Natural stimulus responsive scaffolds/cells for bone tissue engineering : influence of lysozyme upon scaffold degradation and osteogenic differentiation of cultured marrow stromal cells induced by CaP coatings

Abstract

This work proposes the use of nonporous, smart, and stimulus responsive chitosan-based scaffolds for bone tissue engineering applications. The overall vision is to use biodegradable scaffolds based on chitosan and starch that present properties that will be regulated by bone regeneration, with the capability of gradual in situ pore formation. Biomimetic calcium phosphate (CaP) coatings were used as a strategy to incorporate lysozyme at the surface of chitosan-based materials with the main objective of controlling and tailoring their degradation profile as a function of immersion time. To confirm the concept, degradation tests with a lysozyme concentration similar to that incorporated into CaP chitosan-based scaffolds were used to study the degradation of the scaffolds and the formation of pores as a function of immersion time. Degradation studies with lysozyme (1.5 g=L) showed the formation of pores, indicating an increase of porosity (*5–55% up to 21 days) resulting in porous threedimensional structures with interconnected pores. Additional studies investigated the influence of a CaP biomimetic coating on osteogenic differentiation of rat marrow stromal cells (MSCs) and showed enhanced differentiation of rat MSCs seeded on the CaP-coated chitosan-based scaffolds with lysozyme incorporated. At all culture times, CaP-coated chitosan-based scaffolds with incorporated lysozyme demonstrated greater osteogenic differentiation of MSCs, bone matrix production, and mineralization as demonstrated by calcium deposition measurements, compared with controls (uncoated scaffolds). The ability of these CaP-coated chitosan-based scaffolds with incorporated lysozyme to create an interconnected pore network in situ coupled with the demonstrated positive effect of these scaffolds upon osteogenic differentiation of MSCs and mineralized matrix production illustrates the strong potential of these scaffolds for application in bone tissue engineering strategies.The authors would like to acknowledge Dr. Serena Danti. This work was supported by the European NoE EX-PERTISSUES (NMP3-CT-2004-500283), the European STREP HIPPOCRATES (NMP3-CT-2003-505758), and the Portuguese Foundation for Science and Technology (FCT) through POCTI and/or FEDER programs. This work was also supported by a grant from the National Institutes of Health (NIH; R01 DE15164) (A. G. M.) and a Bioengineering Research Partnership with the Baylor College of Medicine through the National Institute of Biomedical Imaging and Bioengineering (NIH Grant 5 R01 EB005173-02). F. K. K. is supported by a training fellowship from the Keck Center Nanobiology Training Program of the Gulf Coast Consortia (NIH Grant 5 T90 DK070121-03)