The procedure for fabricating inverse opal scaffolds include four major steps:4d i
) production of uniform gelatin microspheres with a fluidic device, ii
) crystallization of the gelatin microspheres into a cubic-close packed (ccp) lattice, followed by heat treatment to interconnect the microspheres with surrounding ones and improve the mechanical strength of the lattice, iii
infiltration of PLGA solution into the void space in the gelatin lattice, and iv
) freeze-drying followed by selective removal of the gelatin microspheres with warm water. The inverse opal scaffolds have a uniform pore size and structure determined by the uniformity in size for the gelatin microspheres and the crystalline lattice. To fabricate non-uniform scaffolds, we produced gelatin microspheres with sizes in the range of 73 to 388 µm by gradually changing the flow rate of the continuous phase during the formation of gelatin emulsion in the fluidic device while all other parameters were kept the same as those for the uniform gelatin microspheres. For the uniform gelatin microspheres, heat treatment at 65 °C for 1 h was enough to obtain a robust lattice by inducing fusion between microspheres at their junctions. In contrast, for the lattice composed of non-uniform gelatin microspheres, it easily fell apart into small pieces even though it underwent the same procedure for heat treatment. In this case, a longer time (at least 2 h) of heat treatment was necessary to make the lattice robust enough for handling. This observation suggested that the areas and number of contact junctions between the non-uniform gelatin microspheres were rather small, corresponding to a limited size and number of windows connecting the pores in a non-uniform scaffold.
, show SEM images of PLGA scaffolds derived from the uniform and non-uniform gelatin microspheres, respectively. The scaffold prepared using the non-uniform gelatin microspheres exhibited a non-uniform and randomly arranged pore structure with an average pore size of 202 ± 94 µm, together with a relatively small size and number of interconnecting windows (). By contrast, the inverse opal scaffold showed a uniform pore size of 205 µm with a long-range ordered and well-controlled interconnectivity. The average diameter of the windows (36 ± 22 µm) in the non-uniform scaffold was much smaller than that (66 ± 7 µm) in the inverse opal scaffold because the window size is mainly determined by the smaller one of two adjacent microspheres. Both the pores and the windows serve as pathways for cell migration and nutrient/waste transportation. Therefore, an ideal scaffold should possess a high degree of interconnectivity and a suitable pore size that both enhance the transportation of nutrients/metabolic wastes and prevent pore occlusion during tissue formation. In some cases, the interconnectivity has been shown to have a greater impact than pore size on cell behavior.7
Figure 1 SEM images of (a) an inverse opal scaffold and (b) a non-uniform scaffold, and size distribution of (c) pores and (d) windows (or the holes connecting adjacent pores) in each scaffold. The black and blank bars correspond to the inverse opal and non-uniform (more ...)
To evaluate the diffusion rate of macromolecules through the scaffold, we designed a simple flow cell using two 50-mL centrifuge tubes and a connector (). Fluorescein isothiocyanate (FITC)-dextran (Mw
≈ 20k) was employed as a model macromolecule in place of a protein nutrient. The concentration of FITC-dextran passing through the scaffold was determined by collecting the eluent in a sampling tap and measuring its absorbance by UV-vis spectroscopy. Control experiments were also conducted in the absence of any scaffold in the flow cell. The diffusion rate was calculated from the slope by regressing the linear portion of each curve. As shown in , the diffusion rate of FITC-dextran through the inverse opal scaffolds (slope=0.035) was slightly lower than that for the control sample (slope=0.037). By contrast, the diffusion rate through the non-uniform scaffolds (slope=0.022) was much lower than that for the inverse opal scaffolds, together with a lag time of ~2 min, which can be attributed to the dominance of small pores and windows in the scaffolds.8
The high diffusion rate of FITC-dextran confirmed a uniform pore size and well-interconnected pore structure inside the inverse opal scaffolds, suggesting an efficient pathway for the transportation of nutrients and metabolic wastes throughout the scaffolds.8
Note that the broad error ranges for the non-uniform scaffolds also suggest a large difference in pore size and structure among the samples and thus low reproducibility for scaffold fabrication.
Figure 2 (a) Illustration of the cell for testing the diffusion of macromolecules passing through a scaffold. (b) Results of diffusion tests (n=3) for FITC-dextran (Mw = 20k) through the scaffolds placed in the specially designed flow cell. The diffusion rate (more ...)
As many researchers have pointed out,9
a large population of cells concentrated at the perimeter of a scaffold is a major obstacle for generating a uniform tissue with high viability. The cells at the perimeter could exhaust oxygen and nutrients, and thus limit their transportation into the interior of a scaffold, eventually resulting in cell necrosis inside the scaffold. Therefore, an even distribution of cells throughout a scaffold can prevent cell necrosis inside the scaffold and facilitate uniform distribution of soluble signaling molecules for cell-cell communications.10
This issue can be addressed by improving the design of microstructure for a scaffold. To examine the distribution of cells in a scaffold, we seeded fibroblasts into each type of scaffolds. At 7 days of culture, we used 4'-6-diamidino-2-phenylindole (DAPI) to stain the nuclei of the cells in the scaffolds and then sectioned the cell/scaffold constructs using microtome until the middle plane (around 500 µm in depth from the surface into the scaffold) of the construct was exposed. , a and b, show fluorescence micrographs taken from cross-sections at the middle plane of the constructs after 7 days of culture, where blue dots indicate cells in the scaffold. It is clear that the cells were uniformly distributed throughout the inverse opal scaffold, whereas the cells in the non-uniform scaffold showed a poor uniformity. A quantitative analysis of cell numbers from the fluorescence micrographs confirmed a more uniform distribution of cells in the inverse opal scaffold than in the non-uniform scaffold (). The uniform distribution of cells can be attributed to the uniform pores and large windows for the inverse opal scaffold, both of which can facilitate the seeding efficiency, migration of cells, and transportation of nutrients and wastes.
Figure 3 (a, b) Fluorescence micrographs and (c, d) quantitative analyses of cell numbers after 7 days of culture from the middle plane of (a, c) an inverse opal scaffold with a pore size of 211 µm and (b, d) a non-uniform scaffold. The fibroblast/scaffold (more ...)
In general, the differentiation of cells is regulated by the morphology, geometry, and surface properties of a scaffold.11
To elucidate the effects of pore size and uniformity on the differentiation of preosteoblasts, we prepared three kinds of apatite (Ap)-coated PLGA scaffolds: non-uniform scaffolds with an average pore size of slightly over 200 µm and inverse opal scaffolds with pore sizes of 211 and 313 µm, respectively. We seeded preosteoblasts into each type of scaffolds, cultured in an osteogenic medium, and evaluated the amounts of secreted extracellular matrix (ECM) and ALP activity during culture. An Ap-decorated surface12
and a pore size of around 200 µm13
are known as optimal conditions for osteogenic induction. In a previous study,14
we have demonstrated that, for inverse opal scaffolds with uniform pore size and structure, PLGA/hydroxyapatite composite scaffolds coated with apatite were more osteo-inductive than both the PLGA/hydroxyapatite composite scaffolds and pristine PLGA scaffolds.
shows an optical micrograph of a pristine Ap-coated inverse opal scaffold with a pore size of 211 µm. At 28 days of culture, a large amount of mineral was found in this inverse opal scaffold (), whereas very little mineral was observed in the inverse opal scaffold with a pore size of 313 µm () and in the non-uniform scaffold (). Since mineral was observed in the entire inversed opal scaffold with a pore size of 211 µm, it can be concluded that there was uniform differentiation for the preosteoblasts and subsequent secretion of mineral from the cells.
Figure 4 Optical micrographs of (a) a pristine Ap-coated PLGA scaffold with a pore size of 211 µm, (b-d) preosteoblast/scaffold constructs at 28 days of culture: inverse opal scaffolds with pore sizes of (b) 211 and (c) 313 µm, respectively, and (more ...)
shows SEM images of the preosteoblast/scaffold constructs at 28 days of culture. A large amount of complex of inorganic and organic ECM was found inside all the pores of the inverse opal scaffold with a pore size of 211 µm (). By contrast, very little mineral without fibrous organic ECM was deposited on the wall of the inverse opal scaffold with a pore size of 313 µm (). Interestingly, a moderate amount of mixture of inorganic and organic ECM was observed in the large pores (around 200 µm) of the non-uniform scaffold, whereas most of the small pores in the scaffold were occluded by the fibrous organic ECM (). These observations suggested that the Ap-decorated pores with sizes of around 200 µm could facilitate the secretion of both inorganic and organic ECMs. In contrast, the large pores (around 300 µm) were favorable for the secretion of inorganic ECM and the small pores were favorable for the secretion of organic fibrous ECM. Therefore, we can conclude that the Ap-decorated pores with a uniform size of around 200 µm would provide the most favorable microenvironment for the differentiation of preosteoblasts. More importantly, the secretion pattern of the cells could be largely controlled by tailoring the properties of an individual pore that the cells attached to, rather than the bulk properties of a scaffold (e.g., the average pore size). The significant dependence of secretion pattern on the pore size emphasizes the importance of a precise control over the pore size. Similar to our findings, Kuboki et al
. have demonstrated the effect of pore size on the pattern of tissue formation by subcutaneously implanting hydroxyapatite scaffolds with different pore sizes (90–120 and 350 µm in diameter) in rats. Their results showed an initial chondrogenesis and subsequent formation of bone tissue in the scaffolds with small pores and direct bone formation with no cartilage intermediate in the scaffolds with large pores.15
SEM images of the preosteoblast/scaffold constructs at 28 days of culture: inverse opal scaffolds with pore sizes of (a) 211 and (b) 313 µm, respectively, and (c) a non-uniform scaffold. Magnified views are shown in the right column.
We also quantitatively evaluated the differentiation of preosteoblasts in the scaffolds using assays based on ALP activity. ALP is a well-known indicative marker for osteogenic differentiation.16
As shown in , there was no significant difference in ALP activities among the samples at 7 days of culture. However, the inverse opal scaffolds with a pore size of 211 µm showed significantly higher ALP activity than the other scaffolds at 14 and 28 days of culture, while there was still no significant difference in ALP activities between the non-uniform scaffolds and the inverse opal scaffolds with a pore size of 313 µm. The ALP test quantitatively confirmed the better performance of the inverse opal scaffolds with a pore size of around 200 µm for the differentiation of preosteoblasts.
Figure 6 ALP activities of the preosteoblasts cultured in three different types of Ap-coated PLGA scaffolds at 7, 14, and 28 days of culture. The data are presented as mean ± s.d. (n = 3). * indicates significant difference between the two groups (p < (more ...)