Chitosan-Based Inverse Opals: Three-Dimensional Scaffolds with Uniform Pore Structures for Cell Culture.

Abstract

Tissue engineering is a promising approach to the development of biological substitutes that regenerate, replace, maintain, or improve the function of damaged tissues. Among various topics related to tissue engineering, the structures and properties of the scaffolds have been studied extensively in the context of material science and biomedical engineering. There are a number of generic requirements for the scaffold: 1) the material used for fabricating the scaffold must be biocompatible and biodegradable, together with positive responses from the seeded cells; 2) the scaffold should contain a network of pores, and favorably in the form of 3D interconnected architecture; and 3) the scaffold should have proper mechanical properties to suit the specific applications, including the generation of cartilage, bone, artificial blood vessel, among others.[1] In order to generate a well-defined scaffold, numerous methods have been proposed, including emulsion freeze drying,[2] high pressure processing,[3] particulate leaching,[4] gas foaming, [5] phase separation,[6] and electrospinning.[7] However, most of these methods are rather limited in terms of capability and feasibility. For example, the electrospinning method can hardly be extended to fabricate truly 3D scaffolds. Many of the other methods typically lead to the formation of irregular pore sizes, shapes, and structures, as well as poor connectivity. The pore size and structure of a scaffold are known to play a vital role in cell culture because they are responsible for not only the adhesion, migration, and distribution of cells, but also for the exchange of nutrients and metabolite wastes. Despite extensive efforts to control the pore sizes and structures, the issues related to uniformity and interconnectivity are yet to be solved as pointed out by many researchers.[8] In addressing these issues, the inverse opal structure can be considered as an ideal system, which has the most uniform pore size and regular 3D interconnectivity. Several groups have already shown the potential of an inverse opal as scaffold for 3D tissue engineering.[9] However, the used materials, silicate and polyacrylamide, are not biodegradable and can thus limit their potential use in clinical applications. Here we describe a technique for fabricating chitosan inverse opal scaffolds characterized by a biodegradable material, uniform pore size, well-controlled interconnectivity, and nanofibrous texture on the wall surface. We used uniform poly(caprolactone) (PCL) microspheres, prepared using a simple fluidic device, as the template.[10] Chitosan was chosen as a scaffold material because of its unique nanofibrous structure that typically develops during freeze-drying, as well as its nontoxic, anti-microbial, biocompatible, and biodegradable properties. Moreover, chitosan does not need a cross-linking procedure because it is only soluble in an acidic solution. We have also evaluated the potential use of the chitosan inverse opal as 3D scaffolds in the culture of preosteoblastic cells.