Cover
Title Page
List of contributors
Preface
CHAPTER 1: Induced pluripotent stem cell technologies for tissue engineering
1. Overview of iPSCs
2. iPSCs for tissue engineering and regeneration
3. iPSCs as disease study models
4. Preclinical considerations
5. Conclusion and prospects
6. Acknowledgements
7. Author disclosure statement
8. References
CHAPTER 2: Immunomodulation by adult stem cells
1. The effect of ageing on MSC function
2. Allogeneic versus autologous MSCs
3. Licensing of MSCs
4. MSCs and TLRs
5. Adult stem cells and the innate immune system
6. Cells of the innate immune system
7. MSCs and the complement system
8. The adaptive immune response
9. Mediators of immunomodulation
10. The role of cell surface adhesion molecules in MSC‐mediated immunosuppression
11. Extracellular vesicles and immunomodulation
12. Homing
13. MSCs as modulators of the bacterial environment
14. Clinical applications of MSCs
15. Summary
16. Acknowledgements
17. References
CHAPTER 3: Research advances in tissue regeneration by dental pulp stem cells
1. Embryological origins of the stem cell pool in adult dental pulp
2. MSCs in primary dentinogenesis and dentine repair
3. SHEDs, SCAPs, and DPSC
4. Classical markers of a mesenchymal stem cell?
5. Alternative markers for mesenchymal stem cells
6. Using MSC markers to identify potential stem cell niches
7. In vivo analysis of MSCs in dental pulp
8. In vitro analysis of MSCs in dental pulp
9. The study of clonal cell lines versus heterogeneous populations
10. Consideration of the micro‐environment during analysis of MSCs
11. Epigenetics and posttranslational regulation of DPSC behaviour
12. Pulpal repair and regeneration
13. Concluding remarks
14. References
CHAPTER 4: Assessing the potential of mesenchymal stem cells in craniofacial bone repair and regeneration
1. Regeneration of large bone volume
2. Cellular and molecular events of bone healing
3. Current methods for measuring bone healing
4. Role of biomaterials in promoting bone healing
5. MSC‐based therapies
6. Bioactive factors for promoting bone repair and regeneration
7. Bioelectrical stimulation of bone repair processes
8. Influence of loading and biomechanical forces
9. Concluding remarks
10. References
CHAPTER 5: Tissue culture models and approaches for dental tissue regeneration
1. Models for dental tissue regeneration
2. Tissue culture models for periodontal disease and bone destruction
3. Appropriate culture systems for pulpal infection
4. Modelling of dental pulp stem cell behaviour in 3D systems
5. Acknowledgements
6. References
CHAPTER 6: Industrial translation requirements for manufacture of stem cell–derived and tissue‐engineered products
1. The penicillin story…and beyond
2. What is manufacturing scale?
3. Requirements for manufacture
4. An argument for bioreactors
5. Bioreactors for cell therapy
6. Bioreactors for controlling hard tissue formation
7. Process considerations
8. Monitoring the consequence of culture expansion
9. Final product: critical quality attributes
10. Conclusions
11. References
CHAPTER 7: Periodontal tissue engineering
1. Overview of past and current clinical periodontal regeneration techniques
2. Periodontal tissue engineering
3. Cells
4. Regulatory growth factor and differentiation factors
5. Scaffolds
6. Blood supply: Vascularisation and endothelial progenitors
7. Gene therapy
8. Conclusions
9. References
CHAPTER 8: Clinical strategies for dental and periodontal disease management
1. Clinical strategies in regenerative endodontics
2. Vital pulp therapies
3. Nonvital pulp therapies
4. Clinical techniques for periodontal regeneration
5. Concluding remarks
6. References
Index
End User License Agreement

List of Tables

Chapter 01
1. Table 1.1 Neural differentiation protocols of ESCs/iPSCs.
Chapter 03
1. Table 3.1 Multipotentiality towards the formation of mesenchymal connective tissues is a principle characterisation for defining an MSC. Dental pulp stem cells (DPSCs), stem cells from human exfoliated teeth (SHEDs), and stem cells from apical papilla (SCAPs) have all demonstrated variable multipotential characteristics as determined by the appearance of cell surface markers, transcription factors, deposition of extracellular matrix proteins, minerals and fatty acids, and cell morphology, characterising a specified differentiation pathway. Ideally, characterisation of lineage commitment and the differentiated cell should utilise a triplicate of markers tracing the differentiation process. (Modified information from Huang et al., 2009.)
2. Table 3.2 Protein markers used to characterise mesenchymal stem cells. MSCs are now regarded as heterogeneous in nature; therefore, there is no one unifying mesenchymal stem cell marker as markers appear or disappear depending upon their proliferation rate and differentiation potentiality.
c04
1. Table 4.1 The temporal stages of natural bone healing, providing optimal signalling cues directing cellular migration, proliferation, and differentiation leading to efficient bone regeneration of a fully functional tissue.
2. Table 4.2 Protein markers for measuring bone healing.
3. Table 4.3 Key research considerations in the design of therapeutic approaches for the repair and regeneration of bone. Similar issues are relevant for cell‐based and acellular‐based therapies.
Chapter 06
1. Table 6.1 Bioreactor comparisons. Modified from Rodrigues et al. (2011).
2. Table 6.2 Critical quality attributes and their meanings.

List of Illustrations

Chapter 01
1. Figure 1.1 Feasible source of cells or stem cells for transgene‐free iPSC generation and subsequent medical applications. Oral stem cells are the most accessible and easiest cells for the reprogramming process. Use of mRNA, protein, or an excisable vector approach allows generation of transgene‐free iPSCs. Note: Although blood cells appear to be the easiest cell type to obtain, multiple steps are involved in their processing before they are ready for reprogramming, which is very inconvenient.
2. Figure 1.2 Schematic representation of a strategy for whole‐tooth regeneration using iPSCs. The patient’s somatic cells are harvested and reprogrammed into patient‐specific iPSCs, which are then induced to form ectodermal epithelial cells and neural crest‐derived mesenchymal cells. They may be further induced to form odontogenic cells in vitro. The two cell populations are combined by direct contact, mimicking the in vivo arrangement. Interaction of these cells leads to formation of an early‐stage tooth germ. Once transplanted into the edentulous region, the recombinants develop into a functional tooth.
3. Figure 1.3 Embryoid body (EB)‐mediated neurogenesis. iPSC/ESC colonies are lifted into suspension to form EBs, followed by growth in adherent culture in defined media containing N2 supplement and basic fibroblast growth factor (bFGF) and allowed to form neural rosettes (NR). Cells in the NRs express many neural stem cell markers such as Nestin, Musashi‐1, and polysialylated‐neuronal cell adhesion molecule. NRs are then detached and grown in suspension followed by reattachment onto a laminin‐coated dish under neurogenic stimulation for further neural differentiation prior to functional assessment through patch clamp electrophysiology.
4. Figure 1.4 Dual‐SMAD inhibition method for neural differentiation. Under serum‐free conditions, adherent single cell‐cultures of iPSCs/hESCs are treated with Noggin or dorsomorphin (BMP inhibitor) and SB431542 (Activin/Nodal inhibitor) to convert iPSCs/hESCs to largely PAX6‐positive (green) neuroectodermal cells that subsequently form neural rosettes (Ki67, green phosphor‐histone H3, red) following 11 days of differentiation. Neural cells generated express FOXG1 (red) and OTX2 (red), along with PAX6 (green).
5. Figure 1.5 Serum‐free EB‐like (SFEB) method for neural differentiation. iPSC/ESC colonies are dissociated to single cells (2 × 10⁵ cells/mL) and cultured in nonadherent dishes. Cell aggregates form spontaneously in the presence of a ROCK inhibitor. Dkk1 (Wnt inhibitor), LeftyA (Nodal signaling antagonist), and soluble BMPRIA‐Fc (BMP‐4 antagonist) are added to the culture from Day 0 to Day 24. The cell aggregates are then replated en bloc on dishes coated with poly‐D‐lysine, laminin, and fibronectin, and cultured until Day 35 in a neural differentiation medium (Neurobasal + B27 and glutamine). For ventralisation experiments, Shh is added. On Day 35, hESC‐derived neural cells express Bf1 (32.9% ± 2.6%, far right image panel, top). The early embryonic telencephalon is subdivided into the pallial (Bf1+/PAX6+ cortical anlage) and basal (e.g., Nkx2.1+) regions. The majority of Bf1+ cells derived from Y‐27632‐treated hES cells coexpressed PAX6 (95.8 ± 0.7%), whereas Nkx2.1 was detected in only a few Bf1+ cells (1% or less) (far right middle image panel). Shh treatment (Days 15–35) decreased the PAX+ population (23.2% ± 5.3%) and increased the proportion of Nkx2.1+ cells among the Bf1+ cells (41.5% ± 14.5%) (far right bottom image panel).
Chapter 02
1. Figure 2.1 Licensing of MSCs. Environmental cues trigger the recruitment and licensing of MSCs to a proinflammatory (MSC1) or anti‐inflammatory (MSC2) phenotype. MSC1 are essential to the early immune response, recruiting and activating innate and adaptive immune cells in response to microbial products such as lipopolysaccharide and peptidoglycan. Activated lymphocytes and M1 macrophages secreting proinflammatory cytokines such as TNFα and IFNγ (or exposure to viral double‐stranded RNA) switches the MSC profile to an MSC2. Licensed MSC2 secrete anti‐inflammatory cytokines central to reducing lymphocyte proliferation, controlling the inflammatory immune response and switching immune cells to a regulatory phenotype (M2 macrophage and Treg induction) to initiate a repair response.
2. Figure 2.2 MSC crosstalk with macrophages regulates innate and adaptive immunity. (a) Unprimed MSC (uMSC) respond to bacterial stimuli such as lipopolysaccharide and peptidoglycan (PGN) through Toll‐like receptors (TLRs) 2/4 by licensing to a proinflammatory MSC1 phenotype. (b) MSC1 secrete chemokine (C‐C motif) ligand 3 and 12 and CXCL12 to recruit proinflammatory M1 macrophages to the site of inflammation. M1 macrophages in turn secrete (c) proinflammatory cytokines such as TNFα and IFNγ, triggering (d) the phenotypic switch of the MSCs to an anti‐inflammatory MSC2. The secretion of immunomodulatory soluble factors such as indoleamine 2,3‐dioxygenase (IDO) and prostaglandin E2 (PGE2) by MSC2 leads to (e) the repolarisation of M1 macrophages to regulatory M2 macrophages. MSC2 and M2 macrophages subsequently work both directly and indirectly to suppress effector T cell proliferation and neutrophil recruitment, whilst increasing the ratio of Tregs dampening the immune response and enhancing the wound healing process.
3. Figure 2.3 BMMSC and OMLP‐PC interaction with T cells. Photomicrographs of isolated (a) bone marrow MSCs (BMMSCs) and (b) oral mucosal lamina propria progenitor cells (OMLP‐PCs) interacting in direct contact co‐cultures with CD3⁺ T cells in vitro. Co‐incubation of BMMSCs and OMLP‐PCs with isolated T cells suppresses T cell proliferation (Protocol 2.2). Bar = 100 μm.
Chapter 03
1. Figure 3.1 Hierarchy model of stem cell differentiation. The mother stem cell is able to divide for self‐renewal to replace itself or give rise to transit amplifying cells, which eventually give rise to the committed progenitors. The model hypothesis is that early transit amplifying cells rapidly divide to produce large colonies. As cell division continues, proliferative rates decrease and only small progenitor cell colonies are able to form. This is associated with a reduction in multipotentiality as the cells become committed to a specific unipotential lineage and finally differentiate to produce a large number of terminally differentiated, nonmitotic cells.
2. Figure 3.2 Resorbing deciduous tooth providing MSC source for SHEDs (A). The developing roots of the tooth below provide a potential source for SCAPs (B), which may be harvested during tooth eruption prior to closure of the root apices.
3. Figure 3.3 Application of FACS and MACS in the selection of side populations such as STRO‐1, CD146, or CD34‐positive cells. Cells can be dual labelled for the selection of cells, although neither method will provide the sole isolation of cells carrying both markers. Alternatively, the procedure can be repeated for the further selection of an MSC‐positive cell within the isolated side population. FACS can also be used to characterise the presence of MSC markers. For detailed protocols, reader is best referred to manufacturer’s instructions.
Chapter 05
1. Figure 5.1 Histological appearance of a rodent tooth slice cultured for 14 days demonstrating maintenance of cell and tissue architecture and viability. Pulpal cells (p) and the odontoblast cell layer (od) remain viable during culture.
2. Figure 5.2 A culture rat mandible slice after 14 days in culture. Cells and tissues of the alveolar bone (b), periodontal ligament (pdl), and pulp (p) show good viability following culture.
3. Figure 5.3 Loss of cell viability and tissue structure of the periodontal ligament in a culture mandible slice following stimulation with bacterial lipopolysaccharide.
4. Figure 5.4 Attachment of Streptococcus anginosus group bacteria to a tooth slice following 24 hours in culture. Histological staining (a) indicates small microcolonies of bacteria adhering to and invading the tissue (arrows), and this is confirmed through observation of fluorescein diacetate positively stained bacteria (b).
Chapter 06
1. Figure 6.1 Autologous versus allogeneic manufacture affects the scale and the impact on quality control (QC) requirements and relative cost proportion of the whole manufacturing process. For the cartoon left, there would be three manufacturing “runs” for the autologous therapy and individual QC associated with each. Yet for the allogeneic therapy, potentially only one manufacturing run to produce multiple doses of drug substance. Typically, for a batch of cells derived for allogeneic therapy, the cost of performing QC analysis will be significantly lower than for autologous therapy.
2. Figure 6.2 Bioreactor technologies and their utility in cell culture at different scales.
3. Figure 6.3 Diagram representing, in its simplest form, the inputs to a unit operation that affect the output product.
Chapter 07
1. Figure 7.1 Diagrammatic representation of guided tissue regeneration principle involving the use of a barrier membrane to create space and facilitate the selective repopulation of the defect with periodontal ligament cells and osteoblasts at the expense of epithelial cells and gingival fibroblasts.
2. Figure 7.2 Diagrammatic representation of the key components of a tissue‐engineered construct: cells, scaffold, instructive messages, and blood supply.
3. Figure 7.3 Scanning electron microscope images of periodontal ligament fibroblasts grown on (a) hydroxyapatite‐tricalcium phosphate (HA‐TCP), (b) polytetrafluoroethlylene (PTFE), (c) demineralised bone xenograft, and (d) gelatin sponge.
4. Figure 7.4 Biphasic scaffold (a) demonstrating excellent integration between periodontal ligament and bone compartments (b). Tissue‐engineered construct with a periodontal ligament cell sheet placed on periodontal side of biphasic PCL scaffold (c), ready for insertion into periodontal defect (d).
Chapter 08
1. Figure 8.1 Illustration of modulation of the “triad of tissue engineering” by environmental factors, affecting the downstream stem cell fate and regenerated tissue.
2. Figure 8.2 Radiographs and micrograph of tooth #29 treated with a partial pulpotomy and direct pulp capping using MTA. (a) Preoperative radiograph showing a large distal proximal carious lesion. (b) Recall radiograph taken 10 months after the pulpotomy showing absence of pathosis; in addition, the patient was asymptomatic. (c and d) Histologic examination of tooth #29 after extraction for orthodontic reasons reveals absence of inflammation and a thick reparative dentinal bridge as seen in both 10X (c) and 50X (d) magnification.
3. Figure 8.3 Two case reports showing successful clinical outcomes using REPs. Both cases reported immature permanent premolar teeth diagnosed with pulp necrosis. Large radiolucent areas are seen surrounding the apex and roots of both teeth, suggesting advanced apical periodontitis in the preoperative radiographs. Teeth were treated with revascularisation procedures, resulting in complete resolution of signs and symptoms of disease. Complete radiographic healing of apical periodontitis is seen in the postoperative radiographs.
4. Figure 8.4 Composite representation of two retrospective and one prospective study showing percentage change in root width and root length between regenerative endodontic procedures and MTA apexification procedures.
5. Figure 8.5 Complete regeneration of pulp tissue after autologous transplantation of CD31– SP cells with SDF‐1 in the emptied root canal after pulpectomy in dogs. (a, d, and h) Pulp CD31– SP cell transplantation. (b, e, and i) Bone marrow CD31– SP cell transplantation. (c, f, and j) Adipose CD31– SP cell transplantation. (a–g) 14 days, (h–j) 28 days after transplantation. (j) Enhanced matrix formation. (g) Ratio of regenerated area to root canal area. Data are expressed as means ± SD.
6. Figure 8.6 Intraoral clinical view following flap elevation showing significant bone loss due to periodontal disease. In this case, a resorbable collagen membrane in combination with a calcium phosphosilicate (bioglass) bone substitute was utilised for periodontal regeneration to enhance the long‐term prognosis of this maxillary canine.
7. Figure 8.7 Series of intraoral clinical photographs showing (a) 12 mm probing depth in the distal region of the lower left 1st mandibular molar; (b) combination of a two‐wall intrabony defect with a three‐wall component at the base of the defect; (c) placement of a nonresorbable barrier membrane through the buccal aspect to provide space maintenance for regeneration in combination with a bone substitute; and (d) 6‐month reentry surgical procedure. Note the newly formed tissue covering approximately 65% of the original defect volume.

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Library of Congress Cataloging‐in‐Publication Data

Names: Waddington, Rachel (Rachel J.), editor. | Sloan, Alistair J., editor.
Title: Tissue engineering and regeneration in dentistry : current strategies / edited by Rachel J Waddington and Alistair J Sloan.
Description: Chichester, West Sussex, UK ; Ames, Iowa : John Wiley & Sons Inc., 2017. | Includes bibliographical references and index.
Identifiers: LCCN 2016022478 | ISBN 9781118741108 (pbk.) | ISBN 9781118741047 (Adobe PDF) | ISBN 9781118741078 (epub)
Subjects: | MESH: Tissue Engineering–methods | Dentistry–methods | Guided Tissue Regeneration–methods | Stomatognathic Diseases–therapy
Classification: LCC RK320.T47 | NLM WU 100 | DDC 617.6/340592–dc23
LC record available at https://lccn.loc.gov/2016022478

A catalogue record for this book is available from the British Library.

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Cover image: © Andrew Brookes/Gettyimages

List of contributors

Wayne Nishio Ayre
School of Dentistry
Cardiff Institute of Tissue Engineering and Repair
Cardiff University
Cardiff, UK

P. Mark Bartold
Colgate Australian Dental Research Centre
Dental School
University of Adelaide
Adelaide, SA, Australia

Vanessa Chrepa
School of Dentistry
University of Texas Health Science Center
San Antonio, TX, USA

John Colombo
School of Dentistry
University of Utah
Salt Lake City, UT, USA

Lindsay C. Davies
Centre for Hematology and Regenerative Medicine
Karolinska Institutet
Stockholm, Sweden;
School of Dentistry
Cardiff University
Heath Park
Cardiff, UK

David de Silva Thompson
University College London
London, UK

Anibal Diogenes
School of Dentistry
University of Texas Health Science Center
San Antonio, TX, USA

Ikbale El Ayachi
Department of Bioscience Research
College of Dentistry
University of Tennessee Health Science Center
Memphis, TN, USA

Stan Gronthos
School of Medical Sciences
Faculty of Health Sciences
University of Adelaide
Adelaide, SA, Australia
South Australian Health and Medical Research Institute
Adelaide, SA, Australia

George T.‐J. Huang
Department of Bioscience Research
College of Dentistry
University of Tennessee Health Science CenterMemphis, TN, USA

Dietmar W. Hutmacher
Institute of Health and Biomedical Innovation
Queensland University of Technology
Brisbane, Queensland, Australia

Saso Ivanovski
Menzies Health Institute Queensland
School of Dentistry and Oral Health
Griffith University
Gold Coast, Queensland, Australia

S. Quentin Jones
School of Dentistry
Cardiff Institute of Tissue Engineering and Repair
Cardiff University
Cardiff, UK

Katarina Le Blanc
Centre for Hematology and Regenerative Medicine
Karolinska Institutet
Stockholm, Sweden

Ryan Moseley
School of Dentistry
Cardiff Institute of Tissue Engineering and Repair
Cardiff University
Cardiff, UK

Roman Perez
Institute of Tissue Regeneration Engineering (ITREN)
Dankook University
Cheonan, Republic of Korea

Carlotta Peticone
University College London
London, UK

Jessica Roberts
North Wales Centre for Primary Care Research
Bangor University
Wrexham, UK

Nikita B. Ruparel
School of Dentistry
University of Texas Health Science Center
San Antonio, TX, USA

Alastair J. Sloan
School of Dentistry
Cardiff Institute of Tissue Engineering and Repair
Cardiff University
Cardiff, UK

Rachel J. Waddington
School of Dentistry
Cardiff Institute of Tissue Engineering and Repair
Cardiff University
Cardiff, UK

Ivan Wall
University College London
London, UK;
Institute of Tissue Regeneration Engineering (ITREN)
Dankook University
Cheonan, Republic of Korea

Xiao‐Ying Zou
Department of Cariology, Endodontology and Operative Dentistry
Peking University School and Hospital of Stomatology
Beijing, China

Tissue Engineering and Regeneration in Dentistry

Current Strategies

List of contributors

Preface