Oral Biology: Molecular Techniques and Applications

Preface
Contents
Contributors
Part I: Saliva and Other Oral Fluids
Chapter 1: RNA Sequencing Analysis of Saliva exRNA
1 Introduction
2 Available Methods for Saliva Collection, Pre- and Post-processing, for RNA-Sequencing
2.1 Saliva Collection
2.2 RNA Extraction Methods
2.2.1 RNA Quantification and Quality Controls (QCs)
2.3 cDNA Library Construction Methods
2.4 Sequencing and Bioinformatic Analysis of RNA-Seq Data
2.4.1 Library Quality Control and Sequencing
2.4.2 Alignment Settings
2.5 Uniqueness of Salivary RNA-Seq Analysis
References
Chapter 2: Proteome Analysis of Oral Biofluids in Periodontal Health and Disease Using Mass Spectrometry
1 Introduction
2 Materials
2.1 GCF Collection
2.2 Qubit Assay
2.3 FASP
2.4 Software for Proteomic Data Analysis
2.5 Equipment for Proteomic Data Analysis
3 Methods
3.1 Gingival Crevicular Fluid (GCF) Collection
3.2 GCF Supernatant Preparation
3.3 Saliva Supernatant Preparation and Total Protein Quantification
3.4 FASP Digestion for the GCF or Saliva Supernatant
3.5 LC-MS/MS Analysis
3.6 Protein Search, Identification, and Label-Free Quantification
4 Notes
References
Chapter 3: Saliva Diagnosis Using Small Extracellular Vesicles and Salivaomics
1 Introduction
2 Materials
2.1 Saliva Collection and Storage
2.2 2.2 Salivary sEVs Isolation Using SEC Columns
2.3 Salivary Genomic DNA Isolation
2.4 16S rRNA Library Preparation and NGS
2.5 Salivary Total RNA Isolation
2.6 RNA Library Preparation and Sequencing
2.7 Salivary Genomic DNA Isolation
2.8 Salivary gDNA Library Preparation
2.9 Materials for Salivary Proteome
3 Methods
3.1 Unstimulated Whole Saliva Collection and Storage
3.2 Salivary sEVs Isolation Using SEC Columns
3.3 Salivary Genomic DNA Isolation
3.4 16S rRNA Gene Amplification and Sequencing
3.5 Salivary Total RNA Isolation Using Trizol and ABI PicoPure Columns
3.6 Salivary RNA Library Preparation and Illumina RNA-Sequencing
3.7 Salivary gDNA Isolation
3.8 Salivary gDNA Library Preparation (see Fig. 3)
3.9 MeDIP, Library Amplification, and Sequencing
3.10 Salivary Proteome Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)
4 Notes
References
Chapter 4: Antioxidant Micronutrients and Oxidative Stress Biomarkers
1 Introduction
2 Materials
2.1 Determination of Ascorbic Acid and Dehydroascorbic Acid
2.2 Measurement of Protein Carbonyls
2.3 Preparation of White Cells for 8-OHdG Analyses
2.3.1 3% Gelatin in PBS
2.4 Comet Assay
2.5 Serum Preparation
2.6 Plasma Preparation
2.7 Metaphosphoric Acid (See Note 1)
2.8 Collection of Gingival Crevicular Fluid (GCF)
2.9 Collection of Saliva
2.10 Measurement of Lipid Oxidation Products
3 Methods
3.1 Determination of Ascorbic Acid and Dehydroascorbic Acid in Human Plasma
3.2 Carotenoid Analyses (See Notes 5, 6, and 7)
3.3 Measurement of Protein Carbonyls to Detect Oxidative Protein Biomarkers
3.3.1 Preparation of ELISA Standards
3.3.2 Method
3.4 Carbonyl ELISA Method
3.5 8-OHdG Analyses or Comet Assay to Determine Oxidative DNA Damage (See Notes 12 and 13)
3.5.1 Preparation of White Cells (Buffy Coats)
3.5.2 3% Gelatin in PBS (150 Bloom from Pig Skin) for Preparation of Buffy Coats
3.6 Comet Assay to Determine Oxidative DNA Damage (See Note 16)
3.6.1 Day 1
3.6.2 Day 2 (See Note 17)
3.6.3 Day 3
3.7 Serum Preparation to Determine Antioxidant Capacity (See Note 20)
3.8 Determination of Antioxidant Capacity (See Note 22)
3.8.1 Plasma Preparation
3.8.2 33.5% Metaphosphoric Acid; for Plasma Vitamin C Analyses (See Notes 23 and 24)
3.9 Collection of Gingival Crevicular Fluid (GCF) to Measure Antioxidant Activity (See Note 25)
3.10 Collection of Saliva Samples to Measure Antioxidant Activity (See Notes 26, 27 and 28) [20]
3.11 Analysis of Phospholipid Oxidation Products (See Notes 29 and 30)
3.11.1 Lipid Extraction
3.11.2 Lipid Analysis
4 Notes
References
Part II: Molecular Biosciences
Chapter 5: The Oral Microbiota in Health and Disease: An Overview of Molecular Findings
1 Introduction
2 Nucleic Acid Technologies
3 Diversity and Taxonomy of Oral Bacteria
4 Refined Bacterial Taxonomy Associated with Oral Diseases
4.1 Dental Caries
4.2 Halitosis
4.3 Periodontal Disease
4.4 Apical Periodontitis
5 Concluding Remarks
References
Chapter 6: The Long and Short of Genome Sequencing: Using a Hybrid Sequencing Strategy to Sequence Oral Microbial Genomes
1 Introduction
2 Purification of Genomic DNA from Oral Microorganisms
2.1 Growth Media and Incubation Conditions
2.2 Purification of Genomic DNA
3 Post-sequencing Bioinformatics
3.1 Genome Assembly Using Canu
3.2 How Do You Solve a Problem Like ONT? The Need for a Hybrid Sequencing Strategy
3.3 Hybrid Genome Assembly Using SPAdes
3.4 Concluding Remarks
4 Notes
References
Chapter 7: Microbial Community Profiling Using Terminal Restriction Fragment Length Polymorphism (T-RFLP) and Denaturing Gradi...
1 Introduction
2 Materials
2.1 DNA Extraction
2.2 Terminal Restriction Fragment Length Polymorphism
2.2.1 PCR Amplification of the 16S rRNA Gene
2.2.2 T-RFLP Analysis
2.3 Denaturing Gradient Gel Electrophoresis
2.3.1 PCR Amplification of 16S rRNA Gene
2.3.2 DGGE Analysis
3 Methods
3.1 DNA Extraction
3.2 Terminal Restriction Fragment Length Polymorphism
3.2.1 PCR Amplification of 16S rRNA Gene
3.2.2 T-RFLP Analysis for ABI PRISM 310 Genetic Analyser
3.3 Denaturing Gradient Gel Electrophoresis
3.3.1 PCR Amplification of 16S rRNA Gene
3.3.2 DGGE Analysis
4 Notes
References
Chapter 8: Bioinformatic Approaches for Describing the Oral Microbiota
1 Introduction
2 Microbiota Analysis Pipeline #1 Using QIIME2
2.1 Import Data into QIIME2
2.2 Remove Primers
2.3 Merge Paired-End Sequences
2.4 Denoise the Data
2.5 OTU Picking
2.6 Taxonomic Classification of OTUs
2.7 Building a Phylogenetic Tree (Optional)
2.8 Filter Steps
2.9 Normalize Sequence Reads per Sample via Rarefaction
2.10 Core Diversity Analysis with Figures (See Note 7)
3 Microbiota Analysis Pipeline #2 Using R and DADA2
3.1 Importing Data into R
3.2 Quality Filtering
3.3 Merge Paired-End Reads
3.4 Chimera Removal
3.5 Taxonomic Analysis
3.6 Core Diversity Analysis via Phyloseq
3.7 Loading in Sample Metadata
3.8 Basic Analyses
4 Notes
References
Chapter 9: Adhesion of Yeast and Bacteria to Oral Surfaces
1 Introduction
2 Materials
2.1 Radiolabeling of Yeast and Bacterial Cells and Cell Culture
2.2 Blot Overlay Assay to Demonstrate Adhesion of Yeast Cells to Immobilized Proteins
2.3 Adhesion of C. albicans Cells to Saliva-Coated Hydroxyapatite
2.4 Adhesion of Saliva-Treated C. albicans Cells to Epithelial Cells
2.5 Adhesion of C. albicans or S. epidermidis Cells to Saliva-Coated Medical Grade Silicone or to Denture Prosthetic Materials
2.6 Adhesion of S. epidermidis Cells to Denture Prosthetic Materials Under Flow Conditions
2.7 C. albicans Biofilm Formation on Denture Acrylic Flag Strips Suspended in Microtiter Wells
3 Methods
3.1 Radiolabeling of Yeast and Bacterial Cells and Cell Culture
3.1.1 To Prepare Inocula for Pre-culture of Yeast or Bacteria
3.1.2 Preparation of C. albicans Cells Radioactively Labeled with 35S-methionine
3.1.3 Preparation of S. epidermidis Cells Radioactively Labeled with 3H-thymidine
3.2 Blot Overlay Assay to Investigate Adhesion of Yeast Cells to Immobilized Proteins
3.2.1 SDS-PAGE Analysis
3.2.2 Electroblotting
3.2.3 Radiolabeled Yeast Overlay
3.3 Adhesion of C. albicans Cells to Saliva-Coated Hydroxyapatite
3.4 Adhesion of Saliva-Treated C. albicans Cells to Epithelial Cells
3.4.1 Epithelial Cell Monolayers
3.4.2 Adherence Assay Conditions
3.4.3 Confocal Microscopy
3.5 Adhesion of C. albicans or S. epidermidis Cells to Saliva-Coated Medical Grade Silicone or to Denture Prosthetic Materials
3.6 Adhesion of S. epidermidis to Denture Prosthetic Materials Under Flow Conditions
3.6.1 Bacteria: (S. epidermidis)
3.6.2 Preparation of Denture Prosthetic Material Surfaces
3.6.3 Parallel Plate Flow Chamber Set Up
3.6.4 Bacterial Deposition
3.7 C. albicans Biofilm Formation on Denture Acrylic Flag Strips Suspended in Microtiter Wells
3.7.1 Preparation of Inocula for Biofilm Experiments
3.7.2 Fabrication of Acrylic Flag Strips
3.7.3 Coating of Acrylic Flags with Saliva
3.7.4 Initial Deposition of Yeast Cells on Acrylic Flags
3.7.5 Biofilm Formation
3.7.6 Biofilm Quantification Using Crystal Violet (CV) Staining
4 Notes
References
Chapter 10: Quantitative Analysis of Periodontal Pathogens Using Real-Time Polymerase Chain Reaction (PCR)
1 Introduction
2 Materials
2.1 Samples
2.2 Positive Controls (Standard Curve)
2.3 DNA Extraction
2.4 qPCR Amplification
2.5 Multiplex qPCR
3 Methods
3.1 Biosafety Measures in Handling Clinical Samples
3.2 Sample Collection
3.3 Positive Controls (Standard Curve)
3.4 DNA Extraction
3.4.1 DNA Extraction from Pure Cultures
3.4.2 DNA Extraction from GCF Samples
3.4.3 DNA Extraction from Blood Samples
3.4.4 DNA Extraction from Any Other Biological Solid Samples, for Example, Atheromatous Plaques or Brain Samples
3.5 Preparation of Standard Curves for qPCR
3.6 qPCR Assay
3.7 Data Analysis
4 Notes
References
Chapter 11: Methods to Study Antagonistic Activities Among Oral Bacteria
1 Introduction
2 Materials
2.1 Bacteriocin Assay
2.2 Biofilm Assay and Confocal Laser Scanning Microscopy
2.3 H2O2 Detection with Indicator Plates
2.3.1 Enzymatic H2O2 Detection
2.3.2 Nonenzymatic H2O2 Detection
2.4 Isolation and Purification of Bacteriocin
2.5 Derivatization of Lantibiotics
2.6 Cloning and Other Genetic Techniques
3 Methods
3.1 Competition Assay on Plate Culture
3.2 Competition Assay in Biofilms
3.3 H2O2 Production Assay
3.4 Bacteriocin Activity Assay by Deferred Antagonism (Plate Overlay)
3.5 Isolation of Bacteriocin
3.6 Purification of Bacteriocin
3.7 Sequencing of the Purified Bacteriocin
3.8 Isolation of Bacteriocin Structural Genes by Reverse Genetics (See Note 10)
3.9 Mutagenesis via Single and Double Crossover
3.10 Gene Expression Analysis by Reporter Fusions
3.11 Luciferase Assay Using Live Cells
4 Notes
References
Chapter 12: Generation of Multispecies Oral Bacteria Biofilm Models
1 Introduction
2 Materials
3 Methods
3.1 Initial Biofilm Baseline Colonization for the Development of All Different Biofilm Models
3.2 Culture Specifics for the Different Biofilm Models
3.2.1 The Dental Caries Model
3.2.2 The Denture Biofilm Model
3.2.3 The Gingivitis and Periodontitis Biofilm Model
3.2.4 Further Analysis and Utility of the Biofilm Models
4 Notes
References
Chapter 13: Markerless Genome Editing in Competent Streptococci
1 Introduction
2 Materials
2.1 Competence Induction and Transformation
2.2 PCR
2.3 Agarose Gel Electrophoresis
2.4 Primers
3 Methods
3.1 Construction of Markerless Amplicons
3.2 Markerless Transformation Protocol Using XIP in S. mutans
3.3 Markerless Transformation Protocol Using CSP in S. pneumoniae
3.4 Examples of Applications
3.4.1 Example 1. Eight-Basepair Inversion
3.4.2 Example 2. Thirty-Nine-Basepair Deletion
3.4.3 Example 3. Single-Base Substitution in S. mutans
3.4.4 Example 4: Single-Base Substitution in S. pneumoniae
4 Notes
References
Chapter 14: A Protocol to Produce Genetically Edited Primary Oral Keratinocytes Using the CRISPR-Cas9 System
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Plasmid Transformation and Purification
2.3 Transfection and Gene Edition Reagents:
2.4 PCR and Sequencing
3 Methods
3.1 Target Sequence Design
3.2 Plasmid Transformation and Purification
3.2.1 Plasmid Transformation
3.2.2 Plasmid Purification
3.3 Transfection
3.4 Cell Selection and Expansion
3.5 Confirmation of CRISPR-Cas9-Mediated Genetic Edition
4 Notes
References
Chapter 15: Size-Based Method for Enrichment of Circulating Tumor Cells from Blood of Colorectal Cancer Patients
1 Introduction
2 Materials
2.1 Cell Lines and Culture Conditions
2.2 Preparation of 10x Red Blood Cell (RBC) Lysis Buffer
2.3 Description of MetaCell CTC Enrichment Kit
2.4 Preparation of 4% Paraformaldehyde (Fixative)
2.5 Preparation of Permeabilization Buffer (0.2% Triton X-100 in DPBS)
2.6 Antibody Buffer
2.7 Blocking Solution (10% Normal Donkey Serum, 3% BSA, in Permeabilization Buffer)
2.8 Primary Antibody Solution
3 Methods
3.1 Thawing Frozen Cell Lines
3.2 Spiking Experiments to Test the MetaCell Method of Enriching for CTCs
3.3 Immunostaining of CRC Cells Retained on the MetaCell Membrane
3.4 Benchmarking Strategy for MetaCell
3.4.1 Recovery Rate
3.4.2 WBC Depletion Rate
3.5 Patient Sampling and blood processing
4 Notes
References
Chapter 16: Strategy for RNA-Seq Experimental Design and Data Analysis
1 Introduction
2 Materials
3 Methods
3.1 RNA-Sequencing Experimental Design
3.1.1 Single-End or Paired-End
3.1.2 Stranded or Unstranded Library
3.1.3 Power Analysis
3.1.4 Higher Coverage or More Replicates
3.1.5 Number of Replicates
3.1.6 Long vs. Short Reads
3.1.7 Total RNA + Ribodepletion vs. mRNA
3.2 Analysis of RNA-Sequencing Data
3.2.1 Setting Up the Computational Environment and Essential Bioinformatic Tools
3.2.2 Acquisition of Datasets from Publicly Available Sources
3.2.3 Assessment of Sequencing Data Quality, RNA-Seq Library Biases, and Processing
3.2.4 Alignment to the Reference Genome
3.2.5 Quantifying All the Transcripts/Genes in Individual Samples to Report Abundance
3.2.6 Differential Expression Analysis
4 Notes
References
Chapter 17: Characterization of the Expression and Role of Histone Acetylation and Deacetylation in Dental Pulp Cells
1 Introduction
2 Materials
2.1 Histochemical Analysis
2.2 Dental Pulp Cell (DPC) Mineralization Culture
2.3 Western Blotting Analysis
3 Methods
3.1 Immunohistochemical Analysis
3.1.1 Preparation of the Tissue Section
3.1.2 Immunostaining
3.2 Mineralizing Cell Culture of Dental Pulp Cells
3.2.1 Cell Culture and mineralization of Rat DPCs
3.2.2 HDACi Supplementation
3.2.3 Alizarin Red Staining to Evaluate Mineralization
3.3 Western Blot Analysis of HDAC Expression
3.3.1 Cell Lysis
3.3.2 Western Blotting
4 Notes
References
Chapter 18: Genome-Wide Analysis of Periodontal and Peri-implant Cells and Tissues
1 Introduction
2 Materials
2.1 Source Materials
2.1.1 Tissue Samples
2.1.2 Primary Mononuclear Cells Isolated from Patient Blood
2.1.3 Cultured Cells
2.2 Extraction and Purification of Nucleic Acids (and Protein)
2.3 Quantitation and Purity Assessment
2.4 High-Throughput Analysis
2.4.1 Microarray Platforms
2.4.2 Next-Generation Sequencing
3 Methods
3.1 Source Materials
3.1.1 Tissue Samples
3.1.2 Primary Mononuclear Cells Isolated from Patient Blood
3.1.3 Cultured Cells
3.2 Extraction and Purification of Nucleic Acids (and Protein)
3.3 Quantitation and Purity Assessment
3.4 High-Throughput Analysis
3.4.1 Microarray Platforms
3.4.2 Next-Generation Sequencing
4 Notes
References
Chapter 19: Differential Expression, Functional and Machine Learning Analysis of High-Throughput -Omics Data Using Open-Source...
1 Introduction
2 Materials
2.1 Hardware
2.2 Software
2.3 Manifests, Annotations, Genome Files
2.4 Targets File
2.5 Raw Data
3 Methods
3.1 Pre-processing of Array Data
3.2 Pre-processing of Sequencing Data
3.3 Differential Expression Analysis
3.4 Functional Analysis
3.5 Upload to Repositories
3.6 Use of Supervised Learning Algorithms for the Distinction of Formerly Classified Aggressive and Chronic Periodontitis Base...
3.7 Identification of Novel Classes of Periodontitis Based on mRNA Expression Profiles Using Unsupervised Clustering
4 Notes
References
Chapter 20: Micro-RNA Profiling in Dental Pulp Cell Cultures
1 Introduction
2 Materials
2.1 DPC Isolation and Culture
2.2 Mineralizing Cell Culture (see Note 1)
2.3 Epigenetic Modifier (HDACi and DNMTi) Solutions
2.4 miRNA Isolation
2.5 RNA Quality Analysis
2.6 Alizarin Red S Staining and Quantification
2.7 RNA Sequencing ``As a Service´´ and ``In-House´´ Data Analysis
3 Methods
3.1 DPC Isolation
3.2 Sub-culture of Confluent Cells
3.3 Sub-culture of Confluent Cells into 6-Well Plates
3.4 Mineralizing Culture (see Notes 2 and 3)
3.5 miRNA Isolation (see Notes 4 and 5)
3.6 RNA Quality Analysis
3.7 Alizarin Red S Staining of Cell Cultures
3.8 Alizarin Red S Quantification
3.9 RNA Sequencing Analysis
3.10 RNA Sequencing Data Analysis
3.10.1 Experiment Creation
3.10.2 Experimental Design
3.10.3 Interpretations
3.10.4 Quantification
3.10.5 Differential Expression Analysis
3.10.6 Target Gene Prediction (see Note 11)
3.10.7 GO Enrichment and Pathway Analysis (see Note 11)
4 Notes
References
Part III: Cells and Tissues
Chapter 21: Oral Epithelial Cell Culture Model for Studying the Pathogenesis of Chronic Inflammatory Disease
1 Introduction
1.1 Three-Dimensional (3D) Cell Culture Techniques
2 Materials
2.1 Epithelial Cell Culture Media and Reagents
2.2 Cell Culture Methodology (Fig. 5)
2.3 Bacterial Growth (See Note 1)
2.4 Immunocytochemistry of Cells Grown on Multi-well Slides
2.5 High-Throughput Immuno-cytochemistry
2.6 mRNA for Gene Expression
2.7 Polymerase Chain Reaction (PCR)
2.8 Agarose Gel Electrophoresis
3 Methods
3.1 Production and Maintenance of Epithelial Cell Model System
3.2 Bacterial Culture/Growth
3.3 Cell Culture
3.3.1 Cell Passage (See Notes 2 and 3)
3.3.2 Cell Growth on Glass Multi-well Slides
3.3.3 Growing Cells in 96-Well Plates
3.4 Immunocytochemical Analyses of NF-kB
3.4.1 Staining Procedure (See Note 4)
3.4.2 Quantification of Cell Translocation
3.5 High-Throughput Immuno-cytochemistry
3.5.1 Fixing of Cell Monolayer
3.5.2 Staining
3.5.3 Data Acquisition and Analysis
3.6 mRNA for Gene Expression
3.6.1 RNA Isolation
3.6.2 Reverse Transcription (RT)
3.6.3 Concentration and Purification of cDNA
3.6.4 Quantification of RNA and DNA
3.7 Polymerase Chain Reaction
3.8 Agarose Gel Electrophoresis
3.8.1 Preparation of Agarose Gel
3.8.2 Gel Electrophoresis
4 Notes
References
Chapter 22: A Cell Culture Method for the Isolation and Study of Primary Human Dental Pulp Cells
1 Introduction
2 Materials
2.1 Cell Culture Media and Related Solutions
2.2 Immunocytochemistry Reagents
2.3 Equipment
3 Method: (Fig. 1a-d)
3.1 Tooth Collection and Transfer to Tissue Culture Laboratory
3.2 Isolation of Human Dental Pulp Cells: Dental Pulp Explant Method
3.3 Cell Passaging: Trypsinization
3.4 Cell Counting
3.4.1 Calculating Total Number of Cells
3.5 Cell Cryopreservation
3.6 Thawing and Revival of Cryopreserved Cells
3.7 Immunocytochemistry
4 Notes
References
Chapter 23: Culturing Adipose-Derived Stem Cells Under Serum-Free Conditions
1 Introduction
2 Materials
2.1 Cell Culture Components
3 Methods
3.1 Isolating ADSC
3.2 Cell Culture and Serum Free Adaptation
3.3 Cell Passage
3.4 Cryopreservation
4 Notes
References
Chapter 24: Quantitative Real-Time Gene Profiling of Human Alveolar Osteoblasts Using a One-Step System
1 Introduction
2 Materials
2.1 Primary Human Alveolar Osteoblast Isolation and Culture
2.2 Experimental Treatment of Osteoblasts
2.3 Total RNA Extraction and DNase 1 Treatment
2.4 One Step - Reverse Transcription and qPCR
2.5 Data Analysis
3 Methods
3.1 Primary Human Alveolar Osteoblast Isolation and Culture
3.2 Experimental Treatment of Osteoblasts
3.3 Total RNA Recovery in TRIzol
3.4 Total RNA Extraction and DNase I Treatment
3.4.1 Total RNA Extraction
3.4.2 DNase 1 Treatment
3.4.3 Elution
3.5 One-Step Reverse Transcription and qPCR
3.6 Data Analysis
3.6.1 Volcano Plot of Relative Gene Expression
3.6.2 Volcano Plot of Selected Regulation
3.6.3 Graph Demonstrating Expression Levels of an Individual Gene (2-ΔCq)
3.6.4 Graph Demonstrating the Fold Regulation of an Individual Gene (2-ΔΔCq)
4 Notes
References
Chapter 25: Fabrication and Characterization of Decellularized Periodontal Ligament Cell Sheet Constructs
1 Introduction
2 Materials
2.1 Primary Human Periodontal Ligament Cell (hPDLC) Harvesting and Expansion
2.2 Melt Electrospun PCL Carrier Membrane Fabrication
2.3 Cell Sheet Fabrication and Harvesting
2.4 Perfusion Decellularization Components
2.5 Cell Sheet Fixation and Preparation for Immunostaining and Confocal Imaging
2.6 Growth Factor Extraction
3 Methods
3.1 Primary Human Periodontal Ligament Cell (hPDLC) Harvesting and Expansion
3.2 Melt Electrospun PCL Carrier Membrane Fabrication
3.3 Cell Sheet Fabrication and Harvesting
3.4 Cell Sheet Construct Decellularization
3.5 Immunostaining of Cell Sheet Constructs
3.6 Growth Factor Extraction
4 Notes
References
Chapter 26: Immunohistochemistry and Immunofluorescence
1 Introduction
2 Materials
2.1 Tissue and Slide Preparation
2.2 Antigen Retrieval (Heat-Induced Method)
2.3 Antigen Retrieval (Enzyme Digestion Method)
2.4 Immunohistochemistry
2.4.1 Double Immunohistochemistry
2.5 Immunofluorescence
3 Methods
3.1 Tissue and Slide Preparation
3.2 Manual Antigen Retrieval (Heat-Induced) Method
3.3 Antigen Retrieval (Enzyme Digestion Method)
3.4 Preparation of Primary Antibody (Optimization of Antibody)
3.5 Primary Antibody Incubation and Secondary Antibody Detection (Single Immunostaining)
3.6 Qualitative and Quantification Analysis of Immunostaining
3.7 Double Immunostaining Immunohistochemistry
3.8 Immunofluorescence Staining
4 Notes
References
Chapter 27: Characterization, Quantification, and Visualization of Neutrophil Extracellular Traps
1 Introduction
2 Materials
2.1 Isolation of Neutrophils from Human Peripheral Blood by Density Gradient Centrifugation
2.2 Isolation of Neutrophils from Human Peripheral Blood by Negative Selection
2.3 Isolation of Neutrophils from the Human Oral Cavity
2.4 Neutrophil ROS Assays
2.5 NET Assays
2.6 Visualization of NETs by SEM
2.7 Visualization of NETs by Fluorescence Microscopy and HCA
2.8 Neutrophil Activation
3 Methods
3.1 Isolation of Neutrophils from Human Peripheral Blood by Density Gradient Centrifugation
3.2 Isolation of Neutrophils from Human Peripheral Blood by Negative Selection
3.3 Isolation of Neutrophils from the Human Oral Cavity
3.4 Chemiluminescence to Measure Neutrophil ROS
3.5 Quantification of NET-DNA
3.6 Quantification of NET-Bound Components
3.6.1 Production of NETs and NET-Bound Components
3.6.2 Measuring NET-Bound Neutrophil Elastase (NE)
3.6.3 Measuring NET-Bound Myeloperoxidase (MPO)
3.6.4 Measuring NET-Bound Cathepsin G (CG)
3.7 Quantification of NET-Entrapped Bacteria
3.8 Quantification of NET-Mediated Killing of Bacteria
3.9 Quantification of NET Degradation by Human Plasma
3.10 Scanning Electron Microscopy (SEM) of NETs
3.11 Immunofluorescence and Fluorescence Microscopy of NETs
3.12 NET Quantification and Visualization with HCA
3.13 Bacterial Culture
4 Notes
References
Chapter 28: Cell Seeding on 3D Scaffolds for Tissue Engineering and Disease Modeling Applications
1 Introduction
2 Materials
2.1 Primary Human Osteoblasts Cell (hOB) Harvesting and Expansion
2.2 Melt Electrowritten Scaffold Fabrication
2.3 Cell Seeding
2.4 Seeded-Scaffold Culture
2.5 3D Construct Imaging
3 Methods
3.1 Primary Human Osteoblasts Cell (hOB) Harvesting and Expansion
3.2 Melt Electrowritten Scaffold Fabrication
3.3 Surface Modification
3.4 Cell Seeding
3.5 Seeded-Scaffold Culture
3.6 Imaging of 3D Construct
3.6.1 Alizarin Red Staining
3.6.2 Picrosirius Red Staining
3.6.3 Immunostaining for Confocal Microscopy
4 Notes
References
Chapter 29: Workflow for Fabricating 3D-Printed Resorbable Personalized Porous Scaffolds for Orofacial Bone Regeneration
1 Introduction
2 Materials
2.1 Computational Modeling of the Patient Matched Scaffold
2.2 3D Printing of the Patient Matched Scaffold
3 Methods
3.1 Computational Modeling of the Patient Matched Scaffold
3.2 3D Printing of the Patient Matched Scaffold
4 Notes: 3D Printing of the Patient Matched Scaffold
References
Chapter 30: Methacrylated Gelatin as an On-Demand Injectable Vehicle for Drug Delivery in Dentistry
1 Introduction
2 Materials
2.1 Gelatin Methacrylation
2.2 GelMA Purification
2.3 Lyophilization
2.4 Hydrogel Fabrication
3 Methods
3.1 Gelatin Methacrylation
3.2 GelMA Purification
3.3 Lyophilization
3.4 Hydrogel Fabrication
3.4.1 Utilization of the Biodegradability of GelMA for Controlled Delivery of Chlorohexidine
3.4.2 An Antibiotic-Eluting Hydrogel for Oral Infection Ablation
4 Notes
References
Chapter 31: In Vitro Biological Testing of Dental Materials
1 Introduction
1.1 Classification of Dental Materials
1.2 Material Characterization Methods
1.2.1 Chemical Characterization
1.3 In Vitro Biological Testing
2 Materials
2.1 Preparation of Growth Media
2.2 Preparation of Differentiation Medium (DM)
2.3 Cell Seeding and Cell Growth
2.4 Subculturing (Passaging) Cells
2.5 Cell Counting
2.6 Cryopreserving Cells
2.7 Reviving Frozen Cells and Material Sterilization
2.8 Cell Seeding on to the Test Material
2.9 Cell Viability/Cytotoxicity Assay (LIVE/DEAD Assay)
2.10 The MTS Cell Proliferation Assay
2.11 Alkaline Phosphatase (ALP) Assay
3 Methods
3.1 Preparation of Growth Media (GM) (See Note 1)
3.2 Preparation of Differentiation Medium (DM)
3.3 Reviving Frozen Cells, Cell Seeding, and Cell Growth
3.4 Subculturing (Passaging) Cells
3.5 Cell Counting
3.6 Cryopreserving/Freezing Cells
3.7 Dental Material Preparation and Sterilization
3.8 Cell Seeding on to the Dental Material Surface
3.9 Cell Viability/Cytotoxicity Assay (LIVE/DEAD Assay)
3.10 Cell Proliferation (MTS Assay)
3.11 Alkaline Phosphatase (ALP) Assay
4 Notes
References
Index