Scaffold design is important in tissue engineering due to its role in supporting cell attachment and growth, assisting exchanges of nutrients/oxygen and metabolite wastes, and eventually facilitating the formation of a functional tissue/organ. Many parameters need to be considered for scaffold design: pore size, porosity, interconnectivity, surface properties, and mechanical properties, among others.
For bone tissue engineering, hydroxyapatite (HAp), a major inorganic component in natural bone, has been often used due to its osteoinductivity, high mechanical strength, and biocompatibility.1
Therefore, much attention has been paid to the impregnation of a scaffold matrix with HAp nanoparticles and the decoration of a scaffold surface with apatite (Ap) using a simulated body fluid (SBF).2
Many research groups have demonstrated that incorporation of HAp nanoparticles and Ap coating could enhance preosteoblast differentiation and mineral secretion.3
However, most of these studies were limited to the evaluation of gross properties (e.g., overall mineral content). Few studies have discussed the microscopic distribution of mineral throughout the scaffolds. The uniform distribution of cells and extracellular matrix (ECM) in the scaffold with suitable mechanical properties is critical for successful bone tissue engineering because a region devoid of cells and/or ECM might become a defect after the formation of bone.4
Therefore, the scaffold should have a uniform pore structure and induce homogeneous production of the appropriate organic (e.g., collagen) and inorganic (e.g., hydroxyapatite mineral) ECM throughout the scaffold. From this point of view, inverse opal structure is excellent scaffold platform for bone tissue engineering.
We recently demonstrated the fabrication of a chitosan scaffold with an inverse opal structure (i.e., inverse opal scaffold) using a cubic-close packed (ccp) lattice of uniform polymer microspheres as the template.5
However, the chitosan inverse opal scaffold is not appropriate for bone tissue engineering because of its low mechanical strength. Therefore, we further developed three new kinds of inverse opal scaffolds composed of poly(D, L-lactide-co
-glycolide) (PLGA) and HAp: i
) PLGA alone, ii
) HAp-impregnated PLGA (PLGA/HAp), and iii
) Ap-coated PLGA/HAp scaffolds. We hypothesized that the HAp-impregnated inverse opal scaffold could provide a more favorable environment for bone tissue engineering due to its uniformity in porosity and surface/mechanical properties, eventually leading to uniform secretion of organic and inorganic matrix throughout the scaffold. In this work, preosteoblasts (MC3T3-E1), the most commonly used cell line for the study of bone tissue engineering, were seeded into the three types of inverse opal scaffolds and induced to differentiate and secrete mineral. The production of mineral (inorganic ECM) serves as an indicator of osteoblastic differentiation of the preosteoblast.6
To quantitatively evaluate the spatial distribution and amount of secreted mineral, X-ray microcomputed tomography (micro-CT) was used to examine the three-dimensional (3D) density and architecture of mineral in a non-invasive manner.7
To our knowledge, this is the first microscopic evaluation of the spatial distribution of mineral secreted from differentiated preosteoblasts onto inverse opal structures using micro-CT.