It is a challenge to non-invasively visualize in vivo the neovascularization in a three-dimensional (3D) scaffold with high spatial resolution and deep penetration depth. Here we used photoacoustic microscopy (PAM) to chronically monitor neovascularization in an inverse opal scaffold implanted in a mouse model for up to six weeks. The neovasculature was observed to develop gradually in the same mouse. These blood vessels not only grew on top of the implanted scaffold but also penetrated into the scaffold. The PAM system offered a lateral resolution of ~45 μm and a penetration depth of ~3 mm into the scaffold/tissue construct. By using the 3D PAM data, we further quantified the vessel area as a function of time

Cai, Xin

Choi, Sung-Wook

Kim, Chulhong

Li, Li

MacEwan, Matthew R.

Wang, Lihong V.

Xia, Younan

Yao, Junjie

Zhang, Yu

Caltech Authors - Main

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Photoacoustic microscopy of
neovascularization in three-
dimensional porous scaffolds in vivo
Xin  Cai, Yu  Zhang, Li  Li, Sung-Wook  Choi, Matthew R.
MacEwan, et al.
Xin  Cai, Yu  Zhang, Li  Li, Sung-Wook  Choi, Matthew R. MacEwan, Junjie
Yao, Chulhong  Kim, Younan  Xia, Lihong V. Wang, "Photoacoustic
microscopy of neovascularization in three-dimensional porous scaffolds in
vivo," Proc. SPIE 8581, Photons Plus Ultrasound: Imaging and Sensing 2013,
858128 (4 March 2013); doi: 10.1117/12.2005236
Event: SPIE BiOS, 2013, San Francisco, California, United States
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Photoacoustic Microscopy of Neovascularization in Three-Dimensional 
Porous Scaffolds In Vivo 
 
Xin Cai,1 Yu Zhang,2 Li Li,3 Sung-Wook Choi,4 Matthew R. MacEwan,1 Junjie Yao,1  
Chulhong Kim,5 Younan Xia,2 and Lihong V. Wang1,∗ 
 
1Optical Imaging Laboratory, Department of Biomedical Engineering, Washington University in St. 
Louis, St. Louis, Missouri 63130 
2Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and 
Emory University, Atlanta, GA 30332, USA 
3Wellman Center for Photomedicine, Massachusetts General Hospital and Harvard Medical School, 
55 Fruit Street, Boston, MA 02114, USA 
4Department of Biotechnology, The Catholic University of Korea, Bucheon 420-743, Korea 
5Department of Biomedical Engineering, University at Buffalo, The State University of New York, 
Buffalo, NY 14260, USA 
 
ABSTRACT 
It is a challenge to non-invasively visualize in vivo the neovascularization in a three-dimensional (3D) scaffold with high 
spatial resolution and deep penetration depth. Here we used photoacoustic microscopy (PAM) to chronically monitor 
neovascularization in an inverse opal scaffold implanted in a mouse model for up to six weeks. The neovasculature was 
observed to develop gradually in the same mouse. These blood vessels not only grew on top of the implanted scaffold 
but also penetrated into the scaffold. The PAM system offered a lateral resolution of ~45 µm and a penetration depth of 
~3 mm into the scaffold/tissue construct. By using the 3D PAM data, we further quantified the vessel area as a function 
of time.  
 
Keywords: Three-dimensional scaffold; photoacoustic microscopy; tissue engineering; neovascularization; biomedical 
imaging. 
1. INTRODUCTION 
Tissue engineering aims to improve or replace biological functions, leading to applications for wound healing. 
Angiogenesis is one of the most important steps in the process of wound healing. Three-dimensional (3D) scaffolds can 
provide physical supports and adjustable microenvironments for tissue regeneration1 Thus, non-invasive in vivo imaging 
of the vascularization process in scaffolds is critical for optimizing the properties of scaffolds, including biocompatibility, 
biodegradability, mechanical strength, porosity, pore size, and interconnectivity.2 However, a significant difficulty in 
scaffold imaging is the lack of an imaging modality with high resolution, deep penetration, and strong contrast. For 
example, laser-scanning optical microscopy (LSM), including confocal and multi-photon microscopy, has high 
resolution. However, due to strong light scattering, especially in the presence of blood, LSM is typically limited to 
                                                            
∗ Corresponding author: lhwang@biomed.wustl.edu 
Photons Plus Ultrasound: Imaging and Sensing 2013, edited by Alexander A. Oraevsky, Lihong V. Wang,
Proc. of SPIE Vol. 8581, 858128 · © 2013 SPIE · CCC code: 1605-7422/13/$18 · doi: 10.1117/12.2005236
Proc. of SPIE Vol. 8581  858128-1
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several hundred micrometers penetration in tissue. Micro-computed tomography (micro-CT) can penetrate up to several 
centimeters. However, it is ionizing irradiation and provides poor contrast for soft tissue, which limits its in vivo 
applications. So, new non-invasive imaging modalities with high resolution, deep penetration, and strong contrast are 
needed for 3D scaffold-based samples. 
 
Photoacoustic microscopy (PAM) is attractive for imaging scaffolds non-invasively. Recently, some investigations for 
PAM imaging and monitoring of 3D scaffold-based samples have been reported.3-6 PAM detects photoacoustic waves 
generated from absorbed laser irradiation.7 The non-ionizing irradiation in PAM imaging is safer than ionizing X-rays in 
micro-CT. Additionally, hemoglobin, the primary carrier of oxygen in blood, exhibits a strong intrinsic optical 
absorption contrast in the range of visible light.8-9 This unique feature allows label-free PAM to map the vasculature and 
to avoid possible alterations to the hemodynamics caused by exogenous angiographic agents.10  
 
Here, we report the capability of PAM to monitor neovascularization in an inverse opal scaffold implanted in a mouse 
model for up to six weeks. The neovasculature was observed to develop gradually in the same mouse. By using the 3D 
PAM data, we further quantified the vessel area as a function of time. 
 
2. MATERIALS AND METHODS 
2.1 Preparation of inverse opal scaffolds 
Poly(D, L-lactide-co-glycolide) (PLGA) inverse opal scaffolds (lactide/glycolide=75/25, Mw≈66,000-107,000, Sigma-
Aldrich) were fabricated by following our published procedures. 11  Briefly, gelatin (Type A; Sigma-Aldrich) 
microspheres with uniform sizes were produced using a simple microfluidic device. Then, gelatin lattices were obtained 
after thermal fusion of cubic-close-packed gelatin microspheres. Finally, PLGA inverse opal scaffolds were fabricated by 
templating against the gelatin lattices. 
 
2.2 Animals and implantation of scaffolds 
All animal experiments were performed in accordance with protocols approved by the Washington University 
Department of Comparative Medicine and the Animal Studies Committee. Athymic nude mice at the age of 4-5 weeks 
were obtained from Harlan and housed in the animal facility at Washington University. The scaffolds (4 mm diameter × 
1.5 mm height) were sterilized in 70% ethanol for at least 2 h prior to implantation, and then were implanted 
subcutaneously in the ears of the mice. For implantation, firstly, a parasagittal incision was made in the ear root of each 
animal approximately 0.5 cm to the head. Secondly, a subcutaneous pocket was created lateral to the incision by using 
blunt dissection. Then, a scaffold was inserted into the pocket. Finally, the skin incision was closed with 9-0 Ethilon 
suture (Ethicon, Somerville) and secured using VetBond dermal adhesive (3M, St. Paul). During the surgeries and PAM 
experiments, the animals were anesthetized by administration of gaseous isoflurane (2%, Butler Inc., Dublin) and 
aseptically prepared. 
 
2.3 Histology 
At 2, 4 and 6 weeks post-implantation, the scaffolds were explanted, fixed in 3.7% formaldehyde (Sigma-Aldrich), 
dehydrated in a graded ethanol series (70–100%), embedded in paraffin, and sectioned in thickness of 5 µm. Then, the 
samples were stained with hematoxylin/eosin and observed under Nanozoomer (Hamamatsu). 
 
2.4 Photoacoustic microscopy 
The schematic of the system has been reported in previous work.3, 8 For photoacoustic excitation, a dye laser (CBR-D, 
Sirah) pumped by a Nd:YLF laser (INNOSLAB, Edgewave) was employed to provide 7-ns laser pulses with a repetition 
rate up to 5 kHz. For photoacoustic detection, a focused ultrasonic transducer with 50 MHz central frequency (V214-BB-
RM, Olympus NDT) was employed. The optical and ultrasonic foci were configured coaxially and confocally. This 
system could achieve 45 μm lateral resolution, 15 μm axial resolution, and more than 3 mm penetration depth. It took ~5 
min to acquire a 3D image with dimensions of 6 × 6 × 3.75 mm3, which covered the whole scaffold sample. 
 
Proc. of SPIE Vol. 8581  858128-2
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Photoacoustic microscopy of neovascularization in three-dimensional porous scaffolds in vivo

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Photoacoustic microscopy of neovascularization in three-dimensional porous scaffolds in vivo

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