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Biomedical subjects

T H van Kuppevelt

Publications and source records attributed to T H van Kuppevelt.

At least 19 recordsLinked to original sources
PubMed2003

Preparation and evaluation of molecularly-defined collagen-elastin-glycosaminoglycan scaffolds for tissue engineering.

Extracellular matrix components are valuable building blocks for the preparation of biomaterials involved in tissue engineering, especially if their biological, chemical and physical characteristics can be controlled. In this study, isolated type I collagen fibrils, elastin fibres and chondroitin sulphate (CS) were used for the preparation of molecularly-defined collagen-elastin-glycosaminoglycan scaffolds. A total of 12 different scaffolds were prepared with four different ratios of collagen and elastin (1:9, 1:1, 9:1 and 1:0), with and without chemical crosslinking, and with and without CS. Collagen was essential to fabricate coherent, porous scaffolds. Electron microscopy showed that collagen and elastin physically interacted with each other and that elastin fibres were enveloped by collagen. By carbodiimide-crosslinking, amine groups were coupled to carboxylic groups and CS could be incorporated. More CS could be bound to collagen scaffolds (10%) than to collagen-elastin scaffolds (2.4-8.5% depending on the ratio). The attachment of CS increased the water-binding capacity to up to 65%. Scaffolds with a higher collagen content had a higher tensile strength whereas addition of elastin increased elasticity. Scaffolds were cytocompatible as was established using human myoblast and fibroblast culture systems. It is concluded that molecularly-defined composite scaffolds can be composed from individual, purified, extracellular matrix components. Data are important in the design and application of tailor-made biomaterials for tissue engineering.

Biocompatible Materials↗
PubMed2002

Loading of collagen-heparan sulfate matrices with bFGF promotes angiogenesis and tissue generation in rats.

The loading of biocompatible matrices with growth factors offers the opportunity to induce specific cell behavior. The attachment of heparan sulfate (HS) to these matrices may promote the binding, modulation, and sustained release of signaling molecules. In this study, basic fibroblast growth factor (bFGF) was bound to crosslinked collagenous matrices with and without covalently attached HS. The tissue response to these matrices was evaluated after subcutaneous implantation in rats. Attachment of HS to collagen matrices increased the bFGF binding capacity threefold and resulted in a more gradual and sustained release of the growth factor in vitro. bFGF primarily was located at the matrix margins. In vivo, the presence of HS without bFGF resulted in a transient vascularization, predominantly at the matrix periphery. Angiogenesis was further enhanced by combining HS with bFGF. In contrast to collagen-HS and collagen/bFGF matrices, collagen-HS/bFGF matrices remained highly vascularized throughout the matrix during the 10-week implantation period. In addition, these latter matrices revealed an intense and prolonged tissue response and considerably promoted the generation of new tissue. Foreign body reactions were only observed sporadically at this time interval. It is concluded that bFGF loading of collagen-HS matrices has additional value for those tissue-engineering applications that require enhanced angiogenesis and generation of new tissue.

Animals↗
PubMed2002

Crosslinked type II collagen matrices: preparation, characterization, and potential for cartilage engineering.

The limited intrinsic repair capacity of articular cartilage has stimulated continuing efforts to develop tissue engineered analogues. Matrices composed of type II collagen and chondroitin sulfate (CS), the major constituents of hyaline cartilage, may create an appropriate environment for the generation of cartilage-like tissue. In this study, we prepared, characterized, and evaluated type 11 collagen matrices with and without CS. Type II collagen matrices were prepared using purified, pepsin-treated, type II collagen. Techniques applied to prepare type I collagen matrices were found unsuitable for type II collagen. Crosslinking of collagen and covalent attachment of CS was performed using 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide. Porous matrices were prepared by freezing and lyophilization, and their physico-chemical characteristics (degree of crosslinking, denaturing temperature, collagenase-resistance, amount of CS incorporated) established. Matrices were evaluated for their capacity to sustain chondrocyte proliferation and differentiation in vitro. After 7 d of culture, chondrocytes were mainly located at the periphery of the matrices. In contrast to type I collagen, type II collagen supported the distribution of cells throughout the matrix. After 14 d of culture, matrices were surfaced with a cartilagenous-like layer, and occasionally clusters of chondrocytes were present inside the matrix. Chondrocytes proliferated and differentiated as indicated by biochemical analyses, ultrastructural observations, and reverse transcriptase PCR for collagen types I, II and X. No major differences were observed with respect to the presence or absence of CS in the matrices.

Biocompatible Materials↗
PubMed2001

Linkage of chondroitin-sulfate to type I collagen scaffolds stimulates the bioactivity of seeded chondrocytes in vitro.

An increasing amount of interest is focused on the potential use of tissue-engineered articular cartilage implants, for repair of defects in the joint surface. In this perspective, various biodegradable scaffolds have been evaluated as a vehicle to deliver chondrocytes into a cartilage defect. This cell-matrix implant should eventually promote regeneration of the traumatized articular joint surface with hyaline cartilage. Successful regeneration can only be achieved with such a tissue-engineered cartilage implant if the seeded cells reveal an appropriate proliferation rate in the biodegradable scaffold together with the production of a new cartilage-specific extracellular matrix. These metabolic parameters can be influenced by the biochemical composition of a cell-delivery scaffold. Further elucidation of specific cell-matrix interactions is important to define the optimal biochemical composition of a cell-delivery vehicle for cartilage repair. In this in vitro study, we investigated the effect of the presence of cartilage-specific glycosaminoglycans in a type I collagen scaffold on the metabolic activity of seeded chondrocytes. Isolated bovine chondrocytes were cultured in porous type I collagen matrices in the presence and absence of covalently attached chondroitin sulfate (CS) up to 14 days. CS did indeed influence the bioactivity of the seeded chondrocytes. Cell proliferation and the total amount of proteoglycans retained in the matrix, were significantly higher (p < 0.001) in type I collagen scaffolds with CS. Light microscopy showed the formation of a more dense cartilaginous layer at the matrix periphery. Scanning electron microscopy revealed an almost complete surfacing of the initially porous surface of both matrices. Histology and reverse transcriptase PCR for various proteoglycan subtypes suggested a good preservation of the chondrocytic phenotype of the seeded cells during culture. The stimulatory potential of CS on both the cell-proliferation and matrix retention, turns this GAG into an interesting biochemical component of a cell-delivery scaffold for use in tissue-engineering articular cartilage.

Animals↗
PubMed2000

Heparan sulfate heterogeneity in skeletal muscle basal lamina: demonstration by phage display-derived antibodies.

The basal lamina (BL) enveloping skeletal muscle fibers contains different glycoproteins, including proteoglycans. To obtain more information on the glycosaminoglycan moiety of proteoglycans, we have selected a panel of anti-heparan sulfate (HS) antibodies from a semisynthetic antibody phage display library by panning against glycosaminoglycan preparations derived from skeletal muscle. Epitope recognition by the antibodies is strongly dependent on O- and N-sulfation of the heparan sulfate. Immunostaining with these antibodies showed a distinct distribution of heparan sulfate epitopes in muscle basal lamina of various species. Clear differences in staining intensity were observed between neural, synaptic, and extrasynaptic basal laminae. Moreover, temporal and regional changes in abundancy of heparan sulfate epitopes were observed during muscle development both in vitro and in vivo. Taken together, these data suggest a role for specific heparan sulfate domains/species in myogenesis and synaptogenesis. Detailed analysis of the functions of heparan sulfate epitopes in muscle morphogenesis has now become feasible with the isolation of antibodies specific for distinct heparan sulfate epitopes.

Animals↗
PubMed2000

Attachment of glycosaminoglycans to collagenous matrices modulates the tissue response in rats.

Biocompatibility and tissue regenerating capacity are essential characteristics in the design of collagenous biomaterials for tissue engineering. Attachment of glycosaminoglycans (GAGs) to collagen may add to these characteristics by creating an appropriate micro-environment. In this study, porous type I collagen matrices were crosslinked using 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide, in the presence and absence of chondroitin sulfate and heparan sulfate. The tissue response to these matrices was evaluated after subcutaneous implantation in rats. Biocompatibility of the matrices was established by the induction of a transitional inflammatory response, and the generation of new host tissue. Non-crosslinked collagen was gradually resorbed and replaced by collagenous connective tissue. By contrast, crosslinked matrices, with and without GAGs. retained their scaffold integrity during implantation, and supported the interstitial deposition and organization of extracellular matrix. In addition, crosslinking decreased tissue reactions at late time intervals. No calcification in any of the implants was observed. The presence of GAGs preserved porous lamellar matrix structures. Heparan sulfate in particular promoted angiogenesis at weeks 2 and 4, predominantly at the matrix periphery. The almost complete absence of macrophages and giant cells associated with collagen-GAG matrices, after 10 weeks implantation, indicated a reduced foreign body reaction. It is concluded that attachment of GAGs to collagen matrices modulates the tissue response. The potential of these biocompatible scaffolds for tissue engineering is increased by preserving porous matrix integrity. promoting angiogenesis and reducing foreign body reactions.

Animals↗
PubMed2000

Development of tailor-made collagen-glycosaminoglycan matrices: EDC/NHS crosslinking, and ultrastructural aspects.

The many biocharacteristics of glycosaminoglycans (GAGs) make them valuable molecules to be incorporated in collagenous biomaterials. To prepare tailor-made collagen-GAG matrices with a well-defined biodegradability and (bioavailable) GAG content, the crosslinking conditions have to be controlled. Additionally, the ultrastructural location of GAGs in engineered substrates should resemble that of the application site. Using chondroitin sulfate (CS) as a model GAG, these aspects were evaluated. The methodology was then applied for other GAGs. CS was covalently attached to collagen using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). A maximum of about 155 mg CS/g matrix could be immobilized. CS incorporation and bioavailability, as evaluated by interaction with specific antibodies and glycosidases, was dependent on the molar ratio EDC:carboxylic groups of CS. The denaturation temperature could be modulated from 61 to 85 degrees C. The general applicability of EDC/NHS for immobilizing GAGs was demonstrated with dermatan sulfate, heparin, and heparan sulfate. These matrices revealed comparable physico-chemical characteristics, biodegradabilities, and preserved bioavailable GAG moieties. At the ultrastructural level, GAGs appeared as discrete, electron-dense filaments, each filament representing a single GAG molecule. Distribution was independent of GAG type. They were observed throughout the matrix fibers and at the outer sites, and located, either parallel or orthogonally, at the periphery of individual collagen fibrils. Compositional and ultrastructural similarity between matrices and tissue structures like cartilage and basement membranes can be realized after attachment of specific GAG types. It is concluded that EDC/NHS is generally applicable for attachment of GAGs to collagen. Modulation of crosslinking conditions provides matrices with well-defined GAG contents, and biodegradabilities. Ultrastructural similarities between artificially engineered scaffolds and their possible application site may favor the use of specific collagen-GAG matrices in tissue engineering.

Animals↗
PubMed2000

Restoration by vacuum inflation of original alveolar dimensions in small human lung specimens.

Resection of lung specimens results in deflated and distorted lung structures. If no major airway is present (as in the case of small lung specimens from biopsies), lung dimensions cannot be restored by inflation under 25 cmH20. This impedes morphological analysis of the tissue. This report describes a simple and easy procedure to restore alveolar dimensions in deflated small lung specimens. Small human lung samples were inflated using moderate vacuum conditions which are provided by a common water stream-driven vacuum device. Restoration of alveolar dimensions and morphology was evaluated for paraffin-embedded as well as frozen tissue, using morphometric and immunohistological analysis. Vacuum inflation results in restoration of original lung dimensions as judged by light and scanning electron microscopy, and by analysis of the mean linear intercept, and the average length, width, perimeter and surface area. It also results in markedly improved cutting characteristics, allowing reliable sectioning of 2 microm cryosections and achieving high resolution images in immunofluorescence. Vacuum inflation is a simple and easy procedure to restore lung architecture of small human lung specimens/biopsies with a concomitant improvement of cutting characteristics. It allows for correct histological analysis of small specimens which cannot be inflated otherwise.

Adult↗
PubMed1999

Differentiation markers of mouse C2C12 and rat L6 myogenic cell lines and the effect of the differentiation medium.

The differentiation grade of cells in culture is dependent on the composition of the culture medium. Two commonly used myogenic cell lines, mouse C2C12 and rat L6, usually differentiate at a low concentration of horse serum. In this study we compared the effect of horse serum with a medium containing a low percentage of Ultroser G and rat brain extract. The maturation grade was evaluated on the basis of various biochemical, (immuno)histochemical and cell-physiological parameters. Substitution of horse serum by Ultroser G and rat brain extract during the differentiation phase resulted in a higher maturation grade of the myotubes of both cell lines, on the basis of creatine kinase activity and the diameter of the myotubes. In addition, the C2C12 myotubes display cross-striation, contain a higher percentage of creatine kinase muscle-specific isoenzyme MM, show a ninefold increase in acetylcholine receptor (AChR) clusters, form a continuous basement membrane, and have a lower resting cytosolic Ca2+ concentration. L6 myotubes show a fivefold increase in AChR clusters and a twofold increase in the expression of the mRNA of the epsilon-subunit of AChR.C2C12 cells show spontaneous contraction and response of cytosolic Ca2+ to various stimulants in contrast to L6 cells which do not. These studies established that the Ultroser G/brain extract medium leads to a higher differentiation grade of both cell lines, but parameters appropriate for use as differentiation markers appear to differ between both cell lines.

Animals↗
PubMed1999

Preparation and characterization of porous crosslinked collagenous matrices containing bioavailable chondroitin sulphate.

Porous collagen matrices with defined physical, chemical and biological characteristics are interesting materials for tissue engineering. Attachment of glycosaminoglycans (GAGs) may add to these characteristics and valorize collagen. In this study, porous type I collagen matrices were crosslinked using dehydrothermal (DHT) treatment and/or 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC), in the presence and absence of chondroitin sulphate (CS). EDC covalently attaches CS to collagen. DHT crosslinking preserved a porous matrix structure. However, attachment of CS to DHT-treated matrices using EDC, resulted in collapsed surfaces, CS located only at the matrix exterior. EDC crosslinking resulted in a partial matrix collapse. This could be prevented if crosslinking was carried out in the presence of ethanol. Matrix porosity was then preserved. The presence of CS during EDC crosslinking resulted in covalent immobilization of CS throughout the matrix. The amount of CS incorporated was increased if crosslinking was performed in the presence of ethanol. EDC-crosslinked matrices, with and without CS, had increased denaturation temperatures and decreased free amine group contents. The susceptibility of these matrices towards degradation by proteolytic enzymes was diminished. Immobilized CS increased the water-binding capacity and decreased the denaturation temperature and tensile strength. Immobilized CS bound anti-CS antibodies and was susceptible to chondroitinase ABC digestion, demonstrating its bioavailability. The modified matrices were not cytotoxic as was established using human myoblast and fibroblast culture systems. It is concluded that the use of ethanol during EDC crosslinking, offers an elegant means for the preparation of defined porous collagenous matrices containing bioavailable, covalently attached CS.

Biocompatible Materials↗
PubMed1999

Hemoglobin and myoglobin content in muscles of broiler chickens.

The two main heme proteins, hemoglobin and myoglobin, are important factors determining meat quality aspects such as color and hemorrhage. The extent of hemorrhage in muscle tissue can probably be determined by measuring the hemoglobin content. The objective of this study was twofold: 1) to develop a specific and reproducible method to quantify the hemoglobin and myoglobin content in muscle tissue of broiler chickens, and 2) to study the effect of hemorrhage on the hemoglobin content in muscle tissue. We tested several methods to determine the total heme, hemoglobin, and myoglobin content in broiler chicken muscles on their specificity, sensitivity, and reproducibility. Methods based on immunological techniques appeared to be very specific and sensitive. The results obtained applying these methods on muscle tissue extracts were, however, not reproducible due to concentration effects. A combination of spectrophotometric analysis of the total heme protein concentration and measurement of the myoglobin concentration, applying size exclusion chromatography, proved to be a reliable and reproducible method to determine the hemoglobin and myoglobin content in chicken muscles. The total heme, hemoglobin, and myoglobin contents were related to muscle type. Extensive hemorrhage increased the hemoglobin content.

Animals↗
PubMed1999

Structural and functional studies on different human FABP types.

Interaction of various ligands with recombinant proteins of 5 human FABP types was studied by radiochemical and fluorescence procedures. Liver, heart, intestinal and myelin FABP showed a higher affinity for oleic acid than adipocyte FABP. Intestinal and adipocyte FABP had a relatively high Kd value for arachidonic acid. Liver and intestinal FABP showed high affinity for DAUDA in contrast to the other FABP types. ANS was only well bound by liver and adipocyte FABP. Retinol was not bound by any FABP type, retinoic acid only by adipocyte FABP. Data indicate the importance of both electrostatic and hydrophobic interaction for the ligand-FABP binding. The immunological crossreactivity between six human FABP types including epidermal FABP and their respective antibodies raised in rabbit, chicken and mouse appeared to be low and may suggest heterogeneity of protein surface.

Amino Acid Sequence↗
PubMed1998

Generation and application of type-specific anti-heparan sulfate antibodies using phage display technology. Further evidence for heparan sulfate heterogeneity in the kidney.

Detailed analysis of various heparan sulfate (HS) species is seriously hampered by a lack of appropriate tools, such as antibodies. We adopted phage display technology to generate anti-HS antibodies. A "single pot" semisynthetic human antibody phage display library was subjected to four rounds of selection on HS from bovine kidney using panning methodology. Three different phage clones expressing anti-HS single chain variable fragment antibodies (HS4C3, HS4D10, and HS3G8) were isolated, with an amino acid sequence of the complementarity-determining region 3 of GRRLKD (VH3 gene, DP-38), SLRMNGCGAHQ (VH3 gene, DP-42), and YYHYKVN (VH1 gene, DP-8), respectively. The antibodies react with HS and heparin, but not with DNA or other glycosaminoglycans. Kd values for HS are about 0.1 microM. The three antibodies react differently toward various HS preparations and show different staining patterns on rat kidney sections, indicating recognition of different HS molecules. This also holds for two described mouse anti-HS IgMs (JM403 and 10E4; both generated by conventional hybridoma technique) and indicates the presence of at least 5 different HS species in the kidney. O- and N-sulfation are important for binding of HS to HS4C3 and HS3G8. The three single chain antibodies, but not JM403, block a basic fibroblast growth factor binding site of HS. It is concluded that phage display technology presents a powerful technique to generate antibodies specific for HS epitopes. This is the first time this technique has been successfully applied to obtain directly antibodies to (poly)saccharides.

Amino Acid Sequence↗
PubMed1998

Primary structure and high expression of human agrin in basement membranes of adult lung and kidney.

Agrin is a heparan sulfate proteoglycan involved in the development of the neuromuscular junction during embryogenesis. In addition to this well-characterized function, agrin may have additional functions in other tissues and during other stages in development. In this study we present the cDNA sequence of human agrin, and demonstrate a high agrin content in adult basement membranes. The N-terminal domain of human agrin is highly similar to that of chick agrin, suggesting a similar function in laminin binding. The presence of three SGXG sequences supports serine-linked glycosylation of the core protein, two sites being particularly favorable for heparan sulfate attachment. Comparison of levels of agrin mRNA in fetal and adult human tissues showed a remarkable upregulation in adult kidney and lung. In both tissues truncated agrin transcripts were detected, lacking the region that encodes the laminin-binding domain. The high transcription levels in lung and kidney corresponded with the accumulation of agrin in the alveolar and glomerular basement membranes, suggesting a filtration-associated function. These data provide new directions for investigating the role of agrin in its different physiological environments, including the basement membranes of the neuromuscular junction, kidney and lung.

Age Factors↗
PubMed1998

Susceptibility of broiler chickens to hemorrhages in muscles: the effect of stock and rearing temperature regimen.

In this study, the effect of genetic constitution (stock) and rearing temperature on the occurrence of hemorrhages in thighs and breasts of water bath stunned broilers was investigated. Particular attention was given to the relation between the susceptibility for hemorrhages, body composition, and adaptations in blood variables induced by low rearing temperatures. A factorial experiment was performed with five parental stocks, differing with respect to growth rate, feed conversion, and body composition, and two temperature regimens (thermoneutral and below the zone of thermoneutrality). Hemorrhage scores in thighs were dependent on rearing temperature, not on stock, and were highest in broilers reared at low temperatures. In all stocks, high scores in the thighs were accompanied by an increased carbon dioxide pressure and bicarbonate and triiodothyronine concentration of the venous blood, and by an increased relative heart weight, hematocrit, and blood loss at slaughter. Hemorrhage scores in breasts were dependent on stock, confounded with day of scoring. An effect of rearing temperature was present only for the leanest, slow-growing stock having the lowest feed conversion ratio at thermoneutral rearing conditions. Scores in breasts were related neither to body composition nor to metabolic and hemodynamic adaptations to low rearing temperatures. It is concluded that high hemorrhage scores in thighs are related to hemodynamic and metabolic adaptations to an increased need for energy and oxygen caused by low rearing temperatures. Hemorrhage scores are not related to stock-dependent differences.

Adaptation, Physiological↗
PubMed1997

Induction of emphysematous lesions in rat lung by beta-D-xyloside, an inhibitor of proteoglycan synthesis.

The possible involvement of proteoglycans in the pathogenesis of emphysema was studied in rats by a single intratracheal instillation of p-nitrophenyl-beta-D-xylopyranoside (beta-D-xyloside), an inhibitor of proteoglycan synthesis. The first 3 days after instillation are characterized by mild hemorrhages, some infiltration of inflammatory cells, and edema. After 1 wk, lung morphology is normal again. Forty days after instillation, considerable parenchymal destruction has occurred as determined by the mean linear intercept (81 +/- 12 microns versus 57 +/- 5 microns for control [P < 0.001]). Pulmonary fibrosis is not observed. Instillation with p-nitrophenyl-alpha-D-xylopyranoside and p-nitrophenol do not induce parenchymal destruction, indicating the specificity of beta-D-xyloside action. Urinary glycosaminoglycan (GAG) content of the beta-D-xyloside-treated rats is increased 15-fold during the first day after instillation, mainly due to elevated levels of chondroitin sulfate and dermatan sulfate. The increase is correlated to the extent of parenchymal destruction after 40 days (r = 0.68; P < 0.002). At day 2 and thereafter, levels are normal again. A short-term increase in dermatan and chondroitin sulfate content is also observed in serum, bronchoalveolar lavage (BAL) fluid, and lung tissue. Heparan sulfate content is decreased in BAL fluid and lung tissue. Instillation with p-nitrophenyl-alpha-D-xylopyranoside and p-nitrophenol do not induce elevated GAG concentration in urine. We suggest that a disturbance in proteoglycan synthesis accompanied by an increase of (beta-D-xyloside-primed) free GAGs results in loss of stability and integrity of the alveolar wall, leading to parenchymal destruction and emphysematous lesions. beta-D-xyloside treatment may be an alternative experimental method for inducing emphysema.

Animals↗
PubMed1996

Altered composition of urinary heparan sulfate in patients with COPD.

In patients with emphysema the integrity of the extracellular matrix (connective tissue skeleton) is compromised. In this study we analyzed glycosaminoglycans, which are main constituents of this matrix, in urines from patients with chronic obstructive pulmonary disease (COPD)/emphysema. Glycosaminoglycans (GAGs) were purified by anion exchange chromatography and quantified using the 1,9-dimethylmethylene blue assay. Heparan sulfate (HS) was assayed using three different chemical methods: digestion with heparitinase or with nitrous acid and by use of an adapted 1,9-dimethylmethylene blue assay. A specific epitope on the HS molecule, defined by the monoclonal antibody JM403, was determined using an inhibition enzyme immunoassay. In patients with COPD total urinary glycosaminoglycan and HS content were not altered. The JM403 epitope of HS, however, was greatly decreased in patients (0.6 versus 4.1 units/mg creatinine for control subjects, p < 0.0001). A similar pattern was observed when patients with bronchial carcinoma with and without emphysema were compared (0.4 versus 2.4 units/mg creatinine respectively, p < 0.0005). Patients with sarcoidosis did not show a decreased epitope content. These results indicate a structural change or an altered processing of the HS molecule in patients with emphysema. Taking into consideration the importance of HS for the stability of the alveolar extracellular matrix, this change may be associated with the pathogenesis of emphysema.

Adult↗