Analysis of 3‐D microstructure of porous poly(lactide‐glycolide) matrices using confocal microscopy

Abstract

Porous matrices of biodegradable polymers are extensively used as scaffolds in tissue engineering and as drug delivery devices. A critical component of the design, processing, and utility of such polymeric systems concerns the local void microarchitecture. In this study, a novel approach based on confocal fluorescence imaging was employed to visualize and quantify in 3 dimensions (3-D) the individual and population-level void morphology within porous polymeric matrices. Poly(lactic acid-glycolic acid) copolymer matrices were cast to yield void configurations of variable void sizes but constant cumulative voidage. Using confocal microscopy, fluorescently saturated polymer matrices were optically sectioned into serial 2-D images, and 3-D void contours were reconstructed via object discrimination and connectivity analysis. The resultant data was used to quantitate the matrix microstructure and map its evolution following polymer degradation. Under conditions of accelerated degradation, matrix erosion was found to cause a significant change in the disposition of voids; this involves two processes (void formation and void enlargement), the extent of which was influenced by the initial void size and the duration of erosion. By virtue of providing both static and dynamic descriptions of the void morphology in poly(lactic acid-glycolic acid) matrices, this is the first spatiotemporal study of the 3-D microarchitecture of porous, bioerodible tissue analog matrices.

Quantitative architectural description of tissue engineering scaffolds

“…12, by optically sectioning at successive heights. 31 The main limitation is the relatively low z-extent possible, compared with the image dimensions available in the x and y axes. 86 Bagherzadeh et al 73 provided an advance in this technique, by incorporating commercially available quantum dots as part of the scaffold fabrication process from polymer solution.…”

Correlating Micro-CT Imaging with Quantitative Histology

“…Sectioning followed by histology can image the scaffold interior but is destructive, lengthy and only semiquantitative (Ho & Hutmacher, 2006). Fluorescence microscopy can be quantitative when high-throughput approaches are applied, e.g., producing 3-D images with confocal fluorescence microscopy (Tjia & Moghe, 1998). Colorimetric and fluorometric soluble assays are available for cell components, such as enzymes, protein or DNA (Ho & Hutmacher, 2006).…”

“…However, these soluble assays are quantitative but do not provide information on cell distribution. In contrast to these methods, by micro-CT we can non-destructively obtain 3-D images that penetrate deep into the scaffold interior and produce inherently quantitative results (Tjia & Moghe, 1998). Micro-CT currently appears to be the most suitable approach for this task (Ho & Hutmacher, 2006;Mather et al, 2007;Mather et al, 2008;Cancedda et al, 2007).…”

Analysis of polymer grafted inside the porous hydrogel using confocal laser scanning microscopy

“…This problem could be solved by applying confocal laser scanning microscopy (CLSM). CLSM offers advantages over conventional SEM procedures as it does not require special water removal prior to examination that allows for studying the bulk structure of hydrogel samples in their native state [15][16][17]. CLSM is a technique to gain high resolution views of thick specimens by rejection of out of focus scattering.…”

“…This feature of CLSM, known as 'optical sectioning', makes it possible to scan at various x-y planes corresponding to different depths of the sample, and, thus to reconstruct the 3-D region of the specimen. CLSM has been widely used for investigation of the bulk structure of polymer materials [15][16][17]. However to the best of our knowledge there were no reports on the study of polymer grafted on the surfaces in the bulk of porous hydrogel material.…”