Optimisation of perfusion bioreactor for bone tissue growth

Lead Research Organisation: Keele University
Department Name: Inst for Science and Tech in Medicine

Abstract

Bone tissue engineering is an emerging therapy for treating patients undergoing orthopaedic trauma or disease. The core of the method is the growth of bone tissue on a initial artificial porous scaffold which mimics real bone. The growth is achieved by flowing stem cells through the scaffold until it is replaced by bone tissue which closely resembles the patients own bone. However, much optimisation is needed before this therapy can be implemented. One of the important factors is the number of cells that are placed on scaffold at the start of the several week culture period necessary to create the tissue engineered construct. The correct number and location of starting cells placed onto a scaffold is critical in determining the functionality of the resulting construct. This project aims to optimise cell-seeding methods on the scaffolds, by developing an experimentally validated computer model. The validation, and subsequent investigation, will import real experimental geometries into the flow model; these will be achieved using digital data captured by micro tomography and other methods. The modelling component is a vital element of the proposed project; it overcomes (i) the problem of the inaccessibility of experimental data in complex flow geometries and (ii) the high cost of exploring the potential parameter space experimentally. Expertise from both Keele University (in tissue engineering and bioreactor design) and Sheffield Hallam University (in flow modelling techniques) will be utilised synergistically in order to address the project aims in this joint proposal. Cell type, attachment proteins, scaffold geometry/chemistry, media perfusion rates and mixing techniques will all be analysed in order to investigate the optimal method of cell seeding for bone tissue engineering. The optimised flow model, which will also make timely use of the most recent mathematical modelling information available (eg King, 2005), will then be practically tested in a sterile laboratory environment. Biochemical assessment will be undertaken to determine the efficacy of the predicted, optimised methodology. Utilising modelling techniques in this way, it is possible to significantly reduce time and costs that would otherwise be spent in the laboratory optimising these essential parameters for tissue engineering.

Technical Summary

Bone tissue engineering is an emerging therapy for treating patients undergoing orthopaedic trauma or disease. However, much optimisation is needed before this therapy can be implemented. One of the important factors in bone tissue engineering is the configuration of placing the cells on a scaffold at the start of the several week culture period which is necessary to create the tissue-engineered construct. Determining the optimal number and location of the starting cells placed onto a scaffold is critical to the functionality of the resulting construct. This project aims to optimise cell-seeding methods on porous 3D constructs, using a lattice Boltzmann (LB) mathematical modelling technique employing the real experimental geometries which will be digitally captured using micro computed tomography (CT) and other methods. Cell type, attachment proteins, scaffold geometry/chemistry, media perfusion rates and mixing techniques will all be analysed in order to determine the optimal regime of cell perfusion seeding for bone tissue engineering. The modelling component is a vital element of the proposed project; it overcomes (i) the problem of the inaccessibility of experimental data in complex flow geometries and (ii) the high cost of exploring the potential parameter space experimentally. Validation of the optimised theoretical model will be performed experimentally using microCT imaging of both fluid flow and cell seeding amongst other techniques.

Publications

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Balint R (2013) Electrical Stimulation Enhanced Mesenchymal Stem Cell Gene Expression for Orthopaedic Tissue Repair in Journal of Biomaterials and Tissue Engineering

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Cartmell SH (2011) 3D sample preparation for orthopaedic tissue engineering bioreactors. in Methods in molecular biology (Clifton, N.J.)

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Hidalgo-Bastida LA (2012) Modeling and design of optimal flow perfusion bioreactors for tissue engineering applications. in Biotechnology and bioengineering

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Rupani A (2012) Osteoblast activity on carbonated hydroxyapatite. in Journal of biomedical materials research. Part A

Related Projects

Project Reference Relationship Related To Start End Award Value
BB/F013892/1 09/06/2008 08/09/2010 £509,341
BB/F013892/2 Transfer BB/F013892/1 13/09/2010 12/04/2012 £105,637
 
Description The key resources created by the grant are the protocols for microCT methodology for 4D mammalian cells and fluid flow CT imaging. Experiments designed to validate the mathematical model developed by Sheffield-Hallam team included imaging of location of cells in simple materials and geometries as well as fluid flow profile inside a perfusion bioreactor.

The first set was tackled by combining technology available for MRI, super-paramagnetic particles, attached to stem cell membrane to tag the cell using a microCT. MicroCT is used for materials characterization as soft tissue and cells do not absorb x-ray. By attaching these specific magnetic particles we imaged cell deposition at high resolution. This method allowed us to provide data to feed and validate the mathematical as a non-destructive tool. Because contrast agents were not required, the scanned sample can go back into culture and re-scanned when needed to track cell deposition and proliferation.

The fluid flow was imaged with a clinical equipment bought for this grant, the XtremeCT. This equipment, with good resolution for a clinical scanner, provided us with an open scanning space to locate complex systems (bioreactors) and track fluid flow when using contrast agents. This was of fundamental use for the developing of the mathematical model given that these results demonstrated the importance of the gravity factor in the mathematical simulation.

We also adapted a protocol to image super para magnetic particles used in MRI to track live cells using a microCT scanner. This protocol has the potential to follow up live cells in 3D in real time without damaging the samples. This technique, although need more development and investment, could be very useful for tissue engineering and in vitro toxicology screening.

1) Five scientific refereed papers already published and two or more in preparation.
2) Independent project website containing project details, management and software resources developed (https://sites.google.com/site/perfusionbioreactor).
3) The successful collaboration between research partners led to further research proposals to the Furlong Research Charitable Foundation and to BBSRC.
4) The findings and work of this grant have been presented nationally and internationally by the Manchester University and PDRA.
5) A BBSRC-ISIS grant was awarded to initiate contact with a Mexican research team. The PDRA travelled to Mexico City to plan collaborative work on osteo-chondral grafts
6) The PDRA was trained in image analysis and science communication and is now employed following the end of this grant in a permanent post as a Lecturer in Medical Cell Biology at Manchester Metropolitan University.
7) The XtremeCT bought for this grant will be used for basic research projects but also for clinical trials and patient follow up
Exploitation Route The key results of the project are:
1) Validation of mathematical models for perfusion bioreactors for tissue engineering can be completed either by validating the fluid flow inside the perfusion or by validating the cell deposition, proliferation and differentiation inside the construct when seeded using perfusion.
2) Fluid flow was imaged using the XtremeCT and in-house made Nylon bioreactors. The systems were perfused with culture media and then Lugol's iodine solution and scanned in "real time" as the liquids entered the chamber and diffused through the scaffold constructs. Although the image resolution was limited, it was possible to determine that heavy agents, such as iodine solutions, are greatly influenced by gravity, which the mathematical model neglected in the first instance.
3) Perfusion seeding was assessed uni-directionally at 0.003 ml/min, equivalent to the velocity of single cells travelling inside the scaffold pores in a standard perfusion flow rate of 0.01 ml/min. "Simple geometry" experiments were assessed to verify cell adhesion in straight and 90 degrees bended sections, which matched the mathematical model boundaries.
4) Online cell tracking was successfully done by magnetic labelling and microCT. Fluorescent tracking could not be used due to the auto-fluorescent nature of many biomaterials causing false positives. We imaged cells, seeded into tygon tubing pre-coated with fibronectin, by attaching specific super paramagnetic nano-particles to our mesenchymal stem cells prior to seeding. Samples were imaged at high resolution and cell deposition indicated that, contrary to what the mathematical model predicted, the cells attached to the external part of the curvature when the cell suspension changed direction.
5) Although this data also enabled the use of microCT as a non-destructive tool to image 4D constructs (scaffolds and cells) in real time; the contrast-agent free imaging was not optimized due to time and resources constraints.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

URL https://sites.google.com/site/perfusionbioreactor/
 
Description This project has gained data to understand the fluid flow dynamics of the culture media and subsequent wall shear stress effects on cells seeded throughout a porous 3D scaffold and cultured in a perfusion bioreactor. Subsequent BBSRC pathfinder funding and BBSRC follow funding after this grant has allowed international and national collaborations to be established with both academics, industry and clinical contacts to further orthopaedic tissue engineering strategies.
First Year Of Impact 2014
Sector Electronics,Healthcare
Impact Types Societal

 
Description Follow on Funding
Amount £134,000 (GBP)
Funding ID BB/M013545/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2015 
End 09/2016