Optimization of Polycaprolactone-collagen Nanofiber Scaffolds For Tissue Regeneration Utilizing Leucine-rich Repeat-containing G-coupled Protein Receptor Stem Cells
Michael Ruebhausen, M.D.1, Michael W. Neumeister, M.D.1, Ashim Gupta, PhD1, Craig Cady, PhD2, Jonathan Tiessen, B.S.2, Jack Blank, B.S.2, Jaclyn Conway, B.S.2, Kalyani Nair, PhD2.
1Southern Illinois University School of Medicine, Springfield, IL, USA, 2Bradley University, Peoria, IL, USA.
Full-thickness wounds without appropriate skin graft donor sites, such as in large burns, have long been a difficult clinical problem. A biocompatible nanofiber scaffold that can aid in tissue regeneration serves as an ideal solution. To date, many efforts have been made to develop a tissue-engineered graft to heal these wounds, though none have proven to be ideal. In this regard, the fabrication and optimization of the mechanical characteristics associated with bioengineered nanofiber scaffolds is essential for maintaining optimal cellular adhesion and proliferation. Electrospinning provides a flexible and controllable process for the generation of nano-scale fiber matrices for this purpose. Polycaprolactone (PCL) is a biocompatible polymer that has been widely studied for its cellular and biological compatibility and use in electrospinning applications. Collagen has been shown to increase cellular proliferation on coated nanofiber scaffolds. The electrospinning of solutions containing both PCL and collagen has allowed fabrication of hybrid matrices that mimic fiber architecture seen prominently in the dermis. Stem cell integration capable of reestablishing native skin properties has been a shortcoming of several stem cell lines, such as adipose-derived stem cells. Leucine-rich repeat-containing G-protein coupled receptor (LGR5 and LGR6) epithelial stem cells, however, have demonstrated the ability to heal wounds more quickly than controls in addition to re-growing hair in healed wounds. The objective of this study is to quantify and characterize all of the mechanical and physical characteristics associated with the hybrid nanofiber scaffolds fabricated with varying concentrations of collagen and PCL in the electrospun scaffold.
Fiber diameter, porosity, degradation rates, stiffness, and tensile properties were all characterized and compared in nanofiber scaffolds composed of PCL + Col 0, 15, and 35%. Gross cell adhesion and morphology was also evaluated using SEM imaging at day 7 post-seeding. Immunofluorescence (IF) was used to verify maintenance of stemness in LGR5 samples with CD73. Additionally, LGR5 and 6 stem cell proliferation will be quantified at three time points: 7, 14, and 21 days after being seeded at a density of 5000 cells/ 0.5mm scaffold.
Increasing collagen I content improved uniformity in fiber diameter, overall porosity and degradation rates. As expected, fiber stiffness and Young’s modulus decreased with increasing collagen. Gross SEM examination and green fluorescent protein IF showed cell adherence in a non-stressed state on the scaffolds.
Examination of IF showed cell staining for CD73, consistent with maintenance of stemness.
The characterization of these nanofiber scaffolds show promise for making highly repeatable and optimized nanofiber scaffolds for stem cell implementation and use in tissue regeneration. Further studies are underway to evaluate stemness and proliferation in scaffolds with varying collagen content. If stemness is maintained, this hybrid graft presents an exciting development for an in-vivo study for evaluating the graft’s performance against other wound healing methods in full thickness wounds.
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