Multiscale photoacoustic microscopy of single-walled carbon nanotube-incorporated tissue engineering scaffolds

Abstract

Three-dimensional polymeric scaffolds provide structural support and function as substrates for cells and bioactive molecules necessary for tissue regeneration. Noninvasive real-time imaging of scaffolds and/or the process of tissue formation within the scaffold remains a challenge. Microcomputed tomography, the widely used technique to characterize polymeric scaffolds, shows poor contrast for scaffolds immersed in biological fluids, thereby limiting its utilities under physiological conditions. In this article, multiscale photoacoustic microscopy (PAM), consisting of both acoustic-resolution PAM (AR-PAM) and optical-resolution PAM (OR-PAM), was employed to image and characterize single-walled carbon-nanotube (SWNT)-incorporated poly(lactic-co-glycolic acid) polymer scaffolds immersed in biological buffer. SWNTs were incorporated to reinforce the mechanical properties of the scaffolds, and to enhance the photoacoustic signal from the scaffolds. By choosing excitation wavelengths of 570 and 638 nm, multiscale PAM could spectroscopically differentiate the photoacoustic signals generated from blood and from carbon-nanotube-incorporated scaffolds. OR-PAM, providing a fine lateral resolution of 2.6 μm with an adequate tissue penetration of 660 μm, successfully quantified the average porosity and pore size of the scaffolds to be 86.5%±1.2% and 153±15 μm in diameter, respectively. AR-PAM further extended the tissue penetration to 2 mm at the expense of lateral resolution (45 μm). Our results suggest that PAM is a promising tool for noninvasive real-time imaging and monitoring of tissue engineering scaffolds in vitro, and in vivo under physiological conditions.

FIG. 1.

Schematic of the reflection-mode AR-PAM and OR-PAM systems. A Cartesian coordinate is also shown in the drawing. CL, conical lens; CorL, correction lens; MMF, multimode fiber; Obj, objective; OC, optical condenser; PD, photodiode; RAP, right-angle prism; RhP, rhomboid prism; SMF, single-mode fiber; SO, silicone oil; US, ultrasonic transducer; AR-PAM, acoustic-resolution photoacoustic microscopy; OR-PAM, optical-resolution photoacoustic microscopy. Color images available online at www.liebertonline.com/tec

FIG. 2.

(a) A bright-field transmission electron microscopy image of bundled SWNTs on the surface of the substrate. (b) An atomic force microscopy image of two separate SWNTs. (c) A scanning electron microscopy image of a porous PLGA scaffold incorporating SWNTs. The bold yellow arrow indicates the PLGA and the dotted yellow arrow points out the SWNTs incorporated within the scaffold. SWNT, single-walled carbon nanotube. PLGA, poly (D, L-lactide-co-glycolide). Color images available online at www.liebertonline.com/tec

FIG. 3.

PAM and micro-CT images of the PLGA scaffolds incorporating SWNTs. (a) A MAP image of the scaffold by AR-PAM. (b) A MAP image of the scaffold by OR-PAM. (c) An optical microscope image of the scaffold. The common features that can be identified from the images have matching numbers. (d) A 3D depiction of the OR-PAM image. (e) A micro-CT MAP image of the scaffold in dry surrounding. (f ) A micro-CT MAP image of the scaffold in fetal bovine serum. (g) A 3D depiction of the micro-CT image in dry surroundings. (h) A 3D depiction of the micro-CT image in fetal bovine serum. micro-CT, microcomputed tomography; 3D, three dimensional; MAP, maximum amplitude projection. Color images available online at www.liebertonline.com/tec

FIG. 4.

PAM MAP and B-scan images of the SWNT-incorporating scaffold in bovine blood at different wavelengths. (a) AR-PAM MAP image at 570 nm. (b) AR-PAM MAP image at 638 nm. (c) Corresponding B-scan image at the dash line of (a). (d) Corresponding B-scan image at the dash line of (b), which shows the scaffold ∼2 mm in the blood. (e) OR-PAM MAP image at 570 nm. (f ) OR-PAM MAP image at 638 nm. (g) Corresponding B-scan image at the dash line of (e). (h) Corresponding B-scan image at the dash line of (f ). The identified scaffold features are marked by matched numbers, which also correspond to the same parts shown in Figure 3.

FIG. 5.

PAM MAP and B-scan images of the SWNT-incorporating scaffold in chicken breast tissue at different wavelengths. (a) AR-PAM MAP image at 570 nm. (b) AR-PAM MAP image at 638 nm. (c) Corresponding B-scan image at the dashed line of (a). (d) Corresponding B-scan image at the dashed line of (b), which shows the scaffold ∼2 mm in the blood. (e) OR-PAM MAP image at 570 nm. (f ) OR-PAM MAP image at 638 nm. (g) Corresponding B-scan image at the dashed line of (e). (h) Corresponding B-scan image at the dashed line of (f ). The identified scaffold features are marked by matched numbers, which also correspond to the same parts shown in Figure 3.