Acidic macromolecules of mineralized tissues: the controllers of crystal formation

The deposition of minerals within many biological tissues is a controlled process. Among the most active agents that control biological mineralization are a group of unusually acidic proteins and glycoproteins. These can interact specifically with some crystal faces but not others, induce oriented nucleation, or intercalate in a regular manner into the crystal lattice itself.     

Nanoindentation hardness of mineralized tissues

A series elastic and plastic deformation model [Sakai, M., 1999. The Meyer hardness: a measure for plasticity? Journal of Materials Research 14(9), 3630–3639] is used to deconvolute the resistance to plastic deformation from the plane strain modulus and contact hardness parameters obtained in a nanoindentation test. Different functional dependencies of contact hardness on the plane strain modulus are examined. Plastic deformation resistance values are computed from the modulus and contact hardness for engineering materials and mineralized tissues. Elastic modulus and plastic deformation resistance parameters are used to calculate elastic and plastic deformation components, and to examine the partitioning of indentation deformation between elastic and plastic. Both the numerical values of plastic deformation resistance and the direct computation of deformation partitioning reveal the intermediate mechanical responses of mineralized composites when compared with homogeneous engineering materials.     

Multigenerational interstitial growth of biological tissues

This study formulates a theory for multigenerational interstitial growth of biological tissues whereby each generation has a distinct reference configuration determined at the time of its deposition. In this model, the solid matrix of a growing tissue consists of a multiplicity of intermingled porous permeable bodies, each of which represents a generation, all of which are constrained to move together in the current configuration. Each generation’s reference configuration has a one-to-one mapping with the master reference configuration, which is typically that of the first generation. This mapping is postulated based on a constitutive assumption with regard to that generations’ state of stress at the time of its deposition. For example, the newly deposited generation may be assumed to be in a stress-free state, even though the underlying tissue is in a loaded configuration. The mass content of each generation may vary over time as a result of growth or degradation, thereby altering the material properties of the tissue. A finite element implementation of this framework is used to provide several illustrative examples, including interstitial growth by cell division followed by matrix turnover.     

Quench cooling and ice crystal formation in biological tissues

The rates of cooling of tissue packages quenched in liquid nitrogen were investigated using microthermocouples. By assembling tissue packages from a standard 200-μm tissue slice a microthermocouple could be positioned at different depths within the package. Results showed that for a given mass of tissue the rates of cooling at different depths were the same. When the tissue mass was varied the rates of cooling at a fixed depth decreased with increasing tissue mass.The ice crystal formations produced when tissues are quenched in liquid nitrogen were investigated using freeze substitution. Assemblies of rabbit cornea of different thicknesses were quenched in liquid nitrogen and freeze substituted. The size of the ice crystal cavities produced during the quenching increased with increasing tissue mass, exhibiting a saturation size for the larger tissue masses. There was no obvious size distribution of the ice crystal cavities across the thickness of the corneas.The results suggest an “isotherm” model for the quenching conditions used in these experiments, there being small or negligible temperature gradients through the tissue which uniformly cools at a fixed rate.     

Glucagon-initiated human growth hormone release: a comparative study

Human growth hormone (HGH) responses in 20 healthy adults to subcutaneous glucagon, arginine infusion and tolbutamide and insulin hypoglycemia were compared. HGH rose in all four tests. HGH response to glucagon was also studied in 49 patients with suspected pituitary insufficiency, of whom 25 also later received an arginine infusion; an abnormal response to glucagon was the most frequent functional abnormality and often HGH was the only anterior pituitary hormone of which a deficiency was detectable. In seven subjects (two healthy controls and five patients with suspected hypopituitarism) there was a subnormal HGH response to arginine but a normal response to glucagon. It is concluded that glucagon is a simple and effective stimulus to HGH release, equal or superior to arginine, tolbutamide and insulin, and is an important test of anterior pituitary function.     

Microradiographic determination of lead in mineralized tissues

Summary A dichromatic contact microradiographic method is presented for quantitative microanalysis of lead in mineralized tissues utilizing the L absorption edges of lead. Sections of mineralized tissue with a thickness of about 100 are exposed to filtered molybdenum K and copper K radiation. Quantitative localization of lead is possible within small areas of the order of 50 ² with an accuracy of 25 · 10^–12g lead. The method has been applied to studies of long term accumulation of lead in bone tissue.     

Collagenase in mineralized tissues of human teeth

A collagenase cleaving native type I [14C]collagen but inactive against the synthetic substrate Pz-Pro-Leu-Gly-Pro-D-Arg was extracted from mineralized human dental tissue. The enzyme specifically degrades native collagen into characteristic products (3/4) and (1/4). Its apparent molecular mass of 68 kDa is relatively high in comparison with collagenases from other oral tissues. The enzyme is a metalloproteinase inhibited by low concentrations of the chelating agents EDTA, 1, 10-phenanthroline, alpha alpha'-dipyridyl, and not affected by diisopropylfluorophosphate, soybean trypsin inhibitor, and p-chloromercuribenzoate. It is stable to lyophilization and can be stored at-20 degrees C for at least 6 months.     

Osteonectin is a minor component of mineralized connective tissues in rat

Osteonectin is a major glycoprotein of porcine and bovine bones and teeth that is found associated with hydroxylapatite crystal surfaces. From the ability of osteonectin to bind calcium ions, it has been proposed as a possible nucleator of hydroxylapatite crystal formation. Analysis of hydroxylapatite-bound proteins of rat bone and dentine, however, has revealed that osteonectin represents only 2.5 +/- 1.5% of the hydroxylapatite-bound protein in long bones, 0.9 +/- 0.5% in calvariae, and less than 0.1% in incisor dentine of animals of different ages. Further, in vivo pulse-chase studies carried out in young adult rats have shown osteonectin to be synthesized at low levels in these tissues. Similarly, low levels of osteonectin were synthesized by rat calvarial cells in vitro. In contrast, fibroblastic cells from periodontal ligament and gingiva synthesized significantly greater amounts of osteonectin. These studies indicate that the low quantities of osteonectin in rat mineralized tissues are a consequence of low rates of formation rather than being due to rapid turnover. The virtual absence of osteonectin in incisor dentine correlates with the lack of peritubular dentine in rat, whereas the low osteonectin content of rat bones may reflect differences in their structure and biophysical properties compared with bones of larger mammals.     

Notch sensitivity of mammalian mineralized tissues in impact

The toughness of bone is an important feature in preventing it from fracturing. We consider the notch sensitivity in impact, and the associations between brittleness, notch sensitivity and post-yield energy absorption of mammalian mineralized tissues. Specimens of bone-like tissues covering a wide range of mineralization were broken, either notched or un-notched, in impact. The greater the mineral content, the greater was the notch sensitivity. Also, the more brittle tissues dissipated the least post-yield energy and were the most notch sensitive. It is suggested that since antler bone, the least mineralized of all known mammalian mineralized tissues, seems to be notch insensitive in impact, no adaptive purpose would be served by having mineralized tissues of a lower mineralization than antler. This may explain the lower cut-off in mineralization seen in mammals.     

Thomsen-Friedenreich-related carbohydrate antigens in normal adult human tissues: a systematic and comparative study

A broad variety of normal human tissues were examined for the expression of Thomsen-Friedenreich (TF)-related histo-blood group antigens. TF (Galβ1-3GalNAcα1-R), Tn (TF precursor, GalNAcα1-R), sialosyl-Tn (NeuAcα2-6GalNAcα1-R), considered to be useful in cancer diagnosis and immunotherapy, and sialosyl-TF, the cryptic form of TF. These antigens or, more correctly, glycotopcs, were determined by immunohistochemistry with at least two monoclonal antibodies (mAbs) each (except sialosyl-TF) as well as by lectin histochemistry. For a better dissection of sialosyl-TF and TF glycotopes, tissue sections were pretreated with galactose oxidase or the galactose oxidase-Schiff sequence. Staining with mAbs appeared to be more restricted than with the lectins used. Distribution patterns among normal epithelia were different for all four antigens. These antigens were also detected in some non-epithelial tissues. They can be classified in the following sequence according to the frequency of their occurrence in normal tissues: sialosyl-TF> >sialosyl-Tn>Tn>TF. Most of the positively staining sites for TF, Tn, and sialosyl-Tn are located in immunologically privileged areas. The complex results obtained with anti-TF mAbs (after treatment of the tissue sections with sialidase fromVibrio cholerae) and the lectins amaranthin and jacalin revealed a differential distribution of the subtypes of sialosyl-TF [NeuAcα2-3Galβ1-3GalNAcα1-R and Galβ1-3 (NeuAcα2-6)GalNAcα1-R] in normal human tissues. From our data it can be inferred that TF, Tn, and sialosyl-Tn are promising targets for a cancer vaccine.     

Bridges linking gap junction particles extracellularly: a freeze-etching rotary-shadowing study of split junctions

The nature of the interaction between neighboring gap junction particles and the mechanism involved in particle crystallization are still unclear. We describe here interparticle bridge-like structures which could participate in the mechanism of gap junction particle aggregation and pattern determination. Gap junction membranes of rat liver, pulled apart by vascular perfusion with hypertonic sucrose, were freeze-fractured in deionized water, etched at - 100 degrees C for 8 min and rotary-shadowed at a 32 degrees angle. At the extracellular true surface of the junctions (ES-surface), the particles appear as 7.0 to 7.5 nm rings often resolvable into six radially arranged subunits. The particles appear linked to each other by filamentous bridges 1.5 to 2.2 nm thick and approximately 1.5 nm long. Some bridges (single bridges) directly interlink neighboring particles while other bridges (multiple bridges) are joined to a particle at one end and to the other bridges at the other end. Bridges are referred to as double or triple bridges if they result from the interaction of two or three bridges respectively. In particles which can be resolved into six subunits, the bridges appear to bind to the subunit tips. Stereo images indicate that the bridges lay in planes lower than the particle summits. The bridges could be either polypeptide chains of the main gap junction protein or peripheral proteins, but the unlikely possibility that they are a shadowing artifact cannot be entirely ruled out yet.