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Addition of the LGR6+ Epithelial Stem Cell from Skin Results in Enhanced Levels Bone Production and a Progressive Osteogenic Lineage
Denver Matthew Lough, MD, PhD1; Damon S. Cooney, MD, PhD2; Shaun Mendenhall, MD1; Joel Reichensperger, BS1; Lisa Cox, BS1; Nicole Cosenza, MS1; Carrie Harrison, BS1; Michael W. Neumeister, MD1
1Plastic Surgery, Southern Illinois University School of Medicine, Springfield, IL; 2Department of Plastic and Reconstructive Surgery, Johns Hopkins University School of Medicine, Baltimore, MD

Background: Fractures complicated with features of malunion, loss of bone construct, comminution and/or osteonecrosis often result in multiple recurring surgical procedures in order to establish even limited intrinsic stability to a fracture site. However, often in this setting, further damage to bone and surrounding soft tissues secondary to recurring micro-trauma and inflammation subsequently prevent optimal union by keeping the defect itself within the reactive phase of fracture healing without a clear progression into the reparative/regenerative stage where osteogenic induction can occur properly. Here, we propose a model system utilizing the easily isolatable Leucine-rich repeat-containing G-protein coupled receptor 6 epithelial stem cell (Lgr6+ ESC), native to the hair follicle’s follicular bulge, for a new novel type of stem cell-based therapy for bone regeneration within fracture defects.

Methods: GFP expressing LGR6+ epithelial stem cells (ESC) were isolated from C57BL/6(UBC-GFP) mice using FACS gated on CD34, CD73, and LGR6 and were then seeded onto human bone. Viability and attachment was assessed using confocal and scanning electron microscopy and bioluminescence. Immunofluorescence, RT-PCR, and proteome arrays were used to determine osteogenic differentiation.

Results: Following LGR6+ ESC seeding, there was significant up regulation of Osteocalcin, COL1A1, BMP2,4 and 6, EGFR, FGF2 and Osteopontin at both gene and protein levels when compared to controls. Alizarin red staining indicated a quantitative colorimetric increase in the the presence of calcific deposition by these cells suggesting a progressive osteogenic lineage when placed on decellularized bone scaffolding.

Conclusion: From this research, we hope to provide the hand community with an alternative stem cell based therapy for complex fracture repair. Additionally, we hope to provide Orthopedic and Plastic Surgery surgical scientists with a new type of epithelial stem cell for clinical applications in both bone and soft tissue regeneration.


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