Gelatin and Collagen from Sheepskin.

Abattoirs dispose of sheepskins as solid waste due to low price and poor demand for sheepskin leather. In principle, as an alternative to being disposed of in landfill, sheepskins can serve as a source of the protein collagen or the hydrolysis product, gelatin. In this research, sheepskins collected from abattoirs were used as a source of collagen. Three extraction methods were compared: acid extraction, acid with enzymes, and alkali extraction. The extracted material was characterized using scanning electron microscopy (SEM) and Fourier-transform infrared spectroscopy (FTIR), small angle X-ray scattering (SAXS), and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The collagen and gelatin extraction yield ranged from 3.1% to 4.8% with the product purity determined by hydroxyproline, ranging from 7.8% for the alkali process to 59% and 68% for the acid and acid-enzyme processes. SDS PAGE showed that the acid process produced fragments with molecular weights in the range 100 to >250 kDa, while acid-enzyme resulted in smaller fragments, below 30 kDa. The FTIR region of the amide I band at 1800-1550 cm-1, which was used as an indicator of the collagen and gelatin content, showed that the gelatin dominated in the acid extracts, and the alkaline extract contained a large portion of keratin. SAXS was found to be a sensitive method for showing the presence of intact collagen fibrils in materials from all of the extraction methods, albeit at low concentrations. Herein, sheepskin is shown to be a useful source for collagen-gelatin material of varying molecular weights.

Niobium K-Edge X-ray Absorption Spectroscopy of Doped TiO2 Produced from Ilmenite Digested in Hydrochloric Acid.

Niobium doping of TiO2 creates a conductive material with many new energy applications. When TiO2 is precipitated from HCl solutions containing minor Nb, the Nb in solution is quantitatively deposited with the TiO2. Here, we investigate the structure of Nb doped in anatase and rutile produced from ilmenite digested in hydrochloric acid. Nb K-edge X-ray absorption near edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) are used to characterize the environment of 0.08 atom % Nb doped in TiO2. XANES shows clear structural differences between Nb-doped anatase and rutile. EXAFS for Nb demonstrates that Nb occupies a Ti site in TiO2 with no near neighbors of Nb. Hydrolysis of Ti and Nb from acid solution, followed by calcination, leads to a well dispersed doped material, with no segregation of Nb. Production of Nb-doped TiO2 by this method may be able to supply future demand for large quantities of the material and in energy applications where a low cost of production, from readily available natural resources, would be highly desirable.

Structure and Strength of Bovine and Equine Amniotic Membrane.

Thin, strong scaffold materials are needed for surgical applications. New materials are required, particularly those readily available, such as from non-human sources. Bovine amniotic membrane (antepartum) and equine amniotic membrane (postpartum) were characterized with tear and tensile tests. The structural arrangement of the collagen fibrils was determined by small-angle X-ray scattering, scanning electron microscopy, and ultrasonic imaging. Bovine amnion had a thickness-normalized tear strength of 12.6 (3.8) N/mm, while equine amnion was 14.8 (5.3) N/mm. SAXS analysis of the collagen fibril arrangement yielded an orientation index of 0.587 (0.06) and 0.681 (0.05) for bovine and equine, respectively. This may indicate a relationship between more highly aligned collagen fibrils and greater strength, as seen in other materials. Amnion from bovine or equine sources are strong, thin, elastic materials, although weaker than other collagen tissue materials commonly used, that may find application in surgery as an alternative to material from human donors.

Collagen dehydration.

Type I collagen is a ubiquitous structural protein in animal tissues. It is normally present in a hydrated form. However, collagen is very dependent on associated water for its mechanical properties. In skin, where type I collagen is dominant, there is a longstanding concern that the skin and therefore collagen may partially dry out and result in structural degradation. Here we show that dehydration of type I collagen fibrils, using 2-propanol, results in a two-stage dehydration process. Initially, the fibrils do not change length, i.e. the D-period remains constant, but shrinkage occurs within the fibrils by an increase in the gap region and a decrease in the overlap region within a D-band and a shortening of the helical turn distance and fibril diameter. Only with further dehydration does the length of the collagen fibril decrease (a decrease in D-period). This mechanism explains why collagen materials are resistant to gross structural change in the early stages of dehydration and shows why they may then suffer from sudden external shrinkage with further dehydration.

Collagen Extraction from Animal Skin.

Collagen is the most abundant structural protein in animals. It is the major component of skin. It finds uses in cosmetics, medicine, yarn production and packaging. This paper reviews the extraction of collagen from hides of most consumed animals for meat with the focus on literature published since 2000. The different pretreatment and extraction techniques that have been investigated for producing collagen from animal skins are reviewed. Pretreatment by enzymatic, acid or alkaline methods have been used. Extraction by chemical hydrolysis, salt solubilization, enzymatic hydrolysis, ultrasound assisted extraction and other methods are described. Post-extraction purification methods are also explained. This compilation will be useful for anyone wishing to use collagen as a resource and wanting to further improve the extraction and purification methods.

Manganese accumulation in probiotic Lactobacillus paracasei ATCC 55544 analyzed by synchrotron X-ray fluorescence microscopy and impact of accumulation on the bacterial viability following encapsulation.

Lactobacillus spp. are known to accumulate large amounts of inorganic manganese, which protects against oxidative damage by scavenging free radicals. The ability of probiotic L. paracasei ATCC 55544 to maintain viability during long-term ambient storage may be enhanced by this microorganism's ability to accumulate manganese, which may act as a free radical scavenger. To investigate this hypothesis, X-ray fluorescence microscopy (XFM) was employed to determine the changes in the elemental composition of L. paracasei during growth in the MRS medium with or without added manganese. Moreover, manganese uptake by cells as a function of physiological growth state, early log vs. stationary phase was evaluated. The semiquantitative X-ray fluorescence microscopy results revealed that lower levels of manganese accumulation occurred during the early log phase of bacterial growth of L. paracasei cells (0.0064 µg/cm2) compared with the stationary phase cells (0.1355 µg/cm2). L. paracasei cells grown in manganese deficient MRS medium resulted in lower manganese uptake by cells (0.0027 µg/cm2). The L. paracasei cells were further embedded in milk powder matrix using a fluidized-bed drying technique and stored at a water activity (aw) of 0.33 at 25 °C for 15 days. The viability counts of L. paracasei cells grown in MRS medium harvested after 18 h growth and embedded in milk powder matrix retained viability of (9.19 ± 0.12 log CFU/g). No viable L. paracasei cells were observed in the case of embedded L. paracasei cells grown in manganese-deficient MRS medium harvested after 18 h growth or in the case of L. paracasei cells harvested after 4 h when grown in MRS medium. The lower level of manganese accumulation was found to be related to the loss of bacterial viability during storage.

Bovine Meniscus Middle Zone Tissue: Measurement of Collagen Fibril Behavior During Compression.

BACKGROUND: Type I collagen is the major component of the extracellular matrix of the knee's meniscus and plays a central role in that joint's biomechanical properties. Repair and reconstruction of tissue damage often requires a scaffold to assist the body to rebuild. The middle zone of bovine meniscus is a material that may be useful for the preparation of extracellular matrix scaffolds. METHODS: Here, synchrotron-based small-angle X-ray scattering (SAXS) patterns of bovine meniscus were collected during unconfined compression. Collagen fibril orientation, D-spacing, compression distance and force were measured. RESULTS: The collagen fibrils in middle zone meniscal fibrocartilage become more highly oriented perpendicular to the direction of compression. The D-spacing also increases, from 65.0 to 66.3 nm with compression up to 0.43 MPa, representing a 1.8% elongation of collagen fibrils perpendicular to the compression. CONCLUSION: The elasticity of the collagen fibrils under tension along their length when the meniscus is compressed, therefore, contributes to the overall elastic response of the meniscus only under loads that exceed those likely to be experienced physiologically.

Measured collagen fibril response to arterial inflation using SAXS.

Arteries are elastic structures containing both elastin and collagen. While the high content of elastin is understood to be important for the elasticity of arteries with systolic and diastolic pressure pulses, the role of collagen in the elastic properties of arteries is less understood. Here we use small angle X-ray scattering to investigate the changes in arrangement of collagen fibrils and the strain experienced by collagen fibrils as arteries are inflated. Collagen fibrils re-orient to become more aligned in both the annular direction and radially as arteries inflate. With arterial pressures up to 32 kPa there is no observable increase in D-spacing of the collagen fibrils (<0.1%) indicating that there is no extension of straightened fibrils and therefore no change in stress on the collagen fibrils. This is in contrast to tissue such as skin where stress of the tissue may induce strains in collagen fibrils of >6%. In arteries the collagen fibril elasticity (strain at the scale of fibrils) is not the main elastic component of the arterial walls. This indicates that wall elasticity is dominated by other factors such as the structural arrangement of the collagen fibers.

Tropical Keratopathy (Florida Spots) in Cats.

The authors used microscopy and synchrotron-based small-angle X-ray scattering analysis (SAXS) to describe lesions macroscopically typical of tropical keratopathy ("Florida spots") from 6 cats on St Kitts. Microscopically, there were varying degrees of epithelial hyperplasia and thinning of the cornea (by 4% to 18%) due to loss of corneal stroma associated with dense accumulations of collagen in the superficial stroma. The collagen fibrils in lesions were wider and had more variable diameters (39.5 ± 5.0 nm, mean ± SD) than in normal corneas (25.9 ± 3.6 nm; P < .01). There were occasional vacuoles (<1 µm) in the corneal epithelial basement membrane but no evidence of inflammation, edema, stromal neovascularization, fibrosis, acid-fast organisms, or structures suggestive of a fungal organism. SAXS analysis showed collagen fibril diameters and variation in size were greater in stroma containing the lesions compared to normal corneas (48.8 ± 4.5 nm vs 35.5 ± 2.6; P < .05). The d-spacing of collagen in the stroma of lesions and normal corneas was the same, but the average orientation index of collagen in lesions was greater (0.428 ± 0.08 vs 0.285 ± 0.03; P < .05). A survey revealed Florida spots lesions were static over time and became less obvious in only 1 of 6 affected cats adopted on St Kitts and taken to areas in the US where lesions are not reported. An anterior stromal collagen disorder with various degrees of epithelial hyperplasia is the pathologic hallmark of lesions clinically identical to Florida spots in cats from St Kitts.

Acellular dermal matrix collagen responds to strain by intermolecular spacing contraction with fibril extension and rearrangement.

Acellular dermal matrix (ADM) materials are used as scaffold materials in reconstructive surgery. The internal structural response of these materials in load-bearing clinical applications is not well understood. Bovine ADM is characterized by small-angle X-ray scattering while subjected to strain. Changes in collagen fibril orientation (O), degree of orientation as an orientation index (OI) (measured both edge-on and flat-on to the ADM), extension (from d-spacing changes) and changes to intermolecular spacing are measured as a result of the strain and stress in conjunction with mechanical measurements. As is already well established in similar systems, when strained, collagen fibrils in ADM can accommodate the strain by reorienting by up to 50° (as an average of all the fibrils). This reorientation corresponds to the OI increasing from 0.3 to 0.7. Here it is shown that concurrently, the intermolecular spacing between tropocollagen decreases by 10% from 15.8 to 14.3Å, with the fibril diameter decreasing from 400 to 375Å, and the individual fibrils extending by an average of 3.1% (D-spacing from 63.9 to 65.9nm). ADM materials can withstand large strain and high stress due to the combined mechanisms of collagen reorientation, individual fibril extension, sliding and changes in the molecular packing density.

Artificially modified collagen fibril orientation affects leather tear strength.

BACKGROUND: Ovine leather has around half the tear strength of bovine leather and is therefore not suitable for high-value applications such as shoes. Tear strength has been correlated with the natural collagen fibril alignment (orientation index, OI). It is hypothesized that it could be possible to artificially increase the OI of the collagen fibrils and that an artificial increase in OI could increase tear strength. RESULTS: Ovine skins, after pickling and bating, were strained biaxially during chrome tanning. The strain ranged from 2 to 15% of the initial sample length, either uniformly in both directions by 10% or with 3% in one direction and 15% in the other. Once tanned, the leather tear strengths were measured and the collagen fibril orientation was measured using synchrotron-based small-angle X-ray scattering. CONCLUSION: The OI increased as a result of strain during tanning from 0.48 to 0.79 (P = 0.001) measured edge-on and the thickness-normalized tear strength increased from 27 to 43 N mm-1 (P < 0.001) after leather was strained 10% in two orthogonal directions. This is evidence to support a causal relationship between high OI (measured edge-on), highly influenced by thickness, and tear strength. It also provides a method to produce stronger leather. © 2017 Society of Chemical Industry.

Deer leather: analysis of the microstructure affecting pebble.

BACKGROUND: Deer leather has a characteristic pattern, referred to as 'pebble', which is accorded such importance that a lack of it renders a leather defective. Synchrotron-based small-angle X-ray scattering (SAXS), ultrasonic imaging, scanning electron microscopy, and tear tests were used to investigate the structural characteristics of well-pebbled and poorly pebbled cervine leathers. RESULTS: Poorly pebbled leather has a less open structure in the upper grain region than well-pebbled leather. The orientation index (OI) of leather with a poor pebble is less than that of the well-pebbled leather, particularly in the corium. The tear strength is also less for the poorly pebbled leather. CONCLUSIONS: The differences in structure between well- and poorly pebbled cervine leathers are not the same as the structural differences between tight and loose bovine leathers, to which they are sometimes compared. On the contrary, good pebble may reflect an internal structure similar to that of looseness. It is hoped that methods to prevent a reduction in pebbling during the processing of cervine leather may be developed by applying this knowledge of cervine leather's structural characteristics. © 2017 Society of Chemical Industry.

A small angle X-ray scattering study of the structure and development of looseness in bovine hides and leather.

BACKGROUND: Some bovine hides produce poor quality leather, termed loose leather. The structural characteristics of hides and the intermediate processed stages that lead to loose leather are not well understood. In the present study, synchrotron-based small angle X-ray scattering (SAXS) is used to investigate collagen fibril orientation at the different stages of processing (i.e. from hide through to leather) that result in both tight and loose leathers. RESULTS: Tight leather of a relatively isotropic texture has a lower orientation index (OI) than loose leather of a more pronounced stratified texture; conversely, tight pickled hide and wet blue have a higher OI than loose pickled hide and wet blue. There is a greater increase in OI on processing from pickled hide to dry crust (leather) for loose material. This is largely the result of a greater increase in hide thickness prior to pickling for loose hide than tight hide, followed by a greater decrease at the dry crust stage. The collagen fibrils in loose leather and wet blue more readily orient under stress than do those in tight leather. Loose leather has a more pronounced layered structure than tight leather, although this difference is not apparent from SAXS measurements of hide prior to the dry crust stage; it develops during processing. CONCLUSION: The greater swelling of the loose hide during processing disrupts the structure and leads to a more layered collagen arrangement on shrinking at the final dry crust stage. © 2016 Society of Chemical Industry.

Collagen Fibril Intermolecular Spacing Changes with 2-Propanol: A Mechanism for Tissue Stiffness.

Materials composed primarily of collagen are important as surgical scaffolds and other medical devices and require flexibility. However, the factors that control the suppleness and flexibility of these materials are not well understood. Acellular dermal matrix materials in aqueous mixtures of 2-propanol were studied. Synchrotron-based small-angle X-ray scattering was used to characterize the collagen structure and structural arrangement. Stiffness was measured by bend tests. Bend modulus increased logarithmically with 2-propanol concentration from 0.5 kPa in water to 103 kPa in pure 2-propanol. The intermolecular spacing between tropocollagen molecules decreased from 15.3 to 11.4 Å with increasing 2-propanol concentration while fibril diameter decreased from 57.2 to 37.2 nm. D-spacing initially increased from 63.6 to 64.2 nm at 50% 2-propanol then decreased to 60.3 nm in pure 2-propanol. The decrease in intermolecular spacing and fibril diameter are due to removal of water and the collapse of the hydrogen bond structure between tropocollagen molecules causing closer packing of the molecules within a fibril. We speculate this tighter molecular packing may restrict the sliding of collagen within fibrils, and similar disruption of the extended hydration layer between fibrils may lead to restriction of sliding between fibrils. This mechanism for tissue stiffness may be more general.

Collagen Fibril Response to Strain in Scaffolds from Ovine Forestomach for Tissue Engineering.

Scaffold biomaterials are typically applied surgically as reinforcement for weakened or damaged tissue, acting as substrates on which healing tissue can grow. Natural extracellular matrix (ECM) materials consisting mainly of collagen are often used for this purpose, but are anisotropic. Ovine forestomach matrix (OFM) ECM was exposed to increasing strain and synchrotron-based SAXS diffraction patterns and revealed that the collagen fibrils within underwent changes in orientation, orientation index (a measure of isotropy), and extension. Response to the strain depended on the direction the collagen fibrils were oriented. When the ECM was stretched in the direction of collagen fibril orientation, the fibrils become more oriented and begin to take up the strain immediately (as shown by the increased d-spacing). Stretch applied perpendicular to dominant fibril direction caused the fibrils to initially become less oriented as they were pulled away from the original direction, and less force was initially transmitted along the length of the fibrils (i.e., the d-spacing changed less). SAXS analysis of OFM and the starting raw tissue showed there is no difference in the structural arrangement of the collagen fibrils. Understanding the directional structural response of these materials under strain may influence how surgeons select and place the materials in use.

Looseness in bovine leather: microstructural characterization.

BACKGROUND: A substantial proportion of bovine leather production may be of poor quality, with the leather suffering from a characteristic known as looseness. This defect results in a poor visual appearance and greatly reduced value. The structural mechanism of looseness is not well understood. RESULTS: Samples of loose and tight bovine leather are characterized using small-angle X-ray scattering, ultrasonic imaging, and electron microscopy. The density of fibre packing and orientation of the fibrils are analysed. Tensile strength is also measured. Loose leather is characterized by more highly aligned collagen fibrils. This results in a weaker connection between the layers. There is a looser packing of the fibres in loose leather than in tight leather, with more gaps between fibre bundles, particularly in a region in the lower grain. This region is visible with in situ ultrasonic imaging. Loose leather has a higher tensile strength than tight leather. CONCLUSION: While a high degree of collagen fibril alignment is normally associated with strong leather, it has been shown that too much alignment results in loose leather. Understanding the physical basis of looseness is the first step in identifying looseness in hides and learning how to prevent looseness from developing during leather manufacture. © 2015 Society of Chemical Industry.

Changes to collagen structure during leather processing.

As hides and skins are processed to produce leather, chemical and physical changes take place that affect the strength and other physical properties of the material. The structural basis of these changes at the level of the collagen fibrils is not fully understood and forms the basis of this investigation. Synchrotron-based small-angle X-ray scattering (SAXS) is used to quantify fibril orientation and D-spacing through eight stages of processing from fresh green ovine skins to staked dry crust leather. Both the D-spacing and fibril orientation change with processing. The changes in thickness of the leather during processing affect the fibril orientation index (OI) and account for much of the OI differences between process stages. After thickness is accounted for, the main difference in OI is due to the hydration state of the material, with dry materials being less oriented than wet. Similarly significant differences in D-spacing are found at different process stages. These are due also to the moisture content, with dry samples having a smaller D-spacing. This understanding is useful for relating structural changes that occur during different stages of processing to the development of the final physical characteristics of leather.

Collagen Fibril Structure and Strength in Acellular Dermal Matrix Materials of Bovine, Porcine, and Human Origin.

Strength is an important characteristic of acellular dermal matrix (ADM) materials used for surgical scaffolds. Strength depends on the material's structure, which may vary with the source from which the product is produced, including species and animal age. Here, variations in the physical properties and structures of ADM materials from three species are investigated: bovine (fetal and neonatal), porcine, and human materials. Thickness normalized, the bovine materials have a similar strength (tear strength of 75-124 N/m) to the human material (79 N/m), and these are both stronger than the porcine material (43 N/m). Thickness-normalized tensile strengths were similar among all species (18-34 N/mm2 for bovine although higher in fetal material, 18 N/mm2 for human and 21 N/mm2 for porcine). Structure is investigated with synchrotron-based small-angle X-ray scattering (SAXS) for collagen fibril orientation index (OI) and scanning electron microscopy (SEM). SEM reveals a more open structure in bovine ADM than in the porcine and human material. A correlation is found between OI and thickness-normalized tear strength in neonatal bovine material measured with the X-rays edge-on to the sample, but this relationship does not extend across species. The collagen fibril arrangement, viewed perpendicular to the surface, varies between species, with the human material having a unimodal distribution and rather isotropic (OI 0.08), the porcine being strongly bimodal and rather highly oriented (OI 0.61), the neonatal bovine between these two extremes with a bimodal distribution tending toward isotropic (OI 0.14-0.21) and the fetal bovine material being bimodal and less isotropic than neonatal (OI 0.24). The OI varies less through the thickness of the porcine and human materials than through the bovine materials. The similarities and differences in structure may inform the suitability of these materials for particular surgical applications.

Age dependent differences in collagen alignment of glutaraldehyde fixed bovine pericardium.

Bovine pericardium is used for heart valve leaflet replacement where the strength and thinness are critical properties. Pericardium from neonatal animals (4-7 days old) is advantageously thinner and is considered as an alternative to that from adult animals. Here, the structures of adult and neonatal bovine pericardium tissues fixed with glutaraldehyde are characterized by synchrotron-based small angle X-ray scattering (SAXS) and compared with the mechanical properties of these materials. Significant differences are observed between adult and neonatal tissue. The glutaraldehyde fixed neonatal tissue has a higher modulus of elasticity (83.7 MPa) than adult pericardium (33.5 MPa) and a higher normalised ultimate tensile strength (32.9 MPa) than adult pericardium (19.1 MPa). Measured edge on to the tissue, the collagen in neonatal pericardium is significantly more aligned (orientation index (OI) 0.78) than that in adult pericardium (OI 0.62). There is no difference in the fibril diameter between neonatal and adult pericardium. It is shown that high alignment in the plane of the tissue provides the mechanism for the increased strength of the neonatal material. The superior strength of neonatal compared with adult tissue supports the use of neonatal bovine pericardium in heterografts.

Collagen fibril diameter and leather strength.

The main structural component of leather and skin is type I collagen in the form of strong fibrils. Strength is an important property of leather, and the way in which collagen contributes to the strength is not fully understood. Synchrotron-based small angle X-ray scattering (SAXS) is used to measure the collagen fibril diameter of leather from a range of animals, including sheep and cattle, that had a range of tear strengths. SAXS data were fit to a cylinder model. The collagen fibril diameter and tear strength were found to be correlated in bovine leather (r(2) = 0.59; P = 0.009), with stronger leather having thicker fibrils. There was no correlation between orientation index, i.e., fibril alignment, and fibril diameter for this data set. Ovine leather showed no correlation between tear strength and fibril diameter, nor was there a correlation across a selection of other animal leathers. The findings presented here suggest that there may be a different structural motif in skin compared with tendon, particularly ovine skin or leather, in which the diameter of the individual fibrils contributes less to strength than fibril alignment does.