Final Report Summary - DECELL BONE REGEN (Development of an innovative supercritical fluid decellularization technology to produce advanced biocompatible scaffolds for complex musculoskeletal regeneration) The need for organs and tissues to treat end-stage organ failure and debilitating musculoskeletal conditions is a huge health requirement for Europe. Current treatments include organ transplants, bone grafts and tissue engineering and regenerative medicine approaches. Significant hurdles and limitations exist with all approaches: shortage of organ donors leads to patient mortality, autogenous bone grafts are often associated with pain and morbidity and allogeneic bone grafts can fail to vascularize and remodel. Existing use of scaffolds, cells and growth factors can also be problematic with worrying complications of negative host response, remodelling and rejection. A highly skilled UK researcher (Dr. Lisa White) with extensive experience in supercritical fluids will join the world leader in extracellular matrix materials (Dr. Steve Badylak at the University of Pittsburgh, USA) to gain unique skills in decellularization to collaboratively address these issues. In a pioneering step, an innovative supercritical fluid decellularization (SFD) technology will be developed. In the outgoing phase comprehensive progress was made with respect to all the training objectives. The fellow acquired substantial experience and skill in the decellularization of tissues and organs. The fellow applied different decellularization agents, such as detergents, acids and bases, to porcine liver, bladder and small intestine and successfully characterized the extent of decellularization. Metrics for successful decellularization were defined. In collaboration with University of Washington, the fellow led a team in the Badylak laboratory to investigate, for the first time, the effect of detergent based decellularization upon the surface molecular functionality of biological scaffolds. This work provided opportunities to investigate recellularization of primary human cells by examination of apoptosis, proliferation, cell morphology and stratification of cell layers using immunohistochemistry. This quantification of recellularization is key to the successful application of a supercritical fluid decellularization (SFD) technology. In addition, this research will provide useful guidance for commercial organizations and clinical end users on the deleterious effects of residual detergents in biologic scaffolds. This research has been published in Acta Biomaterialia (doi: 10.1016/j.actbio.2016.12.033). The fellow was involved in several projects in the Badylak lab during her outgoing period which provided valuable insight into complex macrophage and host remodelling interactions. In particular, the fellow played a key role in elucidating macrophage responses to ECM hydrogels sterilized by radiation, ethylene oxide and supercritical fluids. The fellow’s expertise in supercritical fluids was utilized in this research and in the development and understanding of the effect of sterilization methods upon the physical and biochemical structures of the hydrogels. A manuscript is currently being prepared and will be submitted in March 2017. In addition, the fellow is a co-author on a manuscript entitled ‘Macrophage Phenotype in Response to ECM Bioscaffolds’ which has been submitted to Seminars in Immunology. This work has also resulted in a provisional patent (PCT 62220409 ‘Non-gelling Soluble Extracellular Matrix with Biological Activity) which has been filed in the US. Upon her return to the UK, the fellow immediately began to utilize the comprehensive training received in decellularization to commence developing a novel SFD technology. The first stage of this process was to determine design constraints and equipment modifications required to adapt existing supercritical equipment to be used for extraction of cellular material. The fellow modified equipment to allow directional flow of supercritical carbon dioxide (scCO2) to facilitate drag to enable nuclear and cellular removal. Internal vessel modifications to permit the use of a liquid entrainer and a baffle to prevent backward tissue movement were also made. Finally, the fellow modified the experimental set up to include extra outlets, to be used in case of blockage, and a collection vessel for cellular material. Health and safety considerations were paramount during the design and equipment modification stages to ensure that aerosols of the tissues were not generated during extraction. Development of the supercritical fluid technology commenced with concerted efforts to determine the key operating variables required to decellularize liver tissue; initial optimization focused on evaluating a range of pressures, temperatures and different material masses and morphology. Although tissue structure was affected by supercritical fluid processing, quantification of DNA in tissue samples indicated that scCO2 alone was not able to remove cellular nuclear material. Further optimization was required and an extended study of pressure cycling (with steady ramps or rapid cycling), pressure ranges, process times, the addition of solvents and packing agents to direct the flow of scCO2 through the tissue were all trialled to define operating conditions for decellularization. A partial degree of success was achieved with the use of ethanol and deionised water in combination with supercritical processing utilizing pressure cycling at elevated P (4000 psi and above) and for extended processing times (>60 minutes). A 15% reduction in DNA was observed. However, these results were only obtained at the end of the project (Month 27). During the early optimization stages (Months 19 - 23) it became apparent that a fully optimized SFD technology would require a much longer period of research and innovation than the 12 month return phase could provide. The fellow successfully sought funding for a PhD studentship to carry on this research; the fellow is now the primary supervisor of an EPSRC Centre for Doctoral Training for Regenerative Medicine PhD Student. Attainment of definitive operating conditions for decellularization of whole livers will be undertaken by the PhD student, supervised by the Marie Curie Fellow. The PhD student will then quantify decellularization of supercritical processed bone materials. Once the supercritical fluid technology is validated with liver and bone the PhD student will undertake research to develop functional recellularized constructs. This research will utilize the extensive skills training and knowledge acquired by the fellow during the outgoing phase. Specifically, the research will benefit from the fellow’s extensive recellularization of decellularized bladders reseeded with human urothelial cells. The SFD process will be utilized to decellularize whole organs to provide functional biological scaffolds with vascular networks, providing promise for whole organ engineering. Application of the SFD technology to bone will produce a powerful ex vivo model that will be utilized to provide essential information on cell response and healing. Finally, this ground-breaking SFD technology will produce advanced biologically compatible scaffolds for the regeneration of complex musculoskeletal defects.
