Distribution Of Glycosaminoglycans Research Articles

A Histological Analysis and Detection of Complement Regulatory Protein CD55 in SARS-CoV-2 Infected Lungs.

A complement imbalance in lung alveolar tissue can play a deteriorating role in COVID-19, leading to acute respiratory distress syndrome (ARDS). CD55 is a transmembrane glycoprotein that inhibits the activation of the complement system at the intermediate cascade level, blocking the activity of the C3 convertase. In our study, lung specimens from COVID-19 and ARDS-positive COVID+/ARDS+ patients were compared with COVID-19 and ARDS-negative COVID-/ARDS- as well as COVID-/ARDS+ patients. Histochemical staining and immunolabeling of CD55 protein were performed. The COVID-/ARDS- specimen showed higher expression and homogeneous distribution of glycosaminoglycans as well as compactly arranged elastic and collagen fibers of the alveolar walls in comparison to ARDS-affected lungs. In addition, COVID-/ARDS- lung tissues revealed stronger and homogenously distributed CD55 expression on the alveolar walls in comparison to the disrupted COVID-/ARDS+ lung tissues. Even though the collapse of the alveolar linings and the accumulation of cellular components in the alveolar spaces were characteristic of COVID+/ARDS+ lung tissues, evaluating CD55 expression could be relevant to understand its relation to the disease. Furthermore, targeting CD55 upregulation as a potential therapy could be an option for post-infectious complications of COVID-19 and other inflammatory lung diseases in the future.

Morphological features of enamel in fluorosis of different degrees of severity

The occurrence of dental fluorosis is facilitated by a violation of enamel mineralization caused by fluorides, which enter the human body in excess during its development and formation and have a toxic effect on enamel blasts. However, the molecular mechanisms involved in the pathogenesis of fluorosis are not fully understood. Enamel formation is a complex process involving cell proliferation and differentiation through epithelial-mesenchymal sequential secretion of matrix proteins, tissue-specific transport of ions including calcium and fluoride, and precipitation and alignment of enamel crystals through interactions between organic and inorganic molecules. Understanding the morphological features of enamel changes during fluoride intoxication of the human body in the endemic region allows us to clearly understand the need for a comprehensive solution to this medical and social problem. The aim was to study the morphological features of enamel in fluorosis in residents of the endemic region of Ukraine, in particular the Poltava region. The work examines different groups of teeth (both intact and affected by fluorosis) removed for orthodontic or clinical indications in men and women aged 17 to 40 years. Morphological signs were studied first on native, and later on histochemically stained sections. It was established that the violation of the structure of the enamel layer of the teeth in mild and severe fluorosis is characterized by both partial and complete violation of the movement of the enamel prisms with signs of destruction. Fragmentation and homogeneity throughout the entire thickness were found in some areas of the enamel. When evaluating histochemically stained sections of teeth affected by fluorosis, it was established that dystrophic changes in the enamel structure and accumulation of acidic glycosaminoglycans in the lesions are more characteristic of mild and moderate forms. Under the conditions of a severe form of fluorosis, complete destruction of the prisms, fragmentation of the lamella, homogenization of areas on the entire enamel layer, which is due to the uneven distribution of acidic mucopolysaccharides, have been established. Morphologically and histochemically dystrophic changes in the areas of the affected enamel are confirmed by uneven distribution and accumulation of acidic glycosaminoglycans. An assessment of the effect of fluoride intoxication on the state of tooth enamel was carried out, which will allow to expand the possibilities of preventive measures for related specialists, as well as to create and develop additional treatment methods that will contribute to the improvement of physical and aesthetic indicators of teeth. dental health.

Conditional Knockout of Glycosaminoglycan Synthesis in Bladder Epithelium Has No Effect on Urinary Voiding in Mice

Glycosaminoglycans (GAGs) on the luminal surface of the bladder epithelium have been suggested to play an important role in barrier function and in protection from infection. There is also a substantial literature that posits a breakdown in the GAG layer may underlie, or be associated with the development of painful bladder syndrome (PBS, aka interstitial cystitis) and indeed, GAG mimetics such as pentosan polysulphate provide symptomatic relief for some patients. However, opinions on the presence and role of GAGs vary, and incontrovertible evidence is lacking. We have taken a unique approach to answering this question by engineering a highly cell-specific genetic mouse model that lacks the obligatory initiating enzyme for GAG synthesis in urothelium. Uroplakin 2-Cre (UP2-Cre) mice were crossed with xylosyltransferase 2-floxed mice to knockout the xylt2 gene in urothelium. RT-PCR confirmed the tissue specificity of the knockout in urothelium but not in bladder smooth muscle. Further, lacZ reporter staining in cryosections demonstrated highly specific expression of the Cre construct in urothelium. Mammals express two xylosyltransferases, XylT1 and XylT2 in different proportions in different tissues. While functionally similar they appear to differ in their substrate specificity and kinetics. qPCR in dissected urothelium demonstrated that XylT2 was the dominant isoform, being expressed at approximately 80-fold higher level than XylT1. Void spot assays (VSA) on filter paper (4 hours) have proven to be a reliable indicator of voiding dysfunction in mice. We conducted VSAs on female Up2 cre/xylt2 fl/fl (n=12) and xylt2 fl/fl (n=11) littermate controls aged 8 – 16 weeks. There was no significant difference in void volumes, number of primary voids, volume/void or number of microvoids (<20 μl) between controls and knockouts, whereas transurethral instillation of protamine sulfate, which injures the apical cell layer, induced pain and a significantly greater number of microvoids, with reduced primary void volumes. We conclude preliminarily, that loss of GAGs in urothelium does not affect bladder function, suggesting that GAGs do not play a role in protecting the suburothelium and adjacent sensory afferents from noxious urine components. Future work will characterize and quantitate the distribution of GAGs within the urothelium to determine whether GAGs are deployed on the luminal surface and, whether they play a role in urothelial repair from injury. NIH grant P20DK097818. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Glycosaminoglycans and collagen fibril distribution at various depths of the corneal stroma of normal and CXL treated rats

Corneal collagen cross-linking (CXL) is widely used to treat keratoconus and ecstatic corneal disorders. The present studies were carried out to investigate the distribution of glycosaminoglycans (GAGs) and collagen fibril (CF) at different depths of the normal and CXL treated corneal stroma of four week old rats 7 days after standard CXL application. Ten Wistar rats’ corneas were used for the study. The epithelium of the cornea from the left eye of each rat was removed and treated with standard CXL application using riboflavin and Ultraviolet-A (UVA) (3 mW/cm2 for 30 min). The cornea from the right eye was used as the control cornea. The cornea was removed from the eye and processed for transmission electron microscopy. A bottom mounted Quemesa camera was used to capture digital images and these images were analysed using iTEM software. In the control cornea, the GAGs area size was not significantly different in the anterior, middle, and posterior stroma. In the CXL treated rats the GAGs area size gradually increased from the anterior to the posterior stroma whereas the spacing between the GAGs gradually decreased. There were very large GAGs present in the posterior stroma of the CXL treated rats. When comparing the control and CXL cornea, the GAGs area in the CXL cornea was significantly higher and inter-GAGs-spacing was smaller than in the control cornea. In the control cornea, the collagen fibrils diameter was higher in the anterior stroma and lowest in the posterior stroma. In the CXL treated cornea, the CF diameter and the interfibrillar spacing gradually decreased from the anterior to the posterior stroma. On comparison between the control and the CXL treated cornea, the interfibrillar spacing was significantly smaller in the CXL treated cornea than the control cornea in the anterior, middle, and posterior stroma but there was no difference in the diameter. The CXL treatment significantly increased the GAGs area and decreased the inter-GAGs-spacing, and inter–CF–spacing. This could be due to the gradual decline in the availability of riboflavin, UVA, and oxygen in the middle and posterior stroma. Further studies are required to investigate the role of keratan sulphate and chondroitin sulphate by using monoclonal antibodies with immunogold technique.

Histopathological and histomorphometric investigation of the effects of different irrigation solutions on Achilles tendon healing in rats.

This study aimed to examine the effect of irrigation fluids containing povidone-iodine (PVP-I), rifampicin (RF), and chlorhexidine gluconate (CHG) used during surgery on healing on a rat Achilles tendon model. Twenty-eight male Sprague-Dawley rats (range, 300 to 400 g) were used in the experiment carried out between November 2022 and December 2022. The rats were divided into PVP-I, RF, CHG, and control groups, with seven rats in each group. Following the tenotomy and repair of the right Achilles tendon, the surgical site was irrigated using PVP-I, RF, CHG, or normal saline (the control group) for 2 min. All rats were sacrificed on the 21st postoperative day. The samples were evaluated histomorphometrically using the scoring system modified by Svensson, Soslowsky, and Cook and histopathologically using the Bonar and Movin classifications. The RF group gave better results in all three scoring systems compared to the control, PVP-I, and CHG groups (p=0.008, p=0.002, and p=0.006, respectively). Cellularity, rounding, and tenocyte morphology showed a significant difference in favor of the RF group (p=0.004). While the distribution of ground substance glycosaminoglycans showed a significant difference in favor of the RF group, there was no significant difference among the other groups (p=0.22). Irrigation solutions containing PVP-I, RF, or CHG show no negative effect on Achilles tendon healing. Moreover, the findings suggest that RF irrigation can accelerate the healing process.

Histological, histochemical and immunohistochemical aspects of the intestine and pancreas of bats of the Phyllostomidae family (Mammalia, Chiroptera)

The present study aims to describe the intestine and pancreas of both Dryadonycteris capixaba and Pygoderma bilabiatum. Furthermore, it aims to identify the enteroendocrine cells that produce serotonin (5-HT), somatostatin (SST), and cholecystokinin (CCK) in the intestine and CCK, SST, glucagon (CGC) and insulin (INS) in the pancreas, relating histological characteristics with feeding habits, besides contributing to basic research on aspects of the diffuse neuroendocrine system of mammals. Histologically, the intestinal wall of the species D. capixaba and P. bilabiatum was similar to that of other mammals. The height of the villi in the small intestine was different among the species studied. In the duodenum and ileum, PAS positive secretion was observed. The duodenal gland present only in P. bilabiatum showed intense reaction for PAS stain, indicating neutral glycosaminoglycans (GAGs). The frequency and distribution of 5-HT Immunoreactive (IR) cells varied among the species. In D. capixaba, these cells were evident only in the ileum (11.8 ± 2.9 μm) and large intestine region. In P. bilabiatum were different among the segments, with the highest frequency in the duodenum (13.3 ± 2.8 μm), jejunum (8.1 ± 2.3 μm) and ileum (10.1 ± 1.9 μm). CCK-IR cells were not observed in D. capixaba. In P. bilabiatum these cells were observed in the ileum (9.5 ± 1.3 μm). SST-IR cells were not found throughout the intestine of the studied species. The pancreas of bats presents islets with differentiated cellular composition. All the antibodies used (CCK, SST, GCG and INS) obtained positive staining in the pancreatic islets of both the species studied. The INS-IR cells marked the entire islet in both species, whereas the CGC-IR cells were visualized throughout the islet in D. capixaba and preferentially in the periphery in P. bilabiatum. CCK and SST-IR cells were located in the peripheral region of the islets in both species. In our study we observed similarities in the intestinal and pancreatic morphology of the studied species, although there were interspecific differences in the distribution of intestinal GAGs and enteroendocrine cells. This suggests that these differences may be more related to the distinct feeding habits of each species than to their phylogeny, as they belong to the same family.

Co-Culture of Mouse Blastocysts on A Human Recellularized Endometrial Scaffold: An In Vitro Model for Future Implantation Studies.

This study evaluates the interaction of mouse blastocysts as a surrogate embryo on a recellularized endometrial scaffold by seeding human endometrial mesenchymal cells (hEMCs). In this experimental study, prepared decellularized human endometrial tissues were characterized by morphological staining, DNA content analysis, and scanning electron microscopic (SEM) analysis. The scaffolds were subsequently recellularized by hEMCs. After seven days of cultivation, the mouse blastocysts were co-cultured on the recellularized scaffolds for 48 hours. Embryo attachment and implantation within these scaffolds were evaluated at the morphological, ultrastructural, molecular, and hormonal levels. There was no morphological evidence of cells and nuclei in the decellularized scaffold. DNA content significantly decreased by 89.92% compared to the control group (P<0.05). Both decellularized and native tissues had similar patterns of collagen bundles and elastin fibers, and glycosaminoglycan (GAGs) distribution in the stroma. After recellularization, the hEMCs attached to the scaffold surface and penetrated different parts of these scaffolds. In the co-cultured group, the embryo attached to the surface of the scaffold after 24 hours and penetrated the recellularized endometrial tissue after 48 hours. We observed multi-layered organoid-like structures formed by hEMC proliferation. The relative expressions of epithelial-related genes, ZO-1 and COL4A1, and SSP1, MMP2, and PRL, as decidualizationrelated genes, were significantly higher in the recellularized group on day 9 in the presence of the embryo compared to the other groups (P<0.05). Beta human chorionic gonadotropin (β-hCG) and prolactin were statistically increased in the recellularized group on day 9 group (P<0.05). hEMCs and mouse embryo co-cultured on a decellularized endometrial scaffold provides an alternative model to study embryo implantation and the earlier stage of embryo development.

MP34-11 URINARY GLYCOSAMINOGLYCANS ARE ASSOCIATED WITH RECURRENT UTI AND UROBIOME ECOLOGY IN POSTMENOPAUSAL WOMEN

You have accessJournal of UrologyCME1 Apr 2023MP34-11 URINARY GLYCOSAMINOGLYCANS ARE ASSOCIATED WITH RECURRENT UTI AND UROBIOME ECOLOGY IN POSTMENOPAUSAL WOMEN Michael L. Neugent, Neha V. Hulyalkar, Philippe E. Zimmern, Kelli L. Palmer, Vladimir Shulaev, and Nicole J. De Nisco Michael L. NeugentMichael L. Neugent More articles by this author , Neha V. HulyalkarNeha V. Hulyalkar More articles by this author , Philippe E. ZimmernPhilippe E. Zimmern More articles by this author , Kelli L. PalmerKelli L. Palmer More articles by this author , Vladimir ShulaevVladimir Shulaev More articles by this author , and Nicole J. De NiscoNicole J. De Nisco More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003268.11AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The composition of urinary glycosaminoglycans (GAGs) is not fully understood. The luminal urothelial GAG layer is likely an important interface between the human host and the resident urobiome, which has been shown to be altered in postmenopausal (PM) women with history of recurrent urinary tract infection (rUTI).1 We sought to profile the urinary GAG distribution and associate urinary GAGs with the ecology of the resident urobiome in a controlled cohort of PM women designed to study rUTI. METHODS: Clean catch midstream urine was collected from a controlled cohort of PM women (n=75) who passed a set of exclusion criteria for an ongoing study of rUTI.1 GAGs were extracted from urine via macromolecular filtration and then enzymatically digested into constituent disaccharides for chondroitin Sulfate (CS), heparin Sulfate (HS), and hyaluronic Acid (HA) following a published protocol.2 The liberated disaccharides were analyzed and quantified via targeted liquid chromatography-mass spectrometry (LC-MS/MS). For whole genome metagenomics, DNA was purified from matched urine and prepared libraries were sequenced on an Illumina NextSeq500 and analyzed for taxonomic enrichment as described previously.1 RESULTS: We find that CS is the major urinary GAG in PM women and women who were experiencing an active UTI exhibited significantly higher urinary CS compared to those with no UTI (Figure 1). Lastly, we find that two urobiome bacterial species, Atopobium vaginae and Blautia obeum exhibit significant negative associations with urinary CS. CONCLUSIONS: Our findings of increased urinary CS during active UTI may be a sign of tissue damage during infection. We find negative associations between bacterial species known to be associated with vaginal dysbiosis and urinary CS levels. These associations may be indicators of bladder dysbiosis and alterations of urothelial GAG composition.

Cationic Carrier Mediated Delivery of Anionic Contrast Agents in Low Doses Enable Enhanced Computed Tomography Imaging of Cartilage for Early Osteoarthritis Diagnosis.

Cartilage tissue exhibits early degenerative changes with onset of osteoarthritis (OA). Early diagnosis is critical as there is only a narrow time window during which therapeutic intervention can reverse disease progression. Computed tomography (CT) has been considered for cartilage imaging as a tool for early OA diagnosis by introducing radio-opaque contrast agents like ioxaglate (IOX) into the joint. IOX, however, is anionic and thus repelled by negatively charged cartilage glycosaminoglycans (GAGs) that hinders its intra-tissue penetration and partitioning, resulting in poor CT attenuation. This is further complicated by its short intra-tissue residence time owing to rapid clearance from joints, which necessitates high doses causing toxicity concerns. Here we engineer optimally charged cationic contrast agents based on cartilage negative fixed charge density by conjugating cartilage targeting a cationic peptide carrier (CPC) and multi-arm avidin nanoconstruct (mAv) to IOX, such that they can penetrate through the full thickness of cartilage within 6 h using electrostatic interactions and elicit similar CT signal with about 40× lower dose compared to anionic IOX. Their partitioning and distribution correlate strongly with spatial GAG distribution within healthy and early- to late-stage arthritic bovine cartilage tissues at 50-100× lower doses than other cationic contrast agents used in the current literature. The use of contrast agents at low concentrations also allowed for delineation of cartilage from subchondral bone as well as other soft tissues in rat tibial joints. These contrast agents are safe to use at current doses, making CT a viable imaging modality for early detection of OA and staging of its severity.

Investigation of Glypican-4 and -6 by Infrared Spectral Imaging during the Hair Growth Cycle

The expression of glypicans in different hair follicle (HF) compartments is still poorly understood. Heparan sulfate proteoglycans (HSPGs) distribution in HF is classically investigated by conventional histology, biochemical analysis, and immunohistochemistry. Our previous study proposed a novel approach to assess hair histology and glypican-1 (GPC1) distribution changes in the HF at different phases of the hair growth cycle using infrared spectral imaging (IRSI). We show in the present manuscript for the first time complementary data on the distribution of glypican-4 (GPC4) and glypican-6 (GPC6) in HF at different phases of the hair growth cycle using IR imaging. Findings were supported by Western blot assays focusing on the GPC4 and GPC6 expression in HFs. Like all proteoglycan features, the glypicans are characterized by a core protein to which sulfated and/or unsulfated glycosaminoglycan (GAG) chains are covalently linked. Our study demonstrates the capacity of IRSI to identify the different HF tissue structures and to highlight protein, proteoglycan (PG), GAG, and sulfated GAG distribution in these structures. The comparison between anagen, catagen, and telogen phases shows the qualitative and/or quantitative evolution of GAGs, as supported by Western blot. Thus, in one analysis, IRSI can simultaneously reveal the location of proteins, PGs, GAGs and sulfated GAGs in HFs in a chemical and label-free manner. From a dermatological point of view, IRSI may constitute a promising technique to study alopecia.

Theranostic Applications of Glycosaminoglycans in Metastatic Renal Cell Carcinoma.

Renal cell carcinoma (RCC) makes up the majority of kidney cancers, with a poor prognosis for metastatic RCC (mRCC). Challenges faced in the management of mRCC, include a lack of reliable prognostic markers and biomarkers for precise monitoring of disease treatment, together with the potential risk of toxicity associated with more recent therapeutic options. Glycosaminoglycans (GAGs) are a class of carbohydrates that can be categorized into four main subclasses, viz., chondroitin sulfate, hyaluronic acid, heparan sulfate and keratan sulfate. GAGs are known to be closely associated with cancer progression and modulation of metastasis by modification of the tumor microenvironment. Alterations of expression, composition and spatiotemporal distribution of GAGs in the extracellular matrix (ECM), dysregulate ECM functions and drive cancer invasion. In this review, we focus on the clinical utility of GAGs as biomarkers for mRCC (which is important for risk stratification and strategizing effective treatment protocols), as well as potential therapeutic targets that could benefit patients afflicted with advanced RCC. Besides GAG-targeted therapies that holds promise in mRCC, other potential strategies include utilizing GAGs as drug carriers and their mimetics to counter cancer progression, and enhance immunotherapy through binding and transducing signals for immune mediators.

Is There Enough Evidence to Support the Role of Glycosaminoglycans and Proteoglycans in Thoracic Aortic Aneurysm and Dissection?-A Systematic Review.

Altered proteoglycan (PG) and glycosaminoglycan (GAG) distribution within the aortic wall has been implicated in thoracic aortic aneurysm and dissection (TAAD). This review was conducted to identify literature reporting the presence, distribution and role of PGs and GAGs in the normal aorta and differences associated with sporadic TAAD to address the question; is there enough evidence to establish the role of GAGs/PGs in TAAD? 75 studies were included, divided into normal aorta (n = 51) and TAAD (n = 24). There is contradictory data regarding changes in GAGs upon ageing; most studies reported an increase in GAG sub-types, often followed by a decrease upon further ageing. Fourteen studies reported changes in PG/GAG or associated degradation enzyme levels in TAAD, with most increased in disease tissue or serum. We conclude that despite being present at relatively low abundance in the aortic wall, PGs and GAGs play an important role in extracellular matrix maintenance, with differences observed upon ageing and in association with TAAD. However, there is currently insufficient information to establish a cause-effect relationship with an underlying mechanistic understanding of these changes requiring further investigation. Increased PG presence in serum associated with aortic disease highlights the future potential of these biomolecules as diagnostic or prognostic biomarkers.

Morphologic and histologic characterization of sheep and porcine TMJ as large animal models for tissue engineering applications

ObjectiveThe aim of this study was to compare and characterize the structural and ultrastructural organization of the temporomandibular joint (TMJ) between two large animal models for use in the development of tissue engineering strategies.Materials and methodsWhole TMJs from sheep and pigs were evaluated with micro-computed tomography (μCT) for morphology and quantitative analyses of bone parameters. Histological examination was performed on the TMJ disc and its attachments to investigate regional distribution of collagen, elastin, and glycosaminoglycans (GAGs).ResultsμCT analyses demonstrate higher bone mineral density (BMD) in the temporal fossa compared to the mandibular condyle in both species, with this variable being significantly higher in sheep than pig. Quantitative morphometry of the trabecular condyle reveals no statistical differences between the species. Histology demonstrates similar structural organization of collagen and elastin between species. Elastin staining was nearly twofold greater in sheep than in the pig disc. Finally, Safranin-O staining for GAGs in the TMJ disc was localized to the intermediate zone in the sheep but was absent from the porcine disc.ConclusionsOur findings show some important differences in the pig and sheep TMJ μCT variables and histology and composition of the disc and discal attachment. These disparities likely reflect differences in masticatory and TMJ functional loading patterns between the two species and provide insights into large animal models towards human applications.Clinical relevanceAs with the established pig model, the sheep is a suitable large animal model for TMJ research such as regenerative strategies, with specific considerations for design parameters appropriate for human-analog applications.

Intramural Distributions of GAGs and Collagen vs. Opening Angle of the Intact Porcine Aortic Wall.

The heterogeneity and contribution of collagen and elastin to residual stresses have been thoroughly studied, but more recently, glycosaminoglycans (GAGs) also emerged as potential regulators. In this study, the opening angle of aortic rings (an indicator of circumferential residual stresses) and the mural distributions of sulfated GAGs (sGAG), collagen, and elastin were quantified in the ascending, aortic arch and descending thoracic regions of 5- to 6-month-old pigs. The opening angle correlated positively with the aortic ring's mean radius and thickness, with good and moderate correlations respectively. The correlations between the sGAG, collagen, elastin, and collagen:sGAG ratio and the opening angle were evaluated to identify aortic compositional factors that could play roles in regulating circumferential residual stresses. The total collagen:sGAG ratio displayed the strongest correlation with the opening angle (r = -0.715, p < 0.001), followed by the total sGAG content which demonstrated a good correlation (r = 0.623, p < 0.001). Additionally, the intramural gradients of collagen, sGAG and collagen:sGAG correlated moderately with the opening angle. We propose that, in addition to the individual role sGAG play through their content and intramural gradient, the interaction between collagen and sGAG should be considered when evaluating circumferential residual stresses in the aorta.

A highly porous type II collagen containing scaffold for the treatment of cartilage defects enhances MSC chondrogenesis and early cartilaginous matrix deposition.

A major challenge in cartilage tissue engineering (TE) is the development of instructive and biomimetic scaffolds capable of driving effective mesenchymal stem cell (MSC) chondrogenic differentiation and robust de novo matrix formation. Type I collagen-based scaffolds are one of the most commonly selected materials given collagen's intrinsic ability to act as an instructive and active biomaterial. However, the chondrogenic potential of these scaffolds does not offer significant improvement over traditional treatments. We propose that taking a biomimetic approach to scaffold development might lead to an improved outcome for enhanced cartilage repair. Therefore, this study aimed to develop innovative type II collagen (CII)-containing scaffolds for enhanced cartilage repair, by incorporating CII and/or hyaluronic acid (HyA) into a type I collagen (CI) framework. Moreover, focus was placed on understanding the potential synergistic effects played by CII in combination with HyA, in terms of MSC chondrogenesis and cartilage-like formation, when both molecules are incorporated into scaffold biomaterials. The newly developed CII-containing scaffold exhibited a highly porous interconnected structure with 99% porosity and similar mechanical properties to previously optimised collagen-based scaffolds. Although all scaffold variants sustained early cartilaginous matrix deposition, the CII-containing scaffolds in the presence of HyA performed best, offering enhanced deposition and distribution of sulphated glycosaminoglycans (sGAG) in vitro by day 28. Taken together, the combination of CII and HyA resulted in the development of a biomimetic scaffold with improved chondrogenic benefits. These simple "off-the-shelf" implants hold great promise to direct enhanced tissue regeneration for the treatment of focal cartilage defects.

The Effect of Ageing and Degeneration on Glycosaminoglycan Concentration in the Intervertebral Disc

Introduction: The correct spatial distribution and high negative charge of glycosaminoglycans (GAGs) within the intervertebral disc (IVD) are responsible for discs water imbibition, proper osmotic pressure, and as such IVD’s physiological swelling behaviors and compressive properties. The aim of this study was to investigate the association of the concentration and distribution of GAG with IVD degeneration as measured by Pfirrmann et al. and Thompson et al. grading systems.&#x0D; Methodology: Full spinal columns (vertebrae L1-S1 and IVD between them) were harvested from fresh cadavers through an anterior dissection. MRI scans were taken of all spinal columns and were assessed using Pfirrmann grading system. All vertebral columns were cut in the midsagittal plane. The level of degeneration was assessed morphologically using Thompson et al. grading system. Samples from five regions of the L5/S1 IVDs were taken for GAG concentration analyses. Standard curve spectrophotometry was utilized for this purpose.&#x0D; Result: One hundred lumbar spine columns (L1-S1) were harvested from cadavers. Radiologic assessment using the Pfirrmann grading system and morphological Thompson grading system classified majority of discs as grade 3 and 4. A total of 478 samples from five regions of L5/S1 IVDs were included in the analysis of GAG content. The samples from the nucleus pulposus showed on average the highest concentration of GAG, although the differences were not statistically significant. The one-way analysis of variance (ANOVA) showed no statistically significant differences in the mean GAG mass between different Pfirrmann grades (F = 1.85, p = 0.13) and between different Thompson grades (F = 1.17, p = 0.33).&#x0D; Conclusion: Our study showed no association between GAG concentration levels and degeneration grade of the IVD as measured by radiological Pfirrmann and morphological Thompson grading systems.

Contrast-Enhanced Micro-Computed Tomography for 3D Visualization and Quantification of Glycosaminoglycans in Different Cartilage Types.

To compare CA4+-enhanced micro-computed tomography (microCT) of bovine articular, meniscal, nasal, and auricular cartilage, each of which possesses a different extracellular matrix (ECM) composition and structure. The diffusion kinetics of CA4+ in different native cartilage types were assessed over 20 hours. The feasibility of CA4+-enhanced microCT to visualize and quantify glycosaminoglycans (GAGs) in these different tissues was tested using safranin-O staining and 1,9-dimethylmethylene blue assay. The diffusion kinetics of CA4+ in auricular cartilage are significantly slower compared with all other cartilage types. Total GAG content per volume correlates to microCT attenuation with an R2 value of 0.79 for all cartilage types. Three-dimensional contrast-enhanced microCT images of spatial GAG distribution reflect safranin-O staining and highlight the differences in ECM structure, with heterogeneous regions with higher GAG concentrations highlighted by the contrast agent. CA4+-enhanced microCT enables assessment of 3-dimensiona distribution and GAG content in different types of cartilage and has promise as an ex vivo diagnostic technique to monitor matrix development in different tissues over time as well as tissue-engineered constructs.

Extracellular Matrix Deposition and Remodeling after Corneal Alkali Burn in Mice.

Corneal transparency relies on the precise arrangement and orientation of collagen fibrils, made of mostly Type I and V collagen fibrils and proteoglycans (PGs). PGs are essential for correct collagen fibrillogenesis and maintaining corneal homeostasis. We investigated the spatial and temporal distribution of glycosaminoglycans (GAGs) and PGs after a chemical injury. The chemical composition of chondroitin sulfate (CS)/dermatan sulfate (DS) and heparan sulfate (HS) were characterized in mouse corneas 5 and 14 days after alkali burn (AB), and compared to uninjured corneas. The expression profile and corneal distribution of CS/DSPGs and keratan sulfate (KS) PGs were also analyzed. We found a significant overall increase in CS after AB, with an increase in sulfated forms of CS and a decrease in lesser sulfated forms of CS. Expression of the CSPGs biglycan and versican was increased after AB, while decorin expression was decreased. We also found an increase in KS expression 14 days after AB, with an increase in lumican and mimecan expression, and a decrease in keratocan expression. No significant changes in HS composition were noted after AB. Taken together, our study reveals significant changes in the composition of the extracellular matrix following a corneal chemical injury.

581 Hair histology and glycosaminoglycans distribution probed by infrared spectral imaging: Focus on heparan sulfate proteoglycan and glypican-1 during hair growth cycle

Heparan sulfate proteoglycans (HSPGs) distribution in hair follicle (HF) is classically investigated by conventional histology, biochemical analysis, and immunohistochemistry. In this study, a novel approach is proposed to assess hair histology and HSPG distribution changes in the HF at different phases of the hair growth cycle using infrared spectral imaging (IRSI). Particularly, we were interested in the expression of glypican-1 in different HF compartments and their potential roles during hair shaft growth, as their roles are still poorly understood. The distribution of HSPGs in HFs was probed by IRSI using the absorption region relevant to sulfation as a spectral marker. Findings were supported by Western immunoblotting and immunohistochemistry assays focusing on the glypican-1 expression and distribution in HFs. This study demonstrates the capacity of IRSI to identify the different HF tissue structures and to highlight protein, proteoglycan (PG), glycosaminoglycan (GAG), and sulfated GAG distribution in these structures. The comparison between anagen, catagen, and telogen phases shows the qualitative and/or quantitative evolution of GAGs, as supported by Western immunoblotting. Thus, IRSI can simultaneously reveal the location of proteins, PGs, GAGs and sulfated GAGs in HFs in a reagent- and label-free manner. From a dermatological point of view, IRSI shows its potential as a promising technique to study alopecia.

Spectral CT imaging of human osteoarthritic cartilage via quantitative assessment of glycosaminoglycan content using multiple contrast agents.

Detection of early osteoarthritis to stabilize or reverse the damage to articular cartilage would improve patient function, reduce disability, and limit the need for joint replacement. In this study, we investigated nondestructive photon-processing spectral computed tomography (CT) for the quantitative measurement of the glycosaminoglycan (GAG) content compared to destructive histological and biochemical assay techniques in normal and osteoarthritic tissues. Cartilage-bone cores from healthy bovine stifles were incubated in 50% ioxaglate (Hexabrix®) or 100% gadobenate dimeglumine (MultiHance®). A photon-processing spectral CT (MARS) scanner with a CdTe-Medipix3RX detector imaged samples. Calibration phantoms of ioxaglate and gadobenate dimeglumine were used to determine iodine and gadolinium concentrations from photon-processing spectral CT images to correlate with the GAG content measured using a dimethylmethylene blue assay. The zonal distribution of GAG was compared between photon-processing spectral CT images and histological sections. Furthermore, discrimination and quantification of GAG in osteoarthritic human tibial plateau tissue using the same contrast agents were demonstrated. Contrast agent concentrations were inversely related to the GAG content. The GAG concentration increased from 25 μg/ml (85 mg/ml iodine or 43 mg/ml gadolinium) in the superficial layer to 75 μg/ml (65 mg/ml iodine or 37 mg/ml gadolinium) in the deep layer of healthy bovine cartilage. Deep zone articular cartilage could be distinguished from subchondral bone by utilizing the material decomposition technique. Photon-processing spectral CT images correlated with histological sections in healthy and osteoarthritic tissues. Post-imaging material decomposition was able to quantify the GAG content and distribution throughout healthy and osteoarthritic cartilage using Hexabrix® and MultiHance® while differentiating the underlying subchondral bone.