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Decellularization to Produce Biological Synovial Extracellular Matrix Scaffolds THESIS Presented in Partial Fulfillment of the R

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Decellularization to Produce Biological Synovial Extracellular Matrix Scaffolds THESIS Presented in Partial Fulfillment of the R Decellularization to Produce Biological Synovial Extracellular Matrix Scaffolds THESIS Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Nathalie Ann Reisbig Graduate Program in Comparative and Veterinary Medicine The Ohio State University 2016 Master's Examination Committee: Alicia Bertone, DVM, PhD, DACVS, DACVSMR (advisor) Maxey Wellman, DVM, MS, PhD, Diplomate ACVP Matthew Brokken, DVM, MSc, DACVS, DACVSMR Abstract The objective of this study was to evaluate four methods to generate a decellularized synovium scaffold (SynECM) for use as a biologic transplant. Villous synovium was harvested and frozen (-80oC) from the femoropatellar and medial femorotibial joints of four adult normal horses <7 years of age. Fresh-thawed equine stifle synovial tissue was decellularized by four methods: 1) 0.1% peracetic acid (PAA) solution (1XPAA), 2) PAA treatment repeated (2XPAA), 3) 1% Triton X followed by DNAse (Triton), and 4) 2M NaCl followed by DNAse (NaCl). Tissue from each method was evaluated for morphology (histologic, scanning electron microscopy), viability (culture and exclusion staining) and decellularization efficiency (presence of residual cells, DNA content, and DNA fragmentation). All four methods resulted in non-viable synovial extracellular matrix scaffolds. Single PAA treatment retained synovium villous matrix integrity but with excess cell residue containing high cellular DNA content and DNA fragments > 25,000 base pairs (bp). 2XPAA treatment also had retained matrix integrity, but low DNA content with short DNA fragments (< 300 bp). The Triton and the NaCl preparations damaged villous structure leaving little to no discernible synovial villi, no identifiable residual cells and short (<300 bp) DNA fragments. The results indicated that two serial treatments of 0.1% PAA was the best method evaluated for obtaining SynECM ii with desirable morphology and low DNA content. An additional advantage of 2XPAA preparation is that PAA sterilizes ECMs during the decellularization process. iii Acknowledgments The author wishes to thank Dr Becky Lovasz for technical assistance. iv Vita 2002................................................................Diploma, Ringstabekk Skole, Oslo, Norway 2004................................................................International Baccalaureate, Oslo, Norway 2012................................................................Vet. Med. Leipzig, Germany 2013 to present ..............................................Resident, School of Veterinary Medicine, The Ohio State University Publications Prospective Randomized Blinded Clinical Trial Evaluating Dental Pulp Treatment for Equine Lameness. Alicia L. Bertone, DVM, PhD, DACVS, DACVSMR; Nathalie Reisbig, MedVet; Allison H. Kilborne, DVM; Navid Salmanzadeh, DVM; Rebecca Lovasz DVM; Mari Kaido, BVSc; Lisa J. Zekas, DVM, DACVR; Matthew Brokken, DVM, MSc, DACVS, DACVSMR; Joy Sizemore BSc; Logan Scheuermann BSc. NAVRMA Abstracts; 164, 2015 Objective Gait Analysis in Naturally Lame Horses. Mari Kaido, BVSc; Allison H. Kilborne, DVM; Joy Sizemore, BSc; Nathalie Reisbig, MedVet; Turi Aarnes, DVM, ACVA; Alicia L. Bertone, DVM, PhD, DACVS, DACVSMR. ACVS Abstracts; 2015. Prospective Randomized Blinded Controlled Clinical Trial for the Injection of Living Dental Pulp Cell Particles for the Treatment of Equine Lameness Conditions Alicia L. Bertone, DVM, PhD, DACVS, DACVSMR; Nathalie Reisbig, MedVet; Allison H. Kilborne, DVM; Navid Salmanzadeh, DVM; Rebecca Lovasz DVM; Mari Kaido, BVSc; Joy Sizemore BSc; Logan Scheuermann BSc; Lisa J. Zekas, DVM, DACVR; Matthew Brokken, DVM, MSc, DACVS, DACVSMR. ACVS Abstracts; 2015. Characterization of Living Synovial Extracellular Matrix Scaffolds for Gene Delivery Reisbig N, Hussein H, Pinnell E, Bertone AL. ACVS Abstracts; 2015. Decellularization to Produce Biological Synovial Extracellular Matrix Scaffolds. NA Reisbig, H Hussein, E Pinnell, AL Bertone. European College of Veterinary Surgeons Annual Abstracts, Berlin, 2015. v Fields of Study Major Field: Comparative and Veterinary Medicine vi Table of Contents Abstract ............................................................................................................................... ii Acknowledgments.............................................................................................................. iv Vita ...................................................................................................................................... v Publications ......................................................................................................................... v Fields of Study ................................................................................................................... vi List of Tables ..................................................................................................................... ix List of Figures ..................................................................................................................... x Chapter 1 Introduction ........................................................................................................ 1 Chapter 2 Materials and Methods ....................................................................................... 5 Decellularization and Creation of Scaffolds (SynECM) ................................................. 6 SynECM Characterization............................................................................................... 8 Quantification of Residual Living Cells ...................................................................... 8 Histological Evaluation ............................................................................................... 8 Scanning Electron Microscopy (SEM) ...................................................................... 10 DNA Isolation and Quantification ............................................................................. 10 vii Gel Electrophoresis .................................................................................................... 10 Data Analysis ................................................................................................................ 11 Chapter 3 Results .............................................................................................................. 12 Chapter 4 Discussion ........................................................................................................ 22 Footnotes ........................................................................................................................... 27 References ......................................................................................................................... 28 viii List of Tables Table 1. The methods used for decellularizing synovium. ................................................. 7 Table 2. Histological criteria scale (0-4) for the decellularized synovium and control ...... 9 Table 3. Overview of results ............................................................................................ 14 ix List of Figures Figure 1. Hematoxylin and eosin staining ........................................................................ 15 Figure 2. Microscopy of hematoxylin-eosin and picrosirius red stained SynECMs. ....... 16 Figure 3. Scanning electron microscopy (280x) of the SynECMs. ................................. 18 Figure 4. Histomorphological scoring of control synovium and SynECMs .................... 19 Figure 5. DNA content of SynECMs ................................................................................ 20 Figure 6. DNA gel electrophoresis of SynECMs.............................................................. 21 x Chapter 1 Introduction Decellularized extracellular matrices (ECMs) have been of intense interest in regenerative research for use as tissue or organ replacement1 and in reconstructive surgical procedures.2 The field of decellularizing ECM has grown and developed to include tissues spanning from decellularized sheaths (skin, esophageal mucosa3, small intestinal submucosa4, human umbilical vein) to full organ decellularization (liver, heart5, lung6). ECM is composed of the molecules secreted by the resident cells in the tissue thereby providing a biologic matrix with the potential to retain the ECM 3-dimensional structure and composition that supports the cell of original phenotype. The ECM functions as a medium for signal transfer to and between cells, influencing the proliferation and migration of resident cells to express the tissue phenotype.7, 8 The ECM is regarded as being in a state of dynamic equilibrium8 that is central to normal tissue and organ development.9 Decellularized ECM with homologous transfer should readily integrate into the natural biologic turnover of ECM for that tissue10. Generating decellularized tissue requires a process that removes cells and antigenic components, yet retains the constituents of 3-D structure and composition, without residual toxicity, such that the ECM promotes the residence and growth of homologous cells that could be transplanted without adverse reaction.11 Numerous studies have shown 1 that the method of decellularization affected the properties of an ECM.12 Fortunately, most ECM components are relatively conserved across mammalian species reducing the risk of a host immune response to allogenic or xenogeneic ECM,13,14 however it has been shown that insufficient
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