Removal Of Glycosaminoglycans Research Articles

The relationship between keratan sulfate glycosaminoglycan density and mechanical stiffening of CXL treatment

The corneal stroma is primarily composed of collagen fibrils, proteoglycans, and glycosaminoglycans (GAGs). It is known that corneal crosslinking (CXL) treatment improves mechanical properties of the cornea. However, the influence of stromal composition on the strengthening effect of CXL procedure has not been thoroughly investigated. The primary objective of the present research was to characterize the effect of keratan sulfate (KS) GAGs on the efficacy of CXL therapy. To this end, the CXL method was used to crosslink porcine corneal samples from which KS GAGs were enzymatically removed by keratanase II enzyme. Alcian blue staining was done to confirm the successful digestion of GAGs and uniaxial tensile experiments were performed for characterizing corneal mechanical properties. The influence of GAG removal and CXL treatment on resistance of corneal samples against enzymatic pepsin degradation was also quantified. It was found that removal of KS GAGs significantly softened corneal tensile properties (P < 0.05). Moreover, the CXL therapy significantly increased the tensile stiffness of GAG-depleted strips (P < 0.05). GAG-depleted corneal buttons were dissolved in the pepsin digestion solution significantly faster than control samples (P < 0.05). The CXL treatment significantly increased the time needed for complete pepsin digestion of GAG-depleted disks (P < 0.05). Based on these observations, we concluded that KS GAGs play a significant role in defining tensile properties and structural integrity of porcine cornea. Furthermore, the stiffening influence of the CXL treatment does not significantly depend on the density of corneal KS GAGs. The findings of the present study provided new information on the relation between corneal composition and CXL procedure mechanical effects.

Assessment of glycosaminoglycan content in bone using Raman spectroscopy.

Glycosaminoglycans (GAGs) are responsible for preserving bone tissue toughness as well as regulating collagen formation and mineralization in the extracellular matrix. However, current methods for characterization of GAGs in bone are destructive, thus unable to capture in situ changes or differences in GAGs between experimental groups. As an alternative, Raman spectroscopy is a non-destructive method and can detect concurrent changes in GAGs and other bone constituents. In this study, we hypothesized that the two most prominent Raman peaks of sulfated GAGs (at ~1066cm-1 and at ~1378cm-1) could be used to detect differences in GAGs content of bone. To test this hypothesis, three experimental models were utilized: an in vitro model (enzymatic removal of GAGs from human cadaver bone), an ex vivo mouse model (biglycan KO vs. WT), and an ex vivo aging model (comparing cadaveric bone samples from young and old donors). All Raman measurements were compared to Alcian blue measurements to confirm the validity of Raman spectroscopy in detecting GAGs changes in bone. Irrespective of different models, it was found that the ~1378cm-1 peak in Raman spectra of bone was uniquely sensitive to changes of GAGs content in bone when normalized with respect to the phosphate phase (~960cm-1); i.e., 1378cm-1/960cm-1 (peak intensity ratio) or 1370-1385cm-1/930-980cm-1 (integrated peak area ratio). In contrast, the 1070cm-1 peak, which includes another major peak of GAGs (1066cm-1), seemed to be compromised to detect changes of GAGs in bone due to concurrent changes of carbonate (CO3) in the similar peak range. This study validates the ability of Raman spectroscopy to detect in situ treatment-, genotype-, and age-related changes in GAG levels of bone matrix.

CHARACTERIZATION OF NATIVE AND DECELLULARIZED PORCINE TENDON UNDER TENSION AND COMPRESSION: A CLOSER LOOK AT GLYCOSAMINOGLYCAN CONTRIBUTION TO TENDON MECHANICS

Decellularised porcine superflexor tendon (pSFT) has been demonstrated to be a suitable scaffold for anterior cruciate ligament reconstruction[1]. While the role of collagen in tendons is well known, the mechanical role of glycosaminoglycans (GAGs) is less clear and may be altered by the decellularisation process.To determine the effects of decellularisation on pSFT GAG content and mechanical function and to investigate the consequences of GAG loss in tensile and compressive loading.pSFTs were decellularised following previous techniques [2]. For GAG removal, native pSFTs were treated with chondroitinase ABC (ChABC; 0.1U/mL, 72h). Cell and GAG removal was validated using histology and quantitative assays. Native, decellularised and ChABC treated groups (n=6) were biomechanically characterised. In tension, specimens underwent stress relaxation and strength testing using previous protocols [1]. Stress relaxation data was fitted to a modified Maxwell-Weichert model to determine time-dependent (E1 &amp; E2) and time-independent moduli (E0). The toe and linear region moduli (Etoe, Elinear), in addition to tensile strength (UTS) and failure strain were determined from strength testing. In compression, specimens underwent confined loading conditions (ramp at 10 s-1 to 10% strain and hold). The aggregate modulus (HA) and zero-strain permeability (k0) were determined using previous techniques [3]. Data was analysed by one-way ANOVA with Tukey post-hoc test to determine significant differences between test groups (p&lt;0.05).Quantitative assays showed no GAG reduction post-decellularisation, but a significant reduction after ChABC treatment. HA was only significantly reduced in the ChABC group. k0 was significantly higher for the ChABC group compared to decellularised. E0 was significantly reduced in the decellularised group compared to native and ChABC groups, while E1 and E2 were not different between groups. Etoe, Elinear, UTS and failure strain were not different between groups.Decellularisation does not affect GAG content or impair mechanical function in pSFT. GAG loss adversely affects pSFT compressive properties, revealing major mechanical contribution under compression, but no significant role under tension.

Glycosaminoglycans modulate microscale mechanics and viscoelasticity in fatigue injured tendons

Tendinopathies are common injuries that typically occur from overuse and fatigue. Treatments target late-stage symptoms with limited success, leading to high rates of reinjury. Early intervention could halt tendinopathy progression to rupture but requires a better understanding of the biomechanical environment associated with early-stage disease. While fatigue injured tendons are further damaged by exercise that is initiated immediately after onset of injury, exercise that is initiated after a brief delay promotes repair. Similar macroscale mechanical properties and collagen damage throughout this delay period suggests that microscale, non-collagenous matrix changes after fatigue injury modulate tendon mechanotransduction and shifts the exercise response from detrimental to reparative. Glycosaminoglycans (GAGs) and proteoglycans (PGs) are increased during chronic tendinopathy, but their role in early-stage disease is unknown. We hypothesized that increased GAGs from fatigue injury modulate viscoelasticity and microscale strains to enable repair from exercise. Various GAG types were increased in the weeks after onset of fatigue injury in the extracellular and pericellular matrices of rat patellar tendons. Enzymatic removal of GAGs from these fatigued tendons increased microscale shear strain, suggesting that GAGs modulate the cell microenvironment after fatigue injury. GAG removal decreased dynamic modulus in the toe region and decreased loss tangent in the linear region of the stress–strain curve in fatigued tendons, suggesting the GAG increase modulates tendon multiscale mechanics and viscoelasticity during fiber uncrimping and fibril sliding and strain transfer. GAGs may influence repair in response to exercise and could serve as a therapeutic target for tendinopathy.

On the Mechanical Roles of Glycosaminoglycans in the Tensile Properties of Porcine Corneal Stroma.

The cornea is primarily composed of collagen fibrils that are embedded in a ground substance rich in proteoglycans and other glycoproteins. It is known that glycosaminoglycan (GAG) side chains of proteoglycans form anti-parallel duplexes between collagen fibrils. The present work was done in order to investigate the mechanical function of GAGs in defining the tensile properties of porcine corneal stroma. Porcine corneal stromal strips dissected from the nasal-temporal direction were divided into control, buffer-treated, and enzyme-treated groups. The samples in the control group were used immediately after dissection. However, the buffer-treated and enzyme-treated samples were, respectively, incubated for 18 hours at 37°C in a buffer solution made up of 100-mM sodium acetate at pH 6.0 or in an enzyme solution containing keratanase II. The Blyscan assay was used to quantify the total GAG content and assess GAG depletion in the samples treated with the enzyme and buffer solutions. Uniaxial tensile tests were also performed to determine the effect of GAG removal on mechanical properties of the cornea. The GAG content in enzyme-treated samples was significantly lower than that of the normal and buffer-treated specimens (P < 0.05). Moreover, GAG-depleted strips showed significantly softer mechanical responses in comparison with the control and buffer samples (P < 0.05). Removing GAGs from the corneal extracellular matrix led to significant tensile property reduction; supporting the hypothesis that there exists a strong correlation between the GAG content and mechanical properties of the corneal stroma.

Removal of GAGs Regulates Mechanical Properties, Collagen Fiber Formation, and Alignment in Tissue Engineered Meniscus.

The complex fibrillar architecture of native meniscus is essential for proper function and difficult to recapitulate in vitro. In the native meniscus, proteoglycan content is low during the development of collagen fibers and progressively increases with aging. In vitro, fibrochondrocytes produce glycosaminoglycans (GAGs) early in culture, in contrast to native tissue, where they are deposited after collagen fibers have formed. This difference in the timing of GAG production hinders the formation of a mature fiber network in such in vitro models. In this study, we removed GAGs from collagen gel-based tissue engineered constructs using chondroitinase ABC (cABC) and evaluated the effect on the formation and alignment of collagen fibers and the subsequent effect on tensile and compressive mechanical properties. Removal of GAGs during maturation of in vitro constructs improved collagen fiber alignment in tissue engineered meniscus constructs. Additionally, removal of GAGs during maturation improved fiber alignment without compromising compressive strength, and this removal improved not only fiber alignment and formation but also tensile properties. The increased fiber organization in cABC-treated groups also appeared to influence the size, shape, and location of defects in these constructs, suggesting that treatment may prevent the propagation of large defects under loading. This data gives another method of modulating the ECM for improved collagen fiber formation and mechanical properties in tissue engineered constructs.

PCV2 Uptake by Porcine Monocytes Is Strain-Dependent and Is Associated with Amino Acid Characteristics on the Capsid Surface.

Porcine circovirus type 2 (PCV2) is associated with several economically important diseases that are described as PCV2-associated diseases (PCVADs). PCV2 is replicating in lymphoblasts, and PCV2 particles are taken up by monocytes without effective replication or complete degradation. Glycosaminoglycans (GAGs) have been demonstrated to be important receptors for PCV2 binding and entry in T-lymphocytes and continuous cell lines. The objective of this study was to determine whether differences exist in viral uptake and outcome among six PCV2 strains from different disease outbreaks in primary porcine monocytes: Stoon-1010 (PCV2a; PMWS), 1121 (PCV2a; abortion), 1147 (PCV2b; PDNS), 09V448 (PCV2d-1; PCVAD with high viral load in lymphoid tissues [PCVADhigh]), DE222-13 (PCV2d-2; PCVADhigh), and 19V245 (PCV2d-2; PCVADhigh). The uptake of PCV2 in peripheral blood monocytes was different among the PCV2 strains. A large number of PCV2 particles were found in the monocytes for Stoon-1010, DE222-13, and 19V245, while a low number was found for 1121, 1147, and 09V448. Competition with, and removal of GAGs on the cell surface, demonstrated an important role of chondroitin sulfate (CS) and dermatan sulfate (DS) in PCV2 entry into monocytes. The mapping of positively/negatively charged amino acids exposed on the surface of PCV2 capsids revealed that their number and distribution could have an impact on the binding of the capsids to GAGs, and the internalization into monocytes. Based on the distribution of positively charged amino acids on PCV2 capsids, phosphacan was hypothesized, and further demonstrated, as an effective candidate to mediate virus attachment to, and internalization in, monocytes. IMPORTANCE PCV2 is present on almost every pig farm in the world and is associated with a high number of diseases (PCV2-associated diseases [PCVADs]). It causes severe economic losses. Although vaccination is successfully applied in the field, there are still a lot of unanswered questions on the pathogenesis of PCV2 infections. This article reports on the uptake difference of various PCV2 strains by peripheral blood monocytes, and reveals the mechanism of the strong viral uptake ability of monocytes of Piétrain pigs. We further demonstrated that: (i) GAGs mediate the uptake of PCV2 particles by monocytes, (ii) positively charged three-wings-windmill-like amino acid patterns on the capsid outer surface are activating PCV2 uptake, and (iii) phosphacan is one of the potential candidates for PCV2 internalization. These results provide new insights into the mechanisms involved in PCVAD and contribute to a better understanding of PCV2 evolution. This may lead to the development of resistant pigs.

Removal of glycosaminoglycans affects the in situ mechanical behavior of extrafibrillar matrix in bone

Previous studies have shown that glycosaminoglycans (GAGs) in bone matrix, coupling with water in bone matrix, may play a significant role in toughening bone tissues. Since GAGs are most likely present only in the extrafibrillar matrix (EFM) of bone, we hypothesized that GAGs in EFM would have a major impact on bone tissue toughness. To confirm this conjecture, we removed GAGs ex vivo from human cadaveric bone samples using a protein deglycosylation mix kit and then examined the in situ mechanical behavior of mineralized collagen fibrils (MCFs) and the surrounding EFM of the samples, using a high-resolution atomic force microscopy (AFM). By testing the bone samples before and after removal of GAGs, we found that under the wet condition removal of GAGs resulted in an increase in the elastic modulus of both EFM and MCFs, whereas a significant decrease in plastic energy dissipation was observed mainly in EFM. In contrast, under the dry condition the removal of GAGs had little effects on the mechanical properties of either MCFs or EFM. These results suggest that both MCFs and EFM contribute to the plastic energy dissipation of bone, whereas in the presence of matrix water removal of GAGs significantly reduces the capacity of EFM in plastic energy dissipation, but not MCFs. In addition, GAGs may affect the elastic modulus of both EFM and MCFs. These findings give rise to new understanding to the underlying mechanism of GAGs in toughening of bone tissues.

Tensile Viscoelastic Properties of the Sclera after Glycosaminoglycan Depletion

ABSTRACT Purpose Fibrillar collagen network and glycosaminoglycans (GAGs) are the primary components of extracellular matrix (ECM) of the sclera. The main goal of this study was to investigate the possible structural roles of GAGs in the scleral tensile properties as a function of preconditioning and displacement rate. Methods Four-step uniaxial stress-relaxation tests were used for characterizing the viscoelastic tensile response of the posterior porcine sclera with and without enzymatic GAG removal. The scleral strips were divided into different groups based on the displacement rate and the presence or absence of a preconditioning step in the loading protocol. The groups were (1) displacement rate of 0.2 mm/min without preconditioning, (2) displacement rate of 1 mm/min without preconditioning, (3) displacement rate of 0.2 mm/min with preconditioning, and (4) displacement rate of 1 mm/min with preconditioning. The peak stress, equilibrium stress, and the equilibrium elastic modulus were calculated for all specimens and compared against each other. Results Increasing the displacement rate from 0.2 mm/min to 1.0 mm/min was found to cause an insignificant change in the equilibrium stress and equilibrium elastic modulus of porcine scleral strips. Removal of GAGs resulted in an overall stiffer tensile behavior independent of the displacement rate in samples that were not preconditioned (P < .05). The behavior of preconditioned samples with and without GAG removal was not significantly different from each other. Conclusions The experimental measurements of the present study showed that GAGs play an important role in the mechanical properties of the posterior porcine sclera. Furthermore, using a preconditioning step in the uniaxial testing protocol resulted in not being able to identify any significant difference in the tensile behavior of GAG depleted and normal scleral strips.

Regional Differences in the Glycosaminoglycan Role in Porcine Scleral Hydration and Mechanical Behavior

PurposeThis study characterized the role of glycosaminoglycans (GAGs) in the hydration, thickness, and biomechanical properties of posterior and anterior porcine sclera.MethodsThe scleral discs and strips were obtained from the anterior and posterior parts of porcine eyes, and their initial hydration and thickness were measured. The anterior and posterior scleral discs were used to show the efficacy of the GAG removal protocol by quantifying their GAG content. The strips were divided into three groups of PBS treatment, buffer treatment, and enzyme treatment in order to assess the effects of different treatment procedures on the thickness, hydration, and viscoelastic properties of the samples. The mechanical properties of the strips were determined by performing uniaxial tensile stress relaxation experiments.ResultsIt was found that the control and buffer groups had insignificant differences in all measured quantities. The samples from the posterior region had a significantly larger GAG content and thickness in comparison with those from anterior region; however, there was an insignificant difference in their hydration. The GAG depletion process decreased the hydration of both anterior and posterior samples significantly (P < 0.05). Furthermore, the mechanical tests showed that the removal of GAGs resulted in stiffer mechanical behavior in both anterior and posterior samples (P < 0.05). In particular, the peak stress and equilibrium stress were significantly larger for the strips in the enzyme treatment group.ConclusionsGAGs and their interaction with the collagen network are important in defining the hydration and mechanical properties of both posterior and anterior sclera.

Repopulation of decellularised articular cartilage by laser-based matrix engraving.

BackgroundIn spite of advances in the treatment of cartilage defects using cell and scaffold-based therapeutic strategies, the long-term outcome is still not satisfying since clinical scores decline years after treatment. Scaffold materials currently used in clinical settings have shown limitations in providing suitable biomechanical properties and an authentic and protective environment for regenerative cells. To tackle this problem, we developed a scaffold material based on decellularised human articular cartilage.MethodsHuman articular cartilage matrix was engraved using a CO2 laser and treated for decellularisation and glycosaminoglycan removal. Characterisation of the resulting scaffold was performed via mechanical testing, DNA and GAG quantification and in vitro cultivation with adipose-derived stromal cells (ASC). Cell vitality, adhesion and chondrogenic differentiation were assessed. An ectopic, unloaded mouse model was used for the assessment of the in vivo performance of the scaffold in combination with ASC and human as well as bovine chondrocytes. The novel scaffold was compared to a commercial collagen type I/III scaffold.FindingsCrossed line engravings of the matrix allowed for a most regular and ubiquitous distribution of cells and chemical as well as enzymatic matrix treatment was performed to increase cell adhesion. The biomechanical characteristics of this novel scaffold that we term CartiScaff were found to be superior to those of commercially available materials. Neo-tissue was integrated excellently into the scaffold matrix and new collagen fibres were guided by the laser incisions towards a vertical alignment, a typical feature of native cartilage important for nutrition and biomechanics. In an ectopic, unloaded in vivo model, chondrocytes and mesenchymal stromal cells differentiated within the incisions despite the lack of growth factors and load, indicating a strong chondrogenic microenvironment within the scaffold incisions. Cells, most noticeably bone marrow-derived cells, were able to repopulate the empty chondrocyte lacunae inside the scaffold matrix.InterpretationDue to the better load-bearing, its chondrogenic effect and the ability to guide matrix-deposition, CartiScaff is a promising biomaterial to accelerate rehabilitation and to improve long term clinical success of cartilage defect treatment.FundingAustrian Research Promotion Agency FFG (“CartiScaff” #842455), Lorenz Böhler Fonds (16/13), City of Vienna Competence Team Project Signaltissue (MA23, #18-08)

On influence of sulfated glycosaminoglycans on tensile properties of posterior sclera

The biomechanical behavior of sclera is conferred by the composition and structure of its extracellular matrix, which is mainly composed of collagen fibers and sulfated glycosaminoglycans (GAGs). Pathological conditions and visual disorders such as glaucoma and myopia could cause significant changes to the mechanical properties and GAG content of sclera. There exists sufficient evidence for the contribution of collagen fibers to the scleral biomechanics; however, possible mechanical roles of GAGs are not fully known. The primary objective of this work was to examine the mechanical function of GAGs through characterizing their effects on the scleral tensile response. For this purpose, chondroitinase ABC was used to deplete GAGs from posterior porcine scleral samples. Comprehensive biochemical and histological analyses were then performed to confirm and quantify GAG removal. Stress-controlled tensile tests with preconditioning were conducted in order to characterize the viscoelastic tensile behavior of treated and untreated specimens. It was found that the enzyme treatment caused a significant thickness reduction but it did not cause any significant change in the tensile properties of sclera. Overall, the findings of this study suggested that alternations in the GAG content of posterior scleral tissue are not important to tensile properties of sclera that are measured by stress-controlled experiments.

Glycosaminoglycans accelerate biomimetic collagen mineralization in a tissue-based in vitro model

Mammalian teeth are attached to the jawbone through an exquisitely controlled mineralization process: unmineralized collagen fibers of the periodontal ligament anchor directly into the outer layer of adjoining mineralized tissues (cementum and bone). The sharp interface between mineralized and nonmineralized collagenous tissues makes this an excellent model to study the mechanisms by which extracellular matrix macromolecules control collagen mineralization. While acidic phosphoproteins, localized in the mineralized tissues, play key roles in control of mineralization, the role of glycosaminoglycans (GAGs) is less clear. As several proteoglycans are found only in the periodontal ligament, it has been hypothesized that these inhibit mineralization of collagen in this tissue. Here we used an in vitro model based on remineralization of mouse dental tissues to determine the role of matrix GAGs in control of mineralization. GAGs were selectively removed from demineralized mouse periodontal sections via enzymatic digestion. Proteomic analysis confirmed that enzymatic GAG removal does not significantly alter protein content. Analysis of remineralized tissue sections by transmission electron microscopy (TEM) shows that GAG removal reduced the rate of remineralization in mineralized tissues compared to the untreated control, while the ligament remained unmineralized. Protein removal with trypsin also reduced the rate of mineralization, but to a lesser extent than GAG removal, despite a much larger effect on protein content. These results indicate that GAGs promote mineralization in mineralized dental tissues rather than inhibiting mineral formation in the ligament, which may have broader implications for understanding control of collagen mineralization in connective tissues.

Detection of Cell Surface Ligands for Human Synovial γδ T Cells.

Lack of understanding of the nature and physiological regulation of γδ T cell ligands has considerably hampered full understanding of the function of these cells. We developed an unbiased approach to identify human γδ T cells ligands by the production of a soluble TCR-γδ (sTCR-γδ) tetramer from a synovial Vδ1 γδ T cell clone from a Lyme arthritis patient. The sTCR-γδ was used in flow cytometry to initially define the spectrum of ligand expression by both human tumor cell lines and certain human primary cells. Analysis of diverse tumor cell lines revealed high ligand expression on several of epithelial or fibroblast origin, whereas those of hematopoietic origin were largely devoid of ligand. This allowed a bioinformatics-based identification of candidate ligands using RNAseq data from each tumor line. We further observed that whereas fresh monocytes and T cells expressed low to negligible levels of TCR-γδ ligands, activation of these cells resulted in upregulation of surface ligand expression. Ligand upregulation on monocytes was partly dependent upon IL-1β. The sTCR-γδ tetramer was then used to bind candidate ligands from lysates of activated monocytes and analyzed by mass spectrometry. Surface TCR-γδ ligand was eliminated by treatment with trypsin or removal of glycosaminoglycans, and also suppressed by inhibition of endoplasmic reticulum-Golgi transport. Of particular interest was that inhibition of glycolysis also blocked TCR-γδ ligand expression. These findings demonstrate the spectrum of ligand(s) expression for human synovial Vδ1 γδ T cells as well as the physiology that regulates their expression.

An investigation of the glycosaminoglycan contribution to biaxial mechanical behaviours of porcine atrioventricular heart valve leaflets.

The atrioventricular heart valve (AHV) leaflets have a complex microstructure composed of four distinct layers: atrialis, ventricularis, fibrosa and spongiosa. Specifically, the spongiosa layer is primarily proteoglycans and glycosaminoglycans (GAGs). Quantification of the GAGs' mechanical contribution to the overall leaflet function has been of recent focus for aortic valve leaflets, but this characterization has not been reported for the AHV leaflets. This study seeks to expand current GAG literature through novel mechanical characterizations of GAGs in AHV leaflets. For this characterization, mitral and tricuspid valve anterior leaflets (MVAL and TVAL, respectively) were: (i) tested by biaxial mechanical loading at varying loading ratios and by stress-relaxation procedures, (ii) enzymatically treated for removal of the GAGs and (iii) biaxially mechanically tested again under the same protocols as in step (i). Removal of the GAG contents from the leaflet was conducted using a 100 min enzyme treatment to achieve approximate 74.87% and 61.24% reductions of all GAGs from the MVAL and TVAL, respectively. Our main findings demonstrated that biaxial mechanical testing yielded a statistically significant difference in tissue extensibility after GAG removal and that stress-relaxation testing revealed a statistically significant smaller stress decay of the enzyme-treated tissue than untreated tissues. These novel findings illustrate the importance of GAGs in AHV leaflet behaviour, which can be employed to better inform heart valve therapeutics and computational models.

Effect of dentin proteoglycans on the stability of resin-dentin bonds against artificial saliva storage

Objective: To investigate the potential effect of proteoglycans (PG) and glycosaminoglycans (GAG) on the stability of resin-dentin bonds against artificial saliva storage. Methods: Seventy-two extracted molars were used to obtain standard dentin bonding surface, and the specimens were etched for 15 s with 37% phosphoric acid and divided into three groups using a table of random number. Then the three groups undergone different incubating procedures as follow: specimens in chondroitinase ABC (C-ABC) group were incubated with C-ABC, specimens in trypsin (TRY) group were incubated with trypsin, and specimens in the control group were incubated with deionized water. All specimens were incubated at 37 ℃ for 48 h in the oscillators. Then specimens in each group were randomly assigned into three subgroups (n=8) as follows: immediate control subgroup, aging subgroups with artificial saliva storage for 6 months and 12 months. Microtensile bond strength (μTBS), fracture mode, bonding interface morphology and nanoleakage were evaluated. Results: Immediately and with artificial saliva storage for 6 months and 12 mouths, the μTBS of TRY group ([49.04±3.57], [37.01±3.21] and [35.27±3.56] MPa) were significantly higher than those in the control group ([40.71±3.32], [28.87±2.34] and [24.20±2.07] MPa) (P<0.05). The immediate μTBS of C-ABC group ([32.94±2.45] MPa) was significantly lower than that of the control group (P<0.05). While with artificial saliva storage for 6 months and 12 mouths, the μTBS of C-ABC group ([26.46±2.45] and [22.50±2.58] MPa) were no differences with those of the control group (P>0.05). The ratio of cohesive fracture increased with the extension of aging time. Some narrow gaps were found in hybrid layer of the control group with artificial saliva storage for 6 months and 12 mouths. Conclusions: Removal of PG increased the μTBS and durable bonds to dentin, while removal of GAG decreased the μTBS, however, it can be of help to create more durable bonds to dentin.

Glycosaminoglycans contribute to extracellular matrix fiber recruitment and arterial wall mechanics.

Elastic and collagen fibers are well known to be the major load-bearing extracellular matrix (ECM) components of the arterial wall. Studies of the structural components and mechanics of arterial ECM generally focus on elastin and collagen fibers, and glycosaminoglycans (GAGs) are often neglected. Although GAGs represent only a small component of the vessel wall ECM, they are considerably important because of their diverse functionality and their role in pathological processes. The goal of this study was to study the mechanical and structural contributions of GAGs to the arterial wall. Biaxial tensile testing was paired with multiphoton microscopic imaging of elastic and collagen fibers in order to establish the structure-function relationships of porcine thoracic aorta before and after enzymatic GAG removal. Removal of GAGs results in an earlier transition point of the nonlinear stress-strain curves [Formula: see text]. However, stiffness was not significantly different after GAG removal treatment, indicating earlier but not absolute stiffening. Multiphoton microscopy showed that when GAGs are removed, the adventitial collagen fibers are straighter, and both elastin and collagen fibers are recruited at lower levels of strain, in agreement with the mechanical change. The amount of stress relaxation also decreased in GAG-depleted arteries [Formula: see text]. These findings suggest that the interaction between GAGs and other ECM constituents plays an important role in the mechanics of the arterial wall, and GAGs should be considered in addition to elastic and collagen fibers when studying arterial function.

The role of glycosaminoglycans in control of collagen biomineralization

Event Abstract Back to Event The role of glycosaminoglycans in control of collagen biomineralization Magdalena Wojtas1, 2, Alexander J. Lausch1 and Eli D. Sone1, 3, 4 1 University of Toronto, Institute of Biomaterials & Biomedical Engineering, Canada 2 Wroclaw University of Technology, Department of Biochemistry, Poland 3 University of Toronto, Department of Materials Science & Engineering, Canada 4 University of Toronto, Faculty of Dentistry, Canada Introduction: Healthy teeth are attached to the jawbone through an exquisitely controlled mineralization process. In periodontitis this attachment is destroyed by bacterial invasion. In order to develop more effective strategies to regenerate the ligament-tooth attachment, it is critical to have a better understanding of the biomineralization of collagen fibrils in the formation of this hard-soft tissue interface. While non-collagenous proteins have been extensively investigated as promoters and inhibitors of collagen mineralization[1] the role of glycosaminoglycans (GAGs) is unclear, notwithstanding some suggestions that GAGs might maintain the ligament in its unmineralized state[2]. The aim of this study was to determine the role of GAGs in collagen mineralization at the periodontal ligament–cementum junction. We used our recently developed tissue based in vitro model, in which the extracellular matrix of demineralized mouse periodontal sections retains sufficient information to direct preferential remineralization of the natively mineralized tissues[3]. Materials and Methods: To determine the role of matrix GAGs in control of mineralization, demineralized mouse periodontal sections were exposed to chondroitinase ABC and hyaluronidase, which digest chondroitin sulfates, and hyaluronan respectively. The GAG removal was quantified using a modified DMMB assay, and polyacrylamide gel electrophoresis (PAGE). Gels were stained with Stains-all method, followed by silver staining to maximize detection. Tissue sections were remineralized as described previously[3] and examined by TEM. Sections digested (D) with chondroitinase ABC and hyaluronidase were compared to the control untreated sections (C). Results and Discussion: Our analysis shows significant removal of GAGs from tissue sections following enzymatic digestion with chondroitinase ABC and hyaluronidase. A DMMB assay, which specifically detects sulfated GAGs, showed an 80% reduction of GAGs compared to control sections (Fig. 1A). PAGE analysis confirmed DMMB results. There were bands observed in control samples, which were not detected in digested samples (Fig. 1B). TEM analysis of remineralized tissue sections has shown that GAG removal reduced the rate of remineralization compared to the control (Fig. 2). The selectivity of remineralization was maintained in all cases; only dentin and cementum remineralized, while the ligament remained unmineralized. These results differ from previous findings where GAG removal caused ligament mineralization in sheep teeth[2]. The acidic character of GAGs implies possible roles in calcium ions binding, nucleation and control of crystal growth. Our preliminary results suggest that GAGs, along with acidic phosphoproteins, are responsible for promoting mineralization in dentin/cementum rather than inhibiting crystal formation in ligament. Conclusion: Our findings indicate that GAGs might play a role in control of nucleation and/or crystal growth during collagen mineralization. This study was supported by the Canadian Institutes of Health Research.; Project supported by Wroclaw Centre of Biotechnology, programme The Leading National Research Centre (KNOW) for years 2014-2018

Coupling Effect of Water and Proteoglycans on the In Situ Toughness of Bone.

Proteoglycans (PGs) are one type of noncollagenous proteins in the extracellular matrix of bone that primarily contain a core protein and glycosaminoglycans (GAGs). GAGs are highly polar and negatively charged, thus have a strong tendency to attract water molecules into the matrix. We hypothesized in this study that PGs in bone play a pivotal role in sustaining the toughness of the tissue only when water is present. To test the hypothesis, we used a novel nanoscratch test to measure the in situ toughness of human cadaveric bone treated with and without peptide-N-glycosidase F (PNGase F), an enzyme that specifically removes the N-linked oligosaccharides of GAGs from core proteins. Cortical bone specimens were prepared from the posterior aspect of mid-diaphyseal femurs of six (n = 6) male human donors between 51.5 ± 5.17 years old. Biochemical and histochemical assays were used to verify whether N-linked oligosaccharides were removed from bone matrix by PNGase F. By testing wet and dehydrated bone specimens, the coupling effect between water and PGs on the in situ toughness of bone was investigated. The two-way ANOVA analyses showed that removal of GAGs had significant effects on the in situ toughness of wet bone samples. In contrast, the removal of GAGs did not show significant effects on the toughness of dry bone. The results of this study, for the first time, suggest that GAGs play a pivotal role in the in situ toughness of bone only when water is present, and vice versa, water functions as a plasticizer in bone only when GAGs are present. © 2015 American Society for Bone and Mineral Research.

Effect of proteoglycans at interfaces as related to location, architecture, and mechanical cues

IntroductionCovalently bound functional GAGs orchestrate tissue mechanics through time-dependent characteristics. ObjectiveThe role of specific glycosaminoglycans (GAGs) at the ligament–cementum and cementum–dentin interfaces within a human periodontal complex were examined. Matrix swelling and resistance to compression under health and modeled diseased states was investigated. Materials and methodsThe presence of keratin sulfate (KS) and chondroitin sulfate (CS) GAGs at the ligament–cementum and cementum–dentin interfaces in human molars (N=5) was illustrated by using enzymes, atomic force microscopy (AFM), and AFM-based nanoindentation. The change in physical characteristics of modeled diseased states through sequential digestion of keratin sulfate (KS) and chondroitin sulfate (CS) GAGs was investigated. One-way ANOVA tests with P<0.05 were performed to determine significant differences between groups. Additionally, the presence of mineral within the seemingly hygroscopic interfaces was investigated using transmission electron microscopy. ResultsImmunohistochemistry (N=3) indicated presence of biglycan and fibromodulin small leucine rich proteoglycans at the interfaces. Digestion of matrices with enzymes confirmed the presence of KS and CS GAGs at the interfaces by illustrating a change in tissue architecture and mechanics. A significant increase in height (nm), decrease in elastic modulus (GPa), and tissue deformation rate (nm/s) of the PDL-C attachment site (215±63–424±94nm; 1.5±0.7–0.4±0.2GPa; 21±7–48±22nm/s), and cementum–dentin interface (122±69–360±159nm; 2.9±1.3–0.7±0.3GPa; 18±4–30±6nm/s) was observed. ConclusionsThe sequential removal of GAGs indicated loss in intricate structural hierarchy of hygroscopic interfaces. From a mechanics perspective, GAGs provide tissue recovery/resilience. The results of this study provide insights into the role of GAGs toward conserved tooth movement in the socket in response to mechanical loads, and modulation of potentially deleterious strain at tissue interfaces.