighbouring dot-like calcium phosphate nuclei were measured and found to lie in the range of 4.0 to 8.5 nm, which is the range in which most of the distances between the collagen cross-bandings appear. The distances between close, parallel calcium phosphate rows within the collagen fibres were found to lie in the range 4.6–8.0 nm which seems to be a good deal shorter than the distances between the typical microholes of the Hodge-Petruska collagen model. Our results and ideas concerning the “active sites” of nucleation also are in agreement with previous amino acid sequence analyses in respect of the occurrence of neighbouring polar amino acids which would be free to bind ions.ZusammenfassungVon Ultradünnschnitten gefriergetrockneten, neugebildeten Dentins der Rattenschneidezähne und von glutaraldehydfixierten Proben der Compacta eines 10 Monate alten Kindes wurden — über Fokusreihen — Aufnahmen bei 20000∶1 und 40000∶1 angefertigt. Die gut fokussierten Aufnahmen wurden auf 200000∶1 bis 600000∶1 nachvergrößert; die Ausbildung der Ca-Phosphatkeime wurde in den beiden Zonen (“hole zone” und „overlapping zone”) der Kollagen-64 nm-Periode beschrieben. Ferner wurden die Abstände zwischen den punktartigen Keimen in den Ca-Phosphat-Nadeln und -Ketten beider Zonen vermessen und der Wahrscheinlichkeitsbereich für die Anzahl der Keime pro Zone (und damit pro Makroperiode) bestimmt. Die Punktkeimabstände lagen im Bereich von 4,0–8,5 nm und die Seitenabstände zwischen dicht benachbarten Ca-Phosphat-Nadeln im Bereich von 4,6–8,0 nm (vgl. Fig. 6). Bei Einsetzung der möglichen Fehlerbeträge unserer Bestimmungen (Anzahl der Keime pro Zone) können sich in der “overlapping zone”3–5 und in der “hole zone”6–8 Keime bilden. Diese Ergebnisse setzten wir in Beziehung: a) zur Anzahl der Querstreifen pro 64 nm-Periode im Kollagen, b) zu den von uns vermessenen Abständen zwischen diesen Querstreifen und c) zu den durch erste Sequenzanalysen erhaltenen Ergebnissen über die direkte Nachbarschaft von polaren, bindungsfähigen Aminosäuren im Kollagen. Es fand sich eine gute Übereinstimmung zwischen unseren Ergebnissen bzw. Schlußfolgerungen und den bisher vorliegenden Bestimmungen am Kollagen.

Electron microprobe investigations into the process of hard tissue formation.

SummaryThe electron microprobe microanalyser has been used to measure the concentrations of Ca, P and S in the predentine of young rat incisors. The specimens were prepared as alcohol fixed embedded ultrathin sections, unfixed vacuum embedded dry cut ultrathin sections and as thin cryostat sections. The results show the influence of preparation on the measured compositions and indicate that Ca is tightly bound to the matrix, whereas P can be easily washed out. Measurements along the dentine-predentine border demonstrated zones of Ca enrichment, the average size of which suggests that the zones could be the prestages of calcospherites. A mineralisation mechanism is discussed in which the high Ca concentration activates pyrophosphatase or ATPase before the onset of nucleation.

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.

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 μm. 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.

Characteristic molar ratios of magnesium, carbon dioxide, calcium and phosphorus in the mineralizing fracture callus and predentine.

SummaryFrom fracture callus in different stages of mineralization the contents of Mg, CO2, Ca and P were determined and compared with those found in predentine. It was found that the Mg- and CO2-contents are high in relation to Ca and P values during the prestages and early stages of mineralization. These relatively high Mg- and CO2-values are connected with high Mg/Ca, Mg/P, CO2/Ca and CO2/P ratios which strongly decrease with the increasing degree of mineralization. These results seem to be part of a general controlling mechanism of the calcification.

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.

Electron microscopical and laser diffraction studies of the nucleation and growth of crystals in the organic matrix of dentine

SummaryThe growing ends of rat incisors were freeze-dried and embedded in methacrylate without contact with any other solution. Dentine from alcohol and formalin fixed human teeth was also embedded in methacrylate. Ultrathin sections were prepared and electron micrographs taken at original magnifications of X20 000 and X40 000. The best focussed pictures from through focus series were selected for photographic enlargement to a total of X200 000. 507 measurements of the distance between dot-like nuclei in the calcium phosphate needles and chains and 536 measurements of the distance between the neighbouring parallel chains and needles were made using a measuring microscope. In addition, the most commonly occurring separation distances between the dot-like nuclei — within individual rows or between neighbouring rows—were measured by laser diffraction of the x20 000 EM negatives.The most commonly occurring range for the distance between the dot-like nuclei and the lateral distance between the rows as determined morphologically was 3.7–6.3 nm. The corresponding value as determined by laser diffraction for the recently formed rat incisor dentine lay in the region 3.0–5.2 nm, whereas the same value reached to 6.5 nm in the case of mature human dentine. The distances between the dot-like nuclei are regarded as representing the distances between active nucleus-inducing centres on a chain-like matrix. From a study of the morphology of the nuclei it is concluded that the plate-like crystallites usually arise through fusion of needle-like rows of dot-like nuclei when these lie close and parallel to one another.ZusammenfassungVon neu gebildetem, gefriergetrocknetem Dentin in Rattenschneidezähnen und von alkohol- und formol-fixiertem, reifem menschlichen Dentin wurden Ultradünnschnitte bei Vergrößerungen 20 000∶1 und 40 000∶1 in Fokusreihen aufgenommen.Die gut fokussierten Aufnahmen des Ratten-Dentins wurden auf nichtschrumpfendem Photopapier und Diafilm-Material 20 0000∶1 nachvergrößert. Mit einem Meßmikroskop wurden 507 Abstände zwischen den punktförmigen Keimen in den Ca-Phosphat-Nadeln und -Ketten sowie 536 Seitenabstände zwischen dicht zusammenliegenden, parallel verlaufenden Nadeln und Ketten bestimmt. Außerdem konnten in beiden Dentin-Arten die am häufigsten auftretenden Abstände beider Abstandsarten durch Anwendung der Laserbeugung auf die bei 20 000∶1 aufgenommenen Filme erhalten werden. Die morphologisch bestimmten Punktkeimabstände und die Seitenabstände lagen vor allem im Bereich von 37–63 Å, die durch Laserbeugung erhaltenen Werte für das neugebildete Dentin im Bereich von 30–52 Å, während sie beim reifen menschlichen Dentin noch bis zu Werten um 65 Å reichten. Wie bei Höhling u. Mitarb. (1970) wurden die Punktkeimabstände als Abstände zwischen den akiven, keiminduzierenden Zentren auf einer kettenartigen Matrix diskutiert. Aus der Morphologie der Keime wurde ferner geschlossen, daß sich hier die blättchenförmigen Kristallite im allgemeinen durch Zusammenwachsen von Nadeln bilden, wenn diese dicht und parallel zusammenliegen.

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.

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 μm. This may represent a circadian pattern of mineralisation corresponding to light microscopically visible incremental lines in dentine.