Alpha Omega Alpha Honor Medical Society

2014 Research Abstract

Optimizing Decellularized Bone as a Scaffold for Novel Stem Cell Therapies for the Alveolar Cleft

Investigator: Justin Morse, University of North Carolina at Chapel Hill School of Medicine

Mentor: John A. van Aalst, MD, MA

Alternatives to iliac bone grafting for the alveolar cleft are being explored to eliminate the need for a secondary surgical site. We developed a swine alveolar cleft model successfully treated with autologous umbilical cord mesenchymal stem cells (UCMSCs) on polymer nanofiber scaffolds. However, lack of volume fill in the cleft defect limits nanofiber scaffold utility. Decellularized porcine maxillary bone is a novel scaffold that may improve volume fill and has optimal architecture for bone regeneration. If effective, decellularized maxilla seeded with UCMSCs may serve as an alternative to iliac bone grafting. This preliminary work evaluates decellularization methods and UCMSC attachment, proliferation, and osteoinduction on decellularized maxilla scaffolds.

Three different decellularization methods were compared on porcine maxilla. All bone received a chemical/enzymatic protocol combined with either 1) agitation, 2) freeze-thaw+agitation, or 3) freeze-thaw+sonication. Samples were cryosectioned and stained with H&E. Mean percent decellularization was calculated by the average nuclear remnants in 3 high power fields (HPF). Remaining DNA was quantified by flourometer. All cell studies were performed on group 3 decellularized scaffolds. To assess attachment, UCMSCs were transduced with lentivirus expressing GFP, seeded, and examined by immunofluorescent microscopy at day 7. Proliferation of UCMSCs compared 3 seeding densities (1, 2, and 4 x 105) and was assessed by colorimetric analysis with Promega CellTiter 96® Proliferation Assay using quantitative absorbance analysis at days 1, 3, 5, and 9. UCMSCs were osteoinduced with standard osteoinduction media. RNA was isolated at day 7 and analyzed by RTPCR for bone morphogenetic protein-2 (BMP2), alkaline phosphatase (ALP), and Osteopontin (OPN) gene expression. Statistical analysis compared different decellularization methods and seeding densities using ANOVA with Kruskal Wallis testing. Wilcoxon Rank Sum testing was used for post hoc testing between groups.

Decellularization resulted in the following percentages of cell removal: agitation 36.86% (SD 14.22); freeze-thaw+agitation 53.96% (SD 3.41); freeze-thaw+sonication 82.51% (SD 5.87), while percent reduction in DNA/bone weight were as follows: agitation 66.7% (SD 5.7), freeze-thaw+agitation 63.4% (SD 11.1), and freeze-thaw+sonication 91.5% (SD 0.01). UCMSCs expressing GFP demonstrated cell attachment and migration throughout the scaffold at day 7. UCMSCs successfully proliferated on scaffolds. A seeding density of 4x105 cells demonstrated the optimum increase in wavelength absorbance. Osteogenic genes BMP2, ALP, and OPN showed RNA upregulation of 479%, 11%, and 38% respectively at day 7.

Group 3 decellularization methods resulted in optimal bone decellularization by H&E and DNA analysis. These scaffolds support UCMSC attachment, migration, and proliferation. Proliferation is optimal at a seeding density of 4x104. RNA upregulation shows successful osteoinduction of UCMSCs demonstrating these scaffolds are suitable for testing in our swine model.

Last modified: 5/29/2015

Updated on May 29, 2015.

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