Microstructure,mechanical, and biomimetic properties of fish scales from Pagrus major

The fish scale of Pagrus major has an orthogonal plywood structure of stratified lamellae, 1-2 microm in thickness, consisting of closely packed 70- to 80-nm-diameter collagen fibers. X-ray diffraction, energy-dispersive X-ray analysis, and infrared spectroscopy indicate that the mineral phase in the scale is calcium-deficient hydroxyapatite containing a small amount of sodium and magnesium ions, as well as carbonate anions in phosphate sites of the apatite lattice. The tensile strength of the scale is high (approximately 90 MPa) because of the hierarchically ordered structure of mineralized collagen fibers. Mechanical failure occurs by sliding of the lamellae and associated pulling out and fracture of the collagen fibers. In contrast, demineralized scales have significantly lower tensile strength (36 MPa), indicating that interactions between the apatite crystals and collagen fibers are of fundamental importance in determining the mechanical properties. Thermal treatment of fish scales to remove the organic components produces remarkable inorganic replicas of the native orthogonal plywood structure of the fibrillary plate. The biomimetic replica produced by heating to 873 K consists of stratified porous lamellae of c-axis-aligned apatite crystals that are long, narrow plates, 0.5-0.6 microm in length and 0.1-0.2 microm in width. The textured inorganic material remains intact when heated to 1473 K, although the size of the constituent crystals increases as a result of thermal sintering.     

Electrochemically-assisted deposition of biomimetic hydroxyapatite-collagen coatings on titanium plate

A biomimetic bone-like composite, made of self-assembled collagen fibrils and carbonate hydroxyapatite nanocrystals, has been performed by an electrochemically-assisted deposition on titanium plate. The electrolytic processes have been carried out using a single type I collagen molecules suspension in a diluted Ca(NO₃)₂ and NH₄H₂PO₄ solution at room temperature and applying a constant current for different periods of time. Using the same electrochemical conditions, carbonate hydroxyapatite nanocrystals or reconstituted collagen fibrils coatings were obtained. The reconstituted collagen fibrils, hydroxyapatite nanocrystals and collagen fibrils/apatite nanocrystals coatings have been characterized chemically, structurally and morphologically, as well as for their ability to bind fibronectin (FN). Fourier Transform Infrared microscopy has been used to map the topographic distribution of the coating components at different times of electrochemical deposition, allowing to single out the individual deposition steps. Moreover, roughness of Ti plate has been found to affect appreciably the nucleation region of the inorganic nanocrystals. Laser scanning confocal microscopy has been used to characterize the FN adsorption pattern on a synthetic biomimetic apatitic phase, which exhibits a higher affinity when it is inter-grown with the collagen fibrils. The results offer auspicious applications in the preparation of medical devices such as biomimetic bone-like composite-coated metallic implants.     

Thermogravimetric analysis of human femur bone

Thermal decomposition analysis of human femur bone has been carried out in the temperature range between 25°C and about 1000°C. In this temperature range, the differential primary weight loss curve yielded four distinct peaks for the femur bone. Each minimum inflexion point was arbitrarily chosen as the temperature at which the thermal decomposition of the component responsible for the peak had been completed. Aided by the thermogravimetric data on tendon collagen, the observed four peaks have been identified as follows: First: dissociation of water from the collagen; second: decomposition of collagen molecule itself; third: residual organic components associated with the collagen; fourth: water loss accompanied with the transformation of inorganic apatite and residual protein that were not decomposed at lower temperature. The fourth peak identified with the crystal transformation has been linked to the transformation ofα-tricalcium phosphate, which is known to have a hexagonal structure, toβ-tricalcium phosphate, which has a rhombohedral structure. This transformation has been observed between 700°C and 780°C which is in accord with the range observed in the transformation of synthetic apatite crystals. The experiments were performed under two different conditions: in normal pressure (in air) and in a reduced pressure of about 30μ Hg. In the average, the total weight loss up to the temperature of about 1000°C was about 35.64% for the heating in air and 36.45% for the heating in the reduced pressure. The weight loss has been carefully analyzed in the different temperature intervals and has been compared with an expected weight loss predicted by three most quoted formulas advanced for the apatite in bone. In addition to this, the conventional thermogravimetric analysis has been carried out in order to retrieve the thermal kinetic decomposition parameters corresponding to each peak. The activation energies corresponding to the first, second, and third peaks have been found to be slightly higher than those of tendon collagen. A detailed analysis showed that the bone contains about 28% protein and about 8% water (apatite crystalline water included).     

Advances in biomimetic collagen mineralisation and future approaches to bone tissue engineering

With an ageing world population and ~20% of adults in Europe being affected by bone diseases, there is an urgent need to develop advanced regenerative approaches and biomaterials capable to facilitate tissue regeneration while providing an adequate microenvironment for cells to thrive. As the main components of bone are collagen and apatite mineral, scientists in the tissue engineering field have attempted in combining these materials by using different biomimetic approaches to favour bone repair. Still, an ideal bone analogue capable of mimicking the distinct properties (i.e., mechanical properties, degradation rate, porosity, etc.) of cancellous bone is to be developed. This review seeks to sum up the current understanding of bone tissue mineralisation and structure while providing a critical outlook on the existing biomimetic strategies of mineralising collagen for bone tissue engineering applications, highlighting where gaps in knowledge exist.     

Biomimetic and bioactive nanofibrous scaffolds from electrospun composite nanofibers

Electrospinning is an enabling technology that can architecturally (in terms of geometry, morphology or topography) and biochemically fabricate engineered cellular scaffolds that mimic the native extracellular matrix (ECM). This is especially important and forms one of the essential paradigms in the area of tissue engineering. While biomimesis of the physical dimensions of native ECM's major constituents (eg, collagen) is no longer a fabrication-related challenge in tissue engineering research, conveying bioactivity to electrospun nanofibrous structures will determine the efficiency of utilizing electrospun nanofibers for regenerating biologically functional tissues. This can certainly be achieved through developing composite nanofibers. This article gives a brief overview on the current development and application status of employing electrospun composite nanofibers for constructing biomimetic and bioactive tissue scaffolds. Considering that composites consist of at least two material components and phases, this review details three different configurations of nanofibrous composite structures by using hybridizing basic binary material systems as example. These are components blended composite nanofiber, core-shell structured composite nanofiber, and nanofibrous mingled structure.     

Bone formation in ectopic and osteogenic tissue induced by a novel BMP-2-related peptide combined with rat tail collagen

Bone morphogenetic proteins (BMPs) play an important role in regulating osteoblast differentiation and subsequent bone formation, mainly evidenced by the induced osteogenic ability of BMP-2 from BMPs. However, BMP-2 alone does not induce the expected efficacy due to its short retention in vivo. In this study, a novel BMP-2-related peptide (designated P24) derived from the “knuckle epitope” of BMP-2 was coupled covalently to type I collagen derived from rat tail and observed under scanning electron microscopy (SEM) in low vacuum mode. The BMP-2-related peptide/collagen composite was implanted in vivo into the pocket of the quadriceps musculature of Sprague-Dawley (SD) rats and then harvested 3 or 6 weeks after surgery. It was found that lyophilized collagen retained a porous network structure with an average inner-diameter of 90 ∼ 160 μm. Based on radiographic evaluation and histological examination, BMP-2-related peptide/collagen induced significant ectopic bone formation compared to that of rat tail collagen alone as a control. Our results indicate collagen served as a good carrier for newly synthesized BMP-2-related peptide and that the BMP-2-related peptide/collagen composite was an effective substitute in bone tissue engineering.     

Osteonectin-derived peptide increases the modulus of a bone-mimetic nanocomposite

Many factors contribute to the toughness of bone including the presence of nano-size apatite crystals, a dense network of collagen fibers, and acidic proteins with the ability to link the mineral phase to the gelatinous collagen phase. We investigated the effect of a glutamic acid (negatively charged) peptide (Glu6), which mimics the terminal region of the osteonectin glycoprotein of bone, on the shear modulus of a synthetic hydrogel/apatite nanocomposite. One end of the synthesized peptide was functionalized with an acrylate group (Ac-Glu6) to covalently attach the peptide to the hydrogel phase of the composite matrix. When microapatite crystals (5 μm diameter) were used, addition of Ac-Glu6 peptide did not affect the modulus of the microcomposite. However, when nanoapatite crystals (100 nm diameter) were used, addition of Ac-Glu6 resulted in significant reinforcement of the shear modulus of the nanocomposite (∼100% in elastic shear modulus). Furthermore, addition of Ac-Gly6 (a neutral glycine sequence) or Ac-Lys6 (a positively charged sequence) did not reinforce the nanocomposite. These results demonstrate that the reinforcement effect of the Glu6 peptide, a sequence in the terminal region of osteonectin, is modulated by the size of the apatite crystals. The findings of this work can be used to develop advanced biomimetic composites for skeletal tissue regeneration.     

Complementary Physical and Mechanical Techniques to Characterise Tooth： A Bone-like Tissue

Bone like tissues are biocomposites comprising an organic matrix （mostly collagen） and a reinforcement phase in the form of mineral crystals （poorly stoichiometric apatite）. The composite properties are a result of the material characteristics of the two phases, their interaction, the relative composition, the orientation and the micro-architecture of the structure. The inherent spatial heterogeneity of these tissues （a result of evolutionary and functional requirements） and their exposure to various environmental and mechanical influences result in highly variable properties on the microscale, which can only be characterised by modern microanalytical methods. We present here results obtained by the complementary use of the modem nanoindentation and micro-X-ray diffraction techniques, which were used to probe the properties and structure of human dentine and enamel of primary molar teeth. The results show that both the addition and the higher organization of mineral within the organic matrix produce stiffer and harder tissue and that the examination of properties within small tissue volumes can be reliably achieved by use of these two methods in parallel. This opens new avenues in the study of biomaterial in general, and for the local characterisation of regions of teeth that suffered bacterial attack, mechanical wear, fluoridisation, chemical bleaching, or dental treatment such as laser ablation or drilling.     

Apatite genesis: A biologically induced or biologically controlled mineral formation process?

Apatite formation from organic matter (ribonucleic acid) and calcium carbonate (cuttlebone) requires intervention of microorganisms. We have attempted to characterize this mineral formation process by locating the alkaline phosphatase and the crystals formed. Alkaline phosphatase, which is important for the liberation of the necessary components, was localized in the periplasmic space of Providencia rettgeri in the same manner as in Escherichia coli. Accordingly, the release of inorganic phosphate and the formation of apatite may occur at this site. However, electron microscope observations revealed the presence of extracellular apatite; moreover, apatite particles that were formed with or without bacteria (with alkaline phosphatase from hydrolyzed ribonucleic acid as phosphorus source) were closely similar in size and appearance. The formation of apatite can thus be qualified as biologically induced mineralization. Nevertheless, a bacterial cell can also act as a nucleator for apatite crystallization, but this would appear exceptional.     

Effects of water on the mechanical properties of gelatin films

The mechanical properties of gelatin films were studied in relation to the effect of water, and compared with those of collagen films. The S-shaped sorption isotherm was separated into an adsorption curve C₁ and dissolution curve C₂. From the C₂ curve, the interaction parameter χ₁ of Flory–Huggins' equation was calculated. The χ₁ of gelatin were larger than those of collagen at low relative humidities (RH), while they coincided with each other at high RH. It was found that a composite curve was made by shifting stress relaxation curves obtained at different humidities along the log time axis. The shift factor for the formation of the composite curve was analyzed by Fujita–Kishimoto's equation, which was based on the free volume theory. The parameter β, which expressed the extent of the contribution of sorbed water to the increase in the free volume of the system, was 0.05 in the range of C₂ from 0 to 0.08 (0–65% RH). This value was much smaller than 0.16 for collagen. The value was 0.16 in the range of C₂ higher than 0.08, which was equal to that of the collagen. Dynamic shear modulus G′, loss modulus G″, and tan δ were determined as functions of RH. The gelatin film extended more than 100% at 73% RH under the very small stress of about 10⁷ dyn/cm². This corresponds to the region where β changes from 0.05 to 0.16, although such a phenomenon was not observed in the collagen film. The wide-angle X-ray pattern of extended gelatin was similar to that of renatured collagen fiber.     

On the relationship between the microstructure of bone and its mechanical stiffness.

A recent study of bone structure shows that the plate-shaped carbonate apatite crystals in individual lamellae are arranged in layers across the lamellae, and that the orientation of these layers are different in alternate lamellae. Based on these findings, a new micromechanical model for the Young's modulus of bone is proposed, which accounts for the anisotropy and geometrical characteristics of the material. The model incorporates the platelet-like geometry of the basic reinforcing unit, the presence of alternating thin and thick lamellae, and the orientations of the crystal platelets in the lamellae. The thin and thick lamellae are modeled as orthotropic composite layers made up of thin rectangular apatite platelets within a collagen matrix, and classical orthotropic elasticity theory is used to calculate the Young's modulus of the lamellae. Bone is viewed as an assembly of such orthotropic lamellae bent into cylindrical structures, and having a constant, alternating angle between successive lamellae. The micromechanical model employs a modified rule-of-mixtures to account for the two types of lamellae. The model provides a curve similar to the published experimental data on the angular dependence of Young's modulus, including a local maximum at an angle between 0 and 90 degrees. A rigorous testing of the model awaits additional experimental data.     

A microstructurally driven model for pulmonary artery tissue

A new constitutive model for elastic, proximal pulmonary artery tissue is presented here, called the total crimped fiber model. This model is based on the material and microstructural properties of the two main, passive, load-bearing components of the artery wall, elastin, and collagen. Elastin matrix proteins are modeled with an orthotropic neo-Hookean material. High stretch behavior is governed by an orthotropic crimped fiber material modeled as a planar sinusoidal linear elastic beam, which represents collagen fiber deformations. Collagen-dependent artery orthotropy is defined by a structure tensor representing the effective orientation distribution of collagen fiber bundles. Therefore, every parameter of the total crimped fiber model is correlated with either a physiologic structure or geometry or is a mechanically measured material property of the composite tissue. Further, by incorporating elastin orthotropy, this model better represents the mechanics of arterial tissue deformation. These advancements result in a microstructural total crimped fiber model of pulmonary artery tissue mechanics, which demonstrates good quality of fit and flexibility for modeling varied mechanical behaviors encountered in disease states.     

Twisted plywood pattern of collagen fibrils in teleost scales: an X-ray diffraction investigation

The distribution and orientation of collagen fibrils, and apatite crystals, in the scales of a bony fish (Leuciscus cephalus) were investigated by X-ray diffraction. The small-angle diffraction patterns obtained with a microfocus scanning setup from most of the examined areas exhibit a distribution of intensity of the collagen reflections according to five preferential orientations, at 36 degrees from one another. It is suggested that the peculiar small-angle X-ray diffraction pattern is due to a plywood arrangement of collagen fibrils in successive layers parallel to the surface of the scale. The fibrils are strictly aligned in each layer and the alignment rotates by 36 degrees in successive layers, according to a discontinuous twist that generates a symmetric plywood pattern. The large spread of the wide-angle reflections does not allow one to distinguish the five directions of orientation in the intensity distribution of the 002 reflection of apatite. However, the patterns recorded from the less ordered regions of the scales display two different orientations of the 002 reflection and allow one to infer a preferential distribution of the apatite crystals with their c-axes parallel to the collagen fibrils. Although much electron microscopic evidence of plywood arrangements in calcified, as well as uncalcified, tissues has been reported, these are the very first diffraction data which unambiguously confirm the presence of these peculiar structures and suggest that this kind of investigation represents a powerful tool with which to study plywood arrangements in biological tissues.     

A new model to simulate the elastic properties of mineralized collagen fibril

Bone, because of its hierarchical composite structure, exhibits an excellent combination of stiffness and toughness, which is due substantially to the structural order and deformation at the smaller length scales. Here, we focus on the mineralized collagen fibril, consisting of hydroxyapatite plates with nanometric dimensions aligned within a protein matrix, and emphasize the relationship between the structure and elastic properties of a mineralized collagen fibril. We create two- and three-dimensional representative volume elements to represent the structure of the fibril and evaluate the importance of the parameters defining its structure and properties of the constituent mineral and collagen phase. Elastic stiffnesses are calculated by the finite element method and compared with experimental data obtained by synchrotron X-ray diffraction. The computational results match the experimental data well, and provide insight into the role of the phases and morphology on the elastic deformation characteristics. Also, the effects of water, imperfections in the mineral phase and mineral content outside the mineralized collagen fibril upon its elastic properties are discussed.     

In vitro reconstruction of full thickness human skin on a composite collagen material

Rapid progress of in vitro techniques in the lastyears enabled the creation of organotypic skin cultures offering newpossibilities in wound treatment. Rebuilding of graft is one of the keyelementsof successful outcome of the procedure.In search for the best scaffold for organotypic skin culture, the novelcomposite xenogenic collagen based material with unique properties has beencreated and used to reconstitute full thickness human skin invitro. Based on our long established technology used for theproduction of collagen dressings for the treatment of burns, this novel,composite material offers excellent growth support of highly biodegradablespongy layer, combined with mechanical strength of collagen membrane. Themodulation of collagen properties was accomplished by consecutive treatmentwithhigh temperature and gamma irradiation. The use of the substrate enabled toobtain organotypic culture that resembles full thickness skin with fibroblastslayer and well-developed multilayer epithelium. Our new material offers easyhandling of obtained graft during surgery along with accelerated cell growth andcontrolled biodegradation of the culture support.     

Osteodentine and vascular osteodentine of Anarhichas lupus (L.)

Summary TEM, SEM and X-ray diffraction analysis demonstrate the heterogeneity of the dentinal tissue of Anarhichas lupus, a vascular osteodentine. The disordered aspect of collagen fibres, incompletely mineralized (the periodical striation being still visible), explains the scattered distribution of crystallites since they are responsible for their arrangement. The low degree of mineralization revealed by the visible collagen striation is confirmed by X-ray diffraction analysis (the crystallinity of vascular osteodentine being much lower than that of the peripheral dental tissue) as well as by high resolution TEM, since no lattice planes could be observed. Osteodentine, supporting bone and proper bone have in common a mineral phase, more or less organized, different from the apatite system.The authors thank Mireille Cottrel-Gengoux for her technical assistance     

The size exclusion characteristics of type I collagen: implications for the role of noncollagenous bone constituents in mineralization

The mineral in bone is located primarily within the collagen fibril, and during mineralization the fibril is formed first and then water within the fibril is replaced with mineral. The collagen fibril therefore provides the aqueous compartment in which mineral grows. Although knowledge of the size of molecules that can diffuse into the fibril to affect crystal growth is critical to understanding the mechanism of bone mineralization, there have been as yet no studies on the size exclusion properties of the collagen fibril. To determine the size exclusion characteristics of collagen, we developed a gel filtration-like procedure that uses columns containing collagen from tendon and bone. The elution volumes of test molecules show the volume within the packed column that is accessible to the test molecules, and therefore reveal the size exclusion characteristics of the collagen within the column. These experiments show that molecules smaller than a 6-kDa protein diffuse into all of the water within the collagen fibril, whereas molecules larger than a 40-kDa protein are excluded from this water. These studies provide an insight into the mechanism of bone mineralization. Molecules and apatite crystals smaller than a 6-kDa protein can diffuse into all water within the fibril and so can directly impact mineralization. Although molecules larger than a 40-kDa protein are excluded from the fibril, they can initiate mineralization by forming small apatite crystal nuclei that diffuse into the fibril, or can favor fibril mineralization by inhibiting apatite growth everywhere but within the fibril.     

In situ mechanical analysis of myofibrillar perturbation and aging on soft, bilayered Drosophila myocardium

Drosophila melanogaster is a genetically malleable organism with a short life span, making it a tractable system in which to study mechanical effects of genetic perturbation and aging on tissues, e.g., impaired heart function. However, Drosophila heart-tube studies can be hampered by its bilayered structure: a ventral muscle layer covers the contractile cardiomyocytes. Here we propose an atomic force microscopy-based analysis that uses a linearized-Hertz method to measure individual mechanical components of soft composite materials. The technique was verified using bilayered polydimethylsiloxane. We further demonstrated its biological utility via its ability to resolve stiffness changes due to RNA interference to reduce myofibrillar content or due to aging in Drosophila myocardial layers. This protocol provides a platform to assess the mechanics of soft biological composite systems and, to our knowledge, for the first time, permits direct measurement of how genetic perturbations, aging, and disease can impact cardiac function in situ.     

<Emphasis Type="Italic">BMP-2</Emphasis> gene-fibronectin-apatite composite layer enhances bone formation

Background  

Safe and efficient gene transfer systems are needed for tissue engineering. We have developed an apatite composite layer including the bone morphogenetic protein-2 (BMP-2) gene and fibronectin (FB), and we evaluated its ability to induce bone formation.     

Speciation and bioavailability of zinc in amended sediments

Abstract

The speciation and bioavailability of zinc (Zn) in smelter-contaminated sediments were investigated as a function of phosphate (apatite) and organic amendment loading rate. Zinc species identified in preamendment sediment were zinc hydroxide-like phases, sphalerite, and zinc sorbed to an iron oxide via X-ray adsorption near edge structure (XANES) spectroscopy. Four months after adding the amendments to the contaminated sediment, hopeite, a Zn phosphate mineral, was identified indicating phosphate was binding and sequestering available Zn and Zn pore water concentrations were decreased at levels of 90% or more. Laboratory experiments indicate organic amendments exhibit a limited effect and may hinder sequestration of pore water Zn when mixed with apatite. The acute toxicity of the sediment Zn was evaluated with Hyalella azteca, and bioaccumulation of Zn with Lumbriculus variegates. The survivability of H. azteca increased as a function of phosphate (apatite) loading rate. In contaminated sediment without apatite, no specimens of H. azteca survived. The bioaccumulation of Zn in L. variegates also followed a trend of decreased bioaccumulation with increased phosphate loading in the contaminated sediment. The research supports an association between Zn speciation and bioavailability.