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The Indian Dental Academy is the Leader in continuing dental education , training dentists in all aspects of dentistry and
offering a wide range of dental certified courses in different formats.


The Indian Dental Academy is the Leader in continuing dental education , training dentists in all aspects of dentistry and
offering a wide range of dental certified courses in different formats.

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Regenerative endodontics / endodontics courses

  1. 1. CONTENTS PAGE NO. 1. Introduction to Regenerative Endodontics 01-06  Definitions  Importance  History 2. Overview of Regenerative Medicine and Endodontics 07-12  Objectives of Regenerative Endodontics  Current strategies for treatment of lost tissue  Need for tissue engineering 3. Pulp -Physiology and Function 13-17  Structure  Nerve innervation  Vascularization  Functions of pulp 4. Major Domains of Regenerative Endodontics  Stem Cells 18-45  Principles of stem cells  General characteristics  Embryonic stem cells  Human dental stem cells  Isolation and characterization of stem cell population  Hierarchical ordering of stem cell niches  Stem cells in dentin regeneration and novel therapies
  2. 2.  Isolation approaches  Size sieved isolation  Stem cell colony cultivation  Magnetic activated cell sorting  Fluorescence activated cell sorting  Regeneration steps in dental tissue  Banking stem cells  Collection  Isolation  Storage o Cryopreservation o Magnetic freezing  Growth Factors 46-60  Role of growth factors in Regenerative Endodontics  Primary effects and interactions of signaling molecule for Regenerative Endodontics  Different growth factors  Platelet derived growth factor  Transforming growth factor b  Bone Morphogenic Protein  Vascular endothelial growth factor  Fibroblast growth factor  Insulin like growth factor  Nerve growth factor  Stromal cell derived factor 1  Different growth factors by various researchers
  3. 3.  Tissue Engineering 61-81  Definition  Stem cells  Scaffolds  Importance  Ideal characteristics  Materials  Morphogens  Materials used in soft and hard tissue engineering applications  Types of Tissue Engineering  Approaches for Tissue Engineering  Conductive approach  Inductive approach  Cell transplantation  Gene therapy  Cell, Tissue and Organ Culture 82-86  Regeneration of dentin pulp complex  Regeneration of entire tooth  Local regeneration from residual dental pulp  Local regeneration from periapical tissue 5. Potential Technologies for Regenerative Endodontics 87-110  Root canal revascularization via blood clotting  Post natal stem cell therapy  Pulp implantation  Scaffold implantation  Injectable scaffold delivery
  4. 4.  Three dimensional cell printing  Gene therapy  Cell therapy  Challenges and future direction  Nerve regeneration  Vascular regeneration  Measurement of clinical outcomes 6. Research Priorities 111-114  Methods to disinfect and shape root canal system  Smear layer removal  Delivery of regenerative endodontic procedures  Reconstruction of bio-tooth from dental pulp stem cells 7. Future perspectives 115-128  Pulp repair and regeneration  Vascularization  Inflammation and regeneration 8. Conclusion 129 9. Bibliography 130-137 Introduction: The goal of modern restorative dentistry is to functionally and cosmetically restore the tooth structure. Natural dental hard tissues, i.e. dentin, enamel and cementum exhibit little or no regenerative capability.1 Till recently, a variety of synthetic materials were developed to restore
  5. 5. the damaged tooth structure. Although these materials have proved to be effective, they do not exhibit the same mechanical and physical properties as naturally formed dentin and enamel. Hence, there is a need for the replacement of the tooth tissue. Tissue engineering is a novel and highly exciting field of research. With tissue engineering techniques, it may be possible to repair damaged tissues or even create replacement organs. Tissue engineering can help in the regeneration of enamel and dentin to restore the lost tooth structure in future. Dental caries remain to be one of the most prevalent young adult and childhood diseases. There are several ways in which one can potentially engineer lost dentin and the dental pulp. The vitality of the dental pulp may be damaged by infection, exposure, trauma and chemicals. They ultimately result in premature tooth loss and therefore, diminish the quality of life. One novel approach to restore the tooth structure is based on biology.2 Millions of teeth are saved each year by root canal therapy. Although the current treatment modalities offer high level of success for many conditions, an ideal form of therapy might consists regenerative approaches in which diseased or necrotic pulp tissues are removed and replaced with healthy pulp tissue to revitalize the teeth. The researchers are working towards this objective. Regenerative Endodontics is the creation and delivery of tissues to replace diseased, missing and traumatized pulp. The regeneration or replacement of oral tissues affected by inherited disorders, trauma and neoplastic or infectious diseases is expected to solve dental problems. Within the next 25 years, unparalleled advances in dentistry and endodontics are set to take place with the availability of artificial teeth, bone, organs and oral tissues as well as the ability to stimulate endodontic regeneration, replace the diseased tissues, produce vaccination against viruses and genetically alter the disease pathogens to help eradicate caries and Periodontitis.
  6. 6. Patient demand for tissue engineering therapy is staggering both in scope and cost. The endodontic speciality may be able to adopt many of these new scientific advances emerging from regenerative medicine, there by developing regenerative endodontic procedures and improving the patient care. Terminologies: Regenerative Endodontics: Regenerative endodontic procedures can be defined as biologically based procedures designed to replace damaged structures, including dentin and root structures, as well as cells of the pulp- dentin complex.1 Tissue engineering: The term Tissue Engineering (TE) was initially defined by the attendees of the first National Science Foundation (NSF) sponsored meeting in 1988 as the “Application of the principles and methods of engineering and life sciences toward fundamental understanding of structure-function relationship in normal and pathological mammalian tissues and the development of biological substitutes for the repair or regeneration of tissue or organ function” (Shalak and Fox, 1988). OR Tissue Engineering is the science of design and manufacture of new tissues to replace lost parts because of disease including cancer and trauma, with the inclusion of certain ingredients like stem cells, morphogens and scaffold (Nakashima et al, 2005). OR As an interdisciplinary field that applies the principles of engineering and the life science towards the development of biological substitutes that restore, maintain or improve tissue function (Langer & Vacanti, 1993).
  7. 7. OR Understanding the principles of tissue growth, and applying this to produce functional replacement tissue for clinical use” (MacArthur and Oreffo). OR Tissue engineering is the employment of biologic therapeutic strategies aimed at the replacement, repair, maintenance, and/or enhancement of tissue function. (Franklin, Murray, Hargreaves JOE, 2008). Biomimetics: It is the science of reconstructing or mimicking natural processes or tissues with the expectation that regeneration will follow. Regeneration: Re-creating what was once formed during embryogenesis or reconstitution of the form and function of injured tissues. Repair: It is the restoration of tissue continuity without its original architecture or function. Stem cells: Stem cells are defined as clonogenic cells capable of both self-renewal and multi-lineage differentiation. Morphogens / Signaling molecules: Morphogens or signaling molecules are proteins that bind to receptors on the cell and induce cellular proliferation and /or differentiation. Scaffold:
  8. 8. A scaffold provides a physicochemical and biological three-dimensional micro environment for cell adhesion, growth, differentiation and migration. Bioreactor: A bioreactor can be defined as any apparatus that attempts to mimic physiological conditions in order to maintain and encourage tissue regeneration. HISTORY:  The regenerative capability of a living creature was recorded as early as 330 BC, when Aristotle observed that a lizard could grow back the lost tip of its tail.
  9. 9.  Hermann (1952) was the first to carry out regenerative endodontic procedure, when he applied calcium hydroxide in vital pulp amputation.3  In 1961, Nygaard-Ostby established a blood clot to use as a scaffold to revascularize tissue within the root canals of teeth.  In 1963, Hematopoietic stem cells giving rise to blood cells were identified in bone marrow.  Concepts of guided tissue and bone regeneration were first published by Melcher in 1976.  Subsequent regenerative dental procedures included guided tissue or guided bone regeneration (GTR, GBR) procedures and distraction osteogenesis (Block et al, 1995).4  Recombinant Human Bone Morphogenic Protein (RHBP) for bone augmentation (Fjuimuraet al, 1995).5  Emdogain for periodontal tissue regeneration (Heijlet al, 1997).6  The application of Platelet Rich Plasma (PRP) for bone augmentation (Kassoliset al, 2000).7  Preclinical trials on the use of Fibroblast Growth Factor 2 (FGF2) for periodontal tissue regeneration.  McCulloch reported the presence of progenitor cells in periodontal ligament of mice in 1985.8  Later, the isolation and identification of multipotent MSCs in human periodontal ligaments were reported in 2004.  In studies in 2006 and 2007, methods to reprogram somatic cells from mice and later in humans, by insertion of genes that reprogrammed the somatic cells and returned them to an embryo like state.
  10. 10.  In January 2011, ADA adapted a new procedure code to allow practitioners to induce bleeding apical bleeding into the root canal in immature permanent teeth with necrotic pulps that have been extripated. An Overview of Regenerative Medicine  Regenerative medicine holds promise for the restoration of tissues and organs damaged by disease, trauma, canceror congenital deformity. Regenerative medicine can be defined as the use of a combination of cells, engineering materials and suitable biochemical factors to improve or replace biological functions in an effort to effect the advancement of medicine. The basis for regenerative medicine is the utilization of tissue engineering therapies.  Probably the first definition of tissue engineering was by Langer and Vacanti, who stated that it was “an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function.” 1  MacArthur and Oreffo defined tissue engineering as “understanding the principles of tissue growth, and applying this to produce functional replacement tissue for clinical use.”9 An Overview of Regenerative Endodontics The development of regenerative endodontic procedure is that although the replaced pulp has potential to revitalize the teeth, it may also become susceptible to further pulp disease and may require retreatment. Regenerative Endodontics comprises of research in:
  11. 11. 1. Adult stem cells 2. Growth factors 3. Organ tissue culture 4. Tissue engineering materials Figure 1: The major domains of research required to develop regenerative endodontic procedures Objectives of Regenerative Endodontics:1  To regenerate pulp-like tissue, ideally, the pulp-dentin complex  Regenerate damaged coronal dentin, such as following a carious exposure  Regenerate resorbed root, cervical or apical dentin. The tissue of interest in regenerative endodontics include
  12. 12.  Dentin  Pulp  Cementum  Periodontal tissues In the future, the scope of regenerative endodontics may be increased to include the replacement of periapical tissues, periodontal ligaments, gingiva, and even whole teeth. This would give patients a clear alternative to the artificial tooth implants that are currently available. Thus, the potential for this area is indeed vast. Over the past century, endodontic therapy has shown a high rate of success in retention of teeth; however, many teeth are not restorable due to various reasons like apical resorption, fractures, incompletely formed roots or carious destruction, also vital pulp therapy is not always predictable. It is these critical issues that have led to the question: What is the ideal replacement of lost tissues? The gold standard to replace an individual’s lost or damaged tissue is the same natural healthy tissue. This standard has led to the concept of Tissue Engineering or Regenerating new tissue from pre-existing tissue i.e. to mimic tooth structure based on biology with application of tissue engineering.1 Over the last two decades, tissue engineering has evolved from science fiction to science. So “Biological solutions to biological problems” is emerging as a new paradigm in medicine and dentistry. Tissue engineering is a contemporary area of applied biomedical research aimed at developing procedures and biomaterials for the fabrication of new tissues to replace damaged tissues and is based on the principles of cell biology, developmental biology and aims to replace dead or dying ones. This may be achieved either by transplanting cells seeded into a porous
  13. 13. material or scaffold having open pores or by relying on in-growth of cells into such a material, which in both cases develops into normal tissue.1 The main requirements for producing an engineered tissue are -Appropriate levels and sequencing of regulatory signals. - Presence and numbers of responsive progenitor cells. -An appropriate cellular matrix or carrier. - Adequate blood supply. Figure 2 Regenerative Endodontics is the creation and delivery of tissues to replace diseased, missing, and traumatized pulp. Regenerative endodontic procedures can be defined as biologically based procedures designed to replace damaged structures, including dentin and root structures, as well as cells of the pulp-dentine complex.
  14. 14. The objectives of regenerative endodontic procedures are to regenerate pulp-like tissue, ideally, the pulp dentine complex; regenerate damaged coronal dentine, such as following a carious exposure; and regenerate resorbed root, cervical or apical dentine. These potential approaches include root canal revascularization, postnatal (adult) stem cell therapy, pulp implantation, scaffold implantation, three-dimensional cell printing, injectable scaffolds and gene therapy. Overall in Regenerative Endodontics, pulp is been built from inside in damaged tooth. Tooth with regenerated pulp will require markedly smaller restoration and will also be able to uphold proprioceptive function of teeth. Every new research comes with hurdles to be overcome. Scientist have to find solution to avoid cell rejection, specialize cell behaviour, long tooth eruption period, identification of appropriate stem cell, expression and regulation of surface protein, longevity of undifferentiated cell, ethical issues. Nevertheless, development of biological approaches for dental reconstruction using stem cell is promising and remains one of the greatest challenges in endodontics for years to come replacing existing treatment modalities in clinical practice. Clearly the future for regenerative and tissue engineering applications to dentistry is one with immense potential, and capable of bringing quantum advances in treatment of our patients. Current strategies for treatment of lost tissue include: 1. Autografts 2. Allografts 3. Synthetic materials (Xenografts)
  15. 15. Although all of these treatment approaches have had successes and have been major advances in medicine, each of them has limitations.10 One of the major shortcomings with autografts, as well as allografts, is the fact that humans do not have significant stores of excess tissue for transplantation. Other restrictions include donor site morbidity, anatomic and structural problems, and elevated levels of resorption during healing. Compounded with this, in the case of allografts, there always exists the possibility of eliciting an immunologic response due to genetic differences, as well as inducing transmissible diseases. Therefore, the gold standard to replace an individual lost or damaged tissue is the same natural healthy tissue, which has led to the concept of regenerating new tissue from pre-existing tissue. Need for tissue engineering:  Most tissue cannot regenerate when injured or diseased.  Even tissues that regenerate spontaneously may not completely do so in large defects (e.g. bone).  Replacement of tissue with permanent implant is very limited. PHYSIOLOGY AND FUNCTIONS OF DENTAL PULP The dental pulp has its genesis in the 6th week of Intra-Uterine life during initiation of tooth development. It forms from cranial neural crest derived ecto-mesenchymal cells which
  16. 16. have migrated cephalically over the developing forebrain and ventrally around the developing foregut. These cells migrate extensively. Some travel down the sides of the head into the maxilla and mandible. During tooth formation aggregates of the neural crest cells, form the individual dental papilla which eventually become dental pulp. The success for endodontic therapy in primary dentition depends largely on complete and proper clinical assessment. For this the clinician should know the difference that happens when a normal tooth becomes infected. Thus it’s obvious that normal tooth physiology and anatomy should be known to know the abnormal.12 Figure 3 Dental pulp may be defined as a special organ with a unique environment of the unyielding dentin surrounding a resistant, resilient soft tissue of mesenchymal origin, which is reinforced with a ground substance. It has a close relationship between its peripheral cell, the odontoblasts and dentin making it a functional entity. It is sometimes referred to as the pulp-dentin complex. The dental pulp has a high circulatory force because of the fluid interchange between capillaries and tissue, which maintain a hydrostatic pressure within this non-compliant chamber. This pressure is called as intra-pulpal pressure that is normally 10mm of Hg and varies with each arterial pulse. Pulp does not have a consistent effective collateral circulation. Principal cells of
  17. 17. the pulps are the odontoblasts, fibroblasts, undifferentiated mesenchymal cells, macrophages and other immune-competent cells. Histologically pulp has four distinct zones - 1. Odontoblastic zone – pulp periphery 2. Cell free zone –Weil`s zone or Weil’s basal layer - Seen beneath the odontoblasts and are prominent in the coronal pulp. 3. Cell– rich zone – cell density is high. 4. Pulp core – presence of major vessels and nerves of the pulp. The approximate number of odontoblasts in coronal dentin is estimated to be 45,000 per sq/mm and lesser in root dentin.12 NERVE INNERVATIONS Dental pulp is richly innervated; it follows the same course as the blood vessels. Each nerve fiber may provide at least eight terminal branches, which again gives to an extensive plexus of nerves in the cell free zone. Thus plexus of nervous is called the Sub-Odontoblastic plexus or plexus of Raschkow. The nerve entering the pulp consists principally of sensory afferents of the trigeminal nerve and sympathetic branches form the superior cervical ganglions. There are two types of nerve fibers.
  18. 18. Figure 4 1) A-delta fibers, which are myelinated fast conducting fibers of diameter 1-6mm and associated with conduction of sharp, localized pain when the dentin is first exposed. 2) C- fibers are myelinated slower conducting and smaller in diameter (2mm) and are associated with conduction of dull diffuse pain. 3) Rouget`s cell or pericytes are seen on the periphery of the capillaries and they supposedly serve as contractile cells capable of reducing the size of vessel lumen. VASCULARIZATION Vascularization of the developing pulp starts during the bell stages, with small branches from the principal vascular trunks of the jaw entering the base of the papilla. The principal vessels enlarge and run through the pulp towards the cuspal regions. Here they give off numerous small branches, which form a bed of venules, arterioles and capillaries in the sub odontoblastic and odontoblastic layers. The vascularity of the odontoblastic layer increases as dentin is progressively laid down, since the odontoblasts retreating onwards through the vascular bed. Some capillaries are found immediately next to the pre-dentinal surface, which some time trapped in the dentine to form capillary loop.Pulpal blood flow is more rapid than in most areas of the body. Pulpal pressure is among the highest of body tissues. Flow of blood in Arteries – 0.3 to 1mm per second; Veins – 0.15mm/ sec and Capillaries – 0.08mm/sec.12
  19. 19. Figure 5 FUNCTIONS OF THE PULP The pulp is the formative organ of the tooth. Encased within the rigid walls of the root canal is the firm, cohesive and resistant unit, the dental pulp organ. The dental pulp carries out the basic functions i.e., 1. Inductive – Cells produce the dentin that surrounds and protects the pulp. The pulp odontoblasts develop the organic matrix and function in its calcification. The pulp also induces the enamel organ to become a particular type of tooth. 2. Formative – The pulp organ cells produce the dentin that surrounds and protects the pulp. The odontoblasts develop the organic matrix and function in its calcification. Through the development of the odontoblasts processes dentin is formed along the tubule wall as well as at the pulp perdentin front. 3. Nutritive – The pulp nourishes the dentin through the odontoblasts and their processes. Dentin is formed along the tubule wall as well as at the pulpal predentin front. 4. Protective – The sensory nerves in the tooth respond with pain to all stimuli such as heat cold pressure operative procedures and chemicals. The nerves also initial reflexes that central
  20. 20. circulation in the pulp this sympathetic function is a reflex providing stimulation to visceral motor fibers terminating on the muscles of blood vessels. 5. Defensive – The pulp is an organ with remarkable restorative abilities. It responds to irritation whether mechanical, thermal chemical or bacterial by producing reparative dentin and mineralizing any affected dentinal tubules.12 STEM CELLS: The term stem cell was proposed for scientific use by Russian histologist Alexander Maksimov in 1908.13 The main principles of stem cells are (1) Self-renewal or the ability to generate at least one daughter cell with characteristics similar to the initiating cell, (2) Multilineage differentiation of a single cell, (3) In vivo functional reconstitution of a given tissue or cell type. GENERAL CHARACTERISTICS 1. Stem cells are undifferentiated embryonic or adult cells that continuously divide. 2. A fundamental property of stem cells is self-renewal or the ability to go through numerous cycles of cell division while maintaining the undifferentiated state.
  21. 21. 3. Stem cells can be either embryonic or adult (postnatal). 4. Embryonic stem cells are isolated from the blastocyst during embryonic development and give rise to the 3 primary germ layers: Ectoderm, Endoderm, and Mesoderm. These cells are totipotent or pluripotent with an unlimited capacity to differentiate and can develop into each of the more than 200 cell types of the adult body. Stem cells are unspecialized cells that have a remarkable ability to develop to many different cell types when needed. Theoretically, they can divide without limit to replenish any other cell type and function as a part of a repair system. Stem cells are known to proliferate over very long periods of time. An important area of research is to understand the signals in an organism that cause a stem cell to proliferate and remain unspecialized until differentiated cells are needed for growth or repair of a specific tissue. Research on stem cells is advancing and needs substantial knowledge about an organism, how it develops from a single cell and how a healthy cell replaces a damaged cell in the adult organism. This is a promising field of science as various studies are still going on and various possibility of cell based therapies to treat diseases which are often referred to as “Regenerative Medicine”, are still in progress. Though stem cells are one of the most fascinating areas of biology today, but like many expanding fields, research on stem cells raises scientific questions as rapidly as it generates new discoveries.
  22. 22. Figure6 Figure7 Stem cells have two important characteristics which make them different from other cells.13 1. They are unspecialized cells that renew themselves for long time through cell division. 2. Under certain physiological conditions they can be induced to become cells with special function such as neurons. Scientists have identified primarily two types of stem cells: 1. Embryonic stem cells (ESCs) 2. Adult stemcells (ASCs).14
  23. 23. EMBRYONIC STEM CELLS Early Embryonic stem cells: The first step in human development occurs when the newly fertilized egg or zygote begins to divide producing a group of stem cells called embryo .these early stem cells are totipotent that possess the ability to become a new individual. Blastocyst embryonic stem cells: Five days after fertilization the embryo forms a ball like structure known as blastocyst. Embryos at the blastocyst contain two types of cells, outer layer of trophoblasts that eventually form placenta. Inner cluster of cells known as inner cell mass that becomes the embryo and then develops into a mature organism. The embryonic stem cells in blastocyst are pluripotent that is having the ability to become almost any kind of cell in the body Fetal stem cells: After 8 weeks of development the embryo is referred as a fetus. By this time it has developed to a human like form. Stem cells in the fetus are responsible for the initial development of all tissues before birth. Umbilical cord stem cells: The umbilical cord is the life line that transports nutrients and oxygen rich blood from the placenta to fetus.Blood from the umbilical cord contains stem cells that are genetically identical
  24. 24. to the new born baby. The umbilical cord stem cells are multipotent that is they can differentiate in to a limited range of cell types. Umbilical cord stem cells can be stored cryogenically after birth for use in future medical therapy. Adult stem cells: This name is rather misleading, because infants and children also have stem cells thus the term post natal stem cells is preferable. These stem cells reside in tissues that have already developed, directing their growth and maintenance throughout the life. These cells are multipotent. Adult stem cells typically generate the cell types of tissue in which they reside. However some experiments over last few years have raised the possibilities of a phenomenon known as plasticity, in which stem cells from one tissue may be able to generate cell types of a completely different tissue.
  25. 25. Figure 8 Researchers have traditionally found the plasticity of embryonic stem cells to be much greater than that of postnatal stem cells, but recent studies indicate that postnatal stem cells are more plastic than first imagined. The plasticity of the stem cell defines its ability to produce cells of different tissues. Post natal stem cells have been found in almost all body tissues including dental tissues.14 Commonly seven types of human dental stem cells have been characterized:15 1. Dental pulp stem cells (DPSCs) 2. Stem cells from human exfoliated deciduous teeth (SHED) 3. Stem cells from apical papilla (SCAP)
  26. 26. 4. Periodontal ligament stem cells (PDLSCs) 5. Periodontal ligament progenitor cells (PDLPs) 6. Dental follicle precursor cells (DFPCs) 7. Dental pulp pluripotent like stem cells (DPPSCs) HUMAN DENTAL STEM CELLS DFPCs Dental follicle precursor cells DPPSCs Dental pulp pluripotent like stem cells DPSCs Dental pulp stems cells PDLSCs Periodontal ligament stem cells PDLPs Periodontal ligament progenitor cells SCAP Stem cells from apical papilla SHED Stem cells from human exfoliated deciduous teeth Progenitor cells: Stem cells generate intermediate cell types before they achieve their fully differentiated state. The intermediate cell is known as precursor or progenitor cell. It is believed that such cells usually differentiate along a particular cellular development pathway. Generally undifferentiated cells are considered to be progenitor cells until their multi- tissue differentiation and self-renewal properties are demonstrated and they become designated as stem cells. Dental pulp stem cells
  27. 27. The dentine - pulp complex has a natural regenerative potential leading to the formation of tertiary dentine. Odontoblasts may survive mild injury, such as attrition or early caries, and secrete a reactionary dentine matrix. However, trauma of greater intensity, such as advanced caries or restorative procedures, may lead to the death of the pre-existing odontoblasts. In response to stimuli at the dentine–pulp interface, new odontoblasts are recruited and differentiate at the site of injury to synthesize a tubular reparative dentine, also sometimes referred to as osteo-dentine. This reparative dentine provides a ‘bridge’ of mineralized tissue immediately below the extensively damaged tissue, in order to preserve pulp vitality. A growing number of studies have now indicated that the post-natal pulp contains several niches of potential progenitor/stem cells, which may have importance in mediating reparative dentine formation. Indeed, progenitor/ stem cell niches are continually being identified in all connective tissues of the body, where they play a fundamental role in wound repair processes. This subset of undifferentiated cells can represent as little as 1% of the total cell population. However, they produce multiple highly differentiated progeny in response to specific extracellular signals. Central to the niche is the ‘true’ adult or ‘mother’ stem cell which displays an infrequent, yet almost unlimited self-renewal. At mitosis, these cells give rise to a renewed mother stem cell and a daughter transit amplifying progenitor cell. These daughter progenitor cells possess a more limited capacity for self-renewal, but are highly proliferative. They also appear to control multi- potentiality, and are capable of following along several cell lineages to ultimately produce terminally differentiated cells such as osteoblasts, odontoblasts, adipocytes, chondrocytes, and neural cells.
  28. 28. Figure 9 The stem cell niche consists of both the ‘true’ adult stem cell surrounded by the transit amplifying progenitor cells. The ‘true’ adult stem cell has the capacity for infrequent, but almost unlimited self-renewal, whereas the daughter transit amplifying cells are highly proliferative and are capable of differentiating along several cell lineages to produce the desired differentiated cell upon stimulation. Within these progenitor/stem cell niches, cell–cell and cell–matrix communication is important in maintaining the status of the stem cell, whereas their responsiveness to external stimuli provides a dynamic system for tissue repair. Stem cell niches in dental pulp:16 Within healthy tissues, the progenitor/stem cell niches usually maintain a quiescent state due to the influence of the environment in which they are found. Injury or trauma, leading to death of the post-mitotic odontoblasts within the dentine matrix, stimulates a cascade of complex and as yet unclarified events, whereby signals are released into the matrix. These signals then cause the progenitor/stem cell population to produce a high proliferative activity and the generation of the terminally differentiated odontoblast cell. The identification of progenitor/stem cell niches is thus
  29. 29. best observed in situ following their activation in response to injury. Studies performed during the 1980s first indicated that the replacement odontoblasts, which synthesized reparative dentine, are derived from undifferentiated mesenchymal cells in the pulp proper. Labelling of cells with tritiated thymidine, following pulp capping, showed an initial proliferation of cells in deep pulpal area below the site of injury, followed by an apparent migration of these cells to the wound site where they undergo further proliferation and differentiation to functional odontoblast-like cells. The source of these replacement odontoblast-like cells (progenitor/stem cell niches) was, and perhaps still, is a subject of some debate. The early studies of Fitzgerald and co-workers proposed that these progenitor cells were derived from a population of fibroblast-like cells. The investigators, however, also noted a potential contribution from daughter cells derived from the perivascular cell population. More recent studies monitoring ex vivo uptake by proliferative cells in response to injury have suggested that the progenitor / stem cell niches reside predominately in the perivascular regions of the pulpal cavity, from where they migrate to the site of injury. However, determination of stem/progenitor cell niches, as determined by elevation in Notch expression, following pulpal injury in vivohas proposed niches additional to the perivascular niche. Notch is purported to be an important signalling molecule which controls stem cell fate. A recent study in rats explored Notch expression following pulp capping. It was found that 1–3 days following pulp capping, Notch 1 expression was increased in the odontoblast and sub- odontoblast layers; Notch 2 expression increased in the pulpal stroma, whereas Notch 1 and 3 increased in cells associated with the perivascular structures. This finding suggests that progenitor/stem cell niches reside in different locations throughout the pulpal tissue. But the responsiveness of each of the progenitor/stem cell niches to injury would appear to vary according to location.1
  30. 30. Several stem/progenitor cell niches may exist in the mature dental pulp. These include a) Undifferentiated mesenchymal cells (so-called sub-odontoblasts) residing in the cell-rich layer close to the existing post-mitotic odontoblasts; b) A perivascular cell population associated with the pulpal vasculature and c) A Notch-2 positive cell population within the central pulpal stroma. Isolation and characterization of stem cell populations in dental pulp:1 The identification and isolation of an odontogenic progenitor population in adult dental pulp were first reported by Gronthos and co-workers in 2000. This group described the identification of dental pulp stem cells (DPSCs) by virtue of their clonogenic abilities, rapid proliferative rates, Figure 10
  31. 31. and capacity to form mineralized tissues both in vitro and in vivo. Subsequent studies isolated single cell colony derived populations of DPSCs which demonstrated multi-potentiality. This meant they were able to form adipocytes and neural precursors in vitro, in addition to dentin-like tissue following transplantation into immune-compromised mice. These studies also demonstrated differences in the odontogenic potential of the single colony-derived populations, with only 67% of cells found capable of forming abundant ectopic dentine in vivo. This may suggest the existence of differing progenitor/stem cell niches within dental pulp, which, although highly proliferative, display a hierarchy for cellular differentiation and multi-potentiality. The cellular characteristics of these DPSCs have been compared with those of bone marrow stem cells. Both dental pulp and bone marrow stem cell populations express similar putative stem cell surface markers, including CD44, CD106, CD146, 3G5, and STRO-1. They both expressed matrix proteins associated with mineral tissue formation, such as alkaline phosphatase, osteocalcin, and osteopontin. Similar expression patterns were also noted for stem cells isolated from periodontal ligament. However, in contrast to bone marrow stem cells, DPSCs have been shown to maintain a 30% higher proliferation rate and a higher growth potential. This higher rate of proliferation has been linked to the increased pulp cell expression of specific cell cycle mediators, namely cyclin-dependant kinase 6 and insulin like growth factor. Transplantation of DPSCs into immune-compromised mice resulted in the formation of a dentine-like tissue, whereas bone marrow stem cells produced a tissue resembling that of lamellar bone. This suggests that inherently different regulatory mechanisms exist within the two stem cell populations, evidenced by the elevated synthesis of dentine specific proteins during the formation of the dentine tissue at the cell transplant sites. Various attempts have subsequently been made to isolate and characterize progenitor/ stem cell populations from adult dental pulp,
  32. 32. with the intention of achieving a more defined clonal population of cells. A mesenchymal stem progenitor population, expressing the cell surface receptor STRO-1, has been isolated from adult dental pulp. The isolation strategy was similar to those previously used for the isolation of bone marrow stem cells. However, the presence of clonogenic cells, with high proliferative capacity, is reported to be much less common in dental pulp compared to bone marrow. These STRO-1 positive cells were found to differentiate down neurogenic, adipogenic, myogenic and chondrogeniclineages, and make a mineralized matrix when cultured in ‘odontogenic’inducing conditions. When cells isolated for STRO-1 were compared with those which were negative for this mesenchymal stem cell marker, only STRO-1 positive cells were capable of differentiating into odontoblasts like cells, indicating the importance of these cells in dentine repair processes. In alternative strategies, subsets of cell populations with progenitor/stem cell characteristics have been selected utilizing the hematopoietic/endothelial marker CD34 and putative stem cell proto-oncogene marker C-kit. By virtue of their infrequent, albeit unlimited self-renewal, attempts have been made to preferentially isolate the ‘true’ or ‘mother’ adult stem cell from porcine dental pulp, by selecting cells which fail to incorporate the DNA binding during cell division. A long proliferative lifespan has been confirmed for these cells, as has their ability to form reparative dentine following autologous transplantation. Primitive cells express high levels of ß1 integrins, which may assist in the constrained localization of stem cell populations through interaction with matrix proteins such as fibronectin. This observation has recently been utilized to obtain a highly proliferative, mesenchymal progenitor population with multi-potentiality from rat dental pulp. Cells expressing the neural crest cell markers of the Eph family have also been localized in mature dental pulp, suggesting these embryonic cells persist in the post-natal tissue. Subsequent characterization of this cell population, by the authors of this
  33. 33. review, has shown the expression of several putative stem cell markers and an ability to differentiate down adipocytic, chondrocytic and osteogenic lineages. Hierarchical ordering of stem cell niches: In view of the known hierarchical order of stem cells in dental pulp, the utilization of different isolation strategies could potentially lead to the isolation of different progenitor/ stem cell populations, which reside in different niches throughout the pulpal tissue.At present, work in this field is in its infancy, and the phenotypic analysis in terms of putative stem cell markers expressed by the various progenitor/stem cell populations is incomplete. Preliminary data have recently been presented which directly compare the expression of several putative stem cell markers of two progenitor cell populations isolated utilizing different stem cell characteristics. These have included LANGFR, which is found on the cell surface of neural crest cells, and ß1 integrin, which is highly expressed by primitive cells. The results provisionally suggest that more than one progenitor/stem cell population exists within mature dental pulp, which differs in their embryonic status, one of neural crest character and one of mesenchymal origin.1 If more than one progenitor stem cell population exists in mature dental pulp, then this may provide an explanation for the observed differences in odontogenic responses following transplantation of single-cell clonal populations. It may also explain differences in proliferative responses and patterns in Notch signaling in response to trauma. The possibility that more than one progenitor stem cell population may exist in mature dental pulp is also supported by the finding that stem cell populations in other tissues, such as primary tooth pulp and periodontal ligament, have differing genotypic and protein expression patterns.
  34. 34. Comparison of human dental-derived progenitor cells:1 It is now well established that bone marrow stromal cells (BMSCs) have the ability to form adherent colonies when plated at reasonably low densities in supplemented growth medium. Studies have demonstrated that each colony is originally derived from the clonal expansion of a single progenitor cell. In addition to these, and stem cell populations derived from adult dental pulp, other so-called mesenchymal stem cell populations have been recently identified from human exfoliated deciduous teeth (SHED cells), and adult periodontal ligament (PDLSCs) by their ability to generate clonogenic adherent colonies when grown and expanded under the same conditions as the BMSCs . Comparison of dental pulp, SHEDs, PDLSCs, and BMSCs has demonstrated that DPSC, SHED, and PDLSCs maintain a higher growth potential compared with BMSCs. However, there are reported differences in their protein and gene expression profile. Interestingly, the SHED cells are distinct from DPSCs by virtue of their higher proliferation rate, increased cell population doublings, and osteoinductive capacity in vivo. In addition to this, although SHED cells are able to differentiate into putative odontoblasts, and are immunoreactive to dentine sialophosphoprotein, they failed to reconstitute a complete dentine–pulp complex which was observed with DPSCs. Such data suggest that SHED cells are distinctly different from DPSCs in regard to odontogenic differentiation and osteogenic induction. It is therefore possible that these SHED cells may represent a population of multipotent stem cells that are more immature than the DPSCs obtained from adult teeth. Although these SHED cells may contain stem/progenitor cells, they may not be a single-cell type, and it is now thought that they may well be a heterogeneous population of cells from the pulp. Stem/progenitor cells in dentine regeneration and novel therapeutics
  35. 35. The identification and characterization of stem/progenitor cells within the dental and craniofacial tissues are now directing applied research into the use of such cells in clinical treatments. Such novel treatment modalities, however, will not be easy to develop due to the complex natural regenerative processes within the injured pulp. It is well understood that uncontrolled inflammation within the pulpal environment negates any natural capacity for repair. This inflammatory response and its progression are an important consideration in harnessing the reparative potential of the tooth and its progenitor cells. Severe injury may lead to odontoblasts cell death at specific foci directly beneath the area of injury, and the reparative dentinogenic response will have implications for the survival of the sub-odontoblasts within the underlying cell-rich zone, possibly one of the stem cell niches. The ability of such cells to migrate to areas of injury from other niches in unaffected areas of the pulp, in order to replace damaged/lost cells, remains an area of ongoing enquiry. The potent mix of bacterial infection and subsequent inflammation will undoubtedly moderate the regenerative process as mentioned earlier. However, it is still unclear how these inflammatory processes affect stem/progenitor cells or the molecular signaling processes responsible for their differentiation. The utilization of post-natal stem cells in clinical applications may be best served by developing materials that stimulate migration of stem/progenitor cells to the site of injury. These would then differentiate into a new generation of odontoblast-like cells when the vitality of the existing post-mitotic odontoblasts is compromised by carious injury or trauma. There is good evidence that such migration occurs in response to pulp capping procedures, with subsequent reparative dentinogenesis and dentine bridge formation. The recruitment of a progenitor cell population to differentiate into a new generation of odontoblast- like cells, leading to reparative dentinogenesis, is a natural reparative response of the dentine–
  36. 36. pulp complex. The finding that dentine bridge formation is stimulated by calcium hydroxide indicates that this process may be exploited clinically. It is tempting to consider the exciting possibilities of clinically directing this aspect of natural regeneration, both by maximizing recruitment of progenitor cells to areas of injury and disease and also through influencing the nature of the cell populations recruited. If the specific chemotactic signals for these cell populations can be determined, this could be harnessed for directed recruitment of these cells, to provide greater specificity and control to the tissue response. It is now clear that growth factors, sequestered within the dentine matrix, influence and direct the processes of reactionary and reparative dentinogenesis. With increasing understanding of pulpal regeneration in recent years, it has been able to suggest more robust hypotheses regarding the molecular and cellular processes responsible for dental regeneration and subsequent novel clinical therapies. Although recruitment of progenitor cells to sites of injury occurs naturally, it may be considered random (given the potential environment of the compromised pulp) and uncontrolled. Although it may be more relevant to try to harness and exploit this natural process, it has been suggested that directed recruitment of progenitor cells might be achieved through local application of enriched populations of cells, either by harvesting cells from nonautologous teeth or autologous exfoliated primary teeth. BMP-2-treated cultured pulp cells and Gdf11- electrotransfected pulp cells have been successfully transplanted to surgically amputated pulps, suggesting a possible therapeutic approach to dental regeneration. Interestingly, the initial regenerated tissue exhibits an osteo-dentine-like appearance, similar to that of reparative dentine (atubular) rather than tubular dentine. It is worth considering that this may be of benefit in dentine bridge formation where an atubular dentine would provide a more effective barrier to bacterial progression during any further carious challenge. However, this may not be entirely
  37. 37. feasible due to the difficulties of obtaining adequate sources and volume of autologous cells to reduce any immune response to the cell transplant. In order to improve yield, cells sourced from non-dental sites have been explored and have been found to be capable of differentiation into odontoblasts in an experimental model system. If such approaches to dental regeneration are to be explored further, tissue banks to source autologous cells will be required. In addition to reparative dentinogenesis, the success of vital pulp therapy also depends on an adequate vascular supply. Local angiogenesis is essential during healing at all wound sites, and pulpal injury and healing are no different. In addition to providing nutrition during the healing process, a good blood supply may also benefit the perivascular progenitor cell niche by increasing perivascular progenitor cell recruitment during regeneration. As discussed earlier, populations of DPSCs express the perivascular cell marker CD146, among other markers, which are co-localized to perivascular sites in the pulp. It is now known that angiogenic growth factors are present among the cocktail of growth factors found within the mature dentine matrix. It would appear that their release after injury may be key to the local up-regulation of angiogenesis at sites of trauma and, thereby indirectly, influence the pool progenitor cells for regeneration.1 Isolation Approaches of DPSCs No stable model has been set up to isolate and purify DPSCs for the lack of specific cell surface markers. The identification of DPSCs mainly depends on the biological characteristics, including small cell volume, vigorous proliferative ability, potent clonogenicity, self-renewal, and multi- differentiation potential.17 Some of the conventional approaches are 1. Size-sieved Isolation
  38. 38. 2. Stem Cell Colony Cultivation 3. Magnetic Activated Cell Sorting 4. Fluorescence Activated Cell Sorting SIZE SIEVED ISOLATION Several studies have demonstrated that small size represents one of the important characteristics of adult stem cells. These small diameter cells have greater viability, proliferative capacity and regenerative capability than those larger ones.18 PROCEDURE -The whole dental pulp tissue is gently separated from dental hard tissues and digested in a solution of 3% collagenase type I for 1 h at 37°C. -The single cell suspension is filtered through a 20 μm strainer to remove those larger differentiated cells. -Then, these smaller cells are seeded onto a 3 μm porous sieve to collect the larger cells. -Those cells on the upper plate surface of 3 μm sieve with a diameter between 3 μm and 20 μm are obtained for further culture and amplification. -Based on this approach, small-sized cell populations containing a high percent of stem cells can be isolated from the single-cell suspension, and thus DPSCs can be preliminary purified.
  39. 39. Figure 11 STEM CELL COLONY CULTIVATION Colony culture is the classical method to purify stem cell from the mixture of heterogeneous cells. Colony culture can be initiated from a disaggregated cell suspension which is made directly from tissues or sub-cultured cells. Human DPSCs from a single cell colony have been extensively studied, manifesting the self-renewal capability and multipotent differentiation. PROCEDURE Firstly, the dental pulp tissue is digested to prepare the single cell suspension. Then, the cell suspension is diluted to 100 cells/ mL and the cells in the suspension are placed into 96-well plates by 0.1 mL/well. The plates are pre-seeded with feeder cells which can provide the sufficient cell density, nutrition, growth factors, and extracellular matrix necessary for the colony
  40. 40. formation. After in vitro incubation at 37°C in 5% CO2 for 1–2 weeks, the single colony cluster containing 50 or more cells is digested and amplified for further experiments. Figure 12 Magnetic Activated Cell Sorting Magnetic activated cell sorting (MACS) is an immune magnetic method used for the separation of stem cell populations based on their surface antigens (CD271, STRO-1, CD34, CD45, and C- Kit). This approach has been widely used for the isolation of many different cell types, including lymphocytes, dendritic cellsand fetal cells from maternal blood, granulocytes, megakaryocytic cells, natural killer cells, T cells, tumor cells, epithelial cells, and hematopoietic stem cell.18 PROCEDURE Firstly, dental pulp is isolated and single cell suspension is prepared. Secondly, the suspension is incubated in the test tube with primary monoclonal antibody against specific membrane surface molecule and subsequently with immune-magnetic beads. These
  41. 41. magnetic beads are pre-coated with monoclonal secondary antibody necessary for searching and binding to target cells. Then, the mixture of cells and beads is placed in the magnetic particle concentrator, andcells specifically bound to beads will attach to the test-tube wall in the magnetic field. The bead-free cells in the supernatant are discarded. Finally, target cells bound to beads arecollected when passing through the magnetic field. The resultant cells are re-suspended and cultured for further experiments. To date, several subpopulations including STRO-1+ and CD271+ DPSCs have been successfully isolated by MACS method. Figure 13 Fluorescence Activated Cell Sorting The fluorescence activated cell sorting (FACS) is a convenient and efficient method that can effectively isolate stem cells from cell suspension based on the cell size and fluorescence. Cells are usually stained with one or more fluorescent dyes specific to targeted cell components.
  42. 42. Fluorescence of each cell is quantified as it rapidly transects the laser beam and fluorescence intensity provides the basis for separation of cell subpopulations. Using this method, STRO-1 selected DPSCs can perform the odontblastic differentiation and hard tissue formation under certain conditions. C-kit+/CD34+/ STRO-1+ DPSCs can differentiate into several kinds of ectomesenchyme-derived mature cells.Furthermore, Hoechst 33342-sorted SP cells from dental pulp have the typical features of stem cells, including the self- renewal and multipotent differentiation potential. However, FACS is a highly sophisticated technique which requires expensive equipment and highly-skilled personnel. FACS-sorted cells often undergo physiological stress and decreased viability. Moreover, this method is not appropriate for processing bulk quantities of cells thatwould almost certainly be required for therapeutic applications. Instead, FACS seems to be more suitable for the research or diagnostic analysis of relatively small cellpopulations. REGENERATION OF DENTAL TISSUE Step #1. The size, shape, functions, and aesthetics of the missing or defective tissue, such as a bone defect, needs to be assessed using cone beam microcomputer and radiographs. Step #2. Stem cells, such as dental pulp stem cells from an exfoliated baby tooth,need to be obtained from the host patient or a donor to serve as the buildingblocks for tissue regeneration. Step #3. Stem cells account for a very small percentage of the cells within tissues. The stem cells must be identified using surface markers and be isolated from all the donated cells using fluorescent cell sorting. Step #4. Millions of stem cells are needed to create functional tissues; this requires that they be expanded using cell culture.
  43. 43. Step #5. The activity of the stem cells must be controlled by growth factors during cell culture to ensure that the stem cells differentiate into a useful cell type (eg, bone or periodontal ligament). Step #6. Cells grown in culture lack a 3-dimensional scaffold necessary to function and have the correct size and shape to generate a tissue; therefore the cells need to be seeded onto a scaffold to form a tissue construct that gives the cells the characteristics of a tissue, such as dental pulp stem cells seeded onto polymer and collagen scaffolds to generate replacement pulp tissue. Step #7. The tissue construct is maintained in cell culture until a functional tissue is generated. Step #8. The tissue construct is grafted or implanted into the donor site,where the regenerated tissue is required. STEM CELLS BANKING Collection, Isolation and preservation of Stem cells COLLECTION Banking is a proactive decision made by the parents, so, the first step as informed to them is to put tooth in sterile saline solution and give a call to tooth bank or attending dentist of the bank. The tooth exfoliated should have pulp red in color, indicating that the pulp received blood flow up until the time of removal, which is indicative of cell viability. If the pulp is gray in color, it is likely that blood flow to the pulp has been compromised, and thus, the stem cells are likely necrotic and are no longer viable for recovery. Teeth that become very mobile, through trauma or disease (e.g. Class III or IV mobility), often have a severed blood supply cannot be the candidates for stem cell recovery. This is why recovery of stem cells from primary teeth is preferred after an extraction than the tooth that is “hanging on by a thread” with mobility. Pulpal stem cells should not be harvested from teeth with apical abscesses, tumors or cysts.
  44. 44. The dentist visually inspects the freshly-extracted tooth to confirm the presence of healthy pulpal tissue and the tooth or teeth is transferred into the vial containing a hypotonic phosphate buffered saline solution, which provides nutrients and helps to prevent the tissue from drying out during transport (up to four teeth in the one vial). Placing a tooth into this vial at room temperature induces hypothermia. The vial is then carefully sealed and placed into the thermette a temperature phase change carrier, after which the carrier is then placed into an insulated metal transport vessel. The thermette along with the insulated transport vessel maintains the sample in a hypothermic state during transportation. This procedure is described as Sustentation.19 The viability of the stem cells is both time and temperature sensitive, and careful attention is required to ensure that the sample will remain viable. The time from harvesting to arrival at the processing storage facility should not exceed 40 hours. STEM CELL ISOLATION: When the tooth bank receives the vial, the following protocol is followed. A) Tooth surface is cleaned by washing three times with Dulbecco’s Phosphate Buffered Saline without Ca2+ and Mg2+ (PBSA). B) Disinfection is done with disinfection reagent such as povidone iodine and again washed with PBSA. C) The pulp tissue is isolated from the pulp chamber with a sterile small forceps or dental excavator. Stem cell rich pulp can also be flushed out with salt water from the center of the tooth. D) Contaminated Pulp tissue is placed in a sterile petri dish which was washed at least thrice with PBSA.
  45. 45. E) The tissue is then digested with collagenase Type I and dispase for 1 hour at 37ºC. Trypsin- EDTA can also be used. F) Isolated cells are passed through a 70 um filter to obtain single cell suspensions. G) Then the cells are cultured in a Mesenchymal Stem Cell Medium (MSC) medium which consists of alpha modified minimal essential medium with 2mM glutamine and supplemented with 15% fetal bovine serum (FBS), 0.1 Mm L- ascorbic acid phosphates, 100U/ml penicillin and 100ug/ml streptomycin at 37ºC and 5% CO2 in air. Usually isolated colonies are visible after 24 hrs. H) Different cell lines can be obtained such as odontogenic, adipogenic and neural by making changes in the MSC medium. If cultures are obtained with unselected preparation, colonies of cells with morphology resembling epithelial cells or endothelial cells can be established. Usually cells disappear during course of successive cell passages. If contamination is extensive,three procedures can be performed 1) Retrypsinizing culture for a short time so that only stromal cells are detached because epithelial or endothelial like cells are more strongly attached to culture flask or dish. 2) Changing medium 4-6 hrs after subculture because stromal cells attach to culture surfaceearlier than contaminating cells. 3) Separate stem cells using Fluorosence Activated Cell Sorting (FACS), in which STRO-1 OR CD 146 can be used. This is considered most reliable.19 STEM CELL STORAGE
  46. 46. In the light of present research, either of the following two approaches are used for stem cell storage. a) Cryopreservation b) Magnetic freezing Cryopreservation: It is the process of preserving cells or whole tissues by cooling them to sub-zero temperatures. At these freezing temperatures, biological activity is stopped. SHED can be successfully stored long-term with cryopreservation and still remain viable for use. These cells can be cryopreservedfor an extended period of time, and when needed, carefully thawed to maintain their viability. Cells harvested near end of log phase growth (approximately 80–90% confluent) are best for cryopreservation. The sample is divided into four cryo-tubes and each part is stored in a separate location in cryogenic system so that even in the unlikely event of a problem with one of storage units, there will be another sample available for use. The cells are preserved in liquid nitrogen vapour at a temperature of less than -150ºC. This preserves the cells and maintains their latency and potency. In a vial, 1-2x 106 cells in 1.5 ml of freezingmedium is optimum. Too low or high cell number may decrease recovery rate. Suchánek J et al (2007) established a protocol of Dental Pulp Stem Cells (DPSCs) isolation and to cultivate DPSCs either from adult and exfoliated tooth, and compared thesecells with mesenchymal progenitor cell (MPCs) cultures. In comparison with bone marrow MPCs, DPSCs shared similar biological characteristics and stem cell properties. The resultsproved that the DPSCs and MPCs were highly proliferative, clonogenic cells that can be expanded beyond Hayflick’s limit and remain cytogenetically stable.
  47. 47. Zhang et al (2006) evaluated the differential potential of stem cells from the cryopreserved pulp of human third molars and concluded that the pulp tissue of the third molarmay serve as a suitable source of multipotent stem cells for future tissue engineering strategies and cell-based therapies, even after cryopreservation. Papaccio G et al (2006) studied the differentiation and morpho-functional properties of cells derived from stem cells after long-term cryopreservation to evaluate their potential for long-term storage with a view to subsequent use in therapy. They concluded that dental pulp stemcells and their osteoblast-derived cells can be long-term cryopreserved and may prove to be attractive for clinical applications. Magnetic freezing: Hiroshima University uses magnetic freezing rather than cryogenic freezing. This technology, is called CAS and exploits the little known phenomena that applying even a weak magnetic field to water or cell tissue will lower the freezing point of that body by up to 6-7 degrees Celsius. The idea of CAS is to completely chill an object below freezing point without freezing occurring, thus ensuring, distributed low temperature without the cell wall damage caused by ice expansion and nutrient drainage dueto capillary action, as normally caused by conventional freezing methods. Then, once the object is uniformly chilled, the magnetic field is turned off and the object snap freezes. The Hiroshima University company is the first expression of this new technology. Using CAS, Hiroshima University claims that it can increase the cell survival rate in teeth to ahigh of 83%.This compares to 63% for liquid nitrogen (-196 degrees C), 45% for ultra-cold freezing (-80 degrees C), and just 21.5% for a household freezer (-20 degrees C). Maintaininga CAS system is a lot cheaper than cryogenics and more reliable as well.
  48. 48. GROWTH FACTORS: Role in Regenerative Endodontics The goal of regenerative endodontics is to regain the vitality and functions of dental pulp– dentin complex.Regeneration of dental pulp-dentin–like tissues by cell homing that is orchestrated by growth factor delivery, without cell transplantation, provides one of the tangible pathways toward clinical translation. Growth factors regulate either transplanted cells or endogenously homed cells in dental pulp–dentin regeneration and the knowledge about the growth factors is important in regenerative endodontics.20 Dental pulp–like tissues can regenerate in vivo following the deliveryof dental or nondental stem/progenitor cells.An alternative approach is to orchestrate dental pulp–dentin regeneration by the homing of host endogenous cells relies on growth factor delivery, instead of cell delivery.
  49. 49. Primary effects & interactions of signaling molecules for regenerative Endodontics21 SIGNALLING MOLECULES (GROWTH FACTORS) TARGET CELLS PRIMARY EFFECTS INTERACTIONS PGDF Dental pulp stem cells -Cell proliferation -Dentinematrix synthesis -Odontoblastic differentiation - Dentinogenesis Combined with IGF-1 or dexamethasone, increased cell proliferation Combined with PDGF, increased cell proliferation TGFβ1 Dental pulp cells Dental pulp stem -Cell proliferation -Extracellular matrix synthesis -Odontoblastic differentiation -Dentinogenesis Chemo taxis Combined with FGF2, increased Odontoblastic differentiation
  50. 50. cells BMP2 Dental pulp cells -Odontoblastic differentiation -Dentinogenesis BMP4 Dental pulp cells -Odontoblastic differentiation -Dentinogenesis BMP7 (OP-1) Dental pulp cells -Dentinogenesis BMP11 (GDF11) Dental pulp stem cells -Odontoblastic differentiation -Dentinogenesis VEGF Dental pulp stem cells Odontoblastic differentiation cell proliferation Under osteogenic conditions, increased osteogenic differentiation FGF2 (bFGF) Dental pulp stem Dental pulp cells Chemotaxis Cell proliferation Cell proliferation Dentinogenesis cells Combined with TGFb1, increased odontoblastic
  51. 51. differentiation IGF Dental pulp cells Cell proliferation Odontoblastic Differentiation Combined with PDGF, increased cell proliferation NGF Dental papilla cells Odontoblastic Differentiation Growth factors affect a broad range of cellular activities including migration, proliferation, differentiation, and apoptosis of all dental pulp cells, including stem/progenitor cells. Figure 14
  52. 52. PLATELET-DERIVED GROWTH FACTOR Platelet-derived growth factor (PDGF) is released by platelets, and has potency inpromoting angiogenesis and cell proliferation.22,23PDGF has 4 isoform homodimers AA, BB, CC, and DD, in addition to a heterodimer, PDGF-AB. PDGF dimers bind to 2 cell-surface receptors known as PDGFRa and PDGFRb. The receptors form dimers before binding to different isoforms of PDGF.PDGF-AA, - BB, and -CC bind to PDGFR a/a, whereas PDGF-AB, -BB, -CC, and -DD bind to PDGFR a/b. PDGF-BB and -DD bind to PDGFR b/b. Therefore, the biological effect of PDGF depends on the expression level of PDGFR dimer on target cells.The chemotaxis and proliferation of mesenchymal stem/progenitor cells can be induced by PDGF in the injury site.24 In trauma, hemorrhage is followed by blood-clot formation in dental pulp. Platelets in the blood clot release a-granules containing PDGFs and attract neutrophils and macrophages. These cells play key roles in early wound healing by producing other signaling molecules for the formation of granulation tissues. It inhibits alkaline phosphatase activity (ALP) activity in dental pulp cells in culture. DSP expression is inhibited by PDGF-AA but is enhanced by PDGF-AB and PDGF-BB, although the mineralized tissue formation is inhibited, suggesting diverging effects of PDGFs on odontoblastic differentiation depending on dimeric form. PDGFs enhance the proliferation of fibroblasts in human dental pulp. PDGF-BB may increase the expression of vascular endothelial growth factor (VEGF) in osteoblasts and promotes angiogenesis at thesite of dental pulp injury.
  53. 53. TRANSFORMING GROWTH FACTOR ß The transforming growth factor b (TGFβ) family comprises a group of diverse growthfactors including TGFβ, bone morphogenetic proteins (BMPs), growth/differentiationfactors (GDFs), anti-Mullerian hormone (AMH), activin and nodal. TGFβ is composed of approximately 390 amino acids, which are released mainly from platelets, macrophages, and bone.25 This inactive polypeptide undergoes proteolytic cleavage to create the active C-terminal 112-amino-acid form. The active form of TGFβ dimerizes to form 25-kDa homodimers.The 3 iso-forms present in mammals, TGFb1, TGFb2, and TGFb3, are detected in human dentin. Inactive TGFβ exists as a large latent complex.Afterproteolytic cleavage, the active TGFβ binds to the type II receptor (TGFβRII) and recruits type I receptor (TGFβRI) to dimerize. TGFβRI, in turn, phosphorylates the intracellular proteins SMAD (homologues of Drosophila proteins including Caenorhabditiselegans protein [SMA] and mothers against decapentaplegic [MAD], in particular, SMAD2 and SMAD3. The activated SMAD complex translocate to the nucleus and activates downstream TGFβ gene transcription. The effect of TGFβ is highly variable and dependent on the type of cells and tissues. TGFβ1 regulates a wide range of cellular activities, such as cell migration, cell proliferation, cell differentiation, and extracellular matrix synthesis.TGFb1has been shown to increase cell proliferation and production of the extracellular matrix in dental pulp tissue cultureand promotes odontoblastic differentiation of dental pulp cells. The effect of TGFb1 can be synergistically upregulated by fibroblast growth factor 2 (FGF2)
  54. 54. BONE MORPHOGENETIC PROTEIN BMPs comprise a subgroup of the TGFβ super family and are involved in many biological activities including cell proliferation, differentiation, and apoptosis.26BMPs have strong osteoinductive and chondrogenic effects. BMP2 was discovered by Urist, who showed ectopic bone formation in connective tissues by transplanted demineralized bone. Later some of BMPs were identified, purified and sequenced from proteins extracted from bone.To date, more than 20 BMPs have been identified and characterized, among which GDFs are included. Unlike TGFβ, BMPs are secreted as an active form of 30 to 38-kDa homodimers after proteolytic cleavage of a synthesized form composed of 400 to 525 amino acids. Two TGFβ receptors (type I and type II) are known to be involved in the BMP signaling pathway. The activity of BMPs is regulated by the antagonists of BMPs such as Noggin and Chordin. This modulation of BMP activity by the BMP antagonists may have a critical role in tooth development.27 BMP2, BMP4, BMP7 and BMP11 are of clinical significance because of their role in inducing mineralization. Human recombinant BMP2 stimulates the differentiation of dental pulp cells into odontoblasts, inducing mRNA expression of dentin sialophosphoproteins (DSPPs) and higher ALP activity on BMP2 application, but has no effect on cell proliferation. DSPP expression and odontoblastic differentiation are regulated likely via BMP2-induced signaling by nuclear transcription factor Y. BMP2 also stimulates the differentiation of dental pulp stem/progenitor cells into odontoblasts in vivo and in vitro.28 Human recombinant BMP2 or BMP4 induces dentin formation when used in capping materials over amputated canine pulp.
  55. 55. Osteodentin formation occurs in amputated canine pulps treated with BMPs in collagen matrix. Bovine dental pulp cells treated with BMP2 and BMP4 differentiate into preodontoblasts. BMP7, also known as osteogenic protein 1, promotes dentinformation when placed over amputated dental pulp in macaque teeth. The dentinogenic effect of BMP7 on amputated dental pulp has been shown in several animal models including rats, ferrets, and miniature swine. Dental pulp cells transfected with BMP11, also known as GDF11, yields mineralization.Dentin matrix protein1, ALP, DSPP, enamelysin, and phosphate-regulating gene are highly expressed inBMP11-transfected cells. Transplantation of BMP11-transfected cell pellets induces formation of dentin like tissue on amputated dental pulp in dogs.29 Ultrasound mediated gene delivery of BMP11 stimulates odontoblastic differentiation of dental pulp stem/progenitor cells in vitro and reparative dentin formation in vivo.30 VASCULAR ENDOTHELIAL GROWTH FACTOR VEGF is a heparin-binding protein with specific affinity to endothelial cells, and plays a key role in angiogenesis.31The functions of VEGF involve the proliferation of endothelial cells and their enhanced survival, stimulating neovascularization in the area of injury. The VEGF family includes VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placenta growth factor. Among these isoforms, VEGF-A is the most versatile in function. VEGF-A, also known as vascular permeability factor, promotes cell migration, cell proliferation, vasodilatation, and vascular permeability by binding to 2 tyrosine kinases receptors,
  56. 56. FIBROBLAST GROWTH FACTOR Fibroblast growth factor (FGF) plays key roles in cell migration, proliferation and differentiation during embryonic development32and wound healing.33 Four FGF receptors, FGFR1 through FGFR4, are expressed in humans. Signal transduction is mediated by interaction between FGFs with the ability to bind to heparin sulfate and heparin sulfate proteoglycosan cell surface. FGF2 is a basic FGF, whereas FGF1 is acidic. FGF2 regulates tooth morphogenesis by controlling cell proliferation and differentiation. FGF2 is a potent angiogenic factor that stimulates formation of new blood vessels in the dental pulp along with PDGFand VEGF. Given its role in cell proliferation and angiogenesis, FGF2 acts as an early stimulating factor in formation of granulation tissue during wound healing. FGF2 induces the migration of dental pulp cells. Using a trans-well migration assay,significantly more dental pulp cells are recruited by basic FGF (FGF2) into a 3- dimensional collagen gel than compared with controls without cytokines and BMP7.FGF2 also stimulates the proliferation of dental pulp cells without differentiation,34 whereas FGF2 combined with TGFb1 induces differentiation of dental pulp cells into odontoblast-like cells, and synergistically upregulates the effect of TGFb1 on odontoblast differentiation. INSULIN-LIKE GROWTH FACTOR
  57. 57. Insulin-like growth factors (IGFs) are single-chain polypeptides that have high sequence similarity to pro-insulin. IGFs, comprising IGF-1 and IGF-2, contribute to odontogenesis and dental tissue repair by cell proliferation and differentiation.35 There are 2 known IGF receptors, of which IGF-1R has tyrosine kinase activity that phosphorylates the insulin receptor substrates and activates MAP kinase and the phosphatidylinositol- 3-kinase (PI3K) cascades. However, IGF-2R has no intrinsic kinase activity. IGF-1R binds to both isoforms of IGFs, but IGF-2R binds only to IGF-2. Of the 2 iso-forms, IGF-1, also known as somatomedin C, has potency in growth and differentiation of dental pulp cells. IGF-1 induces proliferation and differentiation of dog dental pulp cells into odontoblast-like cells in serum-free medium.36 IGF-1 with PDGF-BB has a synergistic effect on the proliferation of dental pulp cells in vitro. IGF-1 and IGF-1R have a higher level of expression in dental pulp tissue from teeth with complete root development than from teeth with incomplete root formation, suggesting that IGF- 1 stimulates mineralization and cell differentiation. NERVE GROWTH FACTOR  Nerve growth factors (NGFs), also known as neutrophins, promote the survival andmaintenance of sympathetic and sensory neurons. NGFs bind to 2 receptors, a p75 low-affinity neutrophin receptor (p75 LANR) and a high-affinity tyrosine kinasereceptor (TRK).  NGFs are involved in the survival and differentiation of neuronal and non neuronal cells through high-affinity trkA, but NGFs regulates apoptosis through p75 LANR. The expression of NGF and p75 LANR increases in dental pulp cells at the injury site.
  58. 58.  NGFs play a role in regulating tooth morphogenesis and tooth innervations in rat tooth development.37 STROMAL CELL–DERIVED FACTOR 1 Stromal cell–derived factor 1 (SDF-1), also known as chemokine (C-X-C motif) ligand 12 (CXCL12), is a chemo attractant involved in cell mobilization and homing by binding to the chemokine receptor CXCR4. SDF-1 functions as a chemokine for hematopoietic stem cells, mesenchymal stromal cells and immune cells. SDF-1 stimulates the migration and proliferation of CD31/CD146 side population (SP) cells isolated from porcine tooth germ that are positive for CXCR4 and negative for hematopoietic markers.Furthermore, the CD31 /CD146 SP cells have strong migration and proliferation activity with localized SDF-1 expression in amputated canine dental pulp. Dental pulp–like tissue with capillaries and nerves regenerate in dog teeth following pulp extirpation and autologous transplantation of the CD31 /CD146 SP cells or CD1051 cells with SDF-1 into root canals.38 GROWTH FACTORS STUDIED BY VARIOUS RESEARCHERS: RESEARCHES SUPER FAMILY FAMILY RESULTS OF THE STUDY TRANSFORMING TGFβ1 -They are secreted by odontoblasts and are deposited with in the dentin matrix where they remain protected in an active form through interaction
  59. 59. Smith et al GROWTH FACTOR with other components of the dentin matrix -stimulated increase in tertiary dentin matrix secretion Huojiaet al TGF TGFβ3 Indicated for the first time that TGFβ3 induces ectopic mineralisation through regulation of osteocalcin and type 1 collagen expression in DPSC Beguekiret alal TGF TGFβ1 BMP2 Demonstrated that TGFB1 present in dentin could interact with some component which acts as a modulator of its activity on the initiation of the psychological and functional differentiation of odontoblasts. Nakashima et al TGF TGFβ,BMP2,B MP4 Demonstrated regulatory role of TGFβ, BMP2 and 4 on the gene expression of extracellular matrix proteins and the differentiation of pulp cells into preodontoblasts
  60. 60. Sloan et al TGF BMP 7 BMP7when applied to freshly cut dentin in monkey teeth stimulated tertiary dentin formation Loharaet al TGF BMP2 BMP2 can direct pulp progenitor stem cell differentiation into odontoblasts and result in dentin formation Roberts clerk et al PDGF EGF VEGF PDGF AB EGF PIGF.FGF2 Dentin matrix contains angiogenic growth factors He et al OTHERS TGF FGF-2 TGFβ1 TGFβ1 initiates odontoblastslike differentiation of DPSCS FGF-2 exerts effect on cell proliferation and synergistically up regulates the effects of TGF b1 Ishimatsuet al OTHERS FGF-2 Dentin regeneration on amputed
  61. 61. pulp can be regulated by adjusting the dose of FGF-2 Goncalveset al PDGF rhVEGF Induce an angiogenic response in the pulp. Finding suggests that cells of severed dental pulps are still capable of responding to the angiogenic stimuli mediated by VEGF could be useful in the treatment of dental pulp conditions that require re vascularisation (immediate replantation of avulsed tooth. This hypothesis is under investigation Aranhaet al PDGF VEGF Hypoxia consequent to trauma enhances VEGF expression in DPSCs Li et al OTHERS GH AND IGF-1 GH and IGF-1 induce BMP-2 and 4 LovsChallet al OTHERS RhIGF-1 Rh IGF-1 enhances reparative dentinogenesis in pulp capping of rat molars
  62. 62. He et al DMP1 DMP can induce differentiation of stem cells into odontoblast like cells and stimulate the formation of mineralized tissues Almushaytet al DMP DMP-1 Found in dentin and bone and regulates the mineralisation Prescott et al DMP DMP DMP 1 induces the cyto differentiation of undifferentiated pulp cells to matrix synthesising cells Sobhaniet al Endochondral Bone Matrix Gelatin (ECBMG) Implantation of Ec-BMG in pulp cavity can induce pulp cells, secondary dentin and osteodentin rabbits Iwasaki et al IL6 Mediator of osteoblastic differentiation in periodontal ligament cells Kajiyaet al Antimicrobial peptide LL37 induces the migration of human pulp cells Kim et al Him OxygenaseHO- 1 Odontoblastic differentiation of hDPSCs and growth are positively regulated by HO-1
  63. 63. induction and vice versa Tissue engineering: Definition: Tissue engineering or regenerative medicine has been defined as ‘an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes for the repair or regeneration of tissue or organ function.” Regenerative medicine emphasizes a transition from organ replacement with bio inert or biocompatible, durable materials to a focus on cell-based organ replacement.39 Tissue Engineering comprises of (a)Adult stem/progenitor cells (b)Scaffold (c) Signalling Molecules
  64. 64. Figure 15 Adult Stem Cells -Capable of differentiating into specialized cells. -Able to respond to morphogens by dividing or specializing. Morphogens -Biological factors that regulate stem cells to form the desirable cell type. -5 major families (BMPs, FGFs, Wnts, Hhs, TNF). -BMPs are major morphogen family for tooth regeneration. Scaffold -Provides a biocompatible 3-dimensional structure for cell adhesion and migration -Biological scaffolds (e.g., collagen, glycosaminoglycan) -Artificial scaffolds (e.g., PLA, PGA, PLGA)
  65. 65. ADULT STEM CELLS/PROGENITOR CELLS Unique characteristics of adult stem cells (a) They exist as undifferentiated cells and maintain this phenotype by the environment and/or the adjacent cell populations until they are exposed to and respond to the appropriate signals. (b) They have an ability to self-replicate for prolonged periods. (c) They maintain their multiple differentiation potential throughout the life of the organism. Progenitor cells retain the differentiation potential and high proliferation capability, but have lost the self-replication property unlike stem cells. SCAFFOLD Importance of scaffold in tissue engineering:40 The scaffold provides a physicochemical and biological three-dimensional microenvironment for cell growth and differentiation, promoting cell adhesion, and migration. The scaffold serves as a carrier for morphogen in protein therapy and for cells in cell therapy. IDEAL CHARACTERISTICS OF SCAFFOLDS:40 Several general characteristics and requirements need to be considered for all designs. The scaffold should be/have: (a) Biocompatible; the scaffold should provoke an appropriate biological response in a specific application and prevent any adverse response of the surrounding tissue.
  66. 66. (b)Biodegradable; the scaffold materials should degrade in tandem with tissue regeneration and remodelling of the extracellular matrix (ECM) into smaller non‐toxic substances without interfering with the function of the surrounding tissue. (c)Promote cell attachment, spreading and proliferation; vital for the regulation of cell growth and differentiation. (d)Suitable mechanical strength; its strength should be comparable to in vivo tissue at the site of implantation as evidently, a scaffold requires more flexibility or rigidity depending on the application in e.g. cardiovascular versus bone prostheses. (e)Good transport properties; to ensure sufficient nutrient transport towards the cells and removal of waste products the scaffold should be highly porous with good pore connectivity, however, it should maintain sufficient mechanical strength implying optimization of porosity. (f)Easy to connect to the vascularization system of the host; to ensure good nutrient supply throughout the scaffold post‐implantation, the scaffold should be connected to the natural nutrient supplying system. (g)Suitable surface characteristics; apart from optimal physiochemical properties, research suggests that the introduction of e.g. surface topography into the scaffold improves tissue organization leading to increased tissue function. Natural polymers such as collagen and glycosaminoglycan offer good biocompatibility and bioactivity, and synthetic polymers can elaborate physicochemical features such as degradation rate, microstructure, and mechanical strength. Commonly used synthetic materials are 1. Poly (lactic acid) (PLA),
  67. 67. 2. Poly (glycolic acid) (PGA), and their copolymers, 3. Poly (lactic-co-glycolic acid) (PLGA), 4. Synthetic hydrogels include Poly Ethylene Glycol (PEG) based polymers, and those modified with cell surface adhesion peptides, such as Arginine, Glycine, and Aspartic acid. Scaffolds containing inorganic compounds such as hydroxyapatiteand calcium phosphate are used to enhance bone conductivity. Physicochemical characterisation of scaffolds:40 For the successful achievement of three-dimensional scaffolds and several criteria of characterisation are required. They can be divided into four categories: (1) Morphology (e.g. porosity, pore size, surface area); (2) Mechanical properties (e.g. compressive and tensile strength); (3) Bulk properties (e.g. degradation and its relevant mechanical properties); (4) Surface properties (e.g. surface energy, chemistry, charge). Porosity is defined as the fraction (i.e. percentage) of the total volume occupied by voids.The most widely used methods for measuring porosity are mercury porosimetry, scanning electron microscopy and confocal laser microscopy. Mechanical properties are extremely important when designing tissue-engineered products. To determine the mechanical properties of a porous structure, conventional testinginstruments may be used.
  68. 68. Mechanical tests can be divided into 1. Creep tests. 2. Stress–relaxation tests. 3. Stress–strain tests. 4. Dynamic mechanical tests. It is generally recognized that the adhesion and proliferation of different types of cells on polymeric materials depend largely on surface characteristics such as wettability (hydrophility/ hydrophobicity of surface free energy), chemistry, charge, roughness, and rigidity. In particular, three-dimensional applications of tissue engineering are more important for cell migration, cell proliferation, DNA/RNA synthesis, and phenotype presentation on the scaffold materials. Surface chemistry and charge can be analyzed by electron scanning chemical analysis and streaming potential, respectively. The wettability of the scaffold surface can be measured by the contact angle with static and dynamic methods. Natural polymers for scaffolds Many naturally occurring scaffolds can be used as biomaterials for tissue engineering purposes. One example is the extracellular matrix (ECM), a very complex biomaterial controlling cell function that designs natural and synthetic scaffolds to mimic specific functions. Alginate, Collagens (Gelatin), Fibrins, Albumin, Gluten, Elastin, Fibroin, Hyarulonic acid, Cellulose, Starch, Chitosan (Chitin), Scleroglucan, Elsinan, Pectin (Pectinic acid), Galactan,
  69. 69. Curdlan, Gellan, Levan, Emulsan, Dextran, Pullulan, Heparin, Silk, Chondroitin-6-sulfate, Polyhydroxyalkanoate, etc. Synthetic Polymers for Scaffolds Natural polymers are typically in short supply because they are expensive, suffer from batch-to- batch variation, and are susceptible to cross-contamination from unknown viruses or unwanted diseases. On the contrary, synthetic polymeric biomaterials have easily controlled physicochemical properties and quality, and have no immunogenicity. They can also be processed with various techniques and consistently supplied in large quantities. In order to adjust the physical and mechanical properties of tissue-engineered scaffolds at a desired place in the human body, the molecular structure and molecular weight are easily adjusted during the synthetic process.41 Synthetic polymers are largely divided into two categories: 1. Biodegradable polymers 2. Non biodegradable Polymers  Non bio degradable polymers include - Polyvinyl alcohol (PVA). - Polyhydroxyethylmethacrylate (PHEMA). - Poly N-isopropylacrylamide.  Biodegradable polymers include Family of poly (α-hydroxy esters) such as - Polyglycolide (PGA). - Polylactide (PLA) and its copolymer, Poly lactide-co-glycolide (PLGA).
  70. 70. - Polyphosphazine. - Polyanhydride. - Poly propylenefumeratepolycyanoacrylate. - Poly ε-caprolactone (PCL). - Polydioxanone (PDO) and biodegradeable polyurethanes. Of these two types of synthetic polymers, synthetic biodegradable polymers are preferred for the application of tissue engineered scaffolds because they minimize the chronic foreign body reaction and lead to the formation of completely natural tissue. That is to say, they can form a temporary scaffold for mechanical and biochemical support. Bio ceramics for scaffolds Bio ceramics are biomaterials that are produced by sintering or melting inorganic raw materials to create an amorphous or crystalline solid body, which can be used as an implant. Porous final products are mainly used for scaffolds. The components of ceramics are calcium, silica, phosphorus, magnesium, potassium, and sodium. Bio ceramics used for tissue engineering may be classified as Nonresorbable (relatively inert) E.g.-Alumina, Zirconia, Silicon nitride and Carbons. Bioactive or surface active (semi-inert) E.g.-certain glass ceramics, such as dense hydroxyapatites [9CaO.Ca (OH)2.3P2O5]
  71. 71. Biodegradeable or resorbable (noninert). E.g. - calcium phosphates, aluminium calcium phosphates, coralline, tricalciumphosphates (3CaO.P2O5), zinc calcium phosphorus oxides, zinc sulfate calcium phosphates, ferric calcium phosphorus oxides, and calcium aluminates. Widely used bio ceramics in tissue engineering, Synthetic apatite and calcium phosphate minerals, coral-derived apatite, bioactive glass and demineralised bone particle (DBP) are widely used in hard tissue engineering. Cytokine release system for scaffolds Growth factors, a type of cytokine, are polypeptides that transmit signals to modulate cellular activities and tissue development such as cell patterning, motility, proliferation, aggregation, and gene expression. As in the development of tissue-engineered organs, the regeneration of functional tissue requires the maintenance of cell viability and differentiated function, encouragement of cell proliferation, modulation of the direction and speed of cell migration, and regulation of cellular adhesion. The easiest method for the delivery of growth factors is via injection near the site of cell differentiation and proliferation.40 Disadvantages of this procedure are - Relatively short half-life. - A relatively high molecular weight and size. - Very low tissue penetration. - Potential toxicity at the systemic level.
  72. 72. One promising way to improve the efficacy of this technique is the locally controlled release of bioactive molecules for the desired release period by the impregnation into a scaffold. Through impregnation into a scaffold carrier, protein structure and biological activity can be stabilized to a certain extent, resulting in a prolonged release time at the local site. The duration of cytokine release from a scaffold is controlled by the types of biomaterials used, the loading amount of cytokine, the formulation factors, and the fabrication process. The cytokine release system may be designed for a variety of geometries and configurations, such as scaffold, tube, nose, microsphere, injectable forms, and fiber. Morphogens Morphogens are extracellularly secreted signals governing morphogenesisduring epithelial-mesenchymal interactions. The morphogenetic signalling networks include the five major classes of evolutionarily conserved genes. FIVE DISTINCT MORPHOGEN FAMILIES INVOLVED IN EMBRYONIC TOOTH DEVELOPMENT:42 1. Bone morphogenetic proteins (BMPs), 2. Fibroblast growth factors (FGFs), 3. Wingless- and interrelated proteins (Wnts), 4. Hedgehog proteins (Hhs), 5. Tumor necrotic factor (TNF) BMPs appear to be sufficient for tooth regeneration in adults.
  73. 73. BMP family members are sequentially and repeatedly involved in embryonic tooth development and the interactions between epithelium and mesenchyme are important in tooth development. BMP4 from the epithelium induces the mesenchyme to be odontogenic. BMP2, BMP4, and BMP7signals expressed in the enamel knot influence both epithelial and mesenchymal cells and are responsible for the maintenance of the enamel knot and the subsequent morphogenesis of epithelium. BMP2, BMP4, BMP6, BMP7, and Gdf11are also expressed during odontoblast differentiation BMP4 and BMP5 during ameloblast differentiation. The BMP signalling networks are complex and regulated at three levels.  Extracellular sites.  Cell membrane site.  Intracellular domains. For the BMPs to act, it needs an antagonist BMP antagonists such as noggin, chordin, and follistatin modulate the bioavailability of the morphogens Two transmembrane receptors, type I and type II with serine-threonine kinase activity are expressed in dental pulp. BMP signals are transduced from the plasma membrane to the nucleus through a limited number of Smad proteins, receptor-activated Smads (R-Smads), common mediator Smads (co-Smads), and inhibitory Smads (I-Smads). Many Smad-interacting proteins have been detected and determine the outcome of the signaling. The BMP signals are reiteratively used for communication and signaling between epithelium and mesenchyme. The same signals in different tissues and at different times result in the various
  74. 74. cellular responses because of the histories of the cells determining their competence to respond to the signals.43 Materials frequently applied in Soft Tissue engineering applications. ORIGIN POLYMER(FAMILY) NATURAL -Collagen-component of the extra cellular matrix (ECM) -Fibroin -Gelatine -Polysaccharides-hyaluronic acid, Chitosan, Alginate, Starch SYNTHETIC -Polyesters Most common are poly α-hydroxy acids Poly lactic acid (PLA) and poly glycolic acid (PGA) Polyanhydrides
  75. 75. Materials frequently applied in hard Tissue Engineering applications CLASS OF MATERIAL TYPE Crystalline ceramics -Hydroxyapatite -Tri calcium phosphate -Calcium meta phosphate Amorphous glasses -Silica -Bio glass Composites -Hydroxyapatite,chitosan, -Titanium/calcium phosphate, -Poly vinyl alcohol, and/or boron -Polylactic acid Metals -Stainless steel -Titanium alumina Tissue engineering can be divided into two types: 1. Ex-vivo (Extracorporeal) tissue engineering 2. In-vivo tissue engineering 1. Ex-vivo tissue engineering: Extracorporeal or ex-vivo tissue engineering involves the expansion, differentiation, or modification of progenitor cells in culture, which then organize into functional tissues through cell-cell signaling, biomolecules production, and formation of extracellular matrix. Ex-vivo tissue engineering products may be fully functional at the time of implantation, or more commonly, have the potential for integration and further maturation after implantation. This
  76. 76. engineering requires the harvest of progenitor cells at various stages of differentiation for expansion and maturation on appropriate scaffolds in culture and subsequent implantation. Complications faced: • Progenitor cell availability, • Difficulty in culturing some progenitor cell types, • The need for cellular patterning and topographic control, • The need for providing a microcirculation for the development of larger, biologically meaningful tissues, • Adds significant cost to treatment, • Culture expansion is fraught with the risk of contamination with bacteria or viruses, • Limits the utility of this type of tissue engineering in patients with limited life expectancies. • Moreover, there is evidence that autologous cells can be rendered immunogenic by prolonged in-vitro culture. 1. In vivo or in situ tissue engineering: In-vivo or in-situ tissue engineering depends on the in-vivo proliferation and differentiation of progenitor cells on scaffolds, or on the administration of growth factors that recruit progenitor cells, and enhance their expansion and differentiation. In-vivo tissue engineering using progenitor cells in which the patient acts as his or her own bioreactor obviates cellular patterning and microcirculation, as most progenitor cell-containing constructs self-assemble into histologically recognizable tissues.
  77. 77. As with extracorporeal tissue engineering, techniques using autologous progenitor cell transplantation are limited by the need for progenitor cell harvest, and at times, ex-vivo expansion prior to implantation. This type of in-situ tissue engineering is designed to compensate for a deficiency in the numbers or function of progenitor cells, as may occur with previous irradiation, scarring, or compromised vascularity. When the in-vivo availability of progenitor cells is not a cause for concern, the use of growth factors or other external stimuli to recruit and stimulate proliferation and differentiation of progenitor cells in vivo is an attractive, but complicated, endeavor. Three general approaches for tissue engineering:42 Currently the literature describes three general tissue engineering approaches. These principles are closely related to each other and may be applied to create new tissues. These approaches include: 1. Design and grow human tissues in vitro for later implantation to repair or replace diseased tissues: The most common example is the skin graft, used for the treatment of burns. Skin graft replacements have been grown in tissue culture and used clinically for more than 10 years- in vitro 2. Implantation of cell-containing or cell-free devices that induce the regeneration of functional human tissues: "signal" molecules, e.g. growth factors may be used to assist in biomaterial-guided tissue regeneration. Also, novel polymers have been created and assembled into three-dimensional configurations, to which cells attach and grow to reconstitute tissues. An example is the use of a polymer matrix to form cartilage , in vivo
  78. 78. 3. The development of external devices containing human tissues designed to replace the function of diseased internal tissues: This approach involves establishing primary cell- lines, placing the cells on or within structural matrices and implanting the new system inside the body. Examples of this approach include repair of bone, muscle, tendon and cartilage, endothelial cell-lined vascular grafts and heart valve substitutes, ex vivo. Figure 16 Tissue engineering a multidisciplinary approach: The cell transplantation strategy truly reflects the multidisciplinary nature of tissue engineering, and requires clinician, bioengineer and cell biologist. 1. Clinician: Biopsy of small sample of tissue containing cells of interest. 2. Cell Biologist: multiplies cells and maintain their function.
  79. 79. 3. Bioengineer: Manufactures the tissue, bioreactor and the material onto which the cells will be placed for transplantation. Lastly the clinician transplants the engineered tissue. Polymer scaffold degrades and is remodeled by the host and transplanted cells resulting in complete natural tissue. Figure 17 Preclinical and Clinical Accomplishments of tissue engineering: It can be categorized into four categories – (Figure 18)  Conductive (passive) approaches  Inductive approaches  Cell transplantation  Gene therapy

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