Age-related changes in human meniscal glycosaminoglycans

Introduction With an increased human lifespan, a major challenge is now to ensure a concomitant increase in healthspan. Meniscal damage and degradation are common and are strongly correlated with subsequent osteoarthritis. Indeed, meniscal damage has been identified in about 60% of people over 60. Markers of pathology will facilitate intervention but first require normal age‐related changes to be established. Methods Undamaged vascular and avascular regions of medial and lateral human menisci were comminuted and the tissue extracted into 4‐m GuHCl and subject to associative CsCl density gradient centrifugation. Aggrecan and the small leucine rich PGs (SLRPs) were isolated and their GAG profiles examined by HPAEC fingerprinting, following enzyme depolymerization, and by an NMR spectroscopy. Results and discussion Analysis of aggrecan and the SLRPs show that there is a complex and dynamic pattern of KS, CS and DS abundance and distribution within human menisci, which changes with age. The abundance of SLRPs is higher in the avascular than vascular tissues, however, this is not reflected in the abundance of aggrecan which is present at similar levels in both tissue regions. The data show no other significant differences between medial and lateral and between vascular and avascular tissue regions. Analysis of the sulfation pattern of CS following digestion by ACII lyase, shows that in both aggrecan and SLRPs the 4‐sulfation level falls with age from 20 to 35% in young tissues to 10–20% in older. Subsequent analyses following ABC lyase depolymerization, to include DS, shows very significant change with age from CS + DS 4‐sulfation levels of ca. 40–55% in young tissue to ca. 15–30% in older. The difference between these datasets represents the contribution made by 4‐sulfated DS. Thus, analysis of the difference suggests that DS makes a decreasing contribution to the CS/DS profile with age. Indeed, this is confirmed by an NMR analysis of these samples. Analysis of the resonances in the region 1.95–2.2 p.p.m. (ref to TSP) allows the estimation of the contribution made by DS, CS and KS. These data show that, in aggrecan, the contribution made by DS chains falls from ca. 10% in younger tissues to ca. 2–4% in older tissues. NMR analysis also shows that KS levels fall with age from ca. 15–20% in younger tissues to 5–10% in older tissues. Analysis of the structure of the KS chains shows chains with a structure similar to that of in articular cartilage but that at all ages there are very low levels of fucosylation (ca. 1–5%). Previous studies of age‐related changes in CS/DS and KS structures have shown significant changes in the first 17 years of life, with only modest nonpathological changes after that time. These data from meniscal tissues do not show such a dramatic halting of normal age‐related changes. Indeed, the data show gradual age‐related changes in DS, CS and KS abundance and structure throughout life. These baseline age‐related changes data will now allow the analysis of pathology‐related changes.     

Indentation properties and glycosaminoglycan content of human menisci in the deep zone

Menisci are two crescent shaped fibrocartilaginous structures that provide fundamental load distribution and support within the knee joint. Their unique shape transmits axial stresses (i.e. “body force”) into hoop or radial stresses. The menisci are primarily an inhomogeneous aggregate of glycosaminoglycans (GAGs) supporting bulk compression and type I collagen fibrils sustaining tension. It has been shown that the superficial meniscal layers are functionally homogeneous throughout the three distinct regions (anterior, central and posterior) using a 300 μm diameter spherical indenter tip, but the deep zone of the meniscus has yet to be mechanically characterized at this scale. Furthermore, the distribution and content of GAG throughout the human meniscal cross-section have not been examined. This study investigated the mechanical properties, via indentation, of the human deep zone meniscus among three regions of the lateral and medial menisci. The distribution of GAGs through the cross-section was also documented. Results for the deep zone of the meniscus showed the medial posterior region to have a significantly greater instantaneous elastic modulus than the central region. No significant differences in the equilibrium modulus were seen when comparing regions or the hemijoint. Histological results revealed that GAGs are not present until at least ～600 μm from the meniscal surface. Understanding the role and distribution of GAG within the human meniscus in conjunction with the material properties of the meniscus will aid in the design of tissue engineered meniscal replacements.     

Identification of glycosaminoglycans in human airway secretions

Glycosaminoglycans (GAGs), known to be present in airway mucus, are macromolecules with a variety of structural and biological functions. In the present work, we used fluorophore-assisted carbohydrate electrophoresis (FACE) to identify and relatively quantify GAGs in human tracheal aspirates (HTA) obtained from healthy volunteers. Primary cultures of normal human bronchial epithelial (NHBE) and submucosal gland (SMG) cells were used to assess their differential contribution to GAGs in mucus. Distribution was further assessed by immunofluorescence in human trachea tissue sections and in cell cultures. HTA samples contained keratan sulfate (KS), chondroitin/dermatan sulfate (CS/DS), and hyaluronan (HA), whereas heparan sulfate (HS) was not detected. SMG cultures secreted CS/DS and HA, CS/DS being the most abundant GAGs in these cultures. NHBE cells synthesized KS, HA, and CS/DS. Confocal microscopy showed that KS was exclusively found at the apical border of NHBE cells and on the apical surface of ciliated epithelial cells in tracheal tissues. CS/DS and HA were present in both NHBE and SMG cells. HS was only found in the extracellular matrix in trachea tissue sections. In summary, HTA samples contain KS, CS/DS, and HA, mirroring a mixture of secretions originated in surface epithelial cells and SMGs. We conclude that surface epithelium is responsible for most HA and all KS present in secretions, whereas glands secrete most of CS/DS. These data suggest that, in diseases where the contribution to secretions of glands versus epithelial cells is altered, the relative concentration of individual GAGs, and therefore their biological activities, will also be affected.     

A method for fluorescent HPLC analysis of CS/DS glycosaminoglycans

Introduction Chondroitin and dermatan sulfate (CS/DS) show considerable species, tissue, age and pathology‐related structural heterogeneity. In addition within chains their sulfation patterns are not random. To elucidate their structure/function relationships, methods for complete characterization are required. A method for keratan sulfate (KS) fingerprinting ( Whitham et al. 1999 ) has been extended for the linkage, repeat and chain cap regions of CS/DS, including the acquisition of CS/DS ratios. Methods Chains were depolymerized by 1 U/100 mg of chondroitin ABC endolyase or ACII lyase at 50 mg/ml in 0.1 m ammonium acetate, pH 8 and for 15 h at 37 °C. Alternatively, chains were de‐N‐acetylated by hydrazinolysis at 98 °C for 24 h at 10 mg/1 ml in anhydrous hydrazine with 100 mg/ml hydrazine sulfate. Then, they were depolymerized by 3.9 m sodium nitrite/0.28 m acetic acid at 0 °C for 4 h. Unreduced chains were released from their protein core in 0.5 m LiOH at 4 °C for 12 h. Materials were fluorescently labelled with 2‐AA as previously described (Whitham et al. 1999) and characterized by HPAEC using a Dionex AS4‐SC column at 50 °C and 2 ml/min with constant 15% 1 m NaOH. A 5‐min isocratic period of 85% H₂O/0% 2 m NaCl was followed by a linear gradient of 0–30% 2 m NaCl over 60 min. The oligosaccharides were monitored using a λ_ex of 315 nm and a λ_em of 400 nm. Results and discussion This method resolves repeat region di‐, tri‐ and tetrasaccharides, capping oligosaccharides and linkage regions and can be used to profile known and unknown oligosaccharides. Unsulfated oligosaccharides elute between 2 and 10 min, monosulfated between 7 and 30 min, disulfated between 25 and 40 min and trisulfated between 49 and 54 min. Allied with data on size, oligosaccharide identification is facilitated. Hydrazinolysis/nitrous acid depolymerization of CS/DS chains results in disaccharides from CS with 4‐ or 6‐sulfation and from DS with 4‐sulfation which retain IdoA and GlcA structures and which can be distinguished chromatographically. The methodology was used to examine CS/DS from shark, whale, bovine and human tracheal, articular and meniscal cartilage and cornea. Tracheal cartilages show predominantly 4‐sulfation with porcine sources being more highly 4‐sulfated (ca. 75%) than bovine (ca. 65%). Articular cartilage comprises mainly 6‐sulfated GalNAc (ca. 95% in the adult), while adult meniscal cartilage shows only ca. 85%. Tracheal and articular cartilage aggrecan showed no IdoA; however, it represented ca. 20% of the uronic acids of bovine meniscal aggrecan, showing the presence of DS. Corneal CS/DS has a very low level of 6‐sulfation (<ca. 5%) but shows an equal abundance of unsulfated and 4‐sulfated residues and contains high levels, ca. 50%, of IdoA residues. Shark cartilage shows ca. 75% 6‐sulfation with significant levels of uronic acid 2‐sulfation found only between a 4‐sulfated residue and a 6‐sulfated residue, reflecting sulfotransferase specificity. Shark cartilage contains modest (ca. 1–5%) levels of DS that may be contaminants of preliminary isolation. This method extends a previous method to now allow the complete examination of KS, CS and DS chains by a single rapid chromatographic method.     

A novel keratanase-generated keratan sulphate antibody and its applications

Introduction The sequencing of the genome has provided us with important information regarding the primary structure of many matrix proteins. This in turn has lead to advances in studies of the functions of post‐translational modifications on connective tissue proteoglycans (PGs). Changes in GAG structure with ageing and disease have been well documented ( Thonar et al. 1986 ; Brown et al. 1998 ). However, little is known about the exact sites of and differential substitution of GAGs on the aggrecan core protein and how these substitutions facilitate normal function or the changes seen with disease. The CS : KS ratio of substitution change significantly, with KS levels increasing with age and decreasing with the onset of disease. Objective was to produce monoclonal antibody (MAb) reagents to keratanase (k'ase) generated stub epitopes, that could be used to help identify, characterize and quantify sites of KS substitution on PGs, providing the potential to determine how the arrangement of such substitutions change with development, ageing and pathology. Methods Bovine Nasal Cartilage aggrecan (BNC A1D1) was trypsin digested, generating a range of glycosaminoglycan (GAG) fragments. The sample was then subjected to anion‐exchange and size exclusion chromatography to separate KS from CS fragments. Fractions collected were analysed by SDS‐PAGE and Western blotting. Fractions positive for KS were pooled and kinase digested to expose the KS stub antigens. Immunization and fusions were carried out as previously described ( Nieduszynski et al. 1990 ). Initial screenings were carried out using ELISA. Briefly, 96‐well microtitre plates were coated with the immunizing antigen overnight at 37 °C. The plates were then blocked prior to the addition of hybridoma media for 1–2 h at 37 °C. Binding was detected using an alkaline phosphatase‐conjugated secondary antibody for 1 h at 37 °C prior to the addition of the substrate. Positive wells were further screened by ELISA and SDS‐PAGE using the immunizing antigen, chondroitinase‐digested BNC and an A1D1 BNC preparation to establish the kinase stub specificity of the hybridomas. Further screenings by Western blotting was carried out on positive hybridomas selected. Antigens used included keratanase‐digested bovine corneal KS‐PGs, keratanase‐II‐digested KS‐PGs and a nonkeratanase‐digested corneal KS‐PG sample. Results Screening: Screening identified two positive hybridomas, B‐KS‐I and B‐KS‐II, which were specific for kinase‐generated KS stub. On screening, these antigens showed reactivity specifically for kinase‐digested BNC abc core, with no reactivity to the nonkinased linear KS GAG epitopes. Reactivity to kinase‐digested corneal KS‐PGs indicated that the MAbs generated were indeed to a stub structure in the KS chain and not to some linkage region epitope, amino acid sequence or oligosaccharide present on the core protein. Application: Immunohistochemistry utilizing B‐KS‐I was used to localize KS in a range of tissues along side anti‐KS 5D4. In human articular cartilage engineered grafts, labelling showed B‐KS‐I and 5D4 to have broadly overlapping labelling patterns for KS; however, label for B‐KS‐I had a much more restricted and subtle tissue distribution than that of antibody 5D4. Discussion These new KS stub MAbs have potential to be used in many different areas of research. They may be used in analysis of trypsin‐digested purified aggrecan from cattle joints of different ages to determine sites of KS substitution, which remain common or change with development and ageing. They may also be used in analysis of cartilage explant culture metabolites to assess KS substitution on the aggrecan fragments generated after stimulation of these cultures with cytokines such as IL‐1 or TNF‐α. Collectively it will provide important new information on the changing pattern of KS substitution in connective tissue PGs with development, ageing and the onset of pathology.     

Allergen-induced airway remodeling in brown norway rats: structural and metabolic changes in glycosaminoglycans

Increased proteoglycan (PG) deposition is a feature of airway remodeling in asthma. Glycosaminoglycans (GAGs) mediate many of the biological and mechanical properties of PGs by providing docking sites through their carbohydrate chains to bioactive ligands; therefore, it is imperative to define structural and metabolic changes of GAGs in asthma. Using a Brown Norway (BN) ovalbumin (OVA)-sensitized and -challenged rat model to induce airway remodeling, we found excessive deposition of chondroitin/dermatan (CS/DS)-, heparan (HS), and keratan (KS) sulfate GAGs in the airways and bronchoalveolar lavage cells of OVA-challenged rats. Disaccharide composition of CS/DS of OVA-challenged rats was significantly different compared with saline-treated (SAL) control rats, with increased levels of 0-, 6-, and 4-sulfated disaccharides. Increases in the amount and a change in the proportion of CS/DS versus HS GAGs were noted in OVA-challenged rats. The higher content and sulfation of CS/DS disaccharides was reflected by the increased expression of xylosyltransferase-I, β1,3-glucuronosyltransferase-I, chondroitin-4, and chondroitin-6 sulfotransferase genes and protein expression of xylosyltransferase-I and β1,3-glucuronosyltransferase-I in OVA-challenged rats. Genes encoding the core proteins of the CS/DS and KS-containing PGs, such as versican, biglycan, decorin, and lumican, were overexpressed in OVA-challenged rats. Our results suggest that GAG biosynthetic enzymes may be involved in the altered expression of GAGs in the airways and are potential targets for inhibiting excess PG-GAG deposition and the airway remodeling process in asthma.     

Regional and Zonal Histo‐Morphological Characteristics of the Lapine Menisci

The menisci have crucial weight‐bearing roles in the knee. Regional variations in structure and cellularity of the meniscus have only been minimally investigated. Therefore, the goal of this study was to illustrate the regional cell density, tissue area, and structure of healthy lapine menisci. Skeletally mature Flemish Giant rabbits were used for this study. Upon sacrifice, menisci were removed, fixed in formalin, and cryosectioned. Histological analysis was performed for the detection of sulfated glycosaminoglycans (GAG), collagen Types I and II, cellular density, and tissue area. ANOVA and paired t tests were used for testing of statistical significance. Glycosaminoglycan coverage of the medial meniscus significantly varied between regions, with the anterior region demonstrating significantly more GAG coverage than the posterior region. Inter‐ and intra‐meniscal comparisons revealed variations between zones, with trends that outer zones of the medial menisci had less GAG coverage. Collagen Types I and II had marked characteristics and varying degrees of coverage across regions. Tissue area varied between regions for both medial and lateral menisci. Cellular density was dependent on region in the lateral meniscus. This is the first study to illustrate regional and zonal variation in glycosaminoglycan coverage, size, and cellular density for healthy lapine meniscal tissue. This data provides baseline information for future investigations in meniscal injury models in rabbits. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

The role of cartilage glycosaminoglycan structure in gagCEST

Glycosaminoglycan (GAG) chemical exchange saturation transfer (gagCEST) is a potential method for cartilage quality assessment. The aim of this study was to investigate how the gagCEST effect depends on the types and molecular organization of GAG typically found in articular cartilage. gagCEST was performed on different concentrations of GAG in various forms: free chains of chondroitin sulfate (CS) of different types (‐A and ‐C) and GAG bound to protein in aggregated and nonaggregated aggrecan extracted from calf articular cartilage. The measured magnetization transfer ratio asymmetry (MTR_asym) was compared with known GAG concentrations or GAG concentrations determined through biochemical analysis. The gagCEST effect was assessed through the linear regression coefficient with 95% confidence interval of MTR_asym per GAG concentration. We observed a lower gagCEST effect in phantoms containing a mixture of CS‐A and CS‐C compared with phantoms containing mainly CS‐A. The difference in response corresponds well to the difference in CS‐A concentration. GAG bound in aggrecan from calf articular cartilage, where CS‐A is assumed to be the major type of GAG, produed a similar gagCEST effect as that observed for free CS‐A. The effect was also similar for aggregated (ie, bound to hyaluronic acid) and nonaggregated aggrecan. In conclusion, our results indicate that the aggrecan structure in itself does not impact the gagCEST effect, but that the effect is strongly dependent on GAG type. In phantoms, the current implementation of gagCEST is sensitive to CS‐A while for CS‐C, the main GAG component in mature human articular cartilage, the sensitivity is limited. This difference in gagCEST sensitivity between GAG types detected in phantoms is a strong motivation to also explore the possibility of a similar effect in vivo.     

Two-dimensional NMR spectroscopy of urinary glycosaminoglycans from patients with different mucopolysaccharidoses

Patients with different types of mucopolysaccharidoses (MPS) lack specific lysosomal enzymes, which leads to tissue accumulation and urinary excretion of glycosaminoglycans (GAGs). Since little is known about the molecular composition of the excreted GAG fragments, we used two-dimensional [1H,13C]-correlation nuclear magnetic resonance (NMR) spectroscopy for a detailed analysis of the urinary GAGs of patients with MPS types I, II, IIIA, IVA and VI. The method revealed that the molecular structures of the excreted GAGs, i.e. heparan sulfate (HS), dermatan sulfate (DS), chondroitin sulfate (CS), and keratan sulfate (KS) are clearly distinct for the different MPS types. The chain terminal residues that are the normal substrates for the defective enzymes constitute characteristic sets of signals for each MPS type. The GAG chains show variations in carbohydrate composition and sulfation patterns that can be related to the different MPS types and clinical features. For example, two patients with MPS IIIA (M. Sanfilippo) with signs of CNS degeneration but only mild somatic features excrete a highly sulfated variant of HS, resembling HS in porcine brain, whereas a patient with MPS I (M. Scheie) and two patients with MPS II (M. Hunter), who present primarily with coarse facial features, joint contractures and skeletal deformities excrete a different type of HS with lower sulfation. In another case study, a patient with MPS IVA (M. Morquio), who presented mainly with skeletal dysplasia, excreted not only excessive amounts of KS but also a highly sulfated CS variant, resembling CS in articular cartilage. The high-resolution NMR analysis of urinary GAGs presented here for the first time provides a solid basis for future studies with a larger number of patients to further explore pathogenesis and course of the MPS diseases.     

A novel keratanase-generated keratan sulfate antibody and its application to structural analysis of skeletal and corneal keratan sulfate

Introduction The objective of this study was to make monoclonal antibodies specific for keratanase‐generated neoepitopes in keratan sulfate (KS) and to use them along with existing KS monoclonal antibodies (e.g. 5D4, IB4) to investigate KS sulfation pattern motifs in connective tissue proteoglycans during development, ageing and disease. Methods Bovine nasal cartilage aggrecan (BNC A1D1) was trypsin digested, generating a range of GAG‐peptide fragments. The sample was then subjected to anion‐exchange and size exclusion chromatography to separate KS peptides from CS attachment domain fragments. Fractions were analysed by Western blotting for positive immunoreactivity for KS, then pooled and keratanase digested to generate ‘KS stub’ antigens. Immunization and fusions were carried out as previously described ( Caterson et al. 1983 ; Hughes et al. 1992 ). Screenings involved the use of a range of antigens; including keratanase vs. keratanase II‐digested bovine cartilage aggrecan and bovine corneal KS‐PGs. A new monoclonal antibody, BKS‐I, was identified that specifically recognized a keratanase‐generated neoepitope on both skeletal and corneal KS. This novel monoclonal antibody was used along with existing KS monoclonal antibodies 5D4 and 1B4 to investigate KS structure. Results and discussion Bovine trypsin‐generated aggrecan KS‐peptides were chondroitinase ABC treated and either keratanase or keratanase II treated. The digests were run on SDS‐PAGE and immunolocated with monoclonal antibody 5D4 (that recognizes linear disulfated N‐acetyl lactosamine disaccharide‐containing segments in KS) and the new ‘KS‐stub’ monoclonal antibody BKS‐I. Our results indicated that there was reduced monoclonal antibody 5D4 immunostaining after keratanase pretreatment. However, keratanase II digestion completely removed all 5D4 structural epitopes. In contrast, BKS‐I showed no immunostaining on the untreated KS‐peptides but strong staining on keratanase treated samples and no staining after keratanase II digestion. Similar patterns of immunoreactivity were observed with Western blot analysis of untreated, keratanase treated and keratanase II treated corneal KS‐PGs. Conclusion These data indicate that monoclonal antibody BKS‐I recognizes a nonreducing terminal neoepitope‐containing sulfated N‐acetylglucosamine adjacent to a nonsulfated lactosamine disaccharide. We also conclude that skeletal KS must have a structure with four possible variations opposed to the generic structures, proposed as being made of disulfated disaccharides at the nonreducing end, followed by a series of monosulfated disaccharides at the middle and nonsulfated disaccharides nearer the linkage region. 5D4 staining, observed after keratanase digestion, indicates that there must be a minimum structure of a pentasulfated hexasaccharide remaining on the KS chain ‘stubs’ near the linkage region of skeletal and corneal KS. The BKS‐I monoclonal antibody can be used to demonstrate differential substitution of KS GAG chains in the CS attachment region of cartilage aggrecan with ageing. It has also proven useful for immunohistochemical analyses identifying the sites of KS–PG association with collagen lamellae of cornea.     

Genetic Heterogeneity and Clinical Variability in Musculocontractural Ehlers–Danlos Syndrome Caused by Impaired Dermatan Sulfate Biosynthesis

Bi‐allelic variants in CHST14, encoding dermatan 4‐O‐sulfotransferase‐1 (D4ST1), cause musculocontractural Ehlers–Danlos syndrome (MC‐EDS), a recessive disorder characterized by connective tissue fragility, craniofacial abnormalities, congenital contractures, and developmental anomalies. Recently, the identification of bi‐allelic variants in DSE, encoding dermatan sulfate epimerase‐1 (DS‐epi1), in a child with MC‐EDS features, suggested locus heterogeneity for this condition. DS‐epi1 and D4ST1 are crucial for biosynthesis of dermatan sulfate (DS) moieties in the hybrid chondroitin sulfate (CS)/DS glycosaminoglycans (GAGs). Here, we report four novel families with severe MC‐EDS caused by unique homozygous CHST14 variants and the second family with a homozygous DSE missense variant, presenting a somewhat milder MC‐EDS phenotype. The glycanation of the dermal DS proteoglycan decorin is impaired in fibroblasts from D4ST1‐ as well as DS‐epi1‐deficient patients. However, in D4ST1‐deficiency, the decorin GAG is completely replaced by CS, whereas in DS‐epi1‐deficiency, still some DS moieties are present. The multisystemic abnormalities observed in our patients support a tight spatiotemporal control of the balance between CS and DS, which is crucial for multiple processes including cell differentiation, organ development, cell migration, coagulation, and connective tissue integrity.     

Characterization of proteoglycans and glycosaminoglycans in splenic AA amyloid induced in mink

Amyloidosis of the protein AA type is readily induced in mink using repeated injections of bacterial lipopolysaccharide (LPS). We have characterized splenic proteoglycans/glycosaminoglycans (PGs/GAGs) in mink during amyloidogenesis. Moderate to rich amounts of amyloid exhibiting green birefringence was demonstrated by polarization microscopy of the splenic section stained with Congo red in seven out of eight minks after 10 weeks of LPS-treatment, and a significant increase in the total amount of PGs and GAGs in AA amyloid spleens was observed (two to eight times that in unstimulated animals). Intact PGs as well as free GAGs were extracted, and heparan sulfate (HS) was the most abundant GAG in the amyloid as well as in the control spleens. The GAGs showing the most pronounced increase in the amyloid spleens was of the chondroitin sulfate/dermatan sulfate (CS/DS) type and these were extracted in the form of free GAG chains. We conclude that there is a selective enrichment of PGs/GAGs in extracted splenic amyloid in the mink, which confirms to previous observations in human amyloid as well as in other animal species, supporting their pathogenic significance in the formation of AA amyloid.     

半月板组织工程化修复实验研究及其生物力学问题的讨论

目的 应用组织工程方法在具完全免疫功能的哺乳动物体内修复半月板缺损并讨论其生物力学问题。方法 15只45天龄的长枫杂交仔猪为实验动物，以改良的Klaflsbrun酶消化法从左膝半月板获得的自体纤维软骨细胞在体外扩增至一定数量，在右膝内侧副韧带前方的内侧半月板造成长1cm的全层缺损，分别将PGA-纤维软骨细胞-pluronic复合物、纤维软骨细胞-pluronic复合物和单纯PGA植入缺损，以正常半月板和旷置缺损作为对照，分别于第9周、16周、25周和36周取材。标本采用大体观察、组织学、生物化学和生物力学作为评估指标。结果 PGA-细胞-pluronic复合物在大体形态、组织学结构和压弹性模量(36周为正常组的69．5％)方面均显示形成了最佳的修复组织，并使对应股骨髁软骨的GAG含量(36周为正常组的58．3％)保持相对稳定。结论自体组织工程化纤维软骨在适宜的力学条件下能够修复乃至再造半月板，并且可以防止膝关节的创伤性退变。     

Composite chondroitin-6-sulfate/dermatan sulfate/chitosan scaffolds for cartilage tissue engineering

Conjugating a single glycosaminoglycan (GAG) species such as chondroitin-6-sulfate (CSC) to chitosan is beneficial to chondrocyte culture and extracellular matrix (ECM) production, but whether fabrication of 3D chitosan scaffolds with additional minor GAG species such as dermatan sulfate (DS) further improves the ECM production is unknown. In this study, Response Surface Methodology (RSM) was employed to design CSC/DS/chitosan scaffolds of various formulations for cartilage engineering and to investigate the roles of individual GAG species in cartilage formation. The CSC/DS formulation affected neither the physical properties of scaffolds nor cell adhesion, but influenced cell morphology, GAGs and collagen production and chondrocytic gene expression. The linear effects elucidated by RSM analysis suggested that within the level range higher CSC levels favored GAGs and collagen production, whereas lower DS levels were desired for these responses. Nonetheless, the quadratic effects of DS and two-way interactions between CSC and DS also contributed to the GAGs and collagen production. Accordingly, the optimal formulation, as predicted by RSM and validated by experiments, comprised 2.8 mg CSC and 0.01 mg DS per scaffold. This study confirmed the importance of DS in cartilage tissue engineering and implicated the feasibility of rational CSC/DS/chitosan scaffold design with the aid of RSM.     

Immunohistochemical and histochemical characterization of the mucosubstances of odontogenic myxoma: histogenesis and differential diagnosis.

To discuss the dental origin of odontogenic myxoma and to provide further information for the differential diagnosis between this tumor and myxoid malignant fibrous histiocytoma (MFH) which occasionally occurs in jaw bones, the contents of glycosaminoglycans (GAGs) and proteoglycans (PGs) in the mucosubstances of 15 odontogenic myxomas, 5 myxoid MFH and 3 human fetal tooth germs in the bell stage of development were characterized using histochemical and immunohistochemical methods. Histochemical staining of hyaluronic acid (HA) was undertaken using biotinylated HA binding protein (B-HABP), and immunohistochemical detection was done using a panel of antibodies against chondroitin 6-sulfate (CS-6), chondroitin 4-sulfate (CS-4), dermatan sulfate (DS), keratan sulfate (KS), heparan sulfate (HS), aggrecan, PG-M/versican, decorin and biglycan. In odontogenic myxoma, CS-6, HA and PG-M/versican were observed in the myxomatous matrix of all cases, while KS and HS were seen in none. As for CS-4, DS, aggrecan, decorin and biglycan, only irregular and mild stainings were shown. Consistent and strong positive straining for CS-6, HA and PG-M/versican were seen in dental papilla and provided evidence supporting the origin of this tumor from dental papilla. Except for the constant staining for HA, the myxoid matrix was rarely stained for most GAGs and PGs in myxoid MFH. Immunodetection of CS-6 and PG-M/version with the use of monoclonal antibodies 3-B-3 and 2-B-1 is therefore recommended as a useful tool in differentiating odontogenic myoma from myxoid MFH.     

Molecular recognition and modulation of hepatocyte growth factor activity by heparan and dermatan sulfates

Introduction Hepatocyte growth factor/scatter factor (HGF/SF) is an unusual growth factor in that it binds both heparan sulfate (HS) ( Lyon et al. 1994 ) and dermatan sulfate (DS) ( Lyon et al. 1998 ) glycosaminoglycans (GAGs) with similar high affinities. Both these GAGs act as co‐receptors for HGF/SF in the activation of the Met receptor ( Lyon et al. 2002 ). Our aim was to determine the sequences in HS and DS that specifically interact with and modulate HGF/SF activity. Materials and methods A structurally unique DS, which possesses O‐sulfation at carbon‐6 of the hexosamine residue (and not carbon‐4 as in mammalian DS), was obtained from the sea cucumber, Ascidia nigra. A variety of HS‐ and DS‐like structures were also generated using various chemical modification procedures (specific desulfations and carboxyl reductions). The ability of these various GAG species to compete with cell surface GAGs for HGF/SF binding was tested using radiolabelled HGF/SF and MDCK cells. The modified GAG structures and the A. nigra DS are currently being tested for their ability to act as co‐receptors for the interaction between HGF/SF and Met by studying cell signalling and cellular response assays, using the sulfated GAG‐deficient CHO‐745 cell line. Results Unexpectedly, A. nigra DS was found to bind HGF/SF strongly with a KD of around 1 nm . This interaction is 20‐fold stronger than that of between HGF/SF and mammalian DS, but similar to that of with HS. A. nigra DS also stimulated HGF/SF‐mediated Erk activation and migration in CHO‐745 cells. Studies using the modified GAG species showed that, in the case of HS, 6‐O‐sulfate and N‐sulfate groups are most important for HGF/SF binding. For HGF/SF binding to DS, hexosamine O‐sulfate is most important. HGF/SF was also found to bind 6‐O‐sulfated GAGs more strongly than 4‐O‐sulfated ones. Discussion The data show that there is flexibility in the structures recognized by HGF/SF, and this explains the ability of the growth factor to bind both HS and DS. However, there are still observable preferences in GAG structure, such as 6‐O‐sulfation over 4‐O‐sulfation. Information on HGF/SF‐binding GAG structures is valuable for the design of HGF/SF antagonists that could be useful therapeutically in the treatment of solid tumours where HGF/SF‐Met activity is up‐regulated.     

Change in decorin during aging of rat placenta

By immunohistochemistry, with or without chondroitinases, decorin was found to be distributed in the extracellular matrix of chorionic villi and amnia. The strength of staining intensified with increasing gestational age. Decorin was isolated from the placenta of 13- to 20-day-old pregnant rats and identified by Western blotting, using an antidecorin core protein antibody. The molecular weight of decorin is approximately 100 kDa, whereas the respective figures for the core protein treated with chondroitinase (chase) ABC and with chase B are approximately 40 kDa and 43 kDa. The difference in the molecular weight between the core protein with chase ABC and B suggests that the glycosaminoglycan (GAG)- base structure on the core protein was chondroitin sulfate (CS) without dermatan sulfate (DS). The decorin content and the proportion of CS to DS in GAG increased with age. We concluded that the age-related changes in the GAG chain may be related to specific functional properties and may have a crucial role in placental tissue organization.