Distribution Of Glycosaminoglycans Research Articles

The bio in the ink: cartilage regeneration with bioprintable hydrogels and articular cartilage-derived progenitor cells.

Despite its limited ability to repair, articular cartilage harbors an endogenous population of progenitor cells (ACPCs), that to date, received limited attention in biomaterials and tissue engineering applications. Harnessing the potential of these cells in 3D hydrogels can open new avenues for biomaterial-based regenerative therapies, especially with advanced biofabrication technologies (e.g. bioprinting). This study highlights the potential of ACPCs to generate neo-cartilage in a gelatin-based hydrogel and bioink. The ACPC-laden hydrogel is a suitable substrate for chondrogenesis and data shows it has a bias in directing cells towards a superficial zone phenotype. For the first time, ACPC-hydrogels are evaluated both as alternative for and in combination with chondrocytes and MSCs, using co-cultures and bioprinting for cartilage regeneration in vitro. This study provides important cues on ACPCs, indicating they represent a promising cell source for the next generation of cartilage constructs with increased biomimicry.

Molecular structure of human aortic valve by µSR- FTIR microscopy

Abstract Aortic valve is a part of the heart most frequently affected by pathological processes in humans what constitute a very serious health problem. Therefore, studies of morphology and molecular microstructure of the AV are needed. µSR- FTIR spectroscopy and microscopy represent unique tools to study chemical composition of the tissue and to identify spectroscopic markers characteristic for structural and functional features. Normal AV reveals a multi-layered structure and the compositional and structural changes within particular layers may trigger degenerative processes within the valve. Thus, deep insight into the structure of the valve to understand pathological processes occurring in AV is needed. In order to identify differences between three layers of human AV, tissue sections of macroscopically normal AV were studied using µSR- FTIR spectroscopy in combination with histological and histochemical stainings. Tissue sections deposited onto CaF2 substrates were mapped and representative set of IR spectra collected from fibrosa, spongiosa and ventricularis were analysed by Principal Component Analysis (PCA) in the spectral range between 1850–1000 cm−1 and 3050–2750 cm−1. PCA revealed a layered molecular structure of the valve and it was possible to identify IR bands associated to different tissue parts. Spongiosa layer was well differentiated from other two layers mainly based on IR bands characteristic for the distribution of glycosaminoglycans (GAGs) in the tissue – like 1170 cm−1 (υas(C-O-S)) and 1380 cm−1 (acetyl amino group). Additionally, it was distinguished from fibrosa and ventricularis based on 1085 cm−1 and 1240 cm−1 bands characteristic for GAGs and for carbohydrates- ν(C-O) and ν(C-O-C) respectively and nucleic acids -νsym(PO2−) and νasym(PO2−) respectively, which were less specific for this layer. The use of µSR- FTIR spectroscopy demonstrated co-localization of GAGs and lipids in spongiosa layer what may indicate their contribution in the very early phase of aortic valve calcific degeneration.

Effects of a viscosupplementation therapy on rabbit menisci in an anterior cruciate ligament transection model of osteoarthritis

The aim of this study was to evaluate the morphological, microstructural, and mechanical effects of a viscosupplementation therapy on rabbit menisci at an early stage of osteoarthritis (OA). Anterior cruciate ligament transection (ACLT) was performed in twelve male New-Zealand White rabbits on the right knee joint. Six of these twelve rabbits received a mono intra-articular injection of high molecular weight hyaluronic acid (HA) two weeks after ACLT. Six additional healthy rabbits served as controls. Medial menisci were removed from all right knees (n=18) six weeks after ACLT and were graded macroscopically. Indentation-relaxation tests were performed in the anterior and posterior regions of the menisci. Collagen fiber organization and glycosaminoglycan (GAG) content were assessed by biphotonic confocal microscopy and histology, respectively. Viscosupplementation significantly (p=0.002) improved the surface integrity of the medial menisci compared to the operated non-treated group. Moreover, the injection seems to have an effect on the GAG distribution in the anterior region of the menisci. However, the viscoelastic properties of both operated groups were similar and significantly lower than those of the healthy group, which was explained by their modified collagen fiber organization. They displayed disruption of the tie fibers due to structural alterations of the superficial layers from which they emanate, leading to modifications in the deep zone. To conclude, the viscosupplementation therapy prevents macroscopic lesions of the menisci, but it fails to restore their collagen fiber organization and their viscoelastic properties. This finding supports the role of this treatment in improving the lubrication over the knee.

Influence of oxygen levels on chondrogenesis of porcine mesenchymal stem cells cultured in polycaprolactone scaffolds.

Chondrogenesis of mesenchymal stem cells (MSCs) is known to be regulated by a number of environmental factors, including local oxygen levels. The hypothesis of this study is that the response of MSCs to hypoxia is dependent on the physical and chemical characteristics of the substrate used. The objective of this study was to explore how different modifications to polycaprolactone (PCL) scaffolds influenced the response of MSCs to hypoxia. PCL, PCL-hyaluronic acid (HA), and PCL-Bioglass® (BG) scaffolds were seeded with MSCs derived from bone marrow and cultured for 35 days under normoxic or low oxygen conditions, and the resulting biochemical properties of the MSC laden construct were assessed. Low oxygen tension has a positive effect over cell proliferation and macromolecules biosynthesis. Furthermore, hypoxia enhanced the distribution of collagen and glycosaminoglycans (GAGs) deposition through the scaffold. On the other hand, MSCs displayed certain material dependent responses to hypoxia. Low oxygen tension had a positive effect on cell proliferation in BG and HA scaffolds, but only a positive effect on GAGs synthesis in PCL and HA scaffolds. In conclusion, hypoxia increased cell viability and expression of chondrogenic markers but the cell response was modulated by the type of scaffold used. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1684-1691, 2017.

Cellular and Matrix Response of the Mandibular Condylar Cartilage to Botulinum Toxin.

ObjectivesTo evaluate the cellular and matrix effects of botulinum toxin type A (Botox) on mandibular condylar cartilage (MCC) and subchondral bone.Materials and MethodsBotox (0.3 unit) was injected into the right masseter of 5-week-old transgenic mice (Col10a1-RFPcherry) at day 1. Left side masseter was used as intra-animal control. The following bone labels were intraperitoneally injected: calcein at day 7, alizarin red at day 14 and calcein at day 21. In addition, EdU was injected 48 and 24 hours before sacrifice. Mice were sacrificed 30 days after Botox injection. Experimental and control side mandibles were dissected and examined by x-ray imaging and micro-CT. Subsequently, MCC along with the subchondral bone was sectioned and stained with tartrate resistant acid phosphatase (TRAP), EdU, TUNEL, alkaline phosphatase, toluidine blue and safranin O. In addition, we performed immunohistochemistry for pSMAD and VEGF.ResultsBone volume fraction, tissue density and trabecular thickness were significantly decreased on the right side of the subchondral bone and mineralized cartilage (Botox was injected) when compared to the left side. There was no significant difference in the mandibular length and condylar head length; however, the condylar width was significantly decreased after Botox injection. Our histology showed decreased numbers of Col10a1 expressing cells, decreased cell proliferation and increased cell apoptosis in the subchondral bone and mandibular condylar cartilage, decreased TRAP activity and mineralization of Botox injected side cartilage and subchondral bone. Furthermore, we observed reduced proteoglycan and glycosaminoglycan distribution and decreased expression of pSMAD 1/5/8 and VEGF in the MCC of the Botox injected side in comparison to control side.ConclusionInjection of Botox in masseter muscle leads to decreased mineralization and matrix deposition, reduced chondrocyte proliferation and differentiation and increased cell apoptosis in the MCC and subchondral bone.

Biological function of unique sulfated glycosaminoglycans in primitive chordates.

Glycosaminoglycans with unique sulfation patterns have been identified in different species of ascidians (sea squirts), a group of marine invertebrates of the Phylum Chordata, sub-phylum Tunicata (or Urochordata). Oversulfated dermatan sulfate composed of [4-α-L-IdoA-(2-O-SO3)-1→3-β-D-GalNAc(4-OSO3)-1]n repeating disaccharide units is found in the extracellular matrix of several organs, where it seems to interact with collagen fibers. This dermatan sulfate co-localizes with a decorin-like protein, as indicated by immunohistochemical analysis. Low sulfated heparin/heparan sulfate-like glycans composed mainly of [4-α-L-IdoA-(2-OSO3)-1→4-α-D-GlcN(SO3)-1 (6-O-SO3)-1]n and [4-α-L-IdoA-(2-O-SO3)-1→4-α-D-GlcN(SO3)-1]n have also been described in ascidians. These heparin-like glycans occur in intracellular granules of oocyte assessory cells, named test cells, in circulating basophil-like cells in the hemolymph, and at the basement membrane of different ascidian organs. In this review, we present an overview of the structure, distribution, extracellular and intracellular localization of the sulfated glycosaminoglycans in different species and tissues of ascidians. Considering the phylogenetic position of the subphylum Tunicata in the phylum Chordata, a careful analysis of these data can reveal important information about how these glycans evolved from invertebrate to vertebrate animals.

Biochemical validity of imaging techniques (X-ray, MRI, and dGEMRIC) in degenerative disc disease of the human cervical spine—an in vivo study

Background ContextOn a molecular level, maturation or degeneration of human intervertebral disc is among others expressed by the content of glycosaminoglycans (GAGs). According to the degenerative status, the disc content can differ in nucleus pulposus (NP) and annulus fibrosus (AF), respectively. Research in this area was conducted mostly on postmortem samples. Although several radiological classification systems exist, none includes biochemical features. Therefore, we focused our in vivo study on a widely spread and less expensive imaging technique for the cervical spine and the correlation of radiological patterns to biochemical equivalents in the intervertebral discs. PurposeThe aim of this pilot study was to (1) measure the GAG content in human cervical discs, (2) to investigate whether a topographic biochemical GAG pattern can be found, and (3) whether there is a correlation between imaging data (X-ray and magnetic resonance imaging [MRI] including delayed gadolinium-enhanced MRI of cartilage [dGEMRIC] as a special imaging technique of cartilage) and the biochemical data. Study Design/SampleWe conducted a prospective experimental pilot study. Patient SampleOnly non-responders to conservative therapy were included. All subjects were physically and neurologically examined, and they completed their questionnaires. Outcome MeasuresVisual analogue scale neck and arm, Neck Disability Index score, radiological parameters (X-rays, MRI, dGEMRIC), and the content of GAG in the cervical disc were assessed. MethodsAfter surgical removal of 12 discs, 96 fractions of AF and NP were biochemically analyzed for the GAG content using dimethylmethylene blue assay. ResultsA quantitative pattern of GAGs in the human cervical disc was identified. There were (1) significantly (p<.001) higher values of GAGs (µg GAG/mg tissue) in the NP (169.9 SD 37.3) compared with the AF (132.4 SD 42.2), and (2) significantly (p<.005) higher values of GAGs in the posterior (right/left: 149.9/160.2) compared with the anterior (right/left: 112.0/120.2) part of the AF. Third, we found in dGEMRIC imaging a significantly (p<.008) different distribution of GAGs in the cervical disc (NP 1083.3 ms [SD 248.6], AF 925.9 ms [SD 137.6]). Finally, we found that grading of disc degeneration in X-ray and MRI was significantly correlated with neither AF- nor NP-GAG content. ConclusionsThe GAG content in human cervical discs can be detected in vivo and is subject to a significantly (p<.05) region-specific pattern (AF vs. NP; anterior vs. posterior in the AF). Up to the levels of AF and NP, this is reproducible in MRI in dGEMRIC technique, but not in X-ray or standard MRI sequences. Potentially, the MRI in dGEMRIC technique can be used as a non-invasive in vivo indicator for disc degeneration in the cervical spine.

Sulfated glycosaminoglycans in human vocal fold lamina propria

IntroductionThe distribution, concentration and function of glycosaminoglycans in the various vocal fold tissues are still unclear. ObjectiveTo evaluate the distribution and concentration of sulfated glycosaminoglycans in different layers of the human vocal fold according to gender and age. MethodsWe used 11 vocal folds obtained from cadavers (7 men and 4 women) with no laryngeal lesion, less than 12h after death, and aged between 35 and 98 years. The folds underwent glycosaminoglycans extraction from the cover and ligament, and post-electrophoresis analysis. Data were compared according to the layer, age and gender. ResultsThe concentration of dermatan sulfate was significantly higher in all layers. No differences were observed in the total concentrations of glycosaminoglycans in layers studied according to gender. It is significantly lower in the cover of individuals aged below 60 years. ConclusionDermatan sulfate, chondroitin sulfate, and heparan sulfate were observed in the human vocal folds cover and ligament of both genders, with the concentration of dermatan sulfate being significantly higher in all layers. Glycosaminoglycans concentration on the cover is significantly lower in individuals below 60 years compared with elderly.

Quantitative imaging of excised osteoarthritic cartilage using spectral CT

To quantify iodine uptake in articular cartilage as a marker of glycosaminoglycan (GAG) content using multi-energy spectral CT. We incubated a 25-mm strip of excised osteoarthritic human tibial plateau in 50% ionic iodine contrast and imaged it using a small-animal spectral scanner with a cadmium telluride photon-processing detector to quantify the iodine through the thickness of the articular cartilage. We imaged both spectroscopic phantoms and osteoarthritic tibial plateau samples. The iodine distribution as an inverse marker of GAG content was presented in the form of 2D and 3D images after applying a basis material decomposition technique to separate iodine in cartilage from bone. We compared this result with a histological section stained for GAG. The iodine in cartilage could be distinguished from subchondral bone and quantified using multi-energy CT. The articular cartilage showed variation in iodine concentration throughout its thickness which appeared to be inversely related to GAG distribution observed in histological sections. Multi-energy CT can quantify ionic iodine contrast (as a marker of GAG content) within articular cartilage and distinguish it from bone by exploiting the energy-specific attenuation profiles of the associated materials. • Contrast-enhanced articular cartilage and subchondral bone can be distinguished using multi-energy CT. • Iodine as a marker of glycosaminoglycan content is quantifiable with multi-energy CT. • Multi-energy CT could track alterations in GAG content occurring in osteoarthritis.

Altered interaction and distribution of glycosaminoglycans and growth factors in mucopolysaccharidosis type I bone disease

The mucopolysaccharidoses (MPSs) comprise a group of lysosomal storage disorders characterized by deficient degradation and subsequent accumulation of glycosaminoglycans (GAGs). Progressive bone and joint disease are a major cause of morbidity, and current therapeutic strategies have limited effect on these symptoms. By elucidating pathophysiological mechanisms underlying bone disease, new therapeutic targets may be identified. Longitudinal growth is regulated by interaction between GAGs and growth factors. Because GAGs accumulate in the MPSs, we hypothesized that altered interaction between growth factors and GAGs contribute to the pathogenesis of MPS bone disease. In this study, binding between GAGs from MPS I chondrocytes and fibroblast growth factor 2 (FGF2) was not significantly different from binding of FGF2 to GAGs from control chondrocytes. FGF2 signaling, however, was increased in MPS I chondrocytes after incubation with FGF2, as compared to control chondrocytes. Using bone cultures, we demonstrated decreased growth of WT mouse bones after incubation with FGF2, but no effect on MPS I bone growth. However, MPS I bones showed decreased growth in the presence of GAGs from MPS I chondrocytes. Finally, we demonstrate altered GAG distribution in MPS I chondrocytes, and altered GAG, FGF2 and Indian hedgehog distribution in growth plates from MPS I mice. In summary, our results suggest that altered interaction and distribution of growth factors and accumulated GAGs may contribute to the pathogenesis of MPS bone disease. In the future, targeting growth factor regulation or the interaction between in growth factors and GAGs might be a promising therapeutic strategy for MPS bone disease.

Sulphated glycosaminoglycans and proteoglycans in the developing vertebral column of juvenile Atlantic salmon (Salmo salar).

In the present study, the distribution of sulphated glycosaminoglycans (GAGs) in the developing vertebral column of Atlantic salmon (Salmo salar) at 700, 900, 1100 and 1400 d° was examined by light microscopy. The mineralization pattern was outlined by Alizarin red S and soft structures by Alcian blue. The temporal and spatial distribution patterns of different types of GAGs: chondroitin-4-sulphate/dermatan sulphate, chondroitin-6-sulphate, chondroitin-0-sulphate and keratan sulphate were addressed by immunohistochemistry using monoclonal antibodies against the different GAGs. The specific pattern obtained with the different antibodies suggests a unique role of the different GAG types in pattern formation and mineralization. In addition, the distribution of the different GAG types in normal and malformed vertebral columns from 15 g salmon was compared. A changed expression pattern of GAGs was found in the malformed vertebrae, indicating the involvement of these molecules during the pathogenesis. The molecular size of proteoglycans (PGs) in the vertebrae carrying GAGs was analysed with western blotting, and mRNA transcription of the PGs aggrecan, decorin, biglycan, fibromodulin and lumican by real-time qPCR. Our study reveals the importance of GAGs in development of vertebral column also in Atlantic salmon and indicates that a more comprehensive approach is necessary to completely understand the processes involved.

Use of quantitative MRI for the detection of progressive cartilage degeneration in a mini-pig model of osteoarthritis caused by anterior cruciate ligament transection.

To investigate the progression of cartilage degeneration using delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) and T2 mapping in a mini-pig model of osteoarthritis (OA) caused by anterior cruciate ligament transection (ACLT). Twelve mini-pigs underwent ACLT in the left knee and were monitored by dGEMRIC (T1, gadolinium [Gd]) and T2 mapping after 2, 4, or 6 weeks (n = 4 each). No ACLT surgery was performed in four healthy control mini-pigs, which were also monitored by dGEMRIC and T2 mapping. Cartilage samples from the weight-bearing regions of the left medial femoral condyles were collected for macroscopic, histological, immunohistochemical, and biochemical analysis. Correlations between biochemical contents and T1,Gd and T2 values were evaluated using Pearson correlation analysis. T1,Gd values were gradually reduced and T2 values increased over time. Cartilage surfaces showed roughness at 4 weeks and additional defects at 6 weeks. Glycosaminoglycan (GAG) distribution and content gradually reduced over time (P < 0.05), and collagen distribution and anisotropy were obviously changed at 6 weeks. However, collagen content did not differ significantly among postoperative timepoints. GAG content was positively related to T1,Gd values (r = 0.888; P < 0.001) and negatively related to T2 values (r = -0.865; P < 0.001). Collagen content had no discernible correlation with T1,Gd or T2 values. dGEMRIC and T2 mapping can monitor the progression of cartilage degeneration in a mini-pig model of ACLT-induced OA, permitting early detection of OA.

A superficial hyperechoic band in human articular cartilage on ultrasonography with histological correlation: preliminary observations.

Purpose:To demonstrate the superficial hyperechoic band (SHEB) in articular cartilage by using ultrasonography (US) and to assess its correlation with histological images.Methods:In total, 47 regions of interest (ROIs) were analyzed from six tibial osteochondral specimens (OCSs) that were obtained after total knee arthroplasty. Ultrasonograms were obtained for each OCS. Then, matching histological sections from all specimens were obtained for comparison with the ultrasonograms. Two types of histological staining were used: Safranin-O stain (SO) to identify glycosaminoglycans (GAG) and Masson’s trichrome stain (MT) to identify collagen. In step 1, two observers evaluated whether there was an SHEB in each ROI. In step 2, the two observers evaluated which histological staining method correlated better with the SHEB by using the ImageJ software.Results:In step 1 of the analysis, 20 out of 47 ROIs showed an SHEB (42.6%, kappa=0.579). Step 2 showed that the SHEB correlated significantly better with the topographical variation in stainability in SO staining, indicating the GAG distribution, than with MT staining, indicating the collagen distribution (P<0.05, kappa=0.722).Conclusion:The SHEB that is frequently seen in human articular cartilage on high-resolution US correlated better with variations in SO staining than with variations in MT staining. Thus, we suggest that a SHEB is predominantly related to changes in GAG. Identifying an SHEB by US is a promising method for assessing the thickness of articular cartilage or for monitoring early osteoarthritis.

Glucose gradients influence zonal matrix deposition in 3D cartilage constructs.

Reproducing the native collagen structure and glycosaminoglycan (GAG) distribution in tissue-engineered cartilage constructs is still a challenge. Articular cartilage has a specific nutrient supply and mechanical environment due to its location and function in the body. Efforts to simulate this native environment have been reported through the use of bioreactor systems. However, few of these devices take into account the existence of gradients over cartilage as a consequence of the nutrient supply by diffusion. We hypothesized that culturing chondrocytes in an environment, in which gradients of nutrients can be mimicked, would induce zonal differentiation. Indeed, we show that glucose gradients facilitating a concentration distribution as low as physiological glucose levels enhanced a zonal chondrogenic capacity similar to the one found in native cartilage. Furthermore, we found that the glucose consumption rates of cultured chondrocytes were higher under physiological glucose concentrations and that GAG production rates were highest in 5 mM glucose. From these findings, we concluded that this condition is better suited for matrix deposition compared to 20 mM glucose standard used in a chondrocyte culture system. Reconsidering the culture conditions in cartilage tissue engineering strategies can lead to cartilaginous constructs that have better mechanical and structural properties, thus holding the potential of further enhancing integration with the host tissue.

Osteochondral repair by a novel interconnecting collagen-hydroxyapatite substitute: a large-animal study.

A novel three-dimensional bicomponent substitute made of collagen type I and hydroxyapatite was tested for the repair of osteochondral lesions in a swine model. This scaffold was assembled by a newly developed method that guarantees the strict integration between the organic and the inorganic parts, mimicking the biological tissue between the chondral and the osseous phase. Thirty-six osteochondral lesions were created in the trochlea of six pigs; in each pig, two lesions were treated with scaffolds seeded with autologous chondrocytes (cell+group), two lesions were treated with unseeded scaffolds (cell- group), and the two remaining lesions were left untreated (untreated group). After 3 months, the animals were sacrificed and the newly formed tissue was analyzed to evaluate the degree of maturation. The International Cartilage Repair Society (ICRS) macroscopic assessment showed significantly higher scores in the cell- and untreated groups when compared with the cell+ group. Histological evaluation showed the presence of repaired tissue, with fibroblast-like and hyaline-like areas in all groups; however, with respect to the other groups, the cell- group showed significantly higher values in the ICRS II histological scores for "cell morphology" and for the "surface/superficial assessment." While the scaffold seeded with autologous chondrocytes promoted the formation of a reparative tissue with high cellularity but low glycosaminoglycans (GAG) production, on the contrary, the reparative tissue observed with the unseeded scaffold presented lower cellularity but higher and uniform GAG distribution. Finally, in the lesions treated with scaffolds, the immunohistochemical analysis showed the presence of collagen type II in the peripheral part of the defect, indicating tissue maturation due to the migration of local cells from the surroundings. This study showed that the novel osteochondral scaffold was easy to handle for surgical implantation and was stable within the site of lesion; at the end of the experimental time, all implants were well integrated with the surrounding tissue and no signs of synovitis were observed. The quality of the reparative tissue seemed to be superior for the lesions treated with the unseeded scaffolds, indicating the promising potential of this novel biomaterial for use in a one-stage procedure for osteochondral repair.

Structure of collagen-glycosaminoglycan matrix and the influence to its integrity and stability.

Glycosaminoglycan (GAG) is a chain-like disaccharide that is linked to polypeptide core to connect two collagen fibrils/fibers and provide the intermolecular force in Collagen-GAG matrix (C-G matrix). Thus, the distribution of GAG in C-G matrix contributes to the integrity and mechanical properties of the matrix and related tissue. This paper analyzes the transverse isotropic distribution of GAG in C-G matrix. The angle of GAGs related to collagen fibrils is used as parameters to qualify the GAGs isotropic characteristic in both 3D and 2D rendering. Statistical results included that over one third of GAGs were perpendicular directed to collagen fibril with symmetrical distribution for both 3D matrix and 2D plane cross through collagen fibrils. The three factors tested in this paper: collagen radius, collagen distribution, and GAGs density, were not statistically significant for the strength of Collagen-GAG matrix in 3D rendering. However in 2D rendering, a significant factor found was the radius of collagen in matrix for the GAGs directed to orthogonal plane of Collagen-GAG matrix. Between two cross-section selected from Collagen-GAG matrix model, the plane cross through collagen fibrils was symmetrically distributed but the total percentage of perpendicular directed GAG was deducted by decreasing collagen radius. There were some symmetry features of GAGs angle distribution in selected 2D plane that passed through space between collagen fibrils, but most models showed multiple peaks in GAGs angle distribution. With less GAGs directed to perpendicular of collagen fibril, strength in collagen cross-section weakened. Collagen distribution was also a factor that influences GAGs angle distribution in 2D rendering. True hexagonal collagen packaging is reported in this paper to have less strength at collagen cross-section compared to quasi-hexagonal collagen arrangement. In this work focus is on GAGs matrix within the collagen and its relevance to anisotropy.

A comparison of glycosaminoglycan distributions, keratan sulphate sulphation patterns and collagen fibril architecture from central to peripheral regions of the bovine cornea

This study investigated changes in collagen fibril architecture and the sulphation status of keratan sulphate (KS) glycosaminoglycan (GAG) epitopes from central to peripheral corneal regions. Freshly excised adult bovine corneal tissue was examined as a function of radial position from the centre of the cornea outwards. Corneal thickness, tissue hydration, hydroxyproline content, and the total amount of sulphated GAG were all measured. High and low-sulphated epitopes of keratan sulphate were studied by immunohistochemistry and quantified by ELISA. Chondroitin sulphate (CS) and dermatan sulphate (DS) distributions were observed by immunohistochemistry following specific enzyme digestions. Electron microscopy and X-ray fibre diffraction were used to ascertain collagen fibril architecture. The bovine cornea was 1021±5.42μm thick at its outer periphery, defined as 9–12mm from the corneal centre, compared to 844±8.10μm at the centre. The outer periphery of the cornea was marginally, but not significantly, more hydrated than the centre (H=4.3 vs. H=3.7), and was more abundant in hydroxyproline (0.12 vs. 0.06mg/mg dry weight of cornea). DMMB assays indicated no change in the total amount of sulphated GAG across the cornea. Immunohistochemistry revealed the presence of both high- and low-sulphated epitopes of KS, as well as DS, throughout the cornea, and CS only in the peripheral cornea before the limbus. Quantification by ELISA, disclosed that although both high- and low-sulphated KS remained constant throughout stromal depth at different radial positions, high-sulphated epitopes remained constant from the corneal centre to outer-periphery, whereas low-sulphated epitopes increased significantly. Both small angle X-ray diffraction and TEM analysis revealed that collagen fibril diameter remained relatively constant until the outer periphery was reached, after which fibrils became more widely spaced (from small angle x-ray diffraction analysis) and of larger diameter as they approached the sclera. Depth-profiled synchrotron microbeam analyses showed that, at different radial positions from the corneal centre outwards, fibril diameter was greater superficially than in deeper stromal regions. The interfibrillar spacing was also higher at mid-depth in the stroma than it was in anterior and posterior stromal regions. Collagen fibrils in the bovine cornea exhibited a fairly consistent spacing and diameter from the corneal centre to the 12mm radial position, after which a significant increase was seen. While the constancy of the overall sulphation levels of proteoglycans in the cornea may correlate with the fibrillar architecture, there was no correlation between the latter and the distribution of low-sulphated KS.

GLYCOSAMINOGLYCAN CHANGES IN REGENERATIVE MENISCI OF THE IMMATURE RABBITS — HISTOLOGICAL AND SPECTROPHOTOMETRIC QUANTITATION

Purpose: This study was conducted primarily to compare the contents and distribution of Glycosaminoglycan (GAG) in the normal and regenerated menisci, and partly to elucidate the role of GAG in the meniscus to fulfill its function. Method: A total of 60 immature rabbits, weighing 300–400 gm and aged 4 weeks, were used for the study. Total medial meniscectomy in the right knee joint was performed in all rabbits. The medial menisci of the left knee joints were used as control. After 16, 20, 24 and 28 weeks postoperatively, the amount of GAG in normal and regenerated menisci were measured. For the measurement of quantity and distribution of GAG contents, photon absorptiometry and Safranin O staining method were used. Results: (1) In 48 of the 60 medial meniscectomized knees, menisci were regenerated in various sizes with degenerative changes of joints, especially on tibial side. (2) In normal menisci, GAG was distributed at inner and middle 1/3, while GAG in regenerated menisci was diffusely found in the entire width. (3) The concentration of GAG per 1 mg of regenerated menisci were 4.36 ± 1.68, 4.58 ± 1.14, 4.65 ± 2.21, 4.60 ± 2.06 μg in each 16, 20, 24, and 28 week groups, while those of normal menisci were 9.10 ± 2.09, 9.75 ± 3.97, 11.80 ± 8.44 and 14.51 ± 8.91 μg. (4) In normal menisci, the amount of GAG was gradually increased as rabbit got older. However, there was no statistical differences of quantitative amount of GAG of regenerated menisci among the 16, 20, 24, and 28 weeks groups during the observation periods (p &gt; 0.05). (5) There was a distinctive difference in Safranin O stainability by every 4 μg increase of GAG in both 1 mg of normal and regenerated menisci. Conclusion: This study showed the quantitative change of GAG between normal and regenerated menisci, and comparative analysis of GAG contents in the menisci by biochemical method and histological method were found to be useful tools.

Collagen and Glycosaminoglycan Profiles in the Canine Cervix during Different Stages of the Estrous Cycle and in Open- and Closed-Cervix Pyometra

ABSTRACTThe extracellular matrix of the cervix that comprises collagen, elastin, proteoglycans and glycosaminoglycans (GAGs) is thought to have an essential role in cervical relaxation. This study investigated the proportion of collagen and smooth muscle as well as the GAGs in cervices obtained from healthy bitches at different stages of the estrous cycle and bitches with open- and closed-cervix pyometra. Cervices were collected after ovariohysterectomy. The proportion of collagen to smooth muscle was determined using Masson’s trichrome staining. Alcian blue staining was used to evaluate the relative distribution of cervical GAGs. The proportion of cervical collagen relative to smooth muscle was higher at estrus compared to anestrus (P≤0.05). It was also higher (P≤0.05) in bitches with open- compared to those with closed-cervix pyometra. Overall, hyaluronan (HA) was the predominant GAG in the canine cervix. In the luminal epithelium, the staining intensity for HA was stronger in estrus than in anestrus (P≤0.05), but not in diestrus (P>0.05). On the contrary, the intensity for the combined keratan sulfate (KS) and heparan sulfate (HS) was stronger in anestrus than in estrus and diestrus (P≤0.05). In bitches with pyometra, the staining intensity of the stroma for KS and HS was weaker in open- compared to closed-cervix pyometra (P≤0.05). Collectively, the different profiles of collagen and GAG suggest that the metabolism of both collagen and GAGs in the canine cervix is associated with hormonal statuses during the estrous cycle and cervical patency of bitches with pathological uterine conditions, such as pyometra.

Contrast enhanced CT attenuation correlates with the GAG content of bovine meniscus

We determined whether contrast-enhanced computed tomography (CECT) attenuation obtained using a µCT scanner correlated with the glycosaminoglycan (GAG) content and distribution in ex vivo bovine menisci. Bovine samples were immersed in different concentrations of the contrast agents CA4+ and Ioxaglate, and the µCT images were compared to Safranin-O staining. CA4+ and Ioxaglate diffusion-in kinetics and the correlation between their CECT attenuations and GAG content were investigated. CA4+ and Ioxaglate both reached steady state in the meniscal regions within 95 h, with tau values of 20.6 ± 3.98 and 25.9 ± 3.71 h (mean ± SD), respectively. Both agents diffused preferentially through the proximal and secondarily through the distal surface. The CA4+ CECT attenuation was strongly and positively correlated with the GAG content of the meniscus regions (R(2) = 0.89, p < 0.001) at low concentrations (12 mgI/ml), while the Ioxaglate CECT attenuation was moderately and negatively correlated with the GAG content (R(2) = 0.51, p = 0.03) at 60 mgI/ml. CECT can image ex vivo menisci, and the CA4+, compared to Ioxaglate, enhanced attenuation strongly correlates with the GAG content and distribution in bovine meniscus.