Transmission electron micrographs of fully mineralized turkey leg tendon in cross-section show the ultrastructure to be more complex than has been previously described. The mineral is divided into two regions. Needlelike-appearing crystallites fill the extrafibrillar volume whereas only platelike crystallites are found within the fibrils. When the specimen is tilted through a large angle, some of the needlelike-appearing crystallites are replaced by platelets, suggesting that the needlelike crystallites are platelets viewed on edge. If so, these platelets have their broad face roughly parallel to the fibril surface and thereby the fibril axis, where the intrafibrillar platelets are steeply inclined to the fibril axis. The projection of the intrafibrillar platelets is perpendicular to the fibril axis. The extrafibrillar volume is at least 60% of the total, the fibrils occupying 40%. More of the mineral appears to be extrafibrillar than within the fibrils. Micrographs of the mineralized tendon in thickness show both needlelike-appearing and platelet crystallites. Stereoscopic views show that the needlelike-appearing crystallites do not have a preferred orientation. From the two-dimensional Fourier transform of a selected area of the cross-sectional image, the platelike crystallites have an average dimension of 58 nm. The needlelike-appearing crystallites have an average thickness of 7 nm. The maximum length is at least 90 nm. Atomic force microscopy (AFM) of unstained, unmineralized turkey leg tendon shows collagen fibrils very much like shadow replicas of collagen in electron micrographs. AFM images of the mineralized tendon show only an occasional fibril. Mineral crystallites are not visible. Because the collagen is within the fibrils, the extrafibrillar mineral must be embedded in noncollagenous organic matter. When the tissue is demineralized, the collagen fibrils are exposed. The structure as revealed by the two modalities is a composite material in which each component is itself a composite. Determination of the properties of the mineralized tendon from the properties of its elements is more difficult than considering the tendon to be just mineral-filled collagen.

The organic content of mineralized tissues has been found to decrease with increasing tissue density, from about 60% of the mineral weight in light bone like deer antler to 1 to 2% in hyperdense bone like porpoise petrosal. The ratio of the weight of mineral that can fill the collagen hole zones to the total mineral content can be no greater than 20% for deer antler and decreases to less than 5% for hyperdense bone. Moreover, the dimensions of hydroxyapatite crystallites have been determined by various investigators to be larger than the intermolecular spacing of collagen molecules. Such crystallites can only be fitted within the collagen fibril if collagen molecules are packed differently from the accepted models. Electron micrographs of fish dentin, at a very early stage of mineralization, show the needle-like crystallites lying in dense strips between collagen fibrils and practically no crystallites within the fibrils. A similar pattern of dense strips of crystallites between fibrils can be identified in examples from more advanced stages of mineralization, taken from fish dentin, cat dentin and cow tibia, even though some of the needle-like crystallites are superimposed on the fibril banded pattern. In every instance there are regions of the fibrils where there are no visible needle-like crystallites. Examination of the work of others shows a similar distribution of the mineral component, except that none exactly resemble the micrograph of the earliest stage of fish dentin provided in this report. The collagen banding is observed to be in spatial phase over many fibrils. The needle-like crystallites may be observed to be bunched in phase with the collagen banding and with the same spatial periodicity. The bunching is most obvious in the least densely mineralized specimens. This observation can account for the x-ray and neutron diffraction patterns which shown the axial period of the mineral to be like that of the collagen axial macroperiod and to be in phase with the hole zones of collagen fibrils. These prior studies were interpreted to show that the crystallites must be within the hole zones. Our images are interpreted to show that most of the mineral is outside of the collagen fibrils in the extrafibrillar volume. The interpretation is in agreement with neutron diffraction studies of various mineralized tissues as well as with earlier diffraction studies of mineralized turkey leg tendon and with the calculations of the amount of mineral that can be contained within the collagen of mineralized tissue.

A technique to correlate the ultrastructural distribution of mineral with its organic material in identical sections of mineralized turkey leg tendon (MTLT) and human bone was developed. Osmium or ethanol fixed tissues were processed for transmission electron microscopy (TEM). The mineralized tissues were photographed at high, intermediate, and low magnifications, making note of section features such as fibril geometry, colloidal gold distribution, or section artifacts for subsequent specimen realignment after demineralization. The specimen holder was removed from the microscope, the tissue section demineralized in situ with a drop of 1 N HCl, then stained with 2% aqueous vanadyl sulfate. The specimen holder was reinserted into the microscope, realigned with the aid of the section features previously noted, and rephotographed at identical magnification used for the mineralized sections. A one to one correspondence was apparent between the mineral and its demineralized crystal "ghost" in both MTLT and bone. The fine structural periodic banding seen in unmineralized collagen was not observed in areas that were fully mineralized before demineralization, indicating that the axial arrangement of the collagen molecules is altered significantly during mineralization. Regions that had contained extrafibrillar crystallites stained more intensely than the intrafibrillar regions, indicating that the noncollagenous material surrounded the collagen fibrils. The methodology described here may have utility in determining the spatial distribution of the noncollagenous proteins in bone.

Streptococcus mutans strain IB-1600 was cultivated in Todd-Hewitt broth (THB) or THB supplemented with sucrose (S). Cell mass obtained from THB exhibited a high cell density and negligible glucan-rich extracellular matrix material (EMM), whereas cell mass from 2% S-supplemented THB exhibited widely-spaced cells separated by EMM. The pH-lowering potential of the different cell masses was studied in vivo with an intra-oral enamel demineralization test and rinsing with glucose solution, and in vitro with a model which permits vertical penetration of glucose through the cell mass and pH evaluation at different depths within the cell mass. In vivo, the pH profile of EMM-rich cell mass derived from 2% S-supplemented THB was characterized by a lower pH minimum and a slower return of the pH as compared with THB-derived cell mass. In vitro, an increase in cell mass EMM content was associated with a more rapid initiation and an increase in the rate of pH drop in the depth of the cell masses. Evaluation of the acidogenic potential of the cells in cell masses derived from THB and 2% S-supplemented THB with suspensions of dispersed cell mass and added glucose indicated no difference. The buffering capacity of cell mass derived from 2% S-supplemented THB within the pH range of 6.5-4.0 was greatly reduced as compared with that of THB-derived cell mass, due to the relatively low buffering capacity of EMM. The presence of EMM also appeared to enhance the porosity of the cell mass.(ABSTRACT TRUNCATED AT 250 WORDS)

A technique to correlate the ultrastructural distribution of mineral with its organic material in identical sections of mineralized turkey leg tendon (MTLT) and human bone was developed. Osmium or ethanol fixed tissues were processed for transmission electron microscopy (TEM). The mineralized tissues were photographed at high, intermediate, and low magnifications, making note of section features such as fibril geometry, colloidal gold distribution, or section artifacts for subsequent specimen realignment after demineralization. The specimen holder was removed from the microscope, the tissue section demineralized in situ with a drop of 1 N HCl, then stained with 2% aqueous vanadyl sulfate. The specimen holder was reinserted into the microscope, realigned with the aid of the section features previously noted, and rephotographed at identical magnification used for the mineralized sections. A one to one correspondence was apparent between the mineral and its demineralized crystal "ghost" in both MTLT and bone. The fine structural periodic banding seen in unmineralized collagen was not observed in areas that were fully mineralized before demineralization, indicating that the axial arrangement of the collagen molecules is altered significantly during mineralization. Regions that had contained extrafibrillar crystallites stained more intensely than the intrafibrillar regions, indicating that the noncollagenous material surrounded the collagen fibrils. The methodology described here may have utility in determining the spatial distribution of the noncollagenous proteins in bone.