Heterogeneity of bone lamellar-level elastic moduli

Advances in our ability to assess fracture risk, predict implant success, and evaluate new therapies for bone metabolic and remodeling disorders depend on our understanding of anatomically specific measures of local tissue mechanical properties near and surrounding bone cells. Using nanoindentation, we have quantified elastic modulus and hardness of human lamellar bone tissue as a function of tissue microstructures and anatomic location. Cortical and trabecular bone specimens were obtained from the femoral neck and diaphysis, distal radius, and fifth lumbar vertebra of ten male subjects (aged 40–85 years). Tissue was tested under moist conditions at room temperature to a maximum depth of 500 nm with a loading rate of 10 nm/sec. Diaphyseal tissue was found to have greater elastic modulus and hardness than metaphyseal tissues for all microstructures, whereas interstitial elastic modulus and hardness did not differ significantly between metaphyses. Trabecular bone varied across locations, with the femoral neck having greater lamellar-level elastic modulus and hardness than the distal radius, which had greater properties than the fifth lumbar vertebra. Osteonal, interstitial, and primary lamellar tissues of compact bone had greater elastic moduli and hardnesses than trabecular bone when comparing within an anatomic location. Only femoral neck interstitial tissue had a greater elastic modulus than its osteonal counterpart, which suggests that microstructural distinctions can vary with anatomical location and may reflect differences in the average tissue age of cortical bone or mineral and collagen organization.     

Heterogeneity of pepsin-solubilized human glomerular basement membrane collagen.

The collagen component of isolated glomerular basement membrane ws solubilized by limited pepsin digestion and further purified. The amino acid and carbohydrate composition of the final material was very similar to that described for the alpha1-chains of type IV collagen. However, the presence of several components was detected when the material was analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis either without or after reduction. The molecular weights of the major components in the reduced material were about 140,000, 100,000, 80,000 and less than 65,000 (in the case of five components). The data thus do not support the previous suggestion that the human glomerular basement membrane contains only one type of collagen polypeptide chain. However, part of the heterogeneity may be due to the presence of more than one collagenous part in a procollagen-type polypeptide chain, and an additional reason may lie in a partial degradation of collagenous portions during pepsin digestion.     

Heterogeneity in drug sensitivity of human tumors in the human tumor colony-forming assay

The human tumor colony-forming assay was used to compare chemosensitivity among tumor cells within a primary tumor, between primary tumor and its metastasis, and between different metastases. The results indicate that the reported discrepancies of in vitro and in vivo results in clinical trials with TCFA for predicting of resistance or sensitivity to cytostatic drugs may be due to therapeutic heterogeneity among tumor colony-forming units within a primary tumors and between primary tumor and metastases, and that the results from a metastatic lesion may have more profound implications in planning treatment of other metastatic lesions of the same patient.     

Heterogeneity of blood flow in tibial cortical bone: an experimental investigation using microspheres

The distribution of tibial blood flow was measured by injecting approximately (600-1000) x 10(3) 15 mu microspheres, labelled with either tin-113 (113Sn) or cobalt-57 (57Co) into femoral arteries of five mature greyhounds. The diaphyseal cortex, stripped of periosteum and devoid of marrow, was sawn into 40 pieces (10 transverse sections x 4 anatomical quarters/section). Relative deposition densities of the 113Sn microspheres in 40 pieces of cortex were found. These values, together with their associated masses, proved, from a statistical point of view, that flow rate heterogeneity was substantial in the diaphysis. In particular, for the diaphyseal cortex, distribution of relative deposition densities (flow rates) in six bones was found to be positively-skewed with a relative dispersion ((SD/mean) x 100) of approximately 40%.     

Glycosaminoglycans of human bone tissue

The glycosaminoglycans of femoral cortical bone, medullary calcified tissue and epiphyseal plate in a case of osteopetrosis were isolated and compared with those of normal cortical bone and epiphyseal cartilage. The methods involved papain digestion of the tissue followed by isolation of the glycosaminoglycans by cetylpyridinium precipitation and by chromatographic separation on cetylpyridinium chloride cellulose columns and a Sephadex column.The composition and concentration of the glycosaminoglycans of osteopetrotic cortex differed from those of normal cortical bone at the corresponding age, and were more in accordance with those reported for newborn children. On the other hand, the solubility profiles of cetylpyridinium-chondroitin sulphate from osteopetrotic cortex and epiphyseal cartilage were similar to those of normal cortical bone and cartilage respectively, indicating that the properties of the first-mentioned polysaccharides did not deviate from the normal. In contrast, the solubility profile of the cetylpyridinium-chondroitin sulphate of the medulla was displaced towards eluants of lower salt concentration, because of a lower degree of sulphation. This can be interpreted as a result of degradation of the tissues making up the medullary plug and of the cartilage remnants, rather than the primitive bone trabeculae. The possibility of a connection between such a degradation and the high mineralization of the tissue is considered.

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Zusammenfassung Die Glykosaminoglykane von corticalem Femurknochen, medullärem verkaltem Gewebe und von der Epiphysenplatte wurden bei einem Fall von Osteopetrose isoliert und mit jenen von normalem corticalem Knochen und Epiphysenknorpel verglichen. Die Methode bestand aus einer Papainverdauung des Gewebes gefolgt von der Glykosaminoglykanisolierung mittels Cetylpyridin-Fällung und nachfolgender chromatographischer Trennung auf Cetylpyridinchlorid-Cellulose-Säulen und einer Sephadex-Säule.Die Zusammensetzung und Konzentration der Glykosaminoglykane des Cortex bei Osteopetrose wichen von jenen im normalen corticalen Knochen des entsprechenden Alters ab und stimmten eher mit den Werten überein, wie sie bei Neugeborenen angegeben werden. Andererseits waren die Löslichkeitsprofile des Cetylpyridin-Chondroitinsulfates, erhalten aus Cortex und aus Epiphysenknorpel bei Osteopetrose, denjenigen von normalem corticalem Knochen, resp. Knorpel, ähnlich. Dies weist darauf hin, daß die Eigenschaften der erstgenannten Polysaccharide nicht von der Norm abwichen. Dagegen war das Löslichkeitsprofil des Cetylpyridin-Chondroitinsulfates der Medulla gegen die Eluate mit geringerer Salzkonzentration verschoben, da es weniger Sulfat enthielt. Dies kann als Folge des Abbaus der den Markraum ausfüllenden Gewebe interpretiert werden, und eher als Folge des Abbaus der Knorpelüberreste als des der ursprünglichen Knochentrabekeln. Die Möglichkeit eines Zusammenhanges zwischen einem solchen Abbau und der starken Verkalkung des Gewebes wird in Betracht gezogen.

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Résumé Les glycosaminoglycanes de l'os cortical fémoral, de l'os spongieux et de la métaphyse, dans un cas d'ostéopétrose, sont isolés et comparés avec ceux de l'os cortical normal et du cartilage épiphysaire. Les analyses ont été réalisées après digestion à la papaine, suivie de précipitation par du cetylpyridinium et séparation chromatographique à l'aide de colonnes de cellulose et chlorure de cetylpyridinium et colonne de Sephadex. La composition et la concentration des glycosaminoglycanes du cortex pathologique différent de celles de l'os cortical normal l'âge correspondant. Elles se rapprochent des valeurs trouvées chez l'enfant nouveau-né. Les courbes de solubilité du sulfate de cetylpyridinium-chondroitine de cortex et cartilage épiphysaire pathologiques sont identiques respectivement à celles de l'os cortical et du cartilage normaux, indiquant que les propriétés du premier polysaccharide restent normales. Par contre, la courbe de solubilité du sulfate de cetylpyridinium-chondroitine de la moëlle est déplacée vers les éluants de faible concentration saline, par suite d'un degré faible de sulfatation. Ce fait peut être interprété comme le résultat d'une dégradation des tissus constituant le remplissage médullaire et les restes de cartilage, plutôt que celle des travées osseuses primitives. L'éventualité d'un rapport entre une telle dégradation et la minéralisation élevée du tissu est évoquée.

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This work was reported, in part, at the meeting of Svenska Patologföreningen, April 26th, 1969; see Nord. Med.82, 1610–1611 (1969).     

Heterogeneity of potential for hematogenous metastasis in a human pancreatic carcinoma

Human pancreatic carcinoma, a disease with grave prognosis, frequently metastasizes to the liver, with detrimental consequences for the host. Good models of experimental metastasis for this disease are lacking. We describe a model of hepatic metastasis from the fast-growing variant (FG) of the human pancreatic carcinoma COLO 357. We also show that the slow-growing variant (SG) of COLO 357 lacks the potential for forming hepatic and pulmonary metastases following injection into the spleen of the nude mouse. This expression of heterogeneity of potential for hematogenous metastases can be exploited by pursuing studies aiming at identifying differences between the cells with and without metastatic potential.     

Heterogeneity and modulation of tumor-associated antigens in human glioblastoma cell lines

Seven human glioblastoma cell lines established in vitro from primary tumor explants were studied. A marked heterogeneity of glial fibrillary acidic protein was observed whereas vimentin was uniformly expressed by all cell lines. Indirect immunofluorescence and flow cytofluorometry revealed a heterogeneous distribution of surface GE 2 and CG 12 tumor-associated antigens (TAA's): three cell lines were positive (greater than 69% TAA-positive cells) and three cell lines were negative (less than 9% TAA-positive cells). One cell line (Hu 228) was moderately positive at early culture passages and subsequently acquired a TAA-negative phenotype. The difference in the relative amounts of surface TAA's of the three positive cell lines was less than twofold. In spite of the heterogeneous distribution of surface TAA's, all cell lines exhibited considerable amounts of intracellular TAA. Treatment with phorbol esters and density-dependent growth arrest decreased the percentage of the TAA-positive cells and the amount of cell-surface TAA's in one cell line (Hu 195). Interferon-gamma treatment in vitro increased the percentage of CG 12-positive cells by 12% and the amount of cell-surface CG 12 antigens by 38% as compared to untreated cells. The percentage of TAA-positive cells among phorbol ester-treated cells of the Hu 195 cell line was lowest 48 hours after treatment, but returned to normal values within the next 48 hours. Reduction of 3H-thymidine incorporation preceded the decrease in number of TAA-positive cells by about 18 hours. Two-color fluorescence analysis performed in positive cell lines for simultaneous determination of surface TAA's and deoxyribonucleic acid content or reactivity with the proliferation-associated Ki67 intracellular marker indicated that GE 2 and CG 12 antigens are expressed preferentially by actively proliferating glioma cells. The results of this study indicate the existence of two different phenotypes in cultured human glioblastoma cells: surface TAA-positive/cytosol TAA-positive and surface TAA-negative/cytosol TAA-positive cell populations. In addition, modulation of TAA expression was dependent on the cell-cycle differentiation stage, culture conditions, and proliferative state of the cells.     

Glycosaminoglycans of human bone tissue

Glycosaminoglycans from human femoral compact bone were characterized and their concentrations and patterns were determined in autopsy cases of various ages, newborn to 94 years of age. Cetylpyridinium precipitation and fractionation methods were used. Absence of postmortem changes of bone glycosaminoglycan pattern for as long as 72 hours was checked on dog femoral bone. Chondroitin-4-sulphate was identified as the predominating glycosaminoglycan of human bone; in addition a minor part was tentatively identified as hyaluronic acid. Concentrations, of glycosaminoglycans were inversely related to age in growing subjects, but no change was noted later in life. The glycosaminoglycan pattern had a similar appearance for all of the adult subjects. Only small deviations from this pattern had a similar for young subjects, possibly due to low molecular weight change or low sulphated chondroitin sulphate; and for one old subjects to a higher proportion of hyaluronic acid.The nomenclature of Balazs and Jeanloz (1965) has been followed.     

Heterogeneity of xenografted osteosarcoma: A human sarcoma transplanted into nude mice

Human osteoblastic osteosarcoma transplanted into nude mice developed two different subtypes of nonosteogenic sarcoma: one solid and the other cystic. This could reflect the heterogeneity of osteoblastic osteosarcoma; various tumor regions have different characteristics.     

Heterogeneity in the presence of CD4-like molecules on human spermatozoa

Summary. The object of the present study was to study if there are differences in the presence of CD4-like molecules on human ejaculated spermatozoa in fertile donors and infertile patients (with globozoospermia). Indirect and absorption enzyme-linked immunosorbent assay and indirect immunofluorescence were applied. The enzyme-linked immunosorbent assay data showed that monoclonal anti-human CD4-antibody recognizes an epitope common to the human spermatozoa with normal morphology and round-headed spermatozoa. Localization of the antigenic determinants, identified by anti-human CD4-monoclonal antibody, in the acrosomal region, including equatorial segment, postnuclear cap and tail was determined in normozoospermic samples. A positive reaction was found on the sperm head both in the acrosomal and postacrosomal region of some round-headed spermatozoa in the samples with globozoospermia. The tails of the normozoospermic spermatozoa and of some round-headed spermatozoa were weakly immunopositive. The results of the experiments carried out are evidence of heterogeneity in the presence of CD4-like antigen determinants on human spermatozoa. These data increase the information about the CD4-antigen characteristic of the spermatozoa from fertile donors and infertile patients.     

Heterogeneity of xenografted osteosarcoma: A human sarcoma transplanted into nude mice

Human osteoblastic osteosarcoma transplanted into nude mice developed two different subtypes of nonosteogenic sarcoma: one solid and the other cystic. This could reflect the heterogeneity of osteoblastic osteosarcoma; various tumor regions have different characteristics.     

Quantitative measurements of human bone enzymes

Summary Up to now quantitative analysis of human bone enzymes is a part of osteology which is not evaluated sufficiently. The lack of standardized methods, fresh bone and reliable methods of bone disintegration seem to impede these experiments mainly. The authors have elaborated a complex method for quantitative measurements of human bone enzymes and they describe the practical use of this method. The importance of quantitative bone enzyme measurements is pointed out.

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Zusammenfassung Die quantitative Analyse der Enzyme menschlicher Knochen ist ein noch nicht ausreichend studierter Teil der Osteologie. Das Fehlen von standardisiertem frischem Knochenmaterial und eine bewährte Methode der Knochenzersetzung sind die Haupthindernissse, solche Versuche durchführen zu können. Die Verfasser arbeiteten eine komplexe Methode für Messungen der Enzyme menschlicher Knochen aus. Die Methode and deren Probe wird beschrieben. Die Wichtigkeit der quantitativen Messungen menschlicher Knochenenzyme wird betont.

     

Crystallographic lattice refinement of human bone

Summary X-ray diffraction studies on bone microsamples (human iliac crest of 87 individuals aged 0–90 years) reveal that certain crystallographic parameters such as unit cell volume of bone apatite, and half-width of (002)-reflection are well correlated with age and with type of tissue (corticalis and spongiosa). Similar to inorganic apatite, the lattice parameters of bone apatite are intensely affected by ionic substitutions and vary mainly due to exchange of hydroxyl- and carbonate-apatite and, to a minor extent, of fluor- and chlorapatite.     

Magnesium distribution in human bone

The present study was undertaken to reveal the magnesium distribution in human bone. Sixty human ribs, obtained from subjects aged 10–80 years of age, were used. Transverse sections were prepared from the middle region of the human ribs. Adjacent sections were ground to a thickness of about 1000 m. One section was used for magnesium determination by atomic absorption spectrophotometry, and the other was used for analysis with X-ray microanalysis. Thirty micron thick samples were abraded continuously from the periosteal and the endosteal surfaces by abrasive microsampling, as previously described by Weatherell et al. [3]. Results showed that magnesium concentrations were higher in both the periosteal and endosteal surfaces and did not change with age in general, although it tended to be higher among teenagers and lower over 80 years old.     

Susceptibility of aging human bone to mixed-mode fracture increases bone fragility

Since bone-mass-based measurements have demonstrated limited success in predicting age-related increase in fracture incidence, recent emphasis has been placed on quantification of bone quality. Specifically, material parameters such as strength, stiffness, and toughness have been quantified to characterize bone quality through mechanical testing. This study is the first one to report multiaxial failure characteristics of bone (quality) by conducting fatigue tests on human cortical bone specimens under physiologically relevant loading involving simultaneous application of axial and torsional loading to produce previously reported in vivo shear/normal stress ratios. Our results show that, compared to uniaxial tests, multiaxial fatigue tests show up to a 20-fold reduction in the fatigue life of human cortical bone. More significantly, the susceptibility of mixed-mode failure increases bone fragility in aging human bone. Furthermore, since the magnitude of mixed-mode loading varies with physiological activities, results of this study also suggest that a reduction in the activities involving significant mixed-mode loading may lower the overall fracture incidence among older individuals.