Articular chondrocyte passage number: influence on adhesion, migration, cytoskeletal organisation and phenotype in response to nano- and micro-metric topography

The isolation and culture of articular chondrocytes is a prerequisite of their use in tissue engineering, but prolonged culture and passaging is associated with de-differentiation. In this paper we studied the influence of nanometric and micrometric grooves (85 nm to 8 microm in depth and 2 microm to 20 microm in width) on 1st and 2nd passage ovine chondrocytes since our earlier findings indicate that primary cells are not affected by such features. 1st and 2nd passage chondrocytes cultured on grooved substrata showed a polarisation of cell shape parallel to the groove long axis and F-actin condensations were evident at groove ridge boundaries. An increase in cell migration with increasing groove depth was observed. Both passages of chondrocytes maintained type II collagen expression, but to a lesser degree in 2nd. This study demonstrates that passage number alters the response of chondrocytes to micrometric and nanometric topography, and could be important in ex vivo cartilage engineering.     

Cu(II) and Zn(II) complexes with hyaluronic acid and its sulphated derivative. Effect on the motility of vascular endothelial cells

With the aim of improving the compatibility of biomaterials to be used for the construction of cardiovascular prosthesis, we have designed bioactive macromolecules resulting from chemical modifications of hyaluronic acid (Hyal). The stability constants of Cu(II) and Zn(II) complexes with the sulphated derivative of hyaluronic acid (HyalS3.5) were evaluated. Two different complexes have been found for each metal ion, CuL, Cu(OH)2L and ZnL, Zn(OH)2L (L means the disaccharide unit of the ligands) in aqueous solution at 37 degrees C. The dihydroxo Cu(II) complex was present in high percentage at pH=7.4. On the contrary, the Zn(II) ion was present with a relatively low percentage of both complexes. The ability to stimulate endothelial cell adhesion and migration was evaluated for Hyal, HyalS3.5 and their complexes with Cu(II) and Zn(II) ions. The results revealed that Hyal and [Cu(OH)2HyalS3.5](4.5)- induced cell adhesion, while [ZnHyalS3.5](2.5)- and [Zn(OH)2HyalS3.5](4.5)- inhibited the process. The chemotactic activity of increasing concentrations of the above complexes was also evaluated, demonstrating that [Cu(OH)2HyalS3.5](4.5)- complex at 1 microM concentration was the most active in inducing cell migration. These results have been also strengthened by analysing adherent cell migration in agarose. In conclusion, sulphated hyaluronic acid coordinated to Cu(II) seems to be a promising matrix molecule for the construction of cardiovascular prosthesis.     

Micropatterned surfaces for the control of endothelial cell behaviour

Micropatterned materials were synthesised by photoimmobilising the sulphated hyaluronic acid, adequately functionalised with a photoreactive moiety, on glass substrates. Four different patterns (10, 25, 50 and 100 microns) were obtained. The spectroscopic and microscopic analysis of the microstructured surfaces revealed that the photoimmobilisation process was successful, demonstrating that the photomask was well reproduced on the sample surface. Analysis of endothelial cell behaviour on these micropatterned materials was performed in terms of adhesion, locomotion and orientation. Decreasing the stripe dimensions a more fusiform shape of the adhered endothelial cells was observed. At the same time the cell locomotion and orientation were increased. Furthermore, a photoimmobilisation of stripes of HyalS (10 and 100 microns) was performed on a continuous HyalS layer, in turn immobilised on glass substrate. Being excluded a different chemistry between the stripe and the substrate, the influence of topography on the behaviour of endothelia cells was thus envisaged.     

Role of fibrinogen conformation in platelet activation

Platelet adhesion and activation induced by fibrinogen (Fbg) coating on polysaccharide layers of hyaluronic acid (Hyal) and its sulfated derivative (HyalS) were analyzed. Hyal or HyalS was coated and grafted on the glass substrate using a photolithographic method. The Fbg coating was achieved by two different routes: the immobilization of Fbg by means of covalent bond to the polysaccharide layers and the mere adsorption of Fbg to Hyal and HyalS surfaces. Platelet adhesion and activation to the surfaces were evaluated using, respectively, scanning electron microscopy (SEM) and quantifying the release of Platelet Factor 4 by ELISA. The method used for the coating of the surfaces with the Fbg influenced the platelet response. In fact, platelet adhesion and activation took place on surfaces covered by bound Fbg but not on those containing adsorbed Fbg. To explain this difference, the molecular mechanism involved in the Fbg--platelet interaction was investigated blocking platelet membrane receptors by monoclonal antibodies. Because the interaction between Fbg and the GPIIb/IIIa platelet membrane receptor was the only molecular pathway involved, Fbg conformation after the interaction (adsorption or binding) with the Hyal and the HyalS chains and the role of serum proteins adsorbed on the Fbg containing surfaces were accurately analyzed. Both adsorbed and bound Fbg prevented the adsorption of further serum proteins; consequently, a direct interaction between Fbg and platelets was supposed and the different platelet behavior was ascribed to the different conformational changes that occurred after the adsorption and the chemical binding of the Fbg to the Hyal and HyalS surfaces.     

Immobilisation of sulphated hyaluronan for improved biocompatibility

Hyaluronan (Hyal) was modified by the insertion of sulphate to hydroxyl groups. A series of heparin-like compounds with controllable properties was obtained. The physicochemical and biological behaviours of all these sulphated hyaluronan acids (HyalSx) and their complexes with heavy metal ions (Cu2+ and Zn2+) were investigated. HyalS, derivatives showed a good anticoagulant activity and low platelet aggregation which increased with increasing degree of sulphation. Moreover HyalSx and their Cu2+ complexes were demonstrated to favour the growth of human endothelial cells. However, the utilisation of HyalSx as a material is hindered by its high solubility in physiological solution. Our approach to improve its stability was directed to the synthesis of new HyalSx-based hydrogels and on the preparation of new biocompatible polymeric surfaces obtained through covalent photoimmobilisation of HyalSx. The reaction of primary ovine chondrocytes and B10D2 endothelial cells was studied on both matrices in terms of cell number, F-actin and CD44 receptor immunostaining. Analysis of cell movement showed that the cells respond to HyalSx showing good adhesion and spreading. These results suggest that HyalSx containing materials could be used as biomaterials to aid cartilage repair and vessel endothelisation.     

The effects of continuous and discontinuous groove edges on cell shape and alignment

Nanofabricated model surfaces and digital image analysis of cell shape were used to address the importance of a continuous sharp edge in the alignment of cells to shallow surface grooves. The grooved model surfaces had either continuous or discontinuous edges of various depths (40-400 nm) but identical surface chemistry and groove/ridge dimensions (15 microm wide). Epithelial cells were cultured on the model surfaces for 10 and 24 h. Fluorescence microscopy combined with image analysis were used to quantify cell area and alignment and to make cell shape classifications of individual cells. The degrees of alignment of cells and the percentages of elongated cell classes increased with groove depth on samples with continuous grooves. Two main differences, with regard to cell response, were observed between the continuous and discontinuous grooved surfaces. First, significantly fewer cells aligned to surface grooves with discontinuous edges than to grooves with continuous edges. Second, there were lower percentages of the elongated cell classes on discontinuous grooves than on continuous ones. We concluded that grooved surfaces with continuous edges are more potent in aligning and inducing elongated cells. The results from the present study suggest that a mechanism of alignment involving orientation along a continuous edge is likely.     

Fibroblast cell behavior on bound and adsorbed fibronectin onto hyaluronan and sulfated hyaluronan substrates

The effect of fibronectin protein (Fn) coating onto polysaccharide layers of hyaluronic acid (Hyal) and its sulfated derivative (HyalS) on fibroblast cell adhesion was analyzed. The Hyal or HyalS were coated and grafted on the glass substrate by a photolithographic method. The Fn coating was achieved by two different routes: the immobilization of Fn by covalent bond to the polysaccharide layers and the simple adsorption of Fn onto Hyal and HyalS surfaces. AFM, SEM, and ATR-FTIR techniques were used for the chemical and topographical characterization of the surfaces. According to AFM and SEM data, the surface topography was dependent on the method used to cover the polysaccharide layers with the protein. ATR-FTIR analysis supplied information about the rearrangement of Fn after the interaction (adsorption or binding) with the Hyal and the HyalS. The conformational changes of the Fn were minimal when it was simply adsorbed on HyalS surfaces and larger once bound, whereas on the Hyal layer the protein underwent a bigger conformational change once adsorbed and covalently grafted. Then, the biological characterization was carried out by analyzing the human diploid skin fibroblasts adhesion on these surfaces. The morphology of fibroblasts was evaluated by SEM, whereas the dynamics of fibroblasts movement were recorded by a time-lapse system. Cell variations in area, perimeter, and length were analyzed at 2, 4, and 6 h. It was found that the addition of Fn (covalently bound or merely adsorbed) was fundamental in the promotion of fibroblasts adhesion and spreading. The greatest adhesion occurred onto HyalS layers covered by the adsorbed Fn.     

Response of zonal chondrocytes to extracellular matrix-hydrogels

We investigated the biological response of chondrocytes isolated from different zones of articular cartilage and their cellular behaviors in poly (ethylene glycol)-based (PEG) hydrogels containing exogenous type I collagen, hyaluronic acid (HA), or chondroitin sulfate (CS). The cellular morphology was strongly dependent on the extracellular matrix component of hydrogels. Additionally, the exogenous extracellular microenvironment affected matrix production and cartilage specific gene expression of chondrocytes from different zones. CS-based hydrogels showed the strongest response in terms of gene expression and matrix accumulation for both superficial and deep zone chondrocytes, but HA and type I collagen-based hydrogels demonstrated zonal-dependent cellular responses.     

Evaluating specific adhesion of Plasmodium falciparum-infected erythrocytes to immobilised hyaluronic acid with comparison to binding of mammalian cells

A feature of infection with Plasmodium falciparum is the ability of parasite-infected erythrocytes to adhere to vascular endothelial cells and accumulate in vital organs, associated with severe clinical disease. Hyaluronic acid was recently identified as a receptor for adhesion and has been implicated in mediating the accumulation of parasites in the placenta. Here, we report in vitro assays to measure specific adhesion of infected erythrocytes to hyaluronic acid that is distinct from binding to chondroitin sulphate A, another glycosaminoglycan implicated as a receptor in placental malaria. In this study, specific adhesion of mature stage infected erythrocytes to hyaluronic acid of high purity immobilised on plastic surfaces was abolished by pre-treating hyaluronic acid with a specific hyaluronate lyase from Streptomyces, whereas the same treatment of chondroitin sulphate A had little effect. Adhesion to hyaluronic acid could not be explained by the presence of chondroitin sulphate A or other glycosaminoglycans in the hyaluronic acid preparations. Chinese hamster ovary cells bound in a similar manner in the assays and confirmed that hyaluronic acid was appropriately immobilised for cell adhesion. In contrast to parasites, these cells did not adhere to chondroitin sulphate A. The adsorption of hyaluronic acid onto plastic surfaces was also confirmed by the use of a specific hyaluronic acid-binding protein. Fixing cells with glutaraldehyde at the completion of adhesion assays reduced the number of parasites remaining adherent to hyaluronic acid, but not to chondroitin sulphate A or CD36. These findings have important implications for understanding and evaluating interactions between P. falciparum and hyaluronic acid that may be involved in disease pathogenesis.     

Incorporation of [3H]glucosamine into glycosaminoglycans in different cell culture conditions by rabbit aortic smooth muscle cells

This study sought to elucidate the optimal cell culture conditions for studies concerned with the incorporation of [³H]glucosamine into glycosaminoglycans by rabbit aortic smooth muscle cells. The incorporation of radioactivity into extracellular sulphated glycosaminoglycans was linear for at least 72 h and that into pericellular sulphated glycosaminoglycans for up to 24 h. The incorporation of radiolabel into hyaluronic acid was linear only up to 12 h. In the exponential growth phase the incorporation of [³H]glucosamine into sulphated glycosaminoglycans and hyaluronic acid proved to be less marked than in the stationary growth phase, but the highest values were nevertheless obtained immediately after trypsinisation. When studied in the stationary growth phase, cell density and incorporation of [³H]glucosamine were positively correlated in the case of hyaluronic acid, but in the case of sulphated glycosaminoglycans there was a negative correlation. The serum concentration of the incubation medium and the incorporation of radioactivity into hyaluronic acid were positively related. With sulphated glycosaminoglycans this was the case only after a 7-day preincubation in the different serum concentrations. when incorporation was studied without preincubation, the incorporation of radioactivity into sulphated glycosaminoglycans proved to be negatively associated with the serum concentration of the medium. The environmental pH of the cells was associated with the incorporation of radioactivity into hyaluronic acid and sulphated glycosaminoglycans in that between pH values 6.8 and 7.9 the incorporation of radioactivity increased when the pH of the medium was raised.     

ECM production of primary human and bovine chondrocytes in hybrid PEG hydrogels containing type I collagen and hyaluronic acid

The development of advanced materials that facilitate hyaline cartilage formation and regeneration in aging populations is imperative. Critical to the success of this endeavor is the optimization of ECM production from clinically relevant cells. However, much of the current literature focuses on the investigation of primary bovine chondrocytes from young calves, which differ significantly than osteoarthritic cells from human sources. This study examines the levels of extracellular matrix (ECM) production using various levels of type I collagen and hyaluronic acid in poly(ethylene glycol) dimethacrylate (PEGDM) hydrogels in total knee arthroplasties, compared with the results from bovine chondrocytes. The addition of type 1 collagen in both the presence and absence of low levels of hyaluronic acid increased ECM production and/or retention in scaffolds containing either bovine or human chondrocytes. These findings are supported consistently with colorimetric quantification, whole mount extracellular matrix staining for both cell types, and histological staining for glycoaminoglycans and collagen of human chondrocyte containing samples. While exhibiting similar trends, the relative ECM productions levels for the primary human chondrocytes are significantly less than the bovine chondrocytes which reinforces the need for additional optimization.     

Oriented Schwann cell growth on microgrooved surfaces

Silicon wafers bearing microgrooved surfaces with various groove width, spacing, and depth were fabricated using microlithography. The orientation of rat Schwann cells along the direction of the grooves was measured at 24 h after seeding the cells. When the width/spacing of the grooves was fixed at 10/10 microm, the mean percentage of aligned cells was 12% for grooves of 0.5 microm depth, 15% for those of 1 microm depth, and 26% for those of 1.5 microm depth (P < 0.05). When the depth of grooves was fixed at 1.5 microm, the mean percentage of aligned cells increased from 26% for width/spacing 10/10 microm, to 33% for 10/20 microm or 20/10 microm, and up to 41% for 20/20 microm (P < 0.05). On the surface with grooves of width/spacing/depth = 20/20/1.5 microm and modified by laminin, the alignment at 24 h approached 60%, versus 51% for collagen-coated surface and 41% for uncoated surface (P < 0.05). At 48 h after seeding, about 66% of the cells were aligned on the above laminin-modified surface. The groove depth influenced orientation of Schwann cells significantly. The cell alignment on 20/20/3 microm microgrooved poly(D,L-lactide-co-glycolide) 90:10 (PLGA) surfaces transferred from silicon reached 72% at 48 h and 92% at 72 h (P < 0.05). Coating this surface with laminin enhanced cell alignment only in short term (67% vs. 62% at 24 h, P < 0.05). The cell alignment guided by surface microgrooves was time dependent.     

A comparative study of glycosaminoglycans in cultures of human, normal and malignant glial cells.

The glycosaminoglycans (GAG) of human cultured normal glial and malignant glioma cell lines were studied using 35S-sulphate or 3H-glucosamine as markers. 35S-labelled GAG were assayed by precipitation with cetylpyridinium chloride; 3H-labelled sulphated GAG and 3H-labelled hyaluronic acid were quantitated after separation on a DEAE-cellulos column. The net production of GAG and the distribution, composition and turnover of GAG were similar in all of the normal cell lines tested, but showed a great variability in the malignant cell lines. Most of the glioma cell lines produced more hyaluronic acid and less sulphated GAG than the normal cell lines, but exceptions were noted. The GAG of the trypsin susceptible (pericellular pool of normal glial cells consisted mainly of heparan sulphate with only minor amounts of other GAG. The analogous material of most glioma cells showed hyaluronic acid as the major GAG. Material liberated by trypsin from EDTA-detached cells (membrane fraction) was enriched in heparan sulphate as compared to the entire pericellular pool. Substrate attached material (SAM) left with the plastic dish after EDTA treatment of normal cultures was rich in heparan sulphate, whereas SAM of glioma cells lacked heparan sulphate or showed greatly reduced amounts of this component. Release of newly synthesized GAG to the extracellular medium was a rapid process in the normal cells but was more or less delayed in the glioma cells. The extracellular medium of the malignant glioma cultures was consistently poor in dermatan sulphate, as compared to that of normal cultures.     

Proteoglycan aggregate formation by articular chondrocytes. Decrease in link-protein synthesis during culture.

The synthesis of link-stabilized proteoglycan aggregates by rabbit articular chondrocytes was investigated by [35S]sulphate labelling of primary monolayer cultures maintained for up to 21 days. (1) At all culture times the cells secreted a high-molecular-weight cartilage-type proteoglycan monomer of which 75%-80% formed aggregates with hyaluronic acid. (2) At 2 days of culture all of the aggregates were in link-stabilized form, but by 21 days only 5% were link-stabilized, as shown by displacement of monomers from the aggregate by hyaluronic acid oligosaccharides. (3) The addition of purified link protein to 21-day culture medium increased the proportion of link-stable aggregate from 5% to 70%. (4) Analysis of [3H]serine-labelled proteoglycan aggregates in the medium showed a marked decrease with culture time in the ratio of 3H-labelled link protein to 3H-labelled core protein present. The results suggest that the secretion of proteoglycan monomers and link protein by articular chondrocytes changes independently during prolonged monolayer culture.     

Placentation in the garter snake, Thamnophis sirtalis

The structure and polysaccharide constitution of the jelly capsule of the egg of Rana pipiens is described. Microscopic examination of the jelly capsule revealed the presence of five discrete jelly layers that differed clearly in their response to selected cytochemical tests. These layers were classified as M1-through M5 from the inner to the outermost layer. A sixth layer occasionally could be observed between M3 and M4. All layers contain neutral mucopolysaccharides. In addition layers M1 and M3 contain sulphated mucopolysaccharides, M2 and M4 contain non-sulphated acid mucopolysaccharides, and layer M5 contains both sulphated and non-sulphated acid mucopolysaccharides. M2 may also contain a small quantity of sulphated mucopolysaccharides. The layer that occasionally appears between M3 and M4 is probably an area in which free acidic groups are in higher concentration than in adjacent areas rather than being a discrete jelly layer. Neither hyaluronic acid nor sialic acid was localized by the methods employed. The possible significance of some of these constituents is discussed.     

The synthesis of glycosaminoglycans by cultures of rabbit corneal endothelial and stromal cells.

Confluent monolayer cultures of rabbit corneal endothelial and stromal cells were incubated independently with [35S]sulphate and [3H]glucosamine for 3 days. AFter incubation, labelled glycosaminoglycans were isolated from the growth medium and from a cellular fraction. These glycosaminoglycans were further characterized by DEAE-cellulose column chromatography and by sequential treatment with various glycosamino-glycan-degrading enzymes. Both endothelial and stromal cultures synthesized hyaluronic acid as the principal product. The cell fraction from the stromal cultures, however, had significantly less hyaluronic acid than that from the endothelial cultures. In addition, both types of cells synthesized a variety of sulphated glycosaminoglycans. The relative amounts of each sulphated glycosaminoglycan in the two cell lines were similar, with chondroitin 4-sulphate, chondroitin 6-sulphate and dermatan sulphate as the major components. Heparan sulphate was present in smaller amounts. Keratan sulphate was also identified, but only in very small amounts (1-3%). The presence of dermatan sulphate and the high content of hyaluronic acid are similar to the pattern of glycosaminoglycans seen in regenerating or developing tissues, including cornea.     

Histochemical localization of protein-polysaccharides in renal tissue

The purpose of this study was to investigate the distribution of protein-polysaccharides in the glomerular and non-glomerular regions of the nephron. The techniques used include the digestion of kidney slices with specific polysaccharidases: neuraminidase, hyaluronidase, chondroitinase ABC, and collagenase followed by several cytochemical techniques to identify the glycosaminoglycans and glycoproteins at the light and electron microscope levels. Differential staining of hyaluronic acid and sulphated glycosaminoglycans was accomplished with Alcian Blue at pH 2.5 and pH 0.5, respectively. Sialoproteins were stained with Alcian Blue at pH 2.5. The periodic acid Schiff’s reaction technique was employed for the visualization of collagen. At the electron microscope level the polysaccharides were identified with the periodic acid-chromic acid-silver methenamine reaction. Our results indicated that the major polysaccharide components of the glomerular basement membrane were sialoproteins and collagen, with smaller amounts of hyaluronic acid and various sulphated glycosaminoglycans. Hyaluronidase digestion resulted in partial detachment of epithelial processes from the glomerular basement membrane indicating the hyaluronic acid may have a role in the stability of the attachment of these processes. Tubular basement membranes also contain sialoproteins and sulphated glycosaminoglycans but in considerably lower concentrations than the glomerular basement membrane. Bowman’s capsule appears to contain mostly sulphated glycosaminoglycans and has a lower concentration of sialoproteins and hyaluronic acid.     

Composition and distribution of glycosaminoglycans in cultures of human normal and malignant glial cells.

The glycosaminoglycans of human cultured normal glial and malignant glioma cells were studied. [35S]Sulphate or [3H]glucosamine added to the culture medium was incorporated into glycosaminoglycans; labelled glycosaminoglycans were isolated by DEAE-cellulose chromatography or gel chromatography. A simple procedure was developed for measurement of individual sulphated glycosaminoglycans in cell-culture fluids. In normal cultures the glycosaminoglycans of the pericellular pool (trypsin-susceptible material), the membrane fraction (trypsin-susceptible material of EDTA-detached cells) and the substrate-attached material consisted mainly of heparan sulphate. The intra- and extra-cellular pools showed a predominance of dermatan sulphate. The net production of hyaluronic acid was low. The accumulation of 35S-labelled glycosaminoglycans in the extracellular pool was essentially linear with time up to 72h. The malignant glioma cells differed in most aspects tested. The total production of glycosaminoglycans was much greater owing to a high production of hyaluronic acid and hyaluronic acid was the major cell-surface-associated glycosaminoglycan in these cultures. Among the sulphated glycosaminoglycans chondroitin sulphate, rather than heparan sulphate, was the predominant species of the pericellular pool. This was also true for the membrane fraction and substrate-attached material. Furthermore, the accumulation of extracellular 35S-labelled glycosaminoglycans was initially delayed for several hours and did not become linear with time until after 24 h of incubation. The glioma cells produced little dermatan sulphate and the dermatan sulphate chains differed from those of normal cultures with respect to the distribution of iduronic acid residues. The observed differences between normal glial and malignant glioma cells were not dependent on cell density; rather they were due to the malignant transformation itself.     

Glycosaminoglycan synthesis in skin fibroblasts from patients with osteogenesis imperfecta

Glycosaminoglycans were analysed from skin fibroblasts with osteogenesis imperfecta (OI) IIA and IIB. The content of sulphated glycosaminoglycans was greatly increased over age-matched controls and to a lesser extent with respect to older age control. Dermatan sulphate in comparison with older control was unaltered in the cells of OI IIA and IIB. The concentration of heparan sulphate was higher in the cells than in the medium, whereas hyaluronic acid, chondroitin sulphate and dermatan sulphate content was higher in the medium. The level of hyaluronic acid was greatly elevated in the medium of OI IIB with respect to both controls.     

Effect of hyaluronic acid on human chondrocyte cell lines from articular cartilage

The effects of hyaluronic acid (HA) derivative on the proliferation and metabolism of human chondrocytes were examined. Cells were obtained from cartilage from metatarsal phalangeal joints of 20 adult humans (aged 22-63) and from femoral knee condyles of 10 subjects (aged 22-77). Chondrocytes isolated by collagenase/Dnase digestion were cultured with addition of different doses of HA for 4 weeks. Morphological studies demonstrated that HA enhanced the adhesion of cells to substrate; HA-treated chondrocytes proliferated better than chondrocytes cultured in HA-free medium. This study shows that HA improves in vitro substrate adhesion ability and proliferative activity of human cartilage cells and that the response to the treatment varies on an individual basis.