Insulin is essential for in vitro chondrogenesis of mesenchymal progenitor cells and influences chondrogenesis in a dose-dependent manner.

PURPOSE: Insulin is a commonly used additive in chondrogenic media for differentiating mesenchymal stem cells (MSCs). The indispensability of other bioactive factors like TGF-ß or dexamethasone in these medium formulations has been shown, but the role of insulin is unclear. The purpose of this study was to investigate whether insulin is essential for MSC chondrogenesis and if there is a dose-dependent effect of insulin on MSC chondrogenesis. METHODS: We cultivated human MSCs in pellet culture in serum-free chondrogenic medium with insulin concentrations between 0 and 50 µg/ml and assessed the grade of chondrogenic differentiation by histological evaluation and determination of glycosaminoglycan (GAG), total collagen and DNA content. We further tested whether insulin can be delivered in an amount sufficient for MSC chondrogenesis via a drug delivery system in insulin-free medium. RESULTS: Chondrogenesis was not induced by standard chondrogenic medium without insulin and the expression of cartilage differentiation markers was dose-dependent at insulin concentrations between 0 and 10 µg/ml. An insulin concentration of 50 µg/ml had no additional effect compared with 10 µg/ml. Insulin was delivered by a release system into the cell culture under insulin-free conditions in an amount sufficient to induce chondrogenesis. CONCLUSIONS: Insulin is essential for MSC chondrogenesis in this system and chondrogenic differentiation is influenced by insulin in a dose-dependent manner. Insulin can be provided in a sufficient amount by a drug delivery system. Therefore, insulin is a suitable and inexpensive indicator substance for testing drug release systems in vitro.

Long-term stable fibrin gels for cartilage engineering.

It is essential that hydrogel scaffold systems maintain long-term shape stability and mechanical integrity for applications in cartilage tissue engineering. Within this study, we aimed at the improvement of a commercially available fibrin gel in order to develop a long-term stable fibrin gel and, subsequently, investigated the suitability of the optimized gel for in vitro cartilage engineering. Only fibrin gels with a final fibrinogen concentration of 25mg/ml or higher, a Ca(2+) concentration of 20mm and a pH between 6.8 and 9 were transparent and stable for three weeks, the duration of the experiment. In contrast, when preparing fibrin gels with concentrations out of these ranges, turbid gels were obtained that shrank and completely dissolved within a few weeks. In rheological characterization experiments, the optimized gels showed a broad linear viscoelastic region and withstood mechanical loadings of up to 10,000 Pa. Bovine chondrocytes suspended in the optimized fibrin gels proliferated well and produced the extracellular matrix (ECM) components glycosaminoglycans and collagen type II. When initially seeding 3 million cells or more per construct (5mm diameter, 2mm thick), after 5 weeks of culture, a coherent cartilaginous ECM was obtained that was homogenously distributed throughout the whole construct. The developed fibrin gels are suggested also for other tissue engineering applications in which long-term stable hydrogels appear desirable.

Solid lipid templating of macroporous tissue engineering scaffolds.

Macroporous biodegradable cell carriers (scaffolds) provide the three-dimensional matrix for tissue formation in vitro. In this study, we present the fabrication of macroporous scaffolds with high inter-pore connectivity from different biodegradable polymers using the recently developed solid lipid templating technique. Starting from a polymer solution and solid lipid microparticles, a dispersion is prepared and subsequently transferred into molds, which are finally submerged in warm hexane to precipitate the polymer and extract the porogens. The study shows how to control pore structure, pore size and porosity of the scaffold using this technique. The process parameters dispersion viscosity, porogen size and type of polymer are considered. Limits of viscosity are examined by macroscopic and microstructure evaluation of the scaffolds prepared at different viscosities. An approach to rationalize these data by oscillation rheometry is shown. Pore size can be controlled by porogen particle size and adaptation of the viscosity of the polymer solution. Porosity can be modified by changing the ratio of porogen to polymer. The suitability of the resulting scaffolds was shown using an established cartilage cell culture model.

In vitro and in vivo cartilage engineering using a combination of chondrocyte-seeded long-term stable fibrin gels and polycaprolactone-based polyurethane scaffolds.

The use of either a hydrogel or a solid polymeric scaffold alone is often associated with distinct drawbacks in many tissue engineering applications. Therefore, in this study, we investigated the potential of a combination of long-term stable fibrin gels and polyurethane scaffolds for cartilage engineering. Primary bovine chondrocytes were suspended in fibrin gel and subsequently injected into a polycaprolactone-based polyurethane scaffold. Cells were homogeneously distributed within this composite system and produced high amounts of cartilage-specific extracellular matrix (ECM) components, namely glycosaminoglycans (GAGs) and collagen type II, within 4 weeks of in vitro culture. In contrast, cells seeded directly onto the scaffold without fibrin resulted in a lower seeding efficiency and distinctly less homogeneous matrix distribution. Cell-fibrin-scaffold constructs implanted into the back of nude mice promoted the formation of adequate engineered cartilaginous tissue within the scaffold after 1, 3, and 6 months in vivo, containing evenly distributed ECM components, such as GAGs and collagen. Again, in constructs seeded without fibrin, histology showed an inhomogeneous and, thus, not adequate ECM distribution compared to seeding with fibrin, even after 6 months in vivo. Strikingly, a precultivation for 1 week in vitro elicited similar results in vivo compared to precultivation for 4 weeks; that is, a precultivation for longer than 1 week did not enhance tissue development. The presented composite system is suggested as a promising alternative toward clinical application of engineered cartilaginous tissue for plastic and reconstructive surgery.

Micronization of insulin by high pressure homogenization.

PURPOSE: The aim of this study was to establish the high pressure homogenization of proteins in non-aqueous suspension as an alternative method for classical micronization strategies and to investigate the effect of high pressure on protein stability and bioactivity. METHODS: The influence of drug loading, homogenization pressure and cycles on particle size reduction was investigated by experimental design using a Box Behnken matrix with insulin as a model compound. Particle size measurements were performed by laser light scattering. Protein stability was investigated by HPLC and HPLC-MS analysis and the bioactivity of insulin was tested in a chondrocyte proliferation assay. For investigations into the effect of temperature on protein stability, insulin was micronized in molten lipid at 75 degrees C in one cycle at 1,000 bar. RESULTS: Within one homogenization cycle at 1,500 bar, the particle size of insulin could be reduced from 15.8 to 7.3 microm, six cycles resulted in a particle size of 3.7 microm d(0.5) (50% of the particles are smaller than the indicated value). Evaluation of the response surface diagram revealed that the homogenization pressure had the highest impact on micronization efficiency, followed by the number of homogenization cycles. Protein stability was maintained during the micronization process as well as bioactivity. Micronization at elevated temperature (75 degrees C) had no effect on protein stability. CONCLUSION: High pressure homogenization of protein suspensions can be used as an alternative method for the micronization of proteins without affecting protein stability or bioactivity.