Isolation and Identification of Cryptic Bioactive Regions in Bovine Achilles Tendon Collagen

Several proteins are known to host specific regions within their sequence, that when exposed or excised out proteolytically can display a range of physiological activities quite different from that of the parent protein. Collagen, a class of structural biopolymers and an important component of the extracellular matrix, is now known to harbor several such bioactive peptides which can act as physiological regulators. This study was undertaken to identify such cryptic sites from bovine Achilles tendon collagen and an antioxidative assay was used to screen for bioactivity. Bacterial crude protease was used to hydrolyze collagen and the hydrolysate was subjected to separation through ion-exchange column chromatography. Fractions were screened using conventional antioxidative assays and further purified by gel permeation chromatography. Two biologically active cryptic peptides were obtained displaying high antioxidative properties, E1 and F3. At low concentrations, both peptides displayed higher chelating ability than EDTA and were able to reduce the auto-oxidation of unsaturated fatty acid. The molecular weights of the peptides were found out through column chromatography and Tricine SDS PAGE; both displayed molecular mass below 4?kDa. Overall E1 displayed a comparatively better antioxidative ability than the others and was further characterized by circular dichroism studies and sequencing. A BLAST search of the active peptide sequence revealed that an almost similar peptide also resides in human collagen Type I.     

Advanced Glycation End-Products Reduce Collagen Molecular Sliding to Affect Collagen Fibril Damage Mechanisms but Not Stiffness

Advanced glycation end-products (AGE) contribute to age-related connective tissue damage and functional deficit. The documented association between AGE formation on collagens and the correlated progressive stiffening of tissues has widely been presumed causative, despite the lack of mechanistic understanding. The present study investigates precisely how AGEs affect mechanical function of the collagen fibril – the supramolecular functional load-bearing unit within most tissues. We employed synchrotron small-angle X-ray scattering (SAXS) and carefully controlled mechanical testing after introducing AGEs in explants of rat-tail tendon using the metabolite methylglyoxal (MGO). Mass spectrometry and collagen fluorescence verified substantial formation of AGEs by the treatment. Associated mechanical changes of the tissue (increased stiffness and failure strength, decreased stress relaxation) were consistent with reports from the literature. SAXS analysis revealed clear changes in molecular deformation within MGO treated fibrils. Underlying the associated increase in tissue strength, we infer from the data that MGO modified collagen fibrils supported higher loads to failure by maintaining an intact quarter-staggered conformation to nearly twice the level of fibril strain in controls. This apparent increase in fibril failure resistance was characterized by reduced side-by-side sliding of collagen molecules within fibrils, reflecting lateral molecular interconnectivity by AGEs. Surprisingly, no change in maximum fibril modulus (2.5 GPa) accompanied the changes in fibril failure behavior, strongly contradicting the widespread assumption that tissue stiffening in ageing and diabetes is directly related to AGE increased fibril stiffness. We conclude that AGEs can alter physiologically relevant failure behavior of collagen fibrils, but that tissue level changes in stiffness likely occur at higher levels of tissue architecture.     

N-Phenacylthiazolium Bromide Reduces Bone Fragility Induced by Nonenzymatic Glycation

Nonenzymatic glycation (NEG) describes a series of post-translational modifications in the collagenous matrices of human tissues. These modifications, known as advanced glycation end-products (AGEs), result in an altered collagen crosslink profile which impacts the mechanical behavior of their constituent tissues. Bone, which has an organic phase consisting primarily of type I collagen, is significantly affected by NEG. Through constant remodeling by chemical resorption, deposition and mineralization, healthy bone naturally eliminates these impurities. Because bone remodeling slows with age, AGEs accumulate at a greater rate. An inverse correlation between AGE content and material-level properties, particularly in the post-yield region of deformation, has been observed and verified. Interested in reversing the negative effects of NEG, here we evaluate the ability of n-phenacylthiazolium bromide (PTB) to cleave AGE crosslinks in human cancellous bone. Cancellous bone cylinders were obtained from nine male donors, ages nineteen to eighty, and subjected to one of six PTB treatments. Following treatment, each specimen was mechanically tested under physiological conditions to failure and AGEs were quantified by fluorescence. Treatment with PTB showed a significant decrease in AGE content versus control NEG groups as well as a significant rebound in the post-yield material level properties (p<0.05). The data suggest that treatment with PTB could be an effective means to reduce AGE content and decrease bone fragility caused by NEG in human bone.     

The Effects of Irreversible Electroporation on the Achilles Tendon: An Experimental Study in a Rabbit Model

Background

To evaluate the potential effects of irreversible electroporation ablation on the Achilles tendon in a rabbit model and to compare the histopathological and biomechanical changes between specimens following electroporation ablation and radiofrequency ablation.

Methods

A total of 140 six-month-old male New Zealand rabbits were used. The animals were randomly divided into two groups, 70 in the radiofrequency ablation group and 70 in the electroporation group. In situ ablations were applied directly to the Achilles tendons of rabbits using typical electroporation (1800 V/cm, 90 pulses) and radiofrequency ablation (power control mode) protocols. Histopathological and biomechanical evaluations were performed to examine the effects of electroporation ablation and radiofrequency ablation over time.

Results

Both electroporation and radiofrequency ablation produced complete cell ablation in the target region. Thermal damage resulted in tendon rupture 3 days post radiofrequency ablation. In contrast, electroporation-ablated Achilles tendons preserved their biomechanical properties and showed no detectable rupture at this time point. The electroporation-ablated tendons exhibited signs of recovery, including tenoblast regeneration and angiogenesis within 2 weeks, and the restoration of their integral structure was evident within 12 weeks.

Conclusions

When applying electroporation to ablate solid tumors, major advantage could be that collateral damage to adjacent tendons or ligaments is minimized due to the unique ability of electroporation ablation to target the cell membrane. This advantage could have a significant impact on the field of tumor ablation near vital tendons or ligaments.     

Corneal Collagen Cross-Linking for the Management of Mycotic Keratitis

Purpose

To evaluate the efficiency of corneal collagen cross-linking (CXL) in addition to topical voriconazole in cases with mycotic keratitis.

Design

Retrospective case series in a tertiary university hospital.

Participants

CXL was performed on 13 patients with mycotic keratitis who presented poor or no response to topical voriconazole treatment.

Methods

The clinical features, symptoms, treatment results and complications were recorded retrospectively. The corneal infection was graded according to the depth of infection into the stroma (from grade 1 to grade 3). The visual analogue scale was used to calculate the pain score before and 2 days after surgery.

Main Outcome Measures

Grade of the corneal infection.

Results

Mean age of 13 patients (6 female and 7 male) was 42.4 ± 17.7 years (20–74 years). Fungus was demonstrated in culture (eight patients) or cytological examination (five patients). Seven of the 13 patients (54%) were healed with topical voriconazole and CXL adjuvant treatment in 26 ± 10 days (15–40 days). The remaining six patients did not respond to CXL treatment; they initially presented with higher grade ulcers. Pre- and post-operative pain score values were 8 ± 0.8 and 3.5 ± 1, respectively (p < 0.05).

Conclusions

The current study suggests that adjunctive CXL treatment is effective in patients with small and superficial mycotic ulcers. These observations require further research by large randomized clinical trials.

     

An Investigation of the Immediate Effect of Static Stretching on the Morphology and Stiffness of Achilles Tendon in Dominant and Non-Dominant Legs

AimsThis study was undertaken to investigate the immediate effect of static stretching on normal Achilles tendon morphology and stiffness, and the different effect on dominant and non-dominant legs; and to evaluate inter-operator and intra-operator reliability of using shear-wave elastography in measuring Achilles tendon stiffness.Methods20 healthy subjects (13 males, 7 females) were included in the study. Thickness, cross-sectional area and stiffness of Achilles tendons in both legs were measured before and after 5-min static stretching using grey-scale ultrasound and shear-wave elastography. Inter-operator and intra-operator reliability of tendon stiffness measurements of six operators were evaluated.ResultsResult showed that there was no significant change in the thickness and cross-sectional area of Achilles tendon after static stretching in both dominant and non-dominant legs (p > 0.05). Tendon stiffness showed a significant increase in non-dominant leg (p < 0.05) but not in dominant leg (p > 0.05). The inter-operator reliability of shear-wave elastography measurements was 0.749 and the intra-operator reliability ranged from 0.751 to 0.941.ConclusionShear-wave elastography is a useful and non-invasive imaging tool to assess the immediate stiffness change of Achilles tendon in response to static stretching with high intra-operator and inter-operator reliability.     

Analyses of In Vitro Nonenzymatic Glycation of Normal and Variant Hemoglobins by MALDI-TOF Mass Spectrometry

MALDI-TOF mass spectrometry is used here to differentiate different glycoisoforms of normal and variant hemoglobins (Hbs) in nonenzymatic in vitro glycation. Single, double, and/or multiple glycation of the α-globin, β-globin, and/or γ-globin is observed. Different glycation rates are observed for various Hbs, and the normal Hb A has the slowest rate. Although the Hb A is relatively stable upon condensation with glucose at 37°C, the variants Hb C, Hb E, Hb F, Hb Leiden, and Hb San Diego are less stable. In addition, data reveal that the number of glucose attached/Hb molecule (state of glycation) increases with longer incubation time, higher glucose concentration, and higher temperature. The pH dependence of the state of glycation is more complex and varies for different Hbs. Although pH has little effect on the state of glycation for Hb C, Hb E, and Hb Leiden, it increases for Hb A and Hb F upon changing the pH of the solution from phosphate buffer saline (pH 7.4) to carbonate buffer (pH 10). Results obtained in this study could lead to the inference that the linkage of Hbs with glucose occurs in diabetic conditions in vivo (37°C, ∼neutral pH, ∼0.007 M glucose), and the state of glycation is more severe in the individuals who carry abnormal Hbs.     

Achilles Tendon Reflex in Accidental Hypothermia and Hypothermic Myxoedema

The photomotogram (P.M.G.) of the Achilles tendon reflex was studied in 26 patients with hypothermia (rectal temperature 33·3°C or less), 10 of whom also had myxoedema (serum protein bound iodine 2·8 μg/100 ml or less). No reflex could be elicited in eight (31%) of these patients, including three of those with myxoedema. Hypothermia increases both the contraction and the relaxation times of the reflex, the relaxation phase being particularly prolonged in those with myxoedema. In those patients from whom the reflex was elicited the ratio of the contraction time to the “half-relaxation time” in the P.M.G. was less than unity in six of the seven with myxoedema, and considerably greater than unity in eight of the 11 (73%) who were euthyroid. Thus, analysis of the Achilles tendon reflex P.M.G. correctly predicted the thyroid status in 14 of the 18 hypothermic patients in whom the Achilles tendon reflex was present (78%). The wider use of this rapid test of thyroid function would allow a more rational use of thyroid hormones in hypothermic patients and so lead to a better assessment of their value.     

In Vitro Nonenzymatic Glycation Enhances the Role of Myoglobin as a Source of Oxidative Stress

Metmyoglobin (Mb) was glycated by glucose in a nonenzymatic in vitro reaction. Amount of iron release from the heme pocket of myoglobin was found to be directly related with the extent of glycation. After in vitro glycation, the unchanged Mb and glycated myoglobin (GMb) were separated by ion exchange (BioRex 70) chromatography, which eliminated free iron from the protein fractions. Separated fractions of Mb and GMb were converted to their oxy forms -MbO² and GMbO², respectively. H²O²-induced iron release was significantly higher from GMbO² than that from MbO². This free iron, acting as a Fenton reagent, might produce free radicals and degrade different cell constituents. To verify this possibility, degradation of different cell constituents catalyzed by these fractions in the presence of H²O² was studied. GMbO² degraded arachidonic acid, deoxyribose and plasmid DNA more efficiently than MbO². Arachidonic acid peroxidation and deoxyribose degradation were significantly inhibited by desferrioxamine (DFO), mannitol and catalase. However, besides free iron-mediated free radical reactions, role of iron of higher oxidation states, formed during interaction of H²O² with myoglobin might also be involved in oxidative degradation processes. Formation of carbonyl content, an index of oxidative stress, was higher by GMbO². Compared to MbO², GMbO² was rapidly auto-oxidized and co-oxidized with nitroblue tetrazolium, indicating increased rate of Mb and superoxide radical formation in GMbO². GMb exhibited more peroxidase activity than Mb, which was positively correlated with ferrylmyoglobin formation in the presence of H²O². These findings correlate glycation-induced modification of myoglobin and a mechanism of increased formation of free radicals. Although myoglobin glycation is not significant within muscle cells, free myoglobin in circulation, if becomes glycated, may pose a serious threat by eliciting oxidative stress, particularly in diabetic patients.     

Fibrin Fiber Stiffness Is Strongly Affected by Fiber Diameter,but Not by Fibrinogen Glycation

The major structural component of a blood clot is a mesh of fibrin fibers. Our goal was to determine whether fibrinogen glycation and fibrin fiber diameter have an effect on the mechanical properties of single fibrin fibers. We used a combined atomic force microscopy/fluorescence microscopy technique to determine the mechanical properties of individual fibrin fibers formed from blood plasma. Blood samples were taken from uncontrolled diabetic patients as well as age-, gender-, and body-mass-index-matched healthy individuals. The patients then underwent treatment to control blood glucose levels before end blood samples were taken. The fibrinogen glycation of the diabetic patients was reduced from 8.8 to 5.0 mol glucose/mol fibrinogen, and the healthy individuals had a mean fibrinogen glycation of 4.0 mol glucose/mol fibrinogen. We found that fibrinogen glycation had no significant systematic effect on single-fiber modulus, extensibility, or stress relaxation times. However, we did find that the fiber modulus, Y, strongly decreases with increasing fiber diameter, D, as Y ∝ D^?1.6. Thin fibers can be 100 times stiffer than thick fibers. This is unusual because the modulus is a material constant and should not depend on the sample dimensions (diameter) for homogeneous materials. Our finding, therefore, implies that fibrin fibers do not have a homogeneous cross section of uniformly connected protofibrils, as is commonly thought. Instead, the density of protofibril connections, ρ_Pb, strongly decreases with increasing diameter, as ρ_Pb ∝ D^?1.6. Thin fibers are denser and/or have more strongly connected protofibrils than thick fibers. This implies that it is easier to dissolve clots that consist of fewer thick fibers than those that consist of many thin fibers, which is consistent with experimental and clinical observations.     

Phenotypic Modulation of Hamster Acinar Cells by Culture in Collagen Matrix

The aim of this study was to assess the effect of different culture conditions on the survival and morphological phenotype of cultured acinar cells. Acinar fragments isolated from hamster pancreas were embedded in rat-tail collagen. Four groups were established: Medium 1—5% NuSerum + basic medium (basic MEDIUM = DMEM/F12 supplemented with dexamethasone, 3-isobutyl-2-methylxanthine, and antibiotics); Medium 2—10% NuSerum + basic medium. Medium 3—Medium 2 supplemented with epidermal growth factor and cholera toxin; and Medium 4:—Medium 3 supplemented with soybean trypsin inhibitor. Freshly isolated acinar cells were retrieved morphologically intact. In Medium 1, more than 80% of cells retained a normal histological appearance at 34 days in culture. Immunostaining for amylase was observed at the apical pole of the cells. The remaining cells showed variable degrees of degeneration. In Medium 2, approximately 50% of acinar cells appeared normal at 34 days in culture, while the remainder were severely degenerated. A few cystic structures were also observed. Positive immunostaining for amylase was limited to the cells with a normal histological appearance. The cells grown in Media 3 and 4 had similar courses of morphological changes. After 8 days in culture, most acinar fragments disappeared and were replaced by cystic structures, lined by a single layer of cuboidal cells. Some amylase-positive immunoreactive cells were integral components of the cystic wall. Cellular amylase activity was a function of the different culture media, a more rapid decrease in amylase activity being observed in Media 3 and 4. Uptake of [³H]thymidine did not show any significant differences between the media. It was also found that the ductlike cells cultured in Medium 4 had a limited capacity to redifferentiate into acinar cells. This study shows that the acinar cell phenotype can be maintainedin vitrofor more than 1 month. This study also suggests that ductal-like epithelial structures arise from transformation of acinar cells.     

小切口微创手术治疗新鲜跟腱断裂的临床价值

目的：探讨小切口微创手术治疗新鲜跟腱断裂的一晦床价值。方法：选取我院新鲜闭合性跟腱断裂患者50例,随机分为实验组和对照组各25例。实验组行小切口手术,对照组行常规切口手术。术后对患者进行随访,采用美国足踝协会（AOFAS）推荐的评分标准对患者术后功能恢复情况进行评价,观察并记录完全恢复患者例数、完全恢复时间、小腿最大周长差和术后并发症发生情况。结果：实验组AOFAS评分为（98．6±9．7）分,痊愈率96．00％,痊愈时间（20．2±3．2）周,两侧小腿最大周长差为（0．79＋0．68）cm,共有1例患者出现并发症,并发症发生率8％;而对照组的AOFAS评分为（91．4±11．5）分,痊愈率92．00％,痊愈时间（22．4±3．8）周,两侧小腿最大周长差为（0．91~0．76）cm;共有6例患者出现并发症,并发症的发生率为24％。两组患者的痊愈率、两侧小腿最大周长差比较差异无统计学意义（痊愈率：x2=-0．355,P=0．552;侧小腿最大周长差：t=O．588,P=0．559）;而与对照组比较,实验组AOFAS评分明显升高,完全恢复时间明显缩短,术后并发症的发生率显著降低,差异均有统计学意义（AOFAS评：t=2．393,P=0．021;恢复时间：t=2．150,P=0．037;并发症发生率：xⅫ．153,P=0．042）。结论：小切口手术与常规切口手术治疗新鲜跟腱断裂的疗效相当,但小切口手术术后恢童时间曼短．并发症更少．临床价值相对更高．     

Inhibition of TGF-β-induced Collagen Production in Rat Achilles Tendons by Injection of Tetramethylpyrazine

This article has no abstract.     

Increased C-telopeptide Cross-linking of Tendon Type I Collagen in Fibromodulin-deficient Mice

The controlled assembly of collagen monomers into fibrils, with accompanying intermolecular cross-linking by lysyl oxidase-mediated bonds, is vital to the structural and mechanical integrity of connective tissues. This process is influenced by collagen-associated proteins, including small leucine-rich proteins (SLRPs), but the regulatory mechanisms are not well understood. Deficiency in fibromodulin, an SLRP, causes abnormal collagen fibril ultrastructure and decreased mechanical strength in mouse tendons. In this study, fibromodulin deficiency rendered tendon collagen more resistant to nonproteolytic extraction. The collagen had an increased and altered cross-linking pattern at an early stage of fibril formation. Collagen extracts contained a higher proportion of stably cross-linked α1(I) chains as a result of their C-telopeptide lysines being more completely oxidized to aldehydes. The findings suggest that fibromodulin selectively affects the extent and pattern of lysyl oxidase-mediated collagen cross-linking by sterically hindering access of the enzyme to telopeptides, presumably through binding to the collagen. Such activity implies a broader role for SLRP family members in regulating collagen cross-linking placement and quantity.     

Functional Grading of Mineral and Collagen in the Attachment of Tendon to Bone

Attachment of dissimilar materials is a major challenge because high levels of localized stress may develop at their interfaces. An effective biologic solution to this problem exists at one of nature's most extreme interfaces: the attachment of tendon (a compliant, structural “soft tissue”) to bone (a stiff, structural “hard tissue”). The goal of our study was to develop biomechanical models to describe how the tendon-to-bone insertion derives its mechanical properties. We examined the tendon-to-bone insertion and found two factors that give the tendon-to-bone transition a unique grading in mechanical properties: 1), a gradation in mineral concentration, measured by Raman spectroscopy; and 2), a gradation in collagen fiber orientation, measured by polarized light microscopy. Our measurements motivate a new physiological picture of the tissue that achieves this transition, the tendon-to-bone insertion, as a continuous, functionally graded material. Our biomechanical model suggests that the experimentally observed increase in mineral accumulation within collagen fibers can provide significant stiffening of the partially mineralized fibers, but only for concentrations of mineral above a “percolation threshold” corresponding to formation of a mechanically continuous mineral network within each collagen fiber (e.g., the case of mineral connectivity extending from one end of the fiber to the other). Increasing dispersion in the orientation distribution of collagen fibers from tendon to bone is a second major determinant of tissue stiffness. The combination of these two factors may explain the nonmonotonic variation of stiffness over the length of the tendon-to-bone insertion reported previously. Our models explain how tendon-to-bone attachment is achieved through a functionally graded material composition, and provide targets for tissue engineered surgical interventions and biomimetic material interfaces.     

Lysyl Oxidase Activity Is Required for Ordered Collagen Fibrillogenesis by Tendon Cells

Lysyl oxidases (LOXs) are a family of copper-dependent oxido-deaminases that can modify the side chain of lysyl residues in collagen and elastin, thereby leading to the spontaneous formation of non-reducible aldehyde-derived interpolypeptide chain cross-links. The consequences of LOX inhibition in producing lathyrism are well documented, but the consequences on collagen fibril formation are less clear. Here we used β-aminoproprionitrile (BAPN) to inhibit LOX in tendon-like constructs (prepared from human tenocytes), which are an experimental model of cell-mediated collagen fibril formation. The improvement in structure and strength seen with time in control constructs was absent in constructs treated with BAPN. As expected, BAPN inhibited the formation of aldimine-derived cross-links in collagen, and the constructs were mechanically weak. However, an unexpected finding was that BAPN treatment led to structurally abnormal collagen fibrils with irregular profiles and widely dispersed diameters. Of special interest, the abnormal fibril profiles resembled those seen in some Ehlers-Danlos Syndrome phenotypes. Importantly, the total collagen content developed normally, and there was no difference in COL1A1 gene expression. Collagen type V, decorin, fibromodulin, and tenascin-X proteins were unaffected by the cross-link inhibition, suggesting that LOX regulates fibrillogenesis independently of these molecules. Collectively, the data show the importance of LOX for the mechanical development of early collagenous tissues and that LOX is essential for correct collagen fibril shape formation.     

Distribution of Young's Modulus in Porcine Corneas after Riboflavin/UVA-Induced Collagen Cross-Linking as Measured by Atomic Force Microscopy

Riboflavin/UVA-induced corneal collagen cross-linking has become an effective clinical application to treat keratoconus and other ectatic disorders of the cornea. Its beneficial effects are attributed to a marked stiffening of the unphysiologically weak stroma. Previous studies located the stiffening effect predominantly within the anterior cornea. In this study, we present an atomic force microscopy-derived analysis of the depth-dependent distribution of the Young''s modulus with a depth resolution of 5 µm in 8 cross-linked porcine corneas and 8 contralateral controls. Sagittal cryosections were fabricated from every specimen and subjected to force mapping. The mean stromal depth of the zone with effective cross-linking was found to be 219±67 µm. Within this cross-linked zone, the mean Young''s modulus declined from 49±18 kPa at the corneal surface to 46±17 kPa, 33±11 kPa, 17±5 kPa, 10±4 kPa and 10±4 kPa at stromal depth intervals of 0–50 µm, 50–100 µm, 100–150 µm, 150–200 µm and 200–250 µm, respectively. This corresponded to a stiffening by a factor of 8.1 (corneal surface), 7.6 (0–50 µm), 5.4 (50–100 µm), 3.0 (100–150 µm), 1.6 (150–200 µm), and 1.5 (200–250 µm), when compared to the Young''s modulus of the posterior 100 µm. The mean Young''s modulus within the cross-linked zone was 20±8 kPa (2.9-fold stiffening), while it was 11±4 kPa (1.7-fold stiffening) for the entire stroma. Both values were significantly distinct from the mean Young''s modulus obtained from the posterior 100 µm of the cross-linked corneas and from the contralateral controls. In conclusion, we were able to specify the depth-dependent distribution of the stiffening effect elicited by standard collagen cross-linking in porcine corneas. Apart from determining the depth of the zone with effective corneal cross-linking, we also developed a method that allows for atomic force microscopy-based measurements of gradients of Young''s modulus in soft tissues in general.