Cell-Friendly Chitosan-Xanthan Gum Membranes Incorporating Hydroxyapatite Designed for Periodontal Tissue Regeneration.

In this work, a simple method was proposed to produce dense composite polysaccharide-based membranes to be used for guided tissue and guided bone regeneration. The mucoadhesive polysaccharides chitosan (C) and xanthan gum (X) were used to produce polyelectrolyte-based complex membranes. Hydroxyapatite (HA) was added to the formulation as a potential drug carrier, in C:X:HA mass proportions equal to 1:1:0.4, 1:1:2, and 1:1:10, and also to improve membranes bioactivity and biomimetic properties. FTIR analysis indicated successful incorporation of HA in the membranes and XRD analysis showed that no changes in the HA crystalline structure were observed after incorporation. The residual mass evaluated by TGA was higher for the formulation produced at the proportion 1:1:10. The membranes produced showed asymmetrical surfaces, with distinct roughness. Increasing the HA concentration increased the surface roughness. Greater in vitro proliferation of dental pulp mesenchymal stem cells was observed on the surface of the membrane with 1:1:10 C:X:HA proportion. However, the 1:1:2 formulation showed the most adequate balance of mechanical and biological properties. These results suggest that adding HA to the membranes can influence mechanical parameters as well as cell adhesion and proliferation, supporting the potential application of these materials in regenerative techniques and the treatment of periodontal lesions.

Phosphorylation of chitosan to improve osteoinduction of chitosan/xanthan-based scaffolds for periosteal tissue engineering.

The periosteum is a membrane that surrounds bones, providing essential cellular and biological components for fracture healing and bone repair. Tissue engineered scaffolds able to function as periosteum substitutes can significantly improve bone regeneration in severely injured tissues. Efforts to develop more bioactive and tunable periosteal substitutes are required to improve the success of this tissue engineering approach. In this work, a chemical modification was performed in chitosan, a polysaccharide with osteoconductive properties, by introducing phosphate groups to its structure. The phosphorylated polymer (Chp) was used to produce chitosan-xanthan-based scaffolds for periosteal tissue engineering. Porous and mechanically reinforced matrices were obtained with addition of the surfactant Kolliphor® P188 and the silicone rubber Silpuran® 2130A/B. Scaffolds properties, such as large pore sizes (850-1097 µm), micro-roughness and thickness (0.7-3.5 mm in culture medium), as well as low thrombogenicity compared to standard implantable materials, extended degradation time and negligible cytotoxicity, enable their application as periosteum substitutes. Moreover, the higher adsorption of bone morphogenetic protein mimic (cytochrome C) by Chp-based formulations suggests improved osteoinductivity of these materials, indicating that, when used in vivo, the material would be able to concentrate native BMPs and induce osteogenesis. The scaffolds produced were not toxic to adipose tissue-derived stem cells, however, cell adhesion and proliferation on the scaffolds surfaces can be still further improved. The mineralization observed on the surface of all formulations indicates that the materials studied have promising characteristics for the application in bone regeneration.

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.