Development of a cell enrichment device for bone repair utilising tissue non-specific alkaline phosphatase on the surface of dental pulp stromal cells

The overall aim of this thesis was to develop a minimally manipulative, label-free microfluidic cell separator device which is able to deliver an enriched population of autologous cells, positive for tissue non-specific alkaline phosphatase (TNAP) via cell capture using either antibody or non-antibody protein binding. TNAP is a promineralising cell surface marker and is potentially useful as a marker for isolation of stem cell populations for use in regenerative therapies. For clinical applications, cells would be isolated from bone marrow aspirate or orthopaedic surgical waste within intraoperative time of less than two hours, then paired with an osteoconductive scaffold to provide an alternative treatment option with potentially accelerated bone repair and regeneration. Dental Pulp Stomal Cells (DPSCs) which were used as a model system throughout this work, were shown to express 2.8 ± 1.3 x 10^5 TNAP molecules on the cells’ surface and the number of TNAP molecules per TNAP+ cell was not altered by factors such as passage number, seeding density and cell donor. Following this, a microfluidc cell separation device was designed and developed for the enrichment of TNAP+ cells, by capture and subsequent release of TNAP+ DPSCs via a surface functionalised with anti-TNAP antibodies. The recovered cells demonstrated a TNAP+ enriched population with up to a two fold enrichment of TNAP+ cells. The device also begun to meet the requirements for a minimally manipulated cell separation as minimal antibody could be detected on the surface of the recovered cells. As well, the capture and release mechanism had minimal effect on the cells’ biological characteristics, as the recovered enriched population retained a high viability and retained their osteogenic differentiation potential. The specificity to TNAP on the cells’ surface of previously identified non-antibody TNAP binding proteins, known as Affimers, was investigated for potential use within the cell separation technology. Affimer proteins were identified for expression and purification, and demonstrated specificity to recombinant TNAP protein. However, there was minimal evidence of specificity to TNAP on the cells’ surface and therefore subsequent development of the device utilised an anti-TNAP antibody instead. This thesis demonstrated a novel cell separation technology capable of providing an enriched population of viable TNAP+ cells with no obvious alterations in their biological characteristics. This provides a platform technology for potential future clinical use in bone regenerative therapies.