Dimethyl 3,3'-dithiobispropionimidate: a novel crosslinking reagent for collagen

Crosslinking agents are used for improving the physical properties and durability of collagenous implants, glutaraldehyde (GTA) being the most widely used. Many of these reagents, including GTA, are known to be cytotoxic and to induce calcification. Hence, it is desirable to develop new crosslinking methods for collagen, so that biocompatibility and physical properties are improved. In the present study, dimethyl 3,3' -dithiobispropionimidate (DTBP) has been tried as a novel crosslinking reagent for collagen. Collagen purified from rat tail tendon has been crosslinked with DTBP and GTA. An increase of 22 degrees C in shrinkage temperature is observed for collagen treated with DTBP under optimal conditions. Crosslinking density determination shows that DTBP induces 10 crosslinks per mole, compared to 13 by GTA. While the tensile strength of GTA-treated samples is greater than those treated with DTBP, the latter shows more extensibility. In vitro degradation studies show that both GTA- and DTBP-treated samples are resistant to degradation by collagenase. The biocompatibility of crosslinked collagen samples studied by subcutaneous implantation in rats show that while both GTA- and DTBP-treated collagen do not degrade for up to 4 weeks, ultrastructural and histological studies indicate that DTBP collagen is far more biocompatible than GTA-treated matrices.     

Chemically modified collagen: a natural biomaterial for tissue replacement

Glutaraldehyde crosslinking of native or reconstituted collagen fibrils and tissues rich in collagen significantly reduces biodegradation. Other aldehydes are less efficient than glutaraldehyde in generating chemically, biologically, and thermally stable crosslinks. Tissues crosslinked with glutaraldehyde retain many of the viscoelastic properties of the native collagen fibrillar network which render them suitable for bioprostheses. Implants of collagenous materials crosslinked with glutaraldehyde are subject long-term to calcification, biodegradation, and low-grade immune reactions. We have attempted to overcome these problems by enhancing crosslinking through bridging of activated carboxyl groups with diamines and using glutaraldehyde to crosslink the epsilon-NH2 groups in collagen and the unreacted amines introduced by aliphatic diamines. This crosslinking reduces tissue degradation and nearly eliminates humoral antibody induction. Covalent binding of diphosphonates, specifically 3-amino-1-hydroxypropane-1, 1-diphosphonic acid (3-APD), and chondroitin sulfate to collagen or to the crosslink-enhanced collagen network reduces its potential for calcification. Platelet aggregation is also reduced by glutaraldehyde crosslinking and nearly eliminated by the covalent binding of chondroitin sulfate to collagen. The cytotoxicity of residual glutaraldehyde--leaching through the interstices of the collagen fibrils or the tissue matrix--and of reactive aldehydes associated with the bound polymeric glutaraldehyde can be minimized by neutralization and thorough rinsing after crosslinking and storage in a nontoxic bacteriostatic solution.     

Modification of collagen matrices for enhancing angiogenesis

The vascularization of engineered tissues in many cases does not keep up with the ingrowth of cells. Nutrient and oxygen supply are not sufficient, which ultimately leads to the death of the invading cells. The enhancement of the angiogenic capabilities of engineered tissues therefore represents a major challenge in the field of tissue engineering. The immobilization of angiogenic growth factors may be useful for enhancing angiogenesis. The most potent angiogenic growth factor specific to endothelial cells, vascular endothelial growth factor (VEGF), occurs in several splice variants. The variant with 165 amino acids both has a high angiogenic activity and a high affinity for heparin. We therefore incorporated heparin molecules into collagen matrices by covalently cross-linking them to amino functions on the collagen. Physical binding of VEGF to the heparin may then prevent a rapid clearance from the implant, while the release rate may become coupled to the degradation of the collagen matrix. The modified matrices were characterized by determination of the extent of the heparin immobilization, the in vitro degradation rate by collagenase. For testing the angiogenic properties, non-modified and heparinized collagen specimens were--either loaded with VEGF or non-loaded--subcutaneously implanted on the back of rats. Specimens were explanted after varying periods of implantation, the dry weights and the hemoglobin contents, as well as immunostained histological sections were evaluated: heparinized collagen matrices loaded with VEGF are vascularized to a substantially higher extent as compared to non-modified matrices.     

Characterization of collagen gel solutions and collagen matrices for cell culture

The influence of glutaraldehyde as a crosslinking agent to increase the strength of collagen matrices for cell culture was examined in this study. Collagen solutions of 1% were treated with different concentrations (0-0.2%) of glutaraldehyde for 24 h. The viscoelasticity of the resulting collagen gel solution was measured using dynamic mechanical analysis (DMA), which demonstrated that all collagen gel solutions examined followed the same model pattern. The creep compliance model of Voigt-Kelvin satisfactorily described the change of viscoelasticity expressed by these collagen gel solutions. These crosslinked collagen gel solutions were freeze-dried to form a matrix with a thickness of about 0.2-0.3 mm. The break modulus of these collagen matrices measured by DMA revealed that the higher the degree of crosslinking. the higher the break modulus. The compatibility of fibroblasts isolated from nude mouse skin with these collagen matrices was found to be acceptable at a cell density of 3 x 10(5) cells/cm2 with no contraction, even when using a concentration of glutaraldehyde of up to 0.2%.     

Effects of a novel cell stabilizing reagent on DNA amplification by PCR as compared to traditional stabilizing reagents

Stabilization of nucleated blood cells by cell stabilizing reagent (BCT reagent) present in the Cell-Free DNA BCT™ blood collection device and consequent prevention of cell-free DNA contamination by cellular DNA during sample storage and shipping have previously been reported. This study was conducted to investigate the effect of this novel cell stabilizing reagent on DNA amplification by PCR as compared to traditional cell stabilizing reagents, formaldehyde and glutaraldehyde. A 787 bp long DNA fragment from human glyceraldehydes-3-phosphate dehydrogenase (GAPDH) gene was amplified by PCR and used as model system. DNA samples and blood samples were treated with BCT reagent, 0.1% formaldehyde or 0.1% glutaraldehyde at room temperature. DNA amplification was studied using conventional and real-time quantitative PCR. Results indicate that exposure of DNA to the BCT reagent for up to 14 days had no effect on DNA amplification by PCR as compared to the untreated control DNA. However, there was statistically significant decrease in DNA amplification in the DNA samples treated with formaldehyde and glutaraldehyde. We conclude that the BCT reagent used in Cell-Free DNA BCT blood collection device to prevent cell-free DNA contamination by cellular DNA had no effect on DNA amplification by PCR.     

Semi-synthetic collagen/poloxamine matrices for tissue engineering

Collagen-containing poloxamine hydrogels were produced with the aim of overcoming the low stiffness displayed by collagen gels that are not otherwise chemically crosslinked. Matrices were obtained by functionalization of a four-arm PEO-PPO block copolymer (poloxamine, Tetronic) with methcrylate groups and subsequent free radical polymerization of water solutions of the modified polymer in the presence of collagen. The resulting matrices had a sharp increase in stiffness, when compared to pure collagen gels. For example, whereas collagen had a storage modulus (G') around 70 Pa and a loss modulus (G') of 10 Pa, a crosslinked collagen/poloxamine system containing 8.3% crosslinked poloxamine had G' and G' values of 7400 and 1000 Pa, respectively. HepG2 cells were seeded within the gels before the crosslinking and the viability levels estimated by AlamarBlue assay were between 65% and 91% for systems containing 0.04-0.09 wt% photoinitiator. HepG2 and endothelial cells also adhered to and spread on the surface of the collagen-containing specimens, suggesting their potential utility in tissue engineering.     

Application of collagen matrices for cartilage tissue engineering.

Articular cartilage shows little capacity for self-repair once it has been damaged. The aim of this study was to investigate different collagen matrices regarding their applicability for cartilage tissue engineering. The matrices consist of collagen I and small amounts of elastine, were crosslinked with carbodiimide or glucose. Primary chondrocytes were seeded onto these different collagen matrices and cultured with or without differentiation medium. The viability of the cells was monitored via MTT test. The arrangement of the cells onto the scaffold was investigated by histological staining. Furthermore, extracellular matrix synthesis was studied by immunohistological staining, especially the expression of the typical chondrogenic marker collagen II. Moreover gene expression for collagen type II was analysed by RT-PCR. The chondrocytes showed high viability on all matrices used. The results for the histological staining revealed a three-dimensional arrangement of the chondrocytes in the collagen matrices. Moreover, the matrices also supported chondrogenic differentiation. On the matrix MATRIDERM 2 mm the synthesis of collagen II was stimulated without adding any differentiation supplements to the cell culture medium, as observed by immunohistological staining and by gene expression analysis of collagen II.     

Glyoxal crosslinking of cell-seeded chitosan/collagen hydrogels for bone regeneration

Chitosan and collagen are natural biomaterials that have been used extensively in tissue engineering, both separately and as composite materials. Most methods to fabricate chitosan/collagen composites use freeze drying and chemical crosslinking to create stable porous scaffolds, which subsequently can be seeded with cells. In this study, we directly embedded human bone marrow stem cells (hBMSC) in chitosan/collagen materials by initiating gelation using β-glycerophosphate at physiological temperature and pH. We further examined the use of glyoxal, a dialdehyde with relatively low toxicity, to crosslink these materials and characterized the resulting changes in matrix and cell properties. The cytocompatibility of glyoxal and the crosslinked gels were investigated in terms of hBMSC metabolic activity, viability, proliferation and osteogenic differentiation. These studies revealed that glyoxal was cytocompatible at concentrations below about 1mM for periods of exposure up to 15 h, though the degree of cell spreading and proliferation were dependent on matrix composition. Glyoxal-crosslinked matrices were stiffer and compacted less than uncrosslinked controls. It was further demonstrated that hBMSC can attach and proliferate in three-dimensional matrices composed of 50/50 chitosan/collagen, and that these materials supported osteogenic differentiation in response to stimulation. Such glyoxal-crosslinked chitosan/collagen composite materials may find utility as cell delivery vehicles for enhancing the repair of bone defects.     

Efficacy of polyanionic collagen matrices for bone defect healing

Polyanionic collagen-elastin matrices (PCEMs) possess attractive properties, such as extra negative charges, piezoelectricity, and extra RGD sites, which could make them effective in the treatment of bone defects. Although they are biocompatible with the osteogenesis process, it is unknown if PCEMs could aid in the recovery of bone defects in challenging situations. To evaluate this hypothesis, three PCEMs, differing in their negative charge density, were implanted in rat calvarial defects. Specimens harvested at 3, 7, 15, 30, and 60 days after implantation were evaluated radiographically and histologically. Two matrices were able to sustain the osteogenesis process and quickly recover the lost bone structure. The third, and most electronegative, left matrix remnants amidst the areas of new bone. The control showed bone formation limited to the boundaries of the defect. These results suggest that some PCEMs might become a useful resource in the treatment of bone defects.     

In vitro enhancement of collagen matrix formation and crosslinking for applications in tissue engineering: a preliminary study

The construction of stable engineered tissue depends on the formation of a functional connective tissue produced by cells locally. A major component of connective tissue is collagen. Its deposition into a stable matrix depends on the enzymatic extracellular conversion of procollagen to collagen. This step is very slow in vitro and we hypothesized that this is due to a lack of crowdedness and insufficient excluded volume effect (EVE) in culture media. We used neutral (670 kDa) and negatively charged dextran sulfate (DxS, 500 kDa) to create EVE in cell cultures and to enhance in vitro matrix formation by accelerating procollagen conversion. Biochemical analyses in 2 human fibroblast lines revealed mostly unprocessed procollagen in uncrowded culture medium, whereas in the presence of DxS, procollagen conversion occurred and most of the collagen was associated with the cell layer. Immunocytochemistry confirmed DxS-related collagen deposition that colocalized with fibronectin. The large neutral dextran showed, in identical concentration ranges, no effects that correlated well with its smaller hydrodynamic radius as determined by dynamic light scattering. This predicted a 10 times bigger crowding power of DxS and benchmarks it as a potentially promising crowding agent facilitating the formation of extracellular matrix in vitro.     

Structural mechanism for alteration of collagen gel mechanics by glutaraldehyde crosslinking

Soft collagenous tissues that are loaded in vivo undergo crosslinking during aging and wound healing. Bioprosthetic tissues implanted in vivo are also commonly crosslinked with glutaraldehyde (GA). While crosslinking changes the mechanical properties of the tissue, the nature of the mechanical changes and the underlying microstructural mechanism are poorly understood. In this study, a combined mechanical, biochemical and simulation approach was employed to identify the microstructural mechanism by which crosslinking alters mechanical properties. The model collagenous tissue used was an anisotropic cell-compacted collagen gel, and the model crosslinking agent was monomeric GA. The collagen gels were incrementally crosslinked by either increasing the GA concentration or increasing the crosslinking time. In biaxial loading experiments, increased crosslinking produced (1) decreased strain response to a small equibiaxial preload, with little change in response to subsequent loading and (2) decreased coupling between the fiber and cross-fiber direction. The mechanical trend was found to be better described by the lysine consumption data than by the shrinkage temperature. The biaxial loading of incrementally crosslinked collagen gels was simulated computationally with a previously published network model. Crosslinking was represented by increased fibril stiffness or by increased resistance to fibril rotation. Only the latter produced mechanical trends similar to that observed experimentally. Representing crosslinking as increased fibril stiffness did not reproduce the decreased coupling between the fiber and cross-fiber directions. The study concludes that the mechanical changes in crosslinked collagen gels are caused by the microstructural mechanism of increased resistance to fibril rotation.     

Pexiganan-incorporated collagen matrices for infected wound-healing processes in rat

The use of peptide-based drugs is limited by their rapid degradability and toxicity at high concentration during their therapeutic application. These problems could be managed by the use of a peptide delivery agent for sustained release in the site of action. Collagen is one of the most proven biomaterials of good biocompatibility with an exceptional ligand encapsulating property. In this work, we have shown that pexiganan, an antimicrobial, 22-amino-acid peptide could be incorporated and delivered to the wound-healing site against bacterial strains Pseudomonas aeruginosa and Staphylococcus aureus. The release profiles of pexiganan collagen films with different collagen concentration were studied. The release of pexiganan from 2.5% w/w of collagen film showed a sustainable activity over 72 h with effective antimicrobial concentrations. Pexiganan-incorporated collagen (PIC)-treated groups were compared with open wound (OW)- and collagen film (CF)-treated rats. PIC-treated animals showed a diminishing level of bacterial growth as compared with OW- and CF-treated animals. The biochemical parameters such as hydroxyproline, protein, DNA, uronic acid, hexosamine, SOD, and catalase content in the granulation tissue of the healing wound revealed increased proliferation of cells involved in tissue reconstruction in PIC-treated groups when compared with OW- and CF-treated groups. Furthermore, spectroscopic studies suggested that collagen structure is not perturbed by pexiganan incorporation. This study provides rationale for application of collagen membrane for antimicrobial peptide delivery in infected wounds.     

Glucose oxidase incorporated collagen matrices for dermal wound repair in diabetic rat models: a biochemical study

Impaired wound healing in diabetes is a well-documented phenomenon. Emerging data favor the involvement of free radicals in the pathogenesis of diabetic wound healing. We investigated the beneficial role of the sustained release of reactive oxygen species (ROS) in diabetic dermal wound healing. In order to achieve the sustained delivery of ROS in the wound bed, we have incorporated glucose oxidase in the collagen matrix (GOIC), which is applied to the healing diabetic wound. Our in vitro proteolysis studies on incorporated GOIC show increased stability against the proteases in the collagen matrix. In this study, GOIC film and collagen film (CF) are used as dressing material on the wound of streptozotocin-induced diabetic rats. A significant increase in ROS (p?相似文献   

Alpha-crystallin-incorporated collagen matrices as an aid for dermal wound healing

This study evaluated the effects of noncovalently incorporated crystallin into the collagen matrix for dermal wound-healing processes in rats. Crystallin-incorporated collagen matrix (CIC) showed better healing when compared to wounds treated with collagen matrix (CS) and without collagen (CR). Biochemical parameters and histological analysis revealed that increased wound contraction enhanced cell proliferation and efficient radical scavenging in the CIC group. The higher shrinkage temperature of CIC films when compared to CS groups suggested increased hydrothermal stability for the former material. An in vitro release study of CIC has showed sustained and time-dependent release of crystallin from the collagen matrix. These results demonstrate the possibility of using crystallin as therapeutic protein in the wound-healing process.     

Chondrocyte-seeded collagen matrices implanted in a chondral defect in a canine model

The objective of our study was to evaluate reparative tissues formed in chondral defects in an adult canine model implanted with cultured autologous articular chondrocytes seeded in type I and II collagen–GAG matrices. Two defects were produced in the trochlea grooves of the knees of 21 dogs, with cartilage removed down to the tidemark. This study includes the evaluation of 36 defects distributed among five treatment groups: Group A, type II collagen matrix seeded with autologous chondrocytes under a sutured type II collagen flap; Group B, type I collagen matrices seeded with chondrocytes under a sutured fascia flap; Group C, unseeded type I collagen matrix implanted under a sutured fascia flap; Group D, fascia lata flap alone; and Group E, untreated defects. All animals were killed 15 weeks after implantation. Six other defects were created at the time of death and evaluated immediately after production as ‘acute defect controls’. In three additional defects, unseeded matrices were sutured to the defect and the knee closed and reopened after 30 min to determine if early displacement of the graft was occurring; these defects served as ‘acute implant controls’. The areal percentages of four tissue types in the chondral zone of the original defect were determined histomorphometrically: fibrous tissue (FT); hyaline cartilage (HC); transitional tissue (TT, including fibrocartilage); and articular cartilage (AC). New tissue formed in the remodeling subchondral bone underlying certain defects was also assessed. Bonding of the repair tissue to the subchondral plate and adjacent cartilage, and degradation of the adjacent tissues were evaluated.

There were no significant differences in the tissues filling the original defect area of the sites treated with chondrocyte-seeded type I and type II matrices. Most of the tissue in the area of the original defect in all of the groups was FT and TT. The areal percentage of HC plus AC was highest in group E, with little such tissue in the cell-seeded groups, and none in groups C and D. The greatest total amount of reparative tissue, however, was found in the cell-seeded type II matrix group. Moreover, examination of the reparative tissue formed in the subchondral region of defects treated with the chondrocyte-seeded collagen matrices (Groups A and B) demonstrated that the majority of the tissue was positive for type II collagen and stained with safranin O. These results indicate an influence of the exogenous chondrocytes on the process of chondrogenesis in this site. In all groups with implants (A–D), 30–50% of the FT and TT was bonded to the adjacent cartilage. Little of this tissue (6–22%) was attached to the subchondral plate, which was only about 50% intact. Remarkable suture damage was found in sections from each group in which sutures were used. Harvest sites showed no regeneration of normal articular cartilage, 18 weeks after the biopsy procedure.

Future studies need to investigate other matrix characteristics, and the effects of cell density and incubation of the seeded sponges prior to implantation on the regenerative response.     

Production of ordered collagen matrices for three-dimensional cell culture.

The aim of this study was to produce collagen gels with controlled fibrillar order as matrices for cell culture. Their structural characterization and colonization by human dermal fibroblasts arc presently reported. Ordered matrices are obtained by using the property of type I collagen monomers to self-assemble in liquid crystalline arrays by slow evaporation of acidic solutions at high concentrations. Induction of fibrillogenesis concomittent with the stabilization of the supramolecular order is then obtained, within petri dishes, by gelation of the viscous preparations under ammoniac vapours. For comparison, dermal equivalents, in which collagen compaction depends on fibroblasts contraction, are made according to the method of Bell et al. (Proc. Natl. Acad. Sci. 76(3) (1979) 1274). The fibrillar arrangement of the collagen network in the samples is determined by polarizing optical microscopy and by transmission electron microscopy. Whereas dermal equivalents exhibit heterogeneous distributions of fibrils, two differents types of order are obtained in the stabilized liquid crystalline collagen samples, namely aligned, i.e. nematic, at 20 mg/ml, or crimped, i.e. precholesteric, at 40 mg/ml. The morphology and behaviour of fibroblasts seeded on the surface of the matrices are analysed from day 1 to day 21. The cells are viable, proliferate at the surface of ordered matrices and migrate up to 400 microm in depth. Production of concentrated and ordered collagen matrices provides new perspectives to study the behaviour of cells in a valorized three-dimensional context where the fibrillar organization becomes close to in vivo situations.     

Dense fibrillar collagen matrices: a model to study myofibroblast behaviour during wound healing

Fibroblastic cells play an important part in wound healing. Human dermal fibroblasts seeded onto three-dimensional fibrillar collagen matrices migrate into the collagen network and differentiate into myofibroblasts. In order to evaluate the use of collagen matrices as model systems for studying myofibroblast phenotype during wound healing, myofibroblast behaviour migrating into dense or loose matrices was compared. The effect of collagen concentration on cell morphology, remodelling, proliferation and apoptosis of human myofibroblasts was evaluated. Myofibroblasts within dense collagen matrices (40 mg/ml) were spindle shaped, similar to cells observed during tissue repair. In contrast, cells within loose matrices (5mg/ml) were more rounded. Matrix hydrolysis activities (MT1-MMP and MMP2) did not differ between the two collagen concentrations. The myofibroblast proliferation rate was measured after 24h bromodeoxyuridine incorporation (BrdU). Cells in dense collagen matrices proliferated at a higher rate than cells in loose matrices at each culture time point tested. For example, 40% of cells in dense matrices were replicating compared to 10% of cells in loose matrices after 28 days in culture. Apoptotic cells were only detected in dense matrices from day 21 onwards when cells had already migrated into the collagen network. Taken together, these results show that a high collagen concentration has a stimulatory effect on myofibroblast proliferation and apoptosis, two important events in wound healing. Thus, dense matrices can be used to create controlled conditions to study myofibroblast phenotype.     

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.