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In vivo micro computed tomography of nanocrystal-doped tissue engineered scaffolds Stacey M Forton, Matthew T Latourette, Maciej Parys, Matti Kiupel, Dena Shahriari, Jeff Sakamoto, and Erik M Shapiro ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.5b00476 • Publication Date (Web): 29 Feb 2016 Downloaded from http://pubs.acs.org on March 7, 2016
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ACS Biomaterials Science & Engineering
In vivo micro computed tomography of nanocrystal-doped tissue engineered scaffolds Stacey M. Forton1, Matthew T. Latourette1, Maciej Parys2, Matti Kiupel3, Dena Shahriari4, Jeff S. Sakamoto4 and Erik M. Shapiro1* 1 Department of Radiology, 846 Service Rd, Michigan State University, East Lansing, MI, 48824, USA 2 Department of Small Animal Clinical Sciences, 736 Wilson Rd, Michigan State University, East Lansing, MI, 48824, USA 3 Department of Pathobiology and Diagnostic Investigation, 736 Wilson Rd, Michigan State University, East Lansing, MI, 48824, USA 4 Department of Mechanical Engineering, 2350 Hayward Ave, University of Michigan, Ann Arbor, MI, 48109, USA
Keywords: tissue engineered scaffold; microCT; gadolinium oxide; bismuth; biomedical imaging; Hounsfield units; alginate; agarose
* Corresponding author Erik M. Shapiro, PhD Department of Radiology Michigan State University 846 Service Rd East Lansing, MI 48824 Tel: +1 (517)884-3270; Fax: +1 (517) 432-2849 Email:
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Word count: ~4500
Figures: 7
Tables: 1
Abstract Tissue engineered scaffolds (TES) hold promise for improving the outcome of cell-based therapeutic strategies for a variety of biomedical scenarios, including musculoskeletal injuries, soft tissue repair and spinal cord injury. Key to TES research and development, and clinical use, is the ability to longitudinally monitor TES location, orientation, integrity and microstructure following implantation. Here we describe a strategy for using micro computed tomography (microCT) to visualize TES following implantation into mice. TES were doped with highly radiopaque gadolinium oxide nanocrystals and were implanted into the hind limbs of mice. Mice underwent serial microCT over 23 weeks. TES were clearly visible over the entire time course. Alginate scaffolds underwent a 20% volume reduction over the first 6 weeks, stabilizing over the next 17 weeks. Agarose scaffold volumes were unchanged. TES attenuation was also unchanged over the entire time course, indicating lack of nanocrystals dissolution or leakage. Histology at the implant site showed the presence of very mild inflammation, typical for a mild foreign body reaction. Blood work indicated marked elevation in liver enzymes, and hematology measured significant reduction in white blood cell counts. While extrapolation of the Xray induced effects on hematopoiesis in these mice to humans is not straightforward, clearly this is an area for careful monitoring. Taken together, these data lend strong support that doping TES with radiopaque nanocrystals and performing microCT imaging, represents a possible strategy for enabling serial in vivo monitoring of TES.
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Introduction
A spinal cord injury (SCI) has the potential to be a debilitating and life-changing event. Depending on the location of the injury, SCI may induce serious loss of function including paralysis, loss of sensory and motor control, or loss of autonomic body control of heart rate, blood pressure, breathing, etc. A majority of SCI are traumatic SCIs resulting from falls, motor vehicle accidents, or violence
1
. The World Health
Organization estimates that between 250,000 and 500,000 people worldwide suffer from a SCI each year 2. One approach to treating SCI is regeneration of the spinal cord via cell-based therapies. Cell-based therapies, including various neural stem cell sources 3, induced pluripotent stem cell derived neural stem cells 4,5 and bone marrow derived mesenchymal stem cells 3, have demonstrated potential for providing functional recovery following SCI. Indeed, there are several ongoing clinical trials assessing a variety of cellular therapies 3. Other treatments include the delivery of nerve growth factor
6
or a calpain
inhibitor 7, and can help address issues involved with spinal cord repair such as cell survival or replacement, axon growth and guidance, and synaptic formations 8. Several of these strategies may encourage the regeneration and growth of damaged axons, however this growth, without guidance, can occur in a disorganized fashion
9,10
. For this reason,
tissue engineered scaffolds (TES) are a promising treatment option to linearly guide regenerating axons. Ideally the TES technology should also allow for the integration of stem cells and/or growth factors to stimulate axon growth. Our laboratory (JSS lab) has developed TES that are biocompatible, biodegradable, have sufficient mechanical properties to be durable, and are highly porous
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to allow cellular or axonal penetration
11
. These TES consist of close-packed arrays of
microchannels that range from 50 – 200 µm in diameter with scaffold walls that range from 30 – 100 µm thick. The relatively thin scaffold walls enable high channel lumen volume approaching 60%
9,10,12-14
. The linearity of the microchannels guides axons that
grow in bundles, to distal targets to allow for recapitulation of native nerve tracts 9. It has been demonstrated that incorporating BDNF or NT-3 secreting cells into agarose scaffolds increases the density of penetrating axons and the distance of axonal regeneration across the lesion site 9,10,12-14. There are several characteristics of TES that are important to monitor in both the research and development phase, but also potentially in clinical implementation. These include the location of the TES, the physical integrity of the TES, changes in pore size, and absorption rate of the material
15
. In order to quantitatively measure these
characteristics of TES post-implantation non-invasive, high resolution, 3D imaging techniques are required. Magnetic resonance imaging (MRI) has been primarily used to image biomaterial implants and is advantageous for an in vivo setting due to the lack of ionizing radiation. In general, bio-constructs have been labeled with iron oxide nanoparticles, either directly in the matrix 16,17 or by seeding the matrix with magnetically labeled cells 18, and detection of the matrix/cells is accomplished via imaging sequences sensitive to the magnetic field inhomogeneity caused by the particles themselves. Using these strategies, the grafts can clearly be visualized in vivo, but the large blooming artifact caused by the iron oxide particles can obstruct accurate definition of the construct itself, making it difficult to measure subtle changes in structural parameters. Even so, small animal MRI resolution is generally limited to 100 µm isotropic over large areas of a
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rodent body 19,20, or 35-50 µm over smaller areas such as embryos 21 or mouse legs 22. A higher resolution imaging modality may be required depending on the type of construct. Given the challenges with MRI-based imaging of iron oxide labeled constructs, here we demonstrate the use of micro computed tomography (microCT) to image implanted TES. In vivo microCT systems for rodents enable image resolutions
