Histological staining alters circular dichroism SHG measurements of collagen.

Circular dichroism second harmonic generation microscopy (CDSHG) is a powerful imaging technique, which allows three-dimensional visualization of collagen fibril orientation in tissues. However, recent publications have obtained contradictory results on whether CDSHG can be used to reveal the relative out-of-plane polarity of collagen fibrils. Here we compare CDSHG images of unstained tendon and tendon which has been stained with hematoxylin and eosin. We find significant differences in the CDSHG between these two conditions, which explain the recent contradictory results within the literature.

Modeling collagen fibril self-assembly from extracellular medium in embryonic tendon.

Collagen is a key structural component of multicellular organisms and is arranged in a highly organized manner. In structural tissues such as tendons, collagen forms bundles of parallel fibers between cells, which appear within a 24-h window between embryonic day 13.5 (E13.5) and E14.5 during mouse embryonic development. Current models assume that the organized structure of collagen requires direct cellular control, whereby cells actively lay down collagen fibrils from cell surfaces. However, such models appear incompatible with the time and length scales of fibril formation. We propose a phase-transition model to account for the rapid development of ordered fibrils in embryonic tendon, reducing reliance on active cellular processes. We develop phase-field crystal simulations of collagen fibrillogenesis in domains derived from electron micrographs of inter-cellular spaces in embryonic tendon and compare results qualitatively and quantitatively to observed patterns of fibril formation. To test the prediction of this phase-transition model that free protomeric collagen should exist in the inter-cellular spaces before the formation of observable fibrils, we use laser-capture microdissection, coupled with mass spectrometry, which demonstrates steadily increasing free collagen in inter-cellular spaces up to E13.5, followed by a rapid reduction of free collagen that coincides with the appearance of less-soluble collagen fibrils. The model and measurements together provide evidence for extracellular self-assembly of collagen fibrils in embryonic mouse tendon, supporting an additional mechanism for rapid collagen fibril formation during embryonic development.

Multifilament Collagen Fiber Bundles with Tendon-like Structure and Mechanical Performance.

Collagen multifilament bundles comprised of thousands of monofilaments are prepared by multipin contact drawing of an entangled polymer solution consisting of collagen and poly(ethylene oxide) (PEO). The multifilament bundles are hydrated in graded concentrations of PEO and phosphate buffered saline (PBS) to promote assembly of collagen fibrils within each monofilament while preserving the structure of the multifilament bundle. Multiscale structural characterization reveals that the hydrated multifilament bundle contains properly folded collagen molecules packed in collagen fibrils containing microfibrils, staggered by exactly one-sixth of the microfibril D-band spacing to produce a periodicity of 11 nm. Sequence analysis predicts that in this structure, phenylalanine residues are close enough within and between microfibrils to become ultraviolet C (UVC) crosslinked. In agreement with this analysis, the ultimate tensile strength (UTS) and Young's modulus of the hydrated collagen multifilament bundles crosslinked by UVC radiation increase nonlinearly with total UVC energy to reach values in the range of native tendons without damage to the collagen molecules. This fabrication method recapitulates the structure of a tendon across multiple length scales and offers tunability in tensile properties using only collagen molecules and no other chemical additives in addition to PEO, which is almost entirely removed during the hydration process.

Measuring collagen fibril diameter with differential interference contrast microscopy.

Collagen fibrils, linear arrangements of collagen monomers, 20-500 nm in diameter, comprising hundreds of molecules in their cross-section, are the fundamental structural unit in a variety of load-bearing tissues such as tendons, ligaments, skin, cornea, and bone. These fibrils often assemble into more complex structures, providing mechanical stability, strength, or toughness to the host tissue. Unfortunately, there is little information available on individual fibril dynamics, mechanics, growth, aggregation and remodeling because they are difficult to image using visible light as a probe. The principle quantity of interest is the fibril diameter, which is difficult to extract accurately, dynamically, in situ and non-destructively. An optical method, differential interference contrast (DIC) microscopy has been used to visualize dynamic structures that are as small as microtubules (25 nm diameter) and has been shown to be sensitive to the size of objects smaller than the wavelength of light. In this investigation, we take advantage of DIC microscopy's ability to report dimensions of nanometer scale objects to generate a curve that relates collagen diameter to DIC edge intensity shift (DIC-EIS). We further calibrate the curve using electron microscopy and demonstrate a linear correlation between fibril diameter and the DIC-EIS. Using a non-oil immersion, 40x objective (NA 0.6), collagen fibril diameters between ~100 nm to ~ 300 nm could be obtained with ±11 and ±4 nm accuracy for dehydrated and hydrated fibrils, respectively. This simple, nondestructive, label free method should advance our ability to directly examine fibril dynamics under experimental conditions that are physiologically relevant.

The influence of animal species, gender and tissue on the structural, biophysical, biochemical and biological properties of collagen sponges.

Although collagen type I is extensively used in biomedicine, no study to-date has assessed how the properties of the produced scaffolds are affected as a function of species, gender and tissue from which the collagen was extracted. Herein, we extracted and characterised collagen from porcine and bovine, male and female and skin and tendon tissues and we subsequently fabricated and assessed the structural, biophysical, biochemical and biological properties of collagen sponges. All collagen preparations were of similar purity and free-amine content (p > 0.05). In general, the porcine groups yielded more collagen; had higher (p < 0.05) denaturation temperature and resistance to enzymatic degradation; and lower (p < 0.05) swelling ratio and compression stress and modulus than the bovine groups of the same gender and tissue. All collagen preparations supported growth of human dermal fibroblasts and exhibited similar biological response to human THP-1 monocytes. These results further illustrate the need for standardisation of collagen preparations for the development of reproducible collagen-based devices. Assessment of the physicochemical and biological properties of collagen sponges as a function of animal species (bovine versus porcine), gender (male versus female) and tissue (skin versus tendon).

Molecular characterization of avian reovirus causing tenosynovitis outbreaks in broiler flocks, Iran.

Avian reoviruses (ARVs) cause arthritis, tenosynovitis, retarded growth, and malabsorption syndrome. After a long time of effective prevention and low rates of viral arthritis/ tenosynovitis in Iran, outbreaks of tenosynovitis in broiler flocks have increased in recent years. Lameness, splay legs, high rate of cull birds, poor performance, uneven birds at harvest, and condemnation at processing cause huge economic losses. In this study, ARVs from the tendons of birds from 23 broiler flocks with marked tenosynovitis were characterized, and their genetic relationship was examined. Analysis of the amino acid sequence of Sigma C protein revealed that all ARVs detected in affected broiler flocks shared genetic homogeneity and this suggests that a single genotype is involved in recent outbreaks. This genotype, so-called "Ardehal strain", is grouped in cluster I with vaccine strains. The amino acid sequence similarity between Ardehal and vaccine strains, including S1133, 1733, and 2408 was less than 80%. As the outbreaks have occurred in progenies of vaccinated flocks, it is proposed here that the difference between vaccine and field strains might contribute to the failure of currently available vaccines to induce protective immunity against Ardehal strain and this led to widespread viral tenosynovitis in Iran.

Implementation of a logarithmic division-of-focal-plane polarimeter to quantify changes in collagen alignment at varying levels of illumination.

We examine the impact of illumination, aperture, and sample thickness on two division-of-focal-plane (DoFP) polarimeters, one created using a standard 3 T pixel and the other with a forward-biased, logarithmic pixel. Across all measured metrics the logarithmic DoFP polarimeter was better able to track real-time changes in collagen alignment than the standard DoFP polarimeter.

Orientation Matters: Polarization Dependent IR Spectroscopy of Collagen from Intact Tendon Down to the Single Fibril Level.

Infrared (IR) spectroscopy has been used for decades to study collagen in mammalian tissues. While many changes in the spectral profiles appear under polarized IR light, the absorption bands are naturally broad because of tissue heterogeneity. A better understanding of the spectra of ordered collagen will aid in the evaluation of disorder in damaged collagen and in scar tissue. To that end, collagen spectra have been acquired with polarized far-field (FF) Fourier Transform Infrared (FTIR) imaging with a Focal Plane Array detector, with the relatively new method of FF optical photothermal IR (O-PTIR), and with nano-FTIR spectroscopy based on scattering-type scanning near-field optical microscopy (s-SNOM). The FF methods were applied to sections of intact tendon with fibers aligned parallel and perpendicular to the polarized light. The O-PTIR and nano-FTIR methods were applied to individual fibrils of 100-500 nm diameter, yielding the first confirmatory and complementary results on a biopolymer. We observed that the Amide I and II bands from the fibrils were narrower than those from the intact tendon, and that both relative intensities and band shapes were altered. These spectra represent reliable profiles for normal collagen type I fibrils of this dimension, under polarized IR light, and can serve as a benchmark for the study of collagenous tissues.

Glycation of type I collagen selectively targets the same helical domain lysine sites as lysyl oxidase-mediated cross-linking.

Nonenzymatic glycation of collagen has long been associated with the progressive secondary complications of diabetes. How exactly such random glycations result in impaired tissues is still poorly understood. Because of the slow turnover rate of most fibrillar collagens, they are more susceptible to accumulate time-dependent glycations and subsequent advanced glycation end-products. The latter are believed to include cross-links that stiffen host tissues. However, diabetic animal models have also displayed weakened tendons with reduced stiffness. Strikingly, not a single experimentally identified specific molecular site of glycation in a collagen has been reported. Here, using targeted MS, we have identified partial fructosyl-hydroxylysine glycations at each of the helical domain cross-linking sites of type I collagen that are elevated in tissues from a diabetic mouse model. Glycation was not found at any other collagen lysine residues. Type I collagen in mouse tendons is cross-linked intermolecularly by acid-labile aldimine bonds formed by the addition of telopeptide lysine aldehydes to hydroxylysine residues at positions α1(I)Lys87, α1(I)Lys930, α2(I)Lys87, and α2(I)Lys933 of the triple helix. Our data reveal that site-specific glycations of these specific lysines may significantly impair normal lysyl oxidase-controlled cross-linking in diabetic tendons. We propose that such N-linked glycations can hinder the normal cross-linking process, thus altering the content and/or placement of mature cross-links with the potential to modify tissue material properties.

Assessing collagen fibrils molecular damage after a single stretch-release cycle.

Mechanical testing of connective tissues such as tendons and ligaments can lead to collagen denaturation even in the absence of macroscale damage. The following tensile loading protocols, ramp loading to failure, overloading and release, cyclic overloading and cyclic fatigue loading, all yield molecular damage in rat or bovine tendons. Single collagen fibrils extracted from the positional common digital extensor tendon of the forelimb also show molecular damage after tensile loading to failure. Using fibrils from the same source we assess changes to the molecular and supramolecular structure after tensile stress relaxation at strains between 4 and 22% followed by release. We observe no broken fibril and no significant change in D-band spacing. However, we observe significant binding of a fluorescent collagen hybridizing peptide to the fibrils indicating that collagen denaturation occurs in a strain dependent way for relaxation times between 1 s and 1500 s. We also show that peptide binding is associated with a decrease of the cross-sectional area of the fibrils providing an estimate of the dry volume loss due to molecular denaturation as well as an estimate of the mechanical energy density required, 25-110 MJ m-3. In summary we show that collagen molecular damage can occur in the absence of fibril failure and without visible changes to the supramolecular structure.

Variations in internal structure, composition and protein distribution between intra- and extra-articular knee ligaments and tendons.

Tendons and ligaments play key roles in the musculoskeletal system in both man and animals. Both tissues can undergo traumatic injury, age-related degeneration and chronic disease, causing discomfort, pain and increased susceptibility to wider degenerative joint disease. To date, tendon and ligament ultrastructural biology is relatively under-studied in healthy, non-diseased tissues. This information is essential to understand the pathology of these tissues with regard to function-related injury and to assist with the future development of tissue-engineered tendon and ligament structures. This study investigated the morphological, compositional and extracellular matrix protein distribution differences between tendons and ligaments around the non-diseased canine stifle joint. The morphological, structural characteristics of different regions of the periarticular tendons and ligaments (the intra-articular anterior cruciate ligament, the extra-articular medial collateral ligament, the positional long digital extensor tendon and energy-storing superficial digital flexor tendons) were identified using a novel semi-objective histological scoring analysis and by determining their biochemical composition. Protein distribution of extracellular matrix collagens, proteoglycans and elastic fibre proteins in anterior cruciate ligament and long digital extensor tendon were also determined using immunostaining techniques. The anterior cruciate ligament was found to have significant morphological differences in comparison with the other three tissues, including less compact collagen architecture, differences in cell nuclei phenotype and increased glycosaminoglycan and elastin content. Intra- and interobserver differences of histology scoring resulted in an average score 0.7, indicative of good agreement between observers. Statistically significant differences were also found in the extracellular matrix composition in terms of glycosaminoglycan and elastin content, being more prominent in the anterior cruciate ligament than in the other three tissues. A different distribution of several extracellular matrix proteins was also found between long digital extensor tendon and anterior cruciate ligament, with a significantly increased immunostaining of aggrecan and versican in the anterior cruciate ligament. These findings directly relate to the different functions of tendon and ligament and indicate that the intra-articular anterior cruciate ligament is subjected to more compressive forces, reflecting an adaptive response to normal or increased loads and resulting in different extracellular matrix composition and arrangement to protect the tissue from damage.

Ultrasound scans and dual energy CT identify tendons as preferred anatomical location of MSU crystal depositions in gouty joints.

OBJECTIVE: The present study was performed to localize the articular deposition of monosodium urate (MSU) crystal in joints. We compare the detection efficiencies of dual-energy CT (DECT) and ultrasound scans. METHODS: Analyses by DECT and ultrasound were performed with 184 bilateral joints of the lower limbs of 54 consecutive gout patients. All joints were categorized into (1) knee, (2) ankle, (3) MTP1, and (4) MTP2, and sorted into those with and those without detectable MSU deposition. The comparison of the positive rate between DECT and ultrasound and the agreement was performed using the McNemar test and the Cohen's κ coefficient, respectively. Next, we listed the MSU crystal deposition as assessed by ultrasound between the DECT-positive and -negative joints according to their interior structure. We included tendons, synovia, cartilage, subcutaneous tissue, etc. RESULTS: Among all joints, the percentages with MSU crystal deposition detected by DECT (99/184, 53.8%) and ultrasound (106/184, 57.6%) were comparable (P = 0.530 > 0.05). For MTP1 (21/34, 61.8%; 12/34, 35.3%; P < 0.05) and MTP2-5 (17/34, 50.0%; 10/34, 29.4%, P < 0.05), ultrasound and DECT were more efficient, respectively. The data concordance in 46 of 50 joints (92.00%; κ = 0.769, P < 0.05) for knee; and 27 of 34 joints (79.41%; κ = 0.588, P < 0.05) for MTP2-5 and suggested that tendons were the most frequent anatomical location of MSU crystal deposition. CONCLUSIONS: The tendons are the most frequent anatomical location of MSU crystal depositions. The concordance rate of knee joints and MTP2-5 joints shows good agreement between DECT and ultrasound depending on the location.

More histologic and ultrastructural degenerative signs in the subscapularis tendon and the joint capsule in male patients with shoulder impingement.

PURPOSE: The purpose of the present study was to analyze biopsy samples from the subscapularis tendon and from the joint capsule from male patients with shoulder impingement syndrome (SAIS) and compare them with samples from male patients with post-traumatic recurrent shoulder instability. The hypothesis of the study was that patients with SAIS would have more histologic and ultrastructural degenerative changes in their subscapularis tendon and joint capsule than patients with post-traumatic recurrent shoulder instability. METHODS: Male patients scheduled for surgery, with either subacromial decompression or Bankart reconstruction, were included. Four biopsies from each patient were obtained from the capsule and four from the subscapularis tendon during arthroscopic surgery. The histologic characteristics and the presence of glycosaminoglycans were assessed using the light microscope, and the ultrastructure was assessed using a transmission electron microscope. RESULTS: Eight patients, median age 53 (45-74) years (p < 0.0001), were included in the impingement group, and 12 patients, median age 27 (22-48) years, were included in the instability group. The histologic assessment revealed significantly higher cellularity and total degeneration score in the capsule (p = 0.016 and p = 0.014 respectively) in patients with subacromial impingement compared with the instability patients. The corresponding finding was not made for the subscapularis tendon. The ultrastructural evaluation revealed that the instability patients had more fibrils with a large diameter (indicating less degeneration) in both the subscapularis tendon and the capsule compared with the impingement patients (p < 0.0001). CONCLUSION: Male patients with subacromial impingement have more histologic and ultrastructural degenerative changes in their shoulder compared with patients with post-traumatic recurrent shoulder instability. CLINICAL RELEVANCE: It appears that in patients with subacromial impingement, the whole shoulder joint is affected and not only the subacromial space. It is the opinion of the authors that intra-articular therapeutic injections could be tried more often in these patients. LEVEL OF EVIDENCE: III.

Automated freeze-thaw cycles for decellularization of tendon tissue - a pilot study.

BACKGROUND: Decellularization of tendon tissue plays a pivotal role in current tissue engineering approaches for in vitro research as well as for translation of graft-based tendon restoration into clinics. Automation of essential decellularization steps like freeze-thawing is crucial for the development of more standardized decellularization protocols and commercial graft production under good manufacturing practice (GMP) conditions in the future. METHODS: In this study, a liquid nitrogen-based controlled rate freezer was utilized for automation of repeated freeze-thawing for decellularization of equine superficial digital flexor tendons. Additional tendon specimens underwent manually performed freeze-thaw cycles based on an established procedure. Tendon decellularization was completed by using non-ionic detergent treatment (Triton X-100). Effectiveness of decellularization was assessed by residual nuclei count and calculation of DNA content. Cytocompatibility was evaluated by culturing allogeneic adipose tissue-derived mesenchymal stromal cells on the tendon scaffolds. RESULTS: There were no significant differences in decellularization effectiveness between samples decellularized by the automated freeze-thaw procedure and samples that underwent manual freeze-thaw cycles. Further, we inferred no significant differences in the effectiveness of decellularization between two different cooling and heating rates applied in the automated freeze-thaw process. Both the automated protocols and the manually performed protocol resulted in roughly 2% residual nuclei and 13% residual DNA content. Successful cell culture was achieved with samples decellularized by automated freeze-thawing as well as with tendon samples decellularized by manually performed freeze-thaw cycles. CONCLUSIONS: Automated freeze-thaw cycles performed by using a liquid nitrogen-based controlled rate freezer were as effective as previously described manual freeze-thaw procedures for decellularization of equine superficial digital flexor tendons. The automation of this key procedure in decellularization of large tendon samples is an important step towards the processing of large sample quantities under standardized conditions. Furthermore, with a view to the production of commercially available tendon graft-based materials for application in human and veterinary medicine, the automation of key procedural steps is highly required to develop manufacturing processes under GMP conditions.

Comparison of DNA yield and STR success rates from different tissues in embalmed bodies.

Formalin fixation is commonly used to preserve tissue sections for pathological testing and embalming cadavers for medical dissection or burial. DNA extracted from formalin-fixed tissues may also provide an alternative source of genetic material for medical diagnosis and forensic casework, such as identifying unknown embalmed human remains. Formaldehyde causes DNA damage, chemical modifications, and degradation, thereby reducing the quantity and quality of DNA available for downstream genetic analyses. By comparing the DNA yield, level of DNA degradation, and short tandem repeat (STR) success of various tissue types, this study is the first of its kind to provide some guidance on which samples from embalmed bodies are likely to generate more complete STR profiles. Tissue samples were dissected from three male embalmed cadavers and included bone, cartilage, hair, muscle, internal organs, skin, teeth, and nail clippings. DNA was purified from all samples using the QIAamp® FFPE Tissue Kit (Qiagen), quantified using the QuantiFiler® Trio DNA Quantification kit (Life Technologies), and genotyped using the GlobalFiler® PCR Amplification Kit (Life Technologies). Results of this study showed variation in DNA quantity and STR success between different types of tissues and some variation between cadavers. Overall, bone marrow samples resulted in the highest DNA yields, the least DNA degradation, and greatest STR success. However, several muscle, hair, and nail samples generated higher STR success rates than traditionally harvested bone and tooth samples. A key advantage to preferentially using these tissue samples over bone (and marrow) and teeth is their comparative ease and speed of collection from the cadaver and processing during DNA extraction. Results also indicate that soft tissues affected by lividity (blood pooling) may experience greater exposure to formalin, resulting in more DNA damage and reduced downstream STR success than tissues under compression. Overall, we recommend harvesting from selected muscles (gastrocnemius, rectus femoris, flexor digitorum brevis, masseter, brachioradialis) or fingernails for human identification purposes.

Developing functional musculoskeletal tissues through hypoxia and lysyl oxidase-induced collagen cross-linking.

The inability to recapitulate native tissue biomechanics, especially tensile properties, hinders progress in regenerative medicine. To address this problem, strategies have focused on enhancing collagen production. However, manipulating collagen cross-links, ubiquitous throughout all tissues and conferring mechanical integrity, has been underinvestigated. A series of studies examined the effects of lysyl oxidase (LOX), the enzyme responsible for the formation of collagen cross-links. Hypoxia-induced endogenous LOX was applied in multiple musculoskeletal tissues (i.e., cartilage, meniscus, tendons, ligaments). Results of these studies showed that both native and engineered tissues are enhanced by invoking a mechanism of hypoxia-induced pyridinoline (PYR) cross-links via intermediaries like LOX. Hypoxia was shown to enhance PYR cross-linking 1.4- to 6.4-fold and, concomitantly, to increase the tensile properties of collagen-rich tissues 1.3- to 2.2-fold. Direct administration of exogenous LOX was applied in native cartilage and neocartilage generated using a scaffold-free, self-assembling process of primary chondrocytes. Exogenous LOX was found to enhance native tissue tensile properties 1.9-fold. LOX concentration- and time-dependent increases in PYR content (∼ 16-fold compared with controls) and tensile properties (approximately fivefold compared with controls) of neocartilage were also detected, resulting in properties on par with native tissue. Finally, in vivo subcutaneous implantation of LOX-treated neocartilage in nude mice promoted further maturation of the neotissue, enhancing tensile and PYR content approximately threefold and 14-fold, respectively, compared with in vitro controls. Collectively, these results provide the first report, to our knowledge, of endogenous (hypoxia-induced) and exogenous LOX applications for promoting collagen cross-linking and improving the tensile properties of a spectrum of native and engineered tissues both in vitro and in vivo.

Ordering of protein and water molecules at their interfaces with chitin nano-crystals.

Synchrotron X-ray diffraction was applied to study the structure of biogenic α-chitin crystals composing the tendon of the spider Cupiennius salei. Measurements were carried out on pristine chitin crystals stabilized by proteins and water, as well as after their deproteinization and dehydration. We found substantial shifts (up to Δq/q=9% in the wave vector in q-space) in the (020) diffraction peak position between intact and purified chitin samples. However, chitin lattice parameters extracted from the set of reflections (hkl), which did not contain the (020)-reflection, showed no systematic variation between the pristine and the processed samples. The observed shifts in the (020) peak position are discussed in terms of the ordering-induced modulation of the protein and water electron density near the surface of the ultra-thin chitin fibrils due to strong protein/chitin and water/chitin interactions. The extracted modulation periods can be used as a quantitative parameter characterizing the interaction length.

Is the Collagen Primed for Mineralization in Specific Regions of the Turkey Tendon? An Investigation of the Protein-Mineral Interface Using Raman Spectroscopy.

The tendons in the turkey leg have specific well-defined areas which become mineralized as the animal ages and they are a thoroughly characterized model system for studying the mineralization process of bone. In this study, nondestructive Raman spectroscopic analysis was used to explore the hypothesis that regions of the turkey tendon that are associated with mineralization exhibit distinct and observable chemical modifications of the collagen prior to the onset of mineralization. The Raman spectroscopy features associated with mineralization were identified by probing (on the micrometer scale) the transition zone between mineralized and nonmineralized regions of turkey leg tendons. These features were then measured in whole tendons and identified in regions of tendon which are destined to become rapidly mineralized around 14 weeks of age. The data show there is a site-specific difference in collagen prior to the deposition of mineral, specifically the amide III band at 1270 cm(-1) increases as the collagen becomes more ordered (increased amide III:amide I ratio) in regions that become mineralized compared to collagen destined to remain nonmineralized. If this mechanism were present in materials of different mineral fraction (and thus material properties), it could provide a target for controlling mineralization in metabolic bone disease.

X-ray crystal structure of anhydrous chitosan at atomic resolution.

We determined the crystal structure of anhydrous chitosan at atomic resolution, using X-ray fiber diffraction data extending to 1.17 Å resolution. The unit cell [a = 8.129(7) Å, b = 8.347(6) Å, c = 10.311(7) Å, space group P21 21 21 ] of anhydrous chitosan contains two chains having one glucosamine residue in the asymmetric unit with the primary hydroxyl group in the gt conformation, that could be directly located in the Fourier omit map. The molecular arrangement of chitosan is very similar to the corner chains of cellulose II implying similar intermolecular hydrogen bonding between O6 and the amine nitrogen atom, and an intramolecular bifurcated hydrogen bond from O3 to O5 and O6. In addition to the classical hydrogen bonds, all the aliphatic hydrogens were involved in one or two weak hydrogen bonds, mostly helping to stabilize cohesion between antiparallel chains. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 361-368, 2016.