Development of volume-stable adipose tissue constructs using polycaprolactone-based polyurethane scaffolds and fibrin hydrogels.

Adipose tissue engineering aims at the restoration of soft tissue defects and the correction of contour deformities. It is therefore crucial to provide functional adipose tissue implants with appropriate volume stability. Here, we investigate two different fibrin formulations, alone or in combination with biodegradable polyurethane (PU) scaffolds as additional support structures, with regard to their suitability to generate volume-stable adipose tissue constructs. Human adipose-derived stem cells (ASCs) were incorporated in a commercially available fibrin sealant as well as a stable fibrin hydrogel previously developed by our group. The composite constructs made from the commercially available fibrin and porous poly(ε-caprolactone)-based polyurethane scaffolds exhibited increased volume stability as compared to fibrin gels alone; however, only constructs using the stable fibrin gels completely maintained their size and weight for 21 days. Adipogenesis of ASCs was not impaired by the additional PU scaffold. After induction with a common hormonal cocktail, for constructs with either fibrin formulation, strong adipogenic differentiation of ASCs was observed after 21 days in vitro. Furthermore, upregulation of adipogenic marker genes was demonstrated at mRNA (PPARγ, C/EBPα, GLUT4 and aP2; qRT-PCR) and protein (leptin; ELISA) levels. Stable fibrin/PU constructs were further evaluated in a pilot in vivo study, resulting in areas of well-vascularized adipose tissue within the implants after only 5 weeks. Copyright © 2013 John Wiley & Sons, Ltd.

Poloxamer-based hydrogels hardening at body core temperature as carriers for cell based therapies: in vitro and in vivo analysis.

Cell-based regenerative therapies for bone defects usually employ bone precursor cells seeded on solid scaffolds. Thermosensitive hydrogels that harden at body core temperature are promising alternative cell carriers as they are applicable minimally invasively. We modified Pluronic® P123 with different chain extenders and assessed rheology and biocompatibility of the resulting hydrogels. The best candidate was tested in a rat's femoral defect model. All gels hardened above 25 °C with butane-diisocyanate-hydrogels (BDI-gels) displaying the highest storage moduli. BDI-gels showed the most favourable biocompatibility and did not affect cellular adipogenic or osteogenic differentiation in vitro. Implantation of BDI-hydrogel into femoral defects did not impede bone healing in vivo as evidenced by µCT and histological analysis. We conclude that thermosensitive BDI-gels are promising alternative cell carriers. The gels harden upon injection in vivo without interfering with bone metabolism. Further experiments will assess the gels' capacity to effectively transport living cells into bone defects.

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.

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.