ABSTRACT
Radically cross-linked hydrogels are frequently used as cell carriers due to their excellent biocompatibility and their tissue-like mechanical properties. Through frequent investigation, PEG-based polymers such as oligo(poly(ethylene glycol)fumarate [OPF] have proven to be especially suitable as cell carriers by encapsulating cells during hydrogel formation. In some cases, NaCl or biodegradable gelatin microparticles were added prior to cross-linking in order to provide space for the proliferating cells, which would otherwise stay embedded in the hydrogel matrix. However, all of these immediate cross-linking procedures involve time consuming sample preparation and sterilization directly before cell culture and often show notable swelling after their preparation. In this study, ready to use OPF-hydrogel scaffolds were prepared by gas foaming, freeze drying, individual packing into bags and subsequent γ-sterilization. The scaffolds could be stored and used "off-the-shelf" without any need for further processing prior to cell culture. Thus the handling was simplified and the sterility of the cell carrier was assured. Further improvement of the gel system was achieved using a two component injectable system, which may be used for homogenous injection molding in order to create individually shaped three dimensional scaffolds. In order to evaluate the suitability of the scaffolds for tissue engineering, constructs were seeded with juvenile bovine chondrocytes and cultured for 28 days. Cross-sections of the respective constructs showed an intense and homogenous red staining of GAG with safranin O, indicating a homogenous cell distribution within the scaffolds and the production of substantial amounts of GAG-rich matrix.
Subject(s)
Chondrocytes/drug effects , Hydrogels/chemistry , Polyesters/chemistry , Polyethylene Glycols/chemistry , Tissue Engineering/methods , Tissue Scaffolds/chemistry , Animals , Cattle , Cell Adhesion , Cell Culture Techniques , Cell Proliferation , Chondrocytes/cytology , Cross-Linking Reagents/chemistry , Free Radicals , Glycosaminoglycans/chemistry , Phenazines/chemistryABSTRACT
After trauma, articular cartilage often does not heal due to incomplete bonding of the fractured surfaces. In this study we investigated the ability of chemical cross-linkers to facilitate bonding of articular cartilage, either alone or in combination with a pre-treatment with surface-degrading agents. Articular cartilage blocks were harvested from the femoropatellar groove of bovine calves. Two cartilage blocks, either after pre-treatment or without, were assembled in a custom-designed chamber in partial apposition and subjected to cross-linking treatment. Subsequently, bonding of cartilage was measured as adhesive strength, that is, the maximum force at rupture of bonded cartilage blocks divided by the overlap area. In a first approach, bonding was investigated after treatment with cross-linking reagents only, employing glutaraldehyde, 1-ethyl-3-diaminopropyl-carbodiimide (EDC)/N-hydroxysuccinimide (NHS), genipin, or transglutaminase. Experiments were conducted with or without compression of the opposing surfaces. Compression during cross-linking strongly enhanced bonding, especially when applying EDC/NHS and glutaraldehyde. Therefore, all further experiments were performed under compressive conditions. Combinations of each of the four cross-linking agents with the degrading pre-treatments, pepsin, trypsin, and guanidine, led to distinct improvements in bonding compared to the use of cross-linkers alone. The highest values of adhesive strength were achieved employing combinations of pepsin or guanidine with EDC/NHS, and guanidine with glutaraldehyde. The release of extracellular matrix components, that is, glycosaminoglycans and total collagen, from cartilage blocks after pre-treatment was measured, but could not be directly correlated to the determined adhesive strength. Cytotoxicity was determined for all substances employed, that is, surface degrading agents and cross-linkers, using the resazurin assay. Taking the favourable cell vitality after treatment with pepsin and EDC/NHS and the cytotoxic effects of guanidine and glutaraldehyde into account, the combination of pepsin and EDC/NHS appeared to be the most advantageous treatment in this study. In conclusion, bonding of articular cartilage blocks was achieved by chemical fixation of their surface components using cross-linking reagents. Application of compressive forces and prior modulation of surface structures enhanced cartilage bonding significantly. Enzymatic treatment in combination with cross-linkers may represent a promising addition to current techniques for articular cartilage repair.
