Relationship between bone tissue strain and lattice strain of HAp crystals in bovine cortical bone under tensile loading

Cortical bone is a composite material composed of hydroxyapatite (HAp) and collagen. As HAp is a crystalline structure, an X-ray diffraction method is available to measure the strain of HAp crystals. However, HAp crystals in bone tissue have been known to have the low degree of crystallization. Authors have proposed an X-ray diffraction method to measure the lattice strain of HAp crystals from the diffusive intensity profile due to low crystallinity. The precision of strain measurement was greatly improved by this method. In order to confirm the possibility of estimating the bone tissue strain with measurements of the strain of HAp crystals, this work investigates the relationship between bone tissue strain on a macroscopic scale and the lattice strain of HAp crystals on a microscopic scale. The X-ray diffraction experiments were performed under tensile loading. Strip bone specimens of 40x6x0.8mm in size were cut from the cortical region of a shaft of bovine femur. A stepwise tensile load was applied in the longitudinal direction of the specimen. By detecting the diffracted X-ray beam transmitted through the specimen, the lattice strain was directly measured in the loading direction. As a result, the lattice strain of HAp crystals showed lower value than the bone tissue strain measured by a strain gage. The bone tissue strain was described with the mean lattice strain of the HAp crystals and the elastic modulus.     

Estimating nanoscale deformation in bone by X-ray diffraction imaging method

Knowledge of internal stress-strain in bone tissue is important for clinical diagnosis and remedies. The inorganic mineral phase of apatite crystals in bone composite, because of its crystalline nature, provides a reliable way of measurement through X-ray diffraction system. Use of two-dimensional detector, imaging plate (IP), is considered to expedite the process with much more information, hence, is widely applied in the study of organization, stress, strain, etc. for crystalline substance. The distortion of Debye rings in the image obtained by IP can be directly related to the deformation in lattice plane of the crystals. Since X-ray diffraction method involves measurement at nano-level, proper focus on the extraction of data and corresponding analysis is needed. In the current work, we considered weighted average value of intensity to locate radius vectors along azimuthal direction in the diffracted rings from the primary array of digital data in steps of pixels. The widely applied approaches for profile shift measurement--peak shift and full width at half maximum (FWHM) of a peak, and shift of center of gravity of profile--were compared with a new concept of segmental shift (SS) proposed previously by the authors. We observed reliable and effective outcomes with higher precision in the consideration of SS while using IP as a detector. Our approach in this work for intensity integration and radius vector positioning especially add precision in such applications.     

Elastic properties of collagen in bone determined by measuring the Debye–Waller factor

Force constant values for thermal vibrational motion of a collagen molecule along the helix axis in tendon, completely demineralized bone (CDB), and partially demineralized bone (PDB) were estimated by determining the Debye–Waller factor (DW factor) for the diffracted X-ray intensity from these specimens. The DW factor for nominal value of 0.286 nm meridional diffraction representing a period along the helical axis of a collagen molecule was measured. As the atomic scattering factor of mineral constituents is much larger than that of collagen, it is difficult to detect the diffraction from collagen in bone specimen. Therefore, PDB was used in this study. In order to compare obtained force constant value for CDB with mechanical properties of collagen in the literature, the value was translated into Young's modulus value using the cross-sectional area of a collagen molecule. In the case of collagen in PDB, i.e., collagen with the close presence of HAp mineral particles, as the DW factor of the diffracted intensity by hydroxyapatite (HAp) was considered to be negligible compared with that of collagen, the DW factor determined was interpreted as that of collagen molecule in PDB specimen. The force constant value obtained for collagen in PDB was significantly larger than that of collagen in CDB. This result was thought to be a manifestation of the hardening of collagen matrix in bone by HAp mineral particles and the first straightforward evidence for a difference in collagen properties depending on the presence of HAp mineral particles. The method employed in this study can be utilized for detecting mechanical properties of the individual constituents of composite materials.     

Age changes in the crystallinity of bone mineral and in the disorder of its crystal

The crystallinity of bone mineral and disorder of the crystal at different ages were measured by the X-ray diffraction method of Ruland. Measurements were made on femoral mid-diaphyses of Wistar rat from 2 weeks to 1 year of age. For a given animal age, the crystallinity of bone mineral increases with age, while the overall disorder of the crystal does not vary within experimental accuracy. The increase in the crystallinity with age is attributed to an increase in crystallite size, a decrease in lattice imperfections, or a combination of both effects. It is suggested that lattice imperfections of the first kind more largely contributes to the disorder of bone mineral crystals than those of the second kind.     

Cholesterol monohydrate nucleation in ultrathin films on water

The growth of a cholesterol crystalline phase, three molecular layers thick at the air-water interface, was monitored by grazing incidence x-ray diffraction and x-ray reflectivity. Upon compression, a cholesterol film transforms from a monolayer of trigonal symmetry and low crystallinity to a trilayer, composed of a highly crystalline bilayer in a rectangular lattice and a disordered top cholesterol layer. This system undergoes a phase transition into a crystalline trilayer incorporating ordered water between the hydroxyl groups of the top and middle sterol layers in an arrangement akin to the triclinic 3-D crystal structure of cholesterol x H(2)O. By comparison, the cholesterol derivative stigmasterol transforms, upon compression, directly into a crystalline trilayer in the rectangular lattice. These results may contribute to an understanding of the onset of cholesterol crystallization in pathological lipid deposits.     

Crystallinity of lyophilised carrot cell wall components

The aim of this work was to investigate the effect of removal of cell wall components on the crystallinity of cell walls using X-ray diffraction. Various insoluble cell wall residues were prepared following a sequential extraction of carrot cell wall material. X-ray diffraction patterns were typical of cellulose although there was a possible contribution of pectic polysaccharides to the crystallinity. As more amorphous material was removed to produce a cellulose rich residue, the crystallinity index increased from 12 to 16%, larger than that estimated from cellulose alone. For the last residue treated with 4M KOH, a lower value of crystallinity was found (14%) which resulted from the change of some crystalline domains of cellulose into amorphous regions. Pressing conditions (temperature, water content) have been investigated and did not alter the crystallinity index significantly.     

Effect of Explosion on the Crystalline Polymorphism of Chitin and Chitosan

For identification of how explosion increases the reactivity of chitin and chitosan, changes in the crystalline polymorphism of these polysaccharides were studied by X-ray diffraction measurements. The α-chitin form of chitin did not change after being exploded, but an X-ray diagram of chitosan showed a hydrated crystal of low crystallinity before the explosion, and increased crystallinity of the hydrated form plus a small amount of an anhydrous crystal after the explosion. The improvement of accessibility to both polysaccharides caused by the explosion seemed not to arise from changes in their crystalline polymorphism or crystallinity     

In situ analysis of mineral content and crystallinity in bone using infrared micro-spectroscopy of the nu(4) PO(4)(3-) vibration.

Measurements of bone mineral content and composition in situ provide insight into the chemistry of bone mineral deposition. Infrared (IR) micro-spectroscopy is well suited for this purpose. To date, IR microscopic (including imaging) analyses of bone apatite have centered on the nu(1),nu(3) PO(4)(3-) contour. The nu(4) PO(4)(3-) contour (500-650 cm(-1)), which has been extensively used to monitor the crystallinity of hydroxyapatite in homogenized bone samples, falls in a frequency region below the cutoff of the mercury-cadmium-telluride detectors used in commercial IR microscopes, thereby rendering this vibration inaccessible for imaging studies. The current study reports the first IR micro-spectroscopy spectra of human iliac crest cross sections in the nu(4) PO(4)(3-) spectral regions, obtained with a synchrotron radiation source and a Cu-doped Ge detector coupled to an IR microscope. The acid phosphate (HPO(4)(2-)) content and mineral crystallite perfection (crystallinity) of a human osteon were mapped. To develop spectra-structure correlations, a combination of X-ray powder diffraction data and conventional Fourier transform IR spectra have been obtained from a series of synthetic hydroxyapatite crystals and natural bone powders of various species and ages. X-ray powder diffraction data demonstrate that there is an increase in average crystal size as bone matures, which correlates with an increase in the nu(4) PO(4)(3-) FTIR absorption peak ratio of two peaks (603/563 cm(-1)) within the nu(4) PO(4)(3-) contour. Additionally, the IR results reveal that a band near 540 cm(-1) may be assigned to acid phosphate. This band is present at high concentrations in new bone, and decreases as bone matures. Correlation of the nu(4) PO(4)(3-) contour with the nu(2) CO (3)(2-) contour also reveals that when acid phosphate content is high, type A carbonate content (i.e., carbonate occupying OH(-) sites in the hydroxyapatite lattice) is high. As crystallinity increases and acid phosphate content decreases, carbonate substitution shifts toward occupation of PO(4)(3-) sites in the hydroxyapatite lattice. Thus, IR microscopic analysis of the nu(4) PO(4)(3-) contour provides a straightforward index of both relative mineral crystallinity and acid phosphate concentration that can be applied to in situ IR micro-spectroscopic analysis of bone samples, which are of interest for understanding the chemical mechanisms of bone deposition in normal and pathological states.     

Nanostructure and elastic modulus of single trabecula in bovine cancellous bone

We aimed to investigate the elastic modulus of trabeculae using tensile tests and assess the effects of nanostructure at the hydroxyapatite (HAp) crystal scale on the elastic modulus. In the experiments, 18 trabeculae that were at least 3 mm in length in the proximal epiphysis of three adult bovine femurs were used. Tensile tests were conducted using a small tensile testing device coupled with microscopy under air-dried condition. The c-axis orientation of HAp crystals and the degree of orientation were measured by X-ray diffraction. To observe the deformation behavior of HAp crystals under tensile loading, the same tensile tests were conducted in X-ray diffraction measurements. The mineral content of specimens was evaluated using energy dispersive X-ray spectrometry. The elastic modulus of a single trabecula varied from 4.5 to 23.6 GPa, and the average was 11.5±5.0 GPa. The c-axis of HAp crystals was aligned with the trabecular axis and the crystals were lineally deformed under tensile loading. The ratio of the HAp crystal strain to the tissue strain (strain ratio) had a significant correlation with the elastic modulus (r=0.79; P<0.001). However, the mineral content and the degree of orientation did not vary widely and did not correlate with the elastic modulus in this study. It suggests that the strain ratio may represent the nanostructure of a single trabecula and would determine the elastic modulus as well as mineral content and orientation.     

荔枝核淀粉特性研究

本文采用扫描电镜观察荔枝核淀粉颗粒呈椭圆或多边形。差示扫描量热仪分析荔枝核淀粉的热力学特性表明,其起始温度、峰温度、终止温度和吸热焓值分别为69．04℃、78．79℃、87．08℃和7．5J/g。X射线衍射分析表明荔枝核淀粉呈C型结晶。Brabender粘度分析表明荔枝核淀粉糊化温度为85．3℃,淀粉糊高温稳定。     

X-ray determination of the crystallinity in bone mineral

The X-ray diffraction method of Ruland (Acta Crystallogr. 14 (1961) 1180-1185) used for the crystallinity determination of synthetic polymers was applied to the mineral present in mature rat cortical bone. The results obtained were compared with those obtained by other X-ray methods of Harper and Posner and Wakelin, Virgin and Crystal. It was concluded that the method of Ruland gives a more reliable determination of the crystallinity of bone mineral than other methods.     

The Effect and Osteoblast Signaling Response of Trace Silicon Doping Hydroxyapatite

It is commonly accepted that silicon-doped hydroxyapatite (HAp) can achieve good repair effects for both spinal fusion and bone defect filling. However, the underlying mechanism by which silicon aids such beneficial effects is still not fully understood. Herein, we report on silicon-doped hydroxyapatites with excellent biocompatibility to osteoblast cells and suggest the signaling pathway involved. Non-doped HAp and trace Si-doped HAp (Si/HAp) with Si concentration close to and higher than natural bones were synthesized (i.e., 32, 260, and 2000 ppm Si). The composition, crystal lattice vibration pattern, and morphology of these samples are characterized by X-ray diffraction (XRD), Fourier transform infrared (FTIR), and SEM, respectively. Positive biological activities of these Si-doped HAp materials were demonstrated through a cytotoxicity study and with the MTT and alkaline phosphatase (ALP) activity assays. The Si-doped samples were not toxic to MC3T3-E1 cells. Indeed, osteoblast proliferation measurement illustrated that 2000 ppm Si-doped HAp increased osteoblast proliferation by about 1.6 times compared to non-doped HAp. The ALP assay also proves that the trace Si doping has the function to enhance cell proliferation and differentiation. The ALP assay showed that Si doping also enhanced cell differentiation. QRT-PCR results revealed that Si-doped HAp enhanced osteogenic differentiation of osteoblast cells by upregulating genes such as MAPK3, Fzd1, Wnt1, Lrp6, and BMP2. In conclusion, Si-doped HAp promotes osteoblast proliferation and differentiation by activating the Wnt/β-catenin and MAPK signaling pathways. This work could provide useful information of the beneficial effects of silicon in human bones and provide clues as to the molecular mechanism of the promotive effect of Si-doped HAp biomaterials.     

Comparative analysis of crystallinity changes in cellulose I polymers using ATR-FTIR, X-ray diffraction, and carbohydrate-binding module probes

Cotton fiber cellulose is highly crystalline and oriented; when native cellulose (cellulose I) is treated with certain alkali concentrations, intermolecular hydrogen bonds are broken and Na-cellulose I is formed. At higher alkali concentrations Na-cellulose II forms, wherein intermolecular and intramolecular hydrogen bonds are broken, ultimately resulting in cellulose II polymers. Crystallinity changes in cotton fibers were observed and assigned using attenuated total reflectance Fourier transform infrared (ATR FT-IR) spectroscopy and X-ray diffraction (XRD) subsequent to sodium hydroxide treatment and compared with an in situ protein-binding methodology using cellulose-directed carbohydrate-binding modules (CBMs). Crystallinity changes observed using CBM probes for crystalline cellulose (CBM2a, CBM3a) and amorphous cellulose (CBM4-1, CBM17) displayed close agreement with changes in crystallinity observed with ATR-FTIR techniques, but it is notable that crystallinity changes observed with CBMs are observed at lower NaOH concentrations (2.0 mol dm(-3)), indicating these probes may be more sensitive in detecting crystallinity changes than those calculated using FTIR indices. It was observed that the concentration of NaOH at which crystallinity changes occur as analyzed using the CBM labeling techniques are also lower than those observed using X-ray diffraction techniques. Analysis of crystallinity changes in cellulose using CBMs offers a new and advantageous method of qualitative and quantitative assessment of changes to the structure of cellulose that occur with sodium hydroxide treatment.     

Crystallinity and structure of starch using wide angle X-ray scattering

Wide angle X-ray diffraction was used to evaluate the crystalline fraction of a variety of starches, using preliminary smoothing then an iterative smoothing algorithm to estimate amorphous background scattering. This methodology was then used to determine initial crystallinity and monitor gelation and retrogradation of high amylose thermoplastic starch used to produce film. Retrogradation was monitored over a 5-day period. It was found that the starch film retrograded rapidly over the first 12 h with the film displaying both B-type crystallinity and long range amorphous ordering that were separately quantitatively calculated. Changes in starch films, including complete or partial gelatinization, retrogradation and crystallinity, were all determined through wide angle X-ray diffraction.     

Extraction and characterization of biocompatible hydroxyapatite from fresh water fish scales for tissue engineering scaffold

In bone tissue engineering, porous hydroxyapatite (HAp) is used as filling material for bone defects, augmentation, artificial bone graft and scaffold material. The present paper compares the preparation and characterization of HAp from fish scale (FS) and synthetic body fluid (SBF) solution. Thermo gravimetric analysis, differential thermal analysis, Fourier transform infrared spectroscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM) and particle size analysis of the samples have been performed. The analysis indicates that synthesized HAp consists of sub-micron HAp particle with Ca/P ratio corresponding to FS and SBF 1.62 and 1.71, respectively. MTT assay and quantitative DNA analysis show growth and proliferation of cells over the HA scaffold with the increase in time. The shape and size (morphology) of mesenchymal stem cells after 3 days show a transition from rounded shape to elongated and flattened shape expressing its spreading behavior. These results confirm that HAp bio-materials from fish scale are physico-chemically and biologically equivalent to the chemically synthesized HAp from SBF. Biological HAp, thus, possesses a great potential for conversion of industrial by-product into highly valuable compounds using simple effective and novel processes.     

Electrospun PVA/HAp nanocomposite nanofibers: biomimetics of mineralized hard tissues at a lower level of complexity

Based on the biomimetic approaches the present work describes a straightforward technique to mimic not only the architecture (the morphology) but also the chemistry (the composition) of the lowest level of the hierarchical organization of bone. This technique uses an electrospinning (ES) process with polyvinyl alcohol (PVA) and hydroxyapatite (HAp) nanoparticles. To determine morphology, crystalline structures and thermal properties of the resulting electrospun fibers with the pure PVA and PVA/HAp nanocomposite (NC) before electrospinning various techniques were employed, including transmission electron microscopy (TEM), high-resolution TEM (HR-TEM), scanning electron microscopy (SEM), x-ray diffraction (XRD), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). In addition, FT-IR spectroscopy was carried out to analyze the complex structural changes upon undergoing electrospinning as well as interactions between HAp and PVA. The morphological and crystallographic investigations revealed that the rod-like HAp nanoparticles exhibit a nanoporous morphology and are embedded within the electrospun fibers. A large number of HAp nanorods are preferentially oriented parallel to the longitudinal direction of the electrospun PVA fibers, which closely resemble the naturally mineralized hard tissues of bones. Due to abundant OH groups present in PVA and HAp nanorods, they strongly interact via hydrogen bonding within the electrospun PVA/HAp NC fibers, which results in improved thermal properties. The unique physiochemical features of the electrospun PVA/HAp NC nanofibers prepared by the ES process will open up a wide variety of future applications related to hard tissue replacement and regeneration (bone and dentin), not limited to coating implants.     

Characterizing the decay of ancient Chinese silk fabrics by microbeam synchrotron radiation diffraction

Scanning synchrotron radiation microdiffraction with an approximately 1 x 1 microm(2) beam has been used as a novel method for characterizing the decay of several T'ang dynasty (618-907 AD) silk fabrics. The crystalline fraction could be visualized based on beta-sheet 210 reflection intensities, extracted by recursive peak fits from several thousand diffraction patterns recorded during mesh scans. The azimuthal width of the 210 reflection, which is related to the orientation distribution of the crystalline domains within nanofibrils and the macroscopic orientation of the fibers traversed by the beam, was found to be sensitive to the overall state of decay of the fabric. The fine structure of the histogram of azimuthal width was related to the fiber hierarchical microstructure and the fabric morphology. SAXS/WAXS analysis supports the assumption of an initial loss of the random chain network with decay. At a subsequent state of aging, decay proceeds into the nanofibrils and the silk fibers break up into even smaller fractions.     

XRD studies of chitin-based polyurethane elastomers

Chitin-based polyurethane elastomers (PUEs) were synthesized by step growth polymerization techniques using poly(epsilon-caprolactone) (PCL) varying diisocyanate and chain extender structures. The viscosity average molecular weight (M(v)) of chitin was deduced from the intrinsic viscosity and found; M(v)=6.067 x 10(5). The conventional spectroscopic characterization of the samples with FTIR, (1)H NMR and (13)C NMR were in accordance with proposed PUEs structure. The crystalline behavior of the synthesized polymers were investigated by X-ray diffraction (XRD), differential scanning calorimetery (DSC) and loss tangent curves (tan delta peaks). The observed patterns of the crystalline peaks for the lower angle for chitin in the 2theta range were indexed as 9.39 degrees, 19.72 degrees, 20.73 degrees, 23.41 degrees and 26.39 degrees. Results showed that crystallinity of the synthesized PUEs samples was affected by varying the structure of the diisocyanate and chain extender. Crystallinity decreased from aliphatic to aromatic characters of the diisocyanates used in the final PU. The presence of chitin also favors the formation of more ordered structure, as higher peak intensities was obtained from the PU extended with chitin than 1,4-butane diol (BDO). The value of peak enthalpy (DeltaH) of chitin was found to be 47.13 J g(-1). The higher DeltaH value of 46.35 J g(-1) was found in the samples extended with chitin than BDO (39.73 J g(-1)).     

Two-dimensional lattices of porin diffract to 6 Å resolution

Porin (the product of gene ompF) is an integral membrane protein (M_r 36 500) of the outer membrane of Escherichia coli (strain B^E). The protein has been purified to homogeneity and reconstituted in dimyristoyl-lecithin. Oriented specimen on a flat surface yielded X-ray diffraction pattern, originating from the two-dimensional protein lattice, to a resolution reaching 6 Å. Although these powder rings are broad compared to corresponding diffraction patterns from purple membranes of Halobacterium halobium, porin is the first reconstituted integral membrane protein which shows diffraction to this resolution.