Composite scaffolds of homogeneously mixed esterified hyaluronan (HY) and gelatin (G) were manufactured with variable component compositions (HY100%; HY95%/G5%; HY70%/G30%). The goals of this study were to analyze the produced composite scaffolds using physical and chemical methods, e.g., scanning electron microscopy, IR-spectroscopy, water contact angle, protein assay, and tensile testing as well as to assess the effects of adding gelatin to the composite scaffolds on attachment, proliferation and chondrogenic differentiation of human mesenchymal stem cells. Numbers of attached cells were significantly higher on the composite material compared to pure hyaluronan at different time points of two-dimensional or three-dimensional cell culture (p < 0.02). In composite scaffolds, a significantly greater amount of cartilage-specific extracellular matrix components was deposited after 28 days in culture (glycosaminoglycan: p < 0.001; collagen: p < 0.001) as compared with 100% hyaluronan scaffolds. Additionally, gelatin containing composite scaffolds displayed stronger promotion of collagen type II expression than pure hyaluronan scaffolds. The mechanism, by which gelatin influences cell adhesion, was examined. The effect was inhibited by collagenase treatment of the composites or by addition of α5β1-integrin blocking antibodies to the cell suspension. In summary, the results describe the establishment of a class of composite polymer scaffolds, consisting of esterified hyaluronan and gelatin, which are potentially useful for cell-based tissue engineering approaches using mesenchymal stem cells for chondrogenic differentiation.

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 pretreatments, 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 crosslinkers may represent a promising addition to current techniques for articular cartilage repair.

Basic science study.

Objective. Posttraumatic integration of articular cartilage at fracture sites is essential for mechanical stability of cartilage, and ruptured cartilage is a prerequisite for early osteoarthritis. This study was undertaken to investigate effects on articular cartilage integration mediated by steroid hormones, interleukin-1␤ (IL-1␤), and combinations thereof.Methods. Articular cartilage blocks were cultured in partial apposition for 2 weeks with ascorbic acid, testosterone, 17␤-estradiol, and dehydroepiandrosterone (DHEA), with or without IL-1␤. Mechanical integration was measured as adhesive strength, i.e., the maximum force at rupture of integrated cartilage blocks divided by the overlap area. Glycosaminoglycan content was used to study synthesized extracellular matrix.Results. Culture in medium without supplements did not lead to integration (adhesive strength 0 kPa). With administration of ascorbic acid (100 g/ml), the median adhesive strength was 49 kPa. In comparison with ascorbic acid alone, all steroid hormones induced a strong, concentration-dependent stimulation of integration (with maximum values observed with DHEA at 3 ؋ 10 ؊5 M, testosterone at 10 ؊8 M, and 17␤-estradiol at 10 ؊11 M). For testosterone and 17␤-estradiol, this was also reflected by an increase of glycosaminoglycan content. Adhesive strength was increased with IL-1␤ at 10 pg/ml, but not at 1 pg/ml or 100 pg/ml. In the presence of both IL-1␤ and sex hormones, integration of articular cartilage was reduced.Conclusion. This is the first study to demonstrate that steroid hormones such as 17␤-estradiol, DHEA, and testosterone stimulate articular cartilage integration. This effect is abrogated by low concentrations of IL-1␤. In the absence of IL-1␤ or after neutralization of IL-1␤, steroid hormones might be favorable adjuvant compounds to optimize cartilage integration.

No abstract