LGR6+ Epithelial Stem Cell Augmentation of Fracture Healing
Andrew Klein, BS, Amanda Ross, MD, Ashim Gupta, MS, PhD, Joel Reichensperger, BS, Lauren Hughes, BS, Max Korzec, BS, Michael Neumeister, MD.
SIU Department of Surgery; Institute for Plastic Surgery, Springfield, IL, USA.
Currently, atrophic and hypertrophic non-unions are challenging complications in fracture care. We propose an alternative approach to the current standard of care in fractures based on the isolation of the patient's autologous epithelial stem cells (ESC) which reside in an easily accessible portion of the skin. We hypothesize that LGR6+ ESCs will provide surgeons with an autologous stem cell source to aid in fracture healing and prevention of fracture non-unions.
LGR6+ ESCs 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 isolated stem cells were stained with Alizarin Red, Alcian Blue and Oil Red O stain to confirm multipotency. 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) determined cell morphology, adhesion and growth on both mediums with and without osteogenic induction. Cell proliferation rates of these samples were determined using an MTT assay and analyzed with ANOVA to determine the effect of group, day, and the interaction on mean density. Osteogenic gene marker expression will be assessed using a qPCR array.
We successfully isolated and cultured LGR6+ ESCs from GFP expressing SD rats. We confirmed their multipotency via separate inductions towards osteoblast, chondrocyte and adipocyte lineages visible under light microscopy. The adipo-, osteo- and chondro-inductions were observed at days 12, 26 and 29 respectively. Uninduced and Induced cells were shown on confocal microscopy and SEM to grow, adhere, survive and maintain their respective non-stressed or osteogenic morphology on the surface of TCPS and CCS at days 7, 14, 21, and 28. The MTT assay demonstrated that cells seeded on CCS and TCPS have no significant variation in proliferation rate between coverslip materials for either the induced or uninduced conditions.
We successfully isolated LGR6+ ESCs and demonstrated the multipotency of the isolated ESCs. We determined that LGR6+ cells grown on CCS have similar proliferation rates and morphology to cells grown on the industry standard TCPS for both induced and uninduced conditions. However, we anticipate in further studies that CCS grown cells will have an increased expression of osteogenic markers compared to cells seeded on TCPS. Post-completion of this study, we will evaluate the in-vivo potential of LGR6+ ESCs in augmenting fracture healing in non-stressed (1mm), atrophic non-union (3mm) and hypertrophic (6mm) non-union fracture models.
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