University of Manchester

X-ray imaging of nanoscale tissue engineering scaffolds

Soeren Neubeck
Project Completed

Scientific Case

The proposed project is the first in a series ultimately aiming at revealing the healing of bone defects using an artificial bone graft substitute material in real time. A novel polymer-based material, developed in the group of Prof.S.Downes, has been shown to hold unexpectedly high potential for the repair of bone defects, as e.g. occurring in osteoporosis. The implant material is made by using electro-spinning of a polymer (PCL) und subsequently treating the obtained fibrous scaffold with a chemical. This chemical treatment induces bioactive moieties on the scaffold, which are thought to be crucial for the improved regenerative power of the material. On the way to eventually translating this novel bone graft substitute material into clinic, a series of quintessential studies is needed. Our general strategy, in order to clarify (qualitatively and quantitatively) the regenerative power of our novel material is as follows. First, we want to obtain three dimensional images of the bare polymer scaffold obtained after electro-spinning and after chemical modification. In a second step, a cryogenic sample holder will be employed to facilitate imaging of cells (osteoblasts) pre-seeded on the scaffold, over various days of cell culturing. Imaging of the proliferation of cells in three-dimensions will allow us to assess the biocompatibility of our scaffold in great detail. Additionally, it would demonstrate successful imaging of live cells in their natural state at HMXIF. In the third step, in vitro studies are planned, in which the bioactive implant material is injected into a bone defect. A series of X-ray imaging experiments over an extended time frame, again employing the cryogenic sample holder, will be conducted to obtain images of how new bone forms and fills the defect by virtue of the implant material.
The first experiment of imaging the three-dimensional polymer (PCL) scaffold is fundamental to all later, more advanced studies. It is essential to obtain three dimensional image reconstructions. From these, it is possible to determine the architectural parameters of the scaffold, such as pore size, pore size distribution and pore interconnectivity, all of which control the ability of the material to accommodate cells and allow for cell proliferation. Once these parameters are known, they will serve as a standard in all future experiments outlined above. So far, the polymeric scaffold has been characterised by SEM, which due to its two-dimensional imaging only allows for an estimate of the three-dimensional parameters. A key finding was that the average diameter of the polymer fibres is ~260nm, with a standard deviation of ~150nm.
In order to obtain the desired three-dimensional images, we would like to carry out X-ray imaging using the xradia-nanoCT in the HMXIF, with subsequent tomographic image reconstruction. Given the ~260nm diameter of a typical fibre of the PCL scaffold, the nanoCT will in principle allow us to resolve individual fibres, which is a must in order to obtain the correct architectural parameters. Since the nano-CT can be operated in phase-contrast mode, it will allow us to image the polymer fibres (absorption contrast might be too weak, because of the light elements in the polymer). Two sets of experiments are planned. One is to be conducted on the electrospun PCL scaffold, as produced and after chemical modification. In parallel, we would like to image an artificial polymer scaffold. The reason for this second set of experiments is as follows. From nanoCT experiments carried out by Dr. Rob Bradley on another fibrous scaffold, with similar dimensions (prepared by Dr. Lucy A. Bosworth, Downes Group), it became apparent that the section where two or more fibers overlap is hard to be properly reconstruct due to the edge contours arising from the phase contrast. The main reason lies in the small dimensions of the polymer scaffolds, with characteristic spatial scales between 500nm and 100microns. However, it is crucial for determining the architecture of the pores of the fibrous scaffold to be able to reliably distinguish between e.g. two crossing fibers touching each other or being separated. Following on a discussion with Dr. Bradley, we agreed to study this question on an artificial polymer scaffold with well-defined crossings of the polymer fibers and alternatively well-defined spacings between fibers. In this sense, we would have a “calibration” standard at hand, which might help in reconstructing the 2D radiographic projections on our randomly oriented PCL scaffold fabricated by electro-spinning. Given, our targeted calibration experiment is successful, it will be very helpful for future studies of polymer scaffolds with scaffold length scales below 100microns.
For obtaining a “calibration standard” polymer scaffold, different rapid prototyping techniques are available. The ideal fiber diameter sought for our experiments is around 5microns, which should be possible to be manufactured using two-photon polymerisation. Such a test sample will be bought.
The images and results obtained will provide the basis for the future experiments outlined above and are further expected to constitute parts of interesting publications. In fact, considering the immediate impact of our planned work, there is currently not a single study reporting a 3D reconstructed image on a polymer scaffold with dimensions similar to ours (fibre diameter ~300nm). A very recent study reports on SR microCT experiments on electrospun polystyrene fibers [1]. But it is clearly mentioned in the manuscript that the fibers they studied had diameters between 1micron and 3microns. Thus, if successful, our experiments would be the first of their kind. They would be very exciting, given the overall importance of micro- and nanometre-sized fibrous scaffolds on one hand, and the widespread use of X-ray microCT for three dimensional characterization on the other hand.
For each of the two distinct set of experiments we would like to ask for 10 days of access to the xradia nano-CT, totalling 20 days of access. 10 days are intended to be spent on “trial and error”-experiments on the electrospun PCL scaffold. In the best case, we will get publishable results on the internal three dimensional scaffold architecture. But in any case, we will be able to obtain useful information on imaging parameters and experimental circumstances that have to be considered for successful imaging in later experiments. For the well-defined test scaffolds, 10 days of experiments should be sufficient to gather all relevant information needed on quantifying the intersection of micron and submicron sized polymer fibre networks.
[1] “Characterisation of internal morphologies in electrospun fibers by X-ray tomographic microscopy”, J. V. Nygaard, T. Uyar, M. Chen, P. Cloetens, P. Kingshott and F. Besenbacher, Nanoscale, Vol. 3, (2011), p. 3594.
Polymers

Experiment Design

For the nanofibrous PCL scaffold, we are interested in obtaining three dimensional images from reconstructing X-ray phase contrast projections. The aim is to be able to determine the micro- and nano-architectural features of these scaffolds. Namely, we are interested in the pore size, the pore size distribution and the pore interconnectivity of our electrospun PCL nanofibre scaffold. Thus, it is essential to be able to resolve individual nanofibres. Special attention must be paid to regions where two or more fibres intersect. It has to be clearly determined, whether the fibers in these regions touch each other or are separated by a gap. This question will be studied on artificial polymer "calibration" samples, being well-defined stacks of polymer fibres (diameter around 5microns).

The PCL fibres making up the polymer scaffold have an average diameter of 260nm, with a standard deviation of 150nm. The resolution sought is 50nm.
Scanners and Rigs
20

Sample & Safety

10
Low Hazard

Scan Records

Project Report