Analyzing Structure and Function of Vascularization in Engineered Bone Tissue by Video-rate Intravital Microscopy
and 3D Image Processing
Yonggang Pang1 MD, PhD, Craig M. Neville1 PhD, Brian E. Grottkau1 MD
Department of Orthopaedic Surgery, Massachusetts General Hospital, Boston, MA 02114, USA
Introduction
Vascularization is one of the key challenges in tissue engineering.
The 3D structure and the microcirculation are two fundamental
parameters for evaluating structure and function of vascularization.
However, current imaging approaches are not able to provide both
cellular/subcellular resolution and fast continuous observation. Here
we report novel techniques using video-rate intravital microscopy
and 3D image processing to evaluate vascularization in the
engineered bone tissue, providing both structural and functional
analysis.
Materials and Methods
Video-rate (30 frames/s) intravital confocal microscope was set up
as reported previously 1 and used in all the following assays. As
illustrated in Fig. 1, human mesechymal stem cells (hMSCs)
serving as pericytes and umbilical vein endothelial cells (HUVECs)
were fluorescently labeled with lentivirus encoding eGFP (green)
and dTomato (red). Osteoinduced hMSCs together with labeled
cells were seeded onto PLGA scaffolds and implanted into the
severe combined immunedeficient mice. Whole blood cells were
labeled with Vibrant DiD (blue) and injected in vivo to evaluate
microcirculation. The following image acquisition and processing
techniques were
used : static and
Fig. 1
dynamic 3D
reconstructions for
the micro
architecture
analysis, mosaic
imaging for the
microvascular
network analysis
and single cell tracking for evaluating microcirculation. Custom
developed software in Matlab and Java were used.
Results
Our system and technique provide the following novel features to
characterize structure and function of vascularization in the
engineered bone tissue: 1 3D imaging provides superior
observation that undifferentiated MSCs are pericytes: 3D
imaging clearly demonstrated that in both in vitro and vivo
observation, labeled MSCs served as pericytes which stabilized the
micro vascular network comparing with degraded network without
MSCs (Fig 2). 2. Simultaneous visualization of blood vessel and
cells: using a hybrid mode combining orthogonal and volume 3D
views, we successfully visualized circulating blood cells inside a
solid vessel wall (Fig. 3). 3 Mosaic view for micro vascular
network quantification: Mosaic imaging extended the visual field
and preserved the resolution. By further image processing using
binary and skeletonization, the micro vascular networks were
quantified (Fig. 4). 4 Evaluate microcirculation function by
single blood cell tracking: with fast 30 fps rate, we were able to
capture the dynamic movement of single blood cells and further
analyzed the velocity (Fig. 5).
Fig. 2 A 3D
view provides
more intuitional
information of a
microvascular
network than a
2D view.
2D
3D
Fig. 3 A hybrid view
combining orthogonal and
volume 3D reconstruction
enables visualizing both
vessel wall and blood cells.
(Left and right show
different perspectives.)
Fig. 4 A representative mosaic
image stitched from multiple
single visual field images. Image
skeletonization was applied to
quantify the microvascular
networks.
Fig. 5 A representative
trajectory of labeled blood
cells (blue) generated by
computer assistant
tracking.
Discussion and Conclusions
This is a new approach to provide a novel platform better
understanding the vascularization of engineered tissue. It provides
structural and functional evaluation on the status of vascularization
in engineered tissue.
Reference
1 Tsigkou, Olga, et al. Engineered vascularized bone
grafts. PNAS (2010): 3311-3316.
Acknowledgments
This project was funded partially by Anthony and Constance
Franchi Fund (Grottkau) and NVIDIA Corporation (Pang).
Disclosures
Authors have nothing to disclose.