Interplay of matrix stiffness and stress relaxation in directing osteogenic differentiation of mesenchymal stem cells.

The aim of this study is to investigate the impact of the stiffness and stress relaxation of poly(acrylamide-co-acrylic acid) hydrogels on the osteogenic differentiation of human mesenchymal stem cells (hMSCs). Varying the amount of the crosslinker and the ratio between the monomers enabled the obtainment of hydrogels with controlled mechanical properties, as characterized using unconfined compression and atomic force microscopy (AFM). Subsequently, the surface of the hydrogels was functionalized with a mimetic peptide of the BMP-2 protein, in order to favor the osteogenic differentiation of hMSCs. Finally, hMSCs were cultured on the hydrogels with different stiffness and stress relaxation: 15 kPa - 15%, 60 kPa - 15%, 140 kPa - 15%, 100 kPa - 30%, and 140 kPa - 70%. The cells on hydrogels with stiffnesses from 60 kPa to 140 kPa presented a star-like shape, typical of osteocytes, which has only been reported by our group for two-dimensional substrates. Then, the extent of hMSC differentiation was evaluated by using immunofluorescence and by quantifying the expression of both osteoblast markers (Runx-2 and osteopontin) and osteocyte markers (E11, DMP1, and sclerostin). It was found that a stiffness of 60 kPa led to a higher expression of osteocyte markers as compared to stiffnesses of 15 and 140 kPa. Finally, the strongest expression of osteoblast and osteocyte differentiation markers was observed for the hydrogel with a high relaxation of 70% and a stiffness of 140 kPa.

Evaluating Poly(Acrylamide-co-Acrylic Acid) Hydrogels Stress Relaxation to Direct the Osteogenic Differentiation of Mesenchymal Stem Cells.

The aim of this study is to investigate polyacrylamide-based hydrogels stress relaxation and the subsequent impact on the osteogenic differentiation of human mesenchymal stem cells (hMSCs). Different hydrogels are synthesized by varying the amount of cross-linker and the ratio between the monomers (acrylamide and acrylic acid), and characterized by compression tests. It has been found that hydrogels containing 18% of acrylic acid exhibit an average relaxation of 70%, while pure polyacrylamide gels show an average relaxation of 15%. Subsequently, hMSCs are cultured on two different hydrogels functionalized with a mimetic peptide of the bone morphogenetic protein-2 to enable cell adhesion and favor their osteogenic differentiation. Phalloidin staining shows that for a constant stiffness of 55 kPa, a hydrogel with a low relaxation (15%) leads to star-shaped cells, which is typical of osteocytes, while a hydrogel with a high relaxation (70%) presents cells with a polygonal shape characteristic of osteoblasts. Immunofluorescence labeling of E11, strongly expressed in early osteocytes, also shows a dramatically higher expression for cells cultured on the hydrogel with low relaxation (15%). These results clearly demonstrate that, by fine-tuning hydrogels stress relaxation, hMSCs differentiation can be directed toward osteoblasts, and even osteocytes, which is particularly rare in vitro.

Polysaccharide-based tissue-engineered vascular patches.

Coronary artery and peripheral vascular diseases are the leading cause of morbidity and mortality worldwide and often require surgical intervention to replace damaged blood vessels, including the use of vascular patches in endarterectomy procedures. Tissue engineering approaches can be used to obtain biocompatible and biodegradable materials directed to this application. In this work, dense or porous scaffolds constituted of chitosan (Ch) complexed with alginate (A) or pectin (P) were fabricated and characterized considering their application as tissue-engineered vascular patches. Scaffolds fabricated with alginate presented higher culture medium uptake capacity (up to 17 g/g) than materials produced with pectin. A degradation study of the patches in the presence of lysozyme showed longer-term stability for Ch-P-based scaffolds. Pectin-containing matrices presented higher elastic modulus (around 280 kPa) and ability to withstand larger deformations. Moreover, these materials demonstrated better performance when tested for hemocompatibility, with lower levels of platelet adhesion and activation. Human smooth muscle cells (HSMC) adhered, spread and proliferated better on matrices produced with pectin, probably as a consequence of cell response to higher stiffness of this material. Thus, the outcomes of this study demonstrate that Ch-P-based scaffolds present superior characteristics for the application as vascular patches. Despite polysaccharides are yet underrated in this field, this work shows that biocompatible tridimensional structures based on these polymers present high potential to be applied for the reconstruction and regeneration of vascular tissues.

Comparative study on complexes formed by chitosan and different polyanions: Potential of chitosan-pectin biomaterials as scaffolds in tissue engineering.

Polyelectrolyte complexes of chitosan (Ch) and pectin (Pc) or alginate (Alg) were produced in the presence or absence of the silicone gel Silpuran® 2130 A/B (Sil) and the surfactant Kolliphor® P188 (Kol). Ch-Pc-Kol-based formulations presented higher porosity (up to 83.3%) and thickness (maximum of 2273.5 µm in PBS). Lower water contact angle was observed for Ch-Alg formulations (minimum of 36.8°) and these formulations presented higher swelling and mass loss in PBS (reaching up to 21.7 g/g and 80.4%, respectively). The addition of Sil to the matrices improved their elastic moduli, reaching a maximum of 4-fold change at 40% strain. The use of pectin instead of alginate augmented the elastic moduli, reaching 66 and 4-fold changes for dense and porous formulations, respectively. Pectin-containing scaffolds presented poroviscoelasticity, a typical mechanical feature of many soft tissues. The suitability of the materials for tissue engineering applications was demonstrated in terms of stability upon degradation in culture medium or lysozyme solution, as well as lack of cytotoxicity. This study evidences the potential of Ch-Pc-based materials to be further explored for this purpose, especially to improve the mechanical properties of chitosan-based scaffolds aiming medical applications.

Mechanically-enhanced polysaccharide-based scaffolds for tissue engineering of soft tissues.

Collagen-based materials are probably among the most used class of biomaterials in tissue engineering and regenerative medicine. Although collagen is often privileged for providing a suitable substrate on which cells can be cultured or a matrix in which cells can be dispersed, its mechanical properties represent a major limitation for clinical translation and even for handling of the obtained regenerated tissue. In this work, the combination of polysaccharides chitosan (Ch) and xanthan gum (X) was investigated as an alternative for scaffolds for soft tissue engineering. Moreover, in an attempt to reach a compromise between obtaining highly porous biomaterials while maintaining appropriate mechanical properties, a surfactant (Kolliphor® P188, K) was added to Ch-X matrices to generate pores, while silicone rubber (Silpuran® 2130A/B, S) was used to balance their mechanical properties. Addition of K (10 or 25% w/w) increased the porosity and pore-dimensions, while addition of S improved by up to 156% and 85% the elastic moduli and the elastic behavior of Ch-X-based scaffolds, under both compressive and tensile loads, respectively, at 50% strain. Relaxation tests confirmed that these materials do have a viscoelastic behavior. The presence of S increased thickness and microscale surface roughness and did not affect liquid uptake and stability, thrombogenicity, biodegradation and cytotoxicity of polysaccharide-based scaffolds. In conclusion, this work shows that Ch-X-S porous blends constitute suitable scaffolds for soft tissue engineering.

Mathematical modeling of uniaxial mechanical properties of collagen gel scaffolds for vascular tissue engineering.

Small diameter tissue-engineered arteries improve their mechanical and functional properties when they are mechanically stimulated. Applying a suitable stress and/or strain with or without a cycle to the scaffolds and cells during the culturing process resides in our ability to generate a suitable mechanical model. Collagen gel is one of the most used scaffolds in vascular tissue engineering, mainly because it is the principal constituent of the extracellular matrix for vascular cells in human. The mechanical modeling of such a material is not a trivial task, mainly for its viscoelastic nature. Computational and experimental methods for developing a suitable model for collagen gels are of primary importance for the field. In this research, we focused on mechanical properties of collagen gels under unconfined compression. First, mechanical viscoelastic models are discussed and framed in the control system theory. Second, models are fitted using system identification. Several models are evaluated and two nonlinear models are proposed: Mooney-Rivlin inspired and Hammerstein models. The results suggest that Mooney-Rivlin and Hammerstein models succeed in describing the mechanical behavior of collagen gels for cyclic tests on scaffolds (with best fitting parameters 58.3% and 75.8%, resp.). When Akaike criterion is used, the best is the Mooney-Rivlin inspired model.