LGR6+ Epithelial Stem Cell Augmentation of Fracture Healing
Amanda Josephine Ross, MD; Ashim Gupta, PhD; Joel Reichensperger, BS; Carrie Harrison, BS; Andrew Klein, BS; Michael W. Neumeister, MD
Southern Illinois University School of Medicine, Springfield, IL
Currently, atrophic and hypertrophic non-unions are a challenging complications in fracture care. We propose an alternative approach to the current standard of care in fracture healing by introducing a theory based on the isolation of the patient's own autologous epithelial stem cells (ESC) which reside in an easily accessible portion of the skin. We hypothesize that LGR6+ ESCs will aid in fracture healing and prevention of fracture non-unions.
LGR6+ ESC were isolated from the hair follicle's follicular bulge of green fluorescent expressing (GFP) expressing Sprague Dawley (SD) rats, SD-Tg (UBC-EGFP) 2BalRrcc, using a FACSAria II flow cytometer with LGR6+, CD45- and CD90+ as markers. The multipotency of the isolated stem cells were determined by inducing them towards osteoblasts, chondrocytes and adipocytes. The induction was confirmed using Alizarin Red, Alcian Blue and Oil Red O stain respectively. The isolated cells were seeded on control tissue culture polystyrene (TCPS) and collagen coated coverslips (CCS) and divided into two groups (uninduced and osteo-induced) for further analysis. Confocal microscopy and Scanning Electron Microscopy (SEM) was used to determine cell morphology, adhesion and growth. A Vi-Cell cell counter was used to measure cell viability (or survival). A FluoReporter Blue Fluorometrics dsDNA quantitation kit was used to determine proliferation rate. Finally, a qPCR assay will be performed to determine the osteogenic gene markers expression.
We successfully isolated and cultured LGR6+ ESC from GFP expressing SD rats. We confirmed their multipotency by successfully inducing them towards three lineages. The adipo-, osteo- and chondro-inductions were observed at days 12, 26 and 29 respectively. Isolated cells displayed growth, adherence, survival and maintained non-stressed morphology on the surface of TCPS and CCS after 28 days. Induced cells seeded on CCS demonstrated a lower proliferation rate as compared to controls, possibly due to low mitotic turn over after differentiation. We expect increased expression of osteogenic markers compared to cells seeded on TCPS.
So far, we have successfully isolated LGR6+ ESC and optimized our protocol to ensure repeatability. We showed the multipotency of the isolated ESC as well as adhesion, growth and viability on a collagen scaffold after osteoinduction. Post-completion of this study, we will evaluate the in-vivo potential of LGR6+ ESC in augmenting fracture healing in non-stressed (1mm), hypertrophic non-union (3mm) and atrophic (6mm) non-union fracture models. The successful completion of this study will provide surgeons with an autologous stem cell source to treat fracture non-unions.
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