A screening approach reveals the influence of mineral coating morphology on human mesenchymal stem cell differentiation

“Biomimetic” inorganic coating on biomaterials has been an active area of research with the aim of providing bioactive surfaces that can regulate cell behavior. Previous studies have demonstrated that human mesenchymal stem cell (hMSC) behavior is differentially regulated by the physical and chemical properties of inorganic mineral coatings, indicating that modulation of mineral properties is potentially important in regulating hMSC behavior. However, the lack of an efficient experimental context, in which to study stem cell behavior on inorganic substrates, has made it difficult to systematically study the effects of specific mineral coating parameters on hMSC behavior. In this study, we developed an efficient experimental platform to screen for the effects of mineral coating morphology on hMSC expansion and differentiation. hMSC expansion on mineral coatings was regulated by the micro-scale morphology of these coatings, with greater expansion on small granule-like coatings when compared to plate-like or net-like coatings. In contrast, hMSC osteogenic differentiation was inversely correlated with cell expansion on mineral coatings indicating that mineral coating morphology was a key parameter that regulates hMSC differentiation. The effect of mineral coating morphology on hMSC behavior underlines the utility of this inorganic screening platform to identify optimal coatings for medical devices and bone tissue engineering applications.     

On the role of bone damage in calcium homeostasis

Bone serves as the reservoir of some minerals including calcium. If calcium is needed anywhere in the body, it can be removed from the bone matrix by resorption and put back into the blood flow. During bone remodelling the resorbed tissue is replaced by osteoid which gets mineralized very slowly. Then, calcium homeostasis is controlled by bone remodelling, among other processes: the more intense is the remodelling activity, the lower is the mineral content of bone matrix. Bone remodelling is initiated by the presence of microstructural damage. Some experimental evidences show that the fatigue properties of bone are degraded and more microdamage is accumulated due to the external load as the mineral content increases. That damage initiates bone remodelling and the mineral content is so reduced. Therefore, this process prevents the mineral content of bone matrix to reach very high (non-physiological) values. A bone remodelling model has been used to simulate this regulatory process. In this model, damage is an initiation factor for bone remodelling and is estimated through a fatigue algorithm, depending on the macroscopic strain level. Mineral content depends on bone remodelling and mineralization rate. Finally, the bone fatigue properties are defined as dependent on the mineral content, closing the interconnection between damage and mineral content. The remodelling model was applied to a simplified example consisting of a bar under tension with an initially heterogeneous mineral distribution. Considering the fatigue properties as dependent on the mineral content, the mineral distribution tends to be homogeneous with an ash fraction within the physiological range. If such dependance is not considered and fatigue properties are assumed constant, the homogenization is not always achieved and the mineral content may rise up to high non-physiological values. Thus, the interconnection between mineral content and fatigue properties is essential for the maintenance of bone's structural integrity as well as for the calcium homeostasis.     

Modeling the tensile mechanical behavior of bone along the longitudinal direction

The tensile stress-strain behavior of bone along its longitudinal axis is modeled by using a simple shear-lag theory, wherein, stresses and strains in a unit cell consisting of an organic matrix reinforced by overlapped mineral platelets are computed. It is assumed that loads are transferred between overlapped mineral-platelets by shear in the organic matrix. The mechanical behavior of bone in which the matrix partially or completely debonds from the sides of the overlapped mineral platelets (after an ultimate interfacial shear stress value is exceeded) is modeled. It is shown that the tensile mechanical behavior of bone can be modeled only by assuming little or no debonding of the organic from the mineral. A physical phenomenon that explains the tensile behavior of bone is, after the interfacial shear stress has reached a constant value over the length of the mineral platelets, the collagen molecules/microfibrils (with the associated mineral platelets) move relative to one another. The tensile stress-strain curve of bovine bone is modeled using this model. The theory predicts the mechanical behavior of the tissue in the elastic, yield and post-yield region. The ultimate strain and strengths are not predicted in the present model.     

Mineral Distributions at the Developing Tendon Enthesis

Tendon attaches to bone across a functionally graded interface, “the enthesis”. A gradient of mineral content is believed to play an important role for dissipation of stress concentrations at mature fibrocartilaginous interfaces. Surgical repair of injured tendon to bone often fails, suggesting that the enthesis does not regenerate in a healing setting. Understanding the development and the micro/nano-meter structure of this unique interface may provide novel insights for the improvement of repair strategies. This study monitored the development of transitional tissue at the murine supraspinatus tendon enthesis, which begins postnatally and is completed by postnatal day 28. The micrometer-scale distribution of mineral across the developing enthesis was studied by X-ray micro-computed tomography and Raman microprobe spectroscopy. Analyzed regions were identified and further studied by histomorphometry. The nanometer-scale distribution of mineral and collagen fibrils at the developing interface was studied using transmission electron microscopy (TEM). A zone (∼20 µm) exhibiting a gradient in mineral relative to collagen was detected at the leading edge of the hard-soft tissue interface as early as postnatal day 7. Nanocharacterization by TEM suggested that this mineral gradient arose from intrinsic surface roughness on the scale of tens of nanometers at the mineralized front. Microcomputed tomography measurements indicated increases in bone mineral density with time. Raman spectroscopy measurements revealed that the mineral-to-collagen ratio on the mineralized side of the interface was constant throughout postnatal development. An increase in the carbonate concentration of the apatite mineral phase over time suggested possible matrix remodeling during postnatal development. Comparison of Raman-based observations of localized mineral content with histomorphological features indicated that development of the graded mineralized interface is linked to endochondral bone formation near the tendon insertion. These conserved and time-varying aspects of interface composition may have important implications for the growth and mechanical stability of the tendon-to-bone attachment throughout development.     

Nutrition or detoxification: why bats visit mineral licks of the Amazonian rainforest

Many animals in the tropics of Africa, Asia and South America regularly visit so-called salt or mineral licks to consume clay or drink clay-saturated water. Whether this behavior is used to supplement diets with locally limited nutrients or to buffer the effects of toxic secondary plant compounds remains unclear. In the Amazonian rainforest, pregnant and lactating bats are frequently observed and captured at mineral licks. We measured the nitrogen isotope ratio in wing tissue of omnivorous short-tailed fruit bats, Carollia perspicillata, and in an obligate fruit-eating bat, Artibeus obscurus, captured at mineral licks and at control sites in the rainforest. Carollia perspicillata with a plant-dominated diet were more often captured at mineral licks than individuals with an insect-dominated diet, although insects were more mineral depleted than fruits. In contrast, nitrogen isotope ratios of A. obscurus did not differ between individuals captured at mineral lick versus control sites. We conclude that pregnant and lactating fruit-eating bats do not visit mineral licks principally for minerals, but instead to buffer the effects of secondary plant compounds that they ingest in large quantities during periods of high energy demand. These findings have potential implications for the role of mineral licks for mammals in general, including humans.     

Forming nacreous layer of the shells of the bivalves Atrina rigida and Pinctada margaritifera: an environmental- and cryo-scanning electron microscopy study

A key to understanding control over mineral formation in mollusk shells is the microenvironment inside the pre-formed 3-dimensional organic matrix framework where mineral forms. Much of what is known about nacre formation is from observations of the mature tissue. Although these studies have elucidated several important aspects of this process, the structure of the organic matrix and the microenvironment where the crystal nucleates and grows are very difficult to infer from observations of the mature nacre. Here, we use environmental- and cryo-scanning electron microscopy to investigate the organic matrix structure at the onset of mineralization in the nacre of two mollusk species: the bivalves Atrina rigida and Pinctada margaritifera. These two techniques allow the visualization of hydrated biological materials coupled with the preservation of the organic matrix close to physiological conditions. We identified a hydrated gel-like protein phase filling the space between two interlamellar sheets prior to mineral formation. The results are consistent with this phase being the silk-like proteins, and show that mineral formation does not occur in an aqueous solution, but in a hydrated gel-like medium. As the tablets grow, the silk-fibroin is pushed aside and becomes sandwiched between the mineral and the chitin layer.     

Host-plant acceptance on mineral soil and humus by the pine weevil Hylobius abietis (L.)

1 The pine weevil Hylobius abietis (L.) (Coleoptera, Curculionidae) is an economically important pest of conifer forest regeneration in Europe and Asia. 2 Soil scarification, which usually exposes mineral soil, is widely used to protect seedlings from weevil attack. However, the mechanism behind this protective effect is not yet fully understood. 3 Field experiments were conducted to determine the pine weevil's responses to visual and odour stimuli from seedlings when moving on mineral soil and on undisturbed humus surface. 4 One experiment measured the number of pine weevils approaching seedlings, with and without added host odour, on mineral soil and undisturbed humus. Seedlings with added host odour attracted more weevils on both soil types. Unexpectedly, somewhat more weevils approached seedlings surrounded by mineral soil. 5 In a similar experiment, feeding attacks on seedlings planted directly in the soil were recorded. Only half as many seedlings were attacked on mineral soil as on undisturbed humus. 6 In the first experiment, the weevils were trapped 2.5 cm from the bases of the seedlings' stems, whereas they could reach the seedlings in the experiment where seedlings were planted directly in the soil. We conclude that the pine weevils' decision on whether or not to feed on a seedling is strongly influenced by the surrounding soil type and that this decision is taken in the close vicinity of the seedling. The presence of pure mineral soil around the seedling strongly reduces the likelihood that an approaching pine weevil will feed on it.     

Growth-Regulating Substances and the Growth of Tiller Buds in Barley; Effects of Cytokinins

An hypothesis was set up from which it was predicted that applicationof cytokinin to barley seedlings grown without mineral nutrientswould lead to rapid growth of the coleoptile and first leaftiller buds. Application of cytokinins to the leaves was ineffective,but supplying a number of known cytokinins by steeping the rootsof 4 d old seedlings in solution for 4 h led to significantgrowth of the coleoptile bud. Adenine and cytokinin analogueshad no effect. Supplying cytokinins through the roots also furtherenhanced the growth of buds of plants given mineral nutrients.Cytokinin treatment reduced root dry matter, with small reductionsin mean axis length and number of lateral roots. For plantsnot given mineral nutrients reduction in root weight was compensatedby an increase in weight of the aerial parts; however, for plantssupplied with mineral nutrients this was not so and the lowerroot weight resulted in a smaller total plant dry weight. An interpretation of tiller bud growth in terms of control byinteracting effects of mineral nutrition, assimilate supply,and cytokinin availability is proposed.     

A study of the interface strength between protein and mineral in biological materials

Bone, tooth, mineralized tendon and sea shells are nanocomposites of protein and mineral with superior mechanical properties. As the mineral is so small at nanoscale, the volume fraction of the protein-mineral interface in the bulk materials can be enormously large; therefore, the mechanics of the interface should be critically important for the integrity of these biomaterials. Currently, people do not have a good understanding of the interface between protein and mineral, a hybrid interface between organic and inorganic constituents in biological materials. In this paper, a tension-shear chain (TSC) model is introduced into the Dugdale model for estimating the fracture energy of biomaterials. The strength of the hybrid interface is then studied with a "soft-hard" bi-layer fracture model, by which we find for the first time that the interface strength depends on both the size and geometry of the mineral crystal, and has been highly optimized through the miniaturization of mineral at nanoscale. This study may provide important insights into the mechanics of bone and tooth at small scale for tissue engineering in biomedical applications.     

Effects of Cadmium on Bone Mineral Density in the Distal and Proximal Forearm: Two Female Population Studies in China

Long-term cadmium exposure may cause bone loss in distal or proximal sites in the forearm. In this study, we observed the effects of cadmium on bone mineral density in both distal and proximal sites in the forearm in two female populations. A total of 456 women living in two different areas participated. All of the participants completed a questionnaire, and the bone mineral density was measured in both the distal and proximal forearm by dual-energy X-ray absorptiometry. Urine samples were collected for the determination of urinary cadmium (UCd). UCd levels were significantly higher in the polluted group than the control group. The bone mineral density of the proximal forearm of subjects in polluted group or with high UCd levels was significantly lower than that of subjects in the control group or with low UCd levels. However, regarding bone mineral density of the distal forearm, this trend was only found in subjects living in area A. Our data showed that cortical bone mineral density in the forearm may be more strongly affected by cadmium exposure than trabecular bone mineral density.     

Identification of QTL affecting seed mineral concentrations and content in the model legume <Emphasis Type="Italic">Medicago truncatula</Emphasis>

Increasing the amount of bioavailable micronutrients such as iron and zinc in plant foods for human consumption is an international goal, intended especially for developing countries where micronutrient deficiencies are an ongoing health risk. Legume seeds have the potential to provide the essential nutrients required by humans, but concentrations of several minerals are low when compared to other foods. In order to increase seed mineral concentrations, it is important to understand the genes and processes involved in mineral distribution within the plant. The main objectives of this study were to use a Medicago truncatula recombinant inbred population (Jemalong-6 × DZA 315.16) to determine loci governing seed mineral concentrations, seed mineral content, and average seed weight, and to use these loci to propose candidate genes whose expression might contribute to these traits. Ninety-three lines in 2004 and 169 lines in 2006 were grown for seed harvest and subsequent analysis of seed Ca, Cu, Fe, K, Mg, Mn, P, and Zn concentrations and content. Quantitative trait loci (QTL) cartographer was used to identify QTL using composite interval mapping (CIM). CIM identified 46 QTL for seed mineral concentration, 26 for seed mineral content, and 3 for average seed weight. At least one QTL was detected for each mineral trait, and colocation of QTL for several minerals was found in both years. Results comparing seed weight with seed mineral concentration and content QTL demonstrate that seed size can be an important determinant of seed mineral concentration. The identification, in this model legume, of transgressive segregation for nearly all the minerals suggests that allelic recombination of relevant mineral-related genes in agronomic legumes could be a successful strategy to increase seed mineral concentrations above current levels. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Discovery of a high molecular weight complex of calcium, phosphate, fetuin, and matrix gamma-carboxyglutamic acid protein in the serum of etidronate-treated rats.

In the present study we report the discovery of a novel protein-mineral complex in the serum of rats treated with doses of the bone-active bisphosphonate etidronate that inhibit normal bone mineralization. The composition of this high molecular mass protein-mineral complex consists of about 18% mineral, 80% fetuin, and 2% matrix Gla protein (MGP) by weight, and the presence of the complex in serum after an injection of 8 mg etidronate/100 g of body weight elevates calcium by 1.8-fold (to 4.3 mm), phosphate by 1.6-fold (to 5.6 mm), and MGP by 25-fold (to 12 microg/ml). The serum mineral complex reaches maximal levels at 6 h after subcutaneous injection of etidronate and is subsequently cleared from serum by 24 h. This highly specific complex of fetuin, MGP, and mineral prevents the growth, aggregation, and precipitation of the mineral component, which indicates that the previously reported calcification inhibitory activities of fetuin and MGP may be related to their ability to form stable complexes with nascent mineral nuclei. Treatment with the vitamin K-antagonist warfarin prevents the increase in serum MGP after etidronate injection, which shows that the increase in serum MGP is due to new synthesis and that the gamma-carboxylation of MGP is necessary for its binding to the serum mineral complex.     

Calcium homeostasis: solving the solubility problem

This report summarizes the evidence that the control of the concentration of free calcium ions in body fluids is centered at mineralized bone surfaces. This process involves an increase in the solubility of bone mineral produced by the non-collagenous proteins existing in the bone extracellular fluid (ECF) and on the adjacent surfaces of bone. The result is a basic equilibrium level produced in the absence of parathyroid hormone (PTH), which is well above the solubility of bone mineral. The effect of PTH is to increase the solubility of bone mineral still further, but the mechanism by which the hormone acts is unknown. The lining cells of the bone contain receptors for PTH and can be observed to respond to this hormone, but the relationship between this response and the increased solubility of bone remains to be discovered. Further research in this field is strongly urged.     

Structural study of the calcifying collagen in turkey leg tendons

The calcified turkey leg tendon represents a simple bone-like tissue that is ideally suited to analysis by diffraction methods. In this paper we report some structural studies of the tendon collagen in the uncalcified, fully calcified and partially calcified states. The low-angle meridional X-ray pattern from the uncalcified tendon is very similar to that of the rat tail tendon, and the resulting one-dimensional structure of the collagen fibril exhibits no feature that could be related to its eventual calcification. The structure of the fully calcified tendon, as determined by a combination of X-ray and neutron diffraction analyses, shows that the mineral is associated with the collagen at the level of the hole or gap region. In the calcifying tendon, increases in the amplitudes of the first and second X-ray meridional reflections are correlated with an increase in the mineral content of the collagen. On the basis of simple models, it is shown that this change in the pattern can be explained by a nucleation mechanism of calcification. It is concluded that when collagen becomes calcified the mineral penetrates throughout the fibril and is crystalline in the hole region but amorphous between the collagen molecules. The mechanism of calcification and the mechanical implications of the fully calcified structure are also discussed.     

Two forms of apatite deposited during mineralization of the hen tendon.

Old hen tendon provides a model suitable for the study of calcification in an extracellular matrix. In the present study, we observed the mineralizing substances of hen tendon by scanning electron microscopy of plasma-osmium-coated specimens and by transmission electron microscopy of those processed by a plasma-polymerization film replica method. The mineralizing front area revealed a number of elliptical particles fused to each other and forming rod-like structures oriented parallel to collagen fibrils. The area of advanced mineralization possessed non-mineralizing cavities, in which tendon cells were likely to exist. At this site, we recognized a second form of mineral structure, one in which the crystals had a scale-like morphology and were deposited onto the major first-form mineral component. This crystal form was similar to hydroxyapatite synthesized under wet reaction conditions. These findings strongly suggest that the second form of mineral formed independent of collagen fibrils existed together with the predominant, collagen-dependent form of mineral. We speculate that cell membranes and an extremely slow mineralization process may contribute to the formation of this form of mineral during the mineralization process in the hen tendon.     

Improved methods for the enumeration of heterotrophic bacteria in bottled mineral waters

At this time the European Union regulations require that the heterotrophic plate counts (HPC) of mineral waters be assessed at two recovery temperatures: 22 degrees C for 72 h and 37 degrees C for 24 h. This procedure is time consuming and expensive. Development of new rapid methods for microbiological assessment of the microbial flora in the bottled water is an industry-driven need.The objectives of this work were to develop a method for the HPC that utilises only one recovery temperature and one incubation period and evaluate the use of, the LIVE/DEAD(R) BacLight Bacterial Viability Kit, 5-cyano-2,3-ditotyl tetrazolium chloride (CTC) and impedance methods to enumerate viable bacteria in bottled mineral water.Results showed that incubation at 30 degrees C could be used instead of incubation at 22 degrees C and 37 degrees C. Good correlation exists between counts at 30 degrees C and counts at 22 degrees C (r>0.90) and all the pathogens important in mineral water analyses grow similarly at 30 degrees C and 37 degrees C during 24 h.It was demonstrated that impedance methods might be useful to the mineral water industry as a rapid indicator of microbiological quality of the water.Results obtained with BacLight and CTC were similar to those obtained with plate counts.     

An Experimental Investigation of the Effect of Bacillus megaterium on Apatite Dissolution

Field observations suggest that some mineral dissolution rates can be enhanced by microbial activity indirectly, without direct contact with the mineral surface. A series of apatite dissolution experiments was performed to better understand this rate enhancement process. Far-from equilibrium abiotic apatite dissolution rates, measured in mixed-flow reactors at 25°C were enhanced by increasing concentration of aqueous organic acids and decreasing aqueous phosphate activity, demonstrating the existence of indirect pathways for microbial rate enhancement. Further apatite dissolution experiments were performed in closed-system reactors in the presence of Bacillus megaterium , a common heterotrophic aerobe. Experiments were designed to allow the bacteria to be either in direct contact or indirect contact with the apatite; in the latter case, the microbes were physically separated from the apatite using dialysis bags. Apatite dissolution in indirect contact with Bacillus megaterium was 50 to 900% faster than abiotic controls. Bacterial rate enhancement was, however, 3 to over 10 times lower when Bacillus megaterium was in direct contract versus indirect contact with the apatite surfaces. These results show that (1) bacteria can accelerate rates without being in physical contact with the dissolving mineral, and (2) microbially mediated dissolution may be less effective when bacteria are in direct contact with mineral surfaces. Supression of mineral dissolution is interpreted to stem from the preferential colonization of reactive sites on the mineral surface.     

Streamlined approach for assessing embedded consumption of lithium and cobalt in the United States

In today's complex global supply chains, time and data intensive analyses are required to understand global flows of mineral commodities from mine to consumer, particularly for mineral commodities in products (electronics, automobiles, etc.) that contain multiple parts with many mineral commodities. National and regional analyses require additional time and data to incorporate international trade flows. However, data limitations and time constraints often prohibit global and national material flow analyses for minor metals. Here we present a methodological approach to circumvent these constraints by utilizing readily available industry-level global data from the United Nations Statistics Division and national industrial data to estimate total requirements for a mineral commodity. We apply this approach to lithium and cobalt use in the United States for the year 2018 and distinguish between apparent raw material consumption versus inferred embedded consumption of lithium and cobalt materials in all forms. The results show that more than half of the United States’ total requirements for both lithium and cobalt is in parts and products that were manufactured outside the United States. In large part, this is due to limited US manufacturing capability for lithium-ion battery materials and cells and the United States’ high import reliance for electronics that use those batteries.     

The origin of electrokinetic potentials in bone tissue: the organic phase

The purpose of this study was to determine the relative contributions of the organic and mineral phases of cow cortical bone to its electrokinetic response at room temperature. The technique of particle electrophoresis permitted electrokinetic (zeta) potentials to be calculated and plotted as a function of pH. Control and demineralized bone particles exhibited similar isoelectric points at pH approximately 5.1 (pH at which the zeta potential is zero), well below the isoelectric point of the bone mineral (pH approximately 8.6). In addition, the use of phosphate-containing buffers resulted in a zeta potential sign reversal of the bone mineral but had no effect on both the control and demineralized bone. These key results form the basis from which we suggest that the bone mineral lies within the organic phase (e.g., the mineral is not exposed to the fluid phase) and that the electrokinetic behavior of bone tissue is dominated by its organic ultrastructure.     

Technical note: CT determination of the mineral density of dry bone specimens using the dipotassium phosphate phantom

Recent studies have demonstrated the potential application of computed tomography (CT) in research into bone density. Clinical studies of bone density using CT commonly employ a dipotassium phosphate phantom to calibrate measurements of mineral density. Designed for in vivo studies, the use of this phantom requires that bones be scanned while immersed in and permeated by fluids or soft tissues similar to water in X-ray attenuation coefficient. However, this condition may not always be met in anthropological applications, which often involve rare and fragile specimens. This study compares mineral density values calculated for a sample of bones scanned—at the same sites—in air and in water. The results indicate that, when scanned in air, the mineral density of trabecular bone is dramatically underestimated, while that of cortical bone is slightly overestimated. We present a linear regression equation to correct this error but recommend that, when possible, researchers calculate their own regressions based on their specific scanning conditions. Am J Phys Anthropol 103:557–560, 1997. © 1997 Wiley-Liss, Inc.