Regen Endo

Regenerative Endodontics
CONTENTS
Introduction An overview of regenerative medicine Tissue engineering Key elements for tissue engineering Stem cells Types of stem cells
Early embryonic stem cells Blastocyst embryonic stem cells Foetal stem cells Adult stem cells Umbilical cord stem cells Sources of stem cells
Autogenous Allogenous Xenogenous
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Progenitor cells Pulp stem cells
Dental pulp stem cells (dpscs) Stem cells from human exfoliated deciduous teeth (shed) Stem cells from apical papillae (scap) Periodontal ligament stem cells (pdlscs). Culturing of stem cells Differentiation of stem cells Cell lines Stem cell identification Growth factors Scaffolds
Ideal requirements of a scaffold Types of scaffold Scaffolds for tissue engineering Clinical applications of tissue engineering concepts An overview of potential technologies for regenerative endodontics
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root canal revascularization via blood clotting, postnatal stem cell therapy pulp implantation scaffold implantation Injectable scaffold delivery Three-dimensional cell printing gene delivery. Tooth regeneration by single cell manipulation bioengineered tooth germ by tissue engineering method using scaffolds bioengineered tooth germ by cell aggregation comparison of cell processing methods Development of a novel bioengineering method Development of a bio-engineered tooth in the adult oral environment Identification of cell sources for tooth regeneration Regulation of tooth size and morphology Delivery of regenerative endodontic procedures Barriers to be addressed to permit introduction of regenerative endodontics
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Challenges and future direction Conclusion References
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INTRODUCTION Millions of teeth are saved each year by root canal therapy. Although current root canal treatment modalities offer high levels of success for many conditions, an ideal form of therapy might consist of regenerative approaches in which diseased or necrotic pulp tissues are removed and replaced with healthy pulp tissues to revitalize the teeth. Human organs and tissues are made up of about 200 kinds of cells that develop from stem cells. Stem cells are undifferentiated cells with the ability to differentiate into any tissue type in order to carry out specific tasks. Recently, stem-cell-transplantation therapy has attracted attention as an alternative to organ transplantation due to stem cells abilities to proliferate and differentiate at an engrafted site . Human stem cells, including embryonic stem cells (ES cells) and adult tissue stem cells, are excellent candidates for transplantation therapy . Human ES cells are enormously promising because of their ability to differentiate into specialized embryonic tissues from all germ layers, including the ectoderm, mesoderm and endoderm . Adult stem cells and progenitor cells act as self-repair systems for the body by replication and development into specialized cells. Recently, stem-cell-based therapies, including stem cell transplantation to repair injured tissues, have passed significant milestones with the successful treatment of various refractory conditions, such as Parkinsons disease, leukaemia, spinal injury, cardiac infarction, diabetes and liver diseases .
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An Overview of Regenerative Medicine Regenerative medicine holds promise for the restoration of tissues and organs damaged by disease, trauma, cancer, or congenital deformity. Regenerative medicine can perhaps be best 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 it wasan interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function. MacArthur and Oreffo defined tissue engineering asunderstanding the principles of tissue growth, and applying this to produce functional replacement tissue for clinical use. Thus it can be said that tissue engineering is the employment of biologic therapeutic strategies aimed at the replacement, repair, maintenance, and/or enhancement of tissue function. The changes to the definition of tissue engineering over the years are driven by scientific progress. Although there can be many differing definitions of regenerative medicine, in practice the term has come to represent applications that repair or replace structural and functional tissues, including bone, cartilage, and blood vessels, among organs and tissues . The ultimate goal of regenerative medicine is to develop fully functioning, bioengineered organs to replace damaged organs. However, bioengineering technologies have not yet achieved three-dimensional reconstructions of fully functioning organs, which would result if various types of stem/progenitor cells could be instructed to become complex, specialized Page 6
arrangements of differentiated cells. One of the most promising techniques for organ reconstruction is a cell culturing method that uses biodegradable scaffolds into which dissociated cells are seeded, re-aggregate and adopt the shape of the scaffold. But, it is still experimental. During embryonic development, almost all organs, including the teeth, arise from various types of organ germ. These are induced by reciprocal interactions between epithelial and mesenchymal cells. An alternative to the scaffold strategy is to develop fully functioning organs from organ germ by manipulating developmental programs involving epithelial mesenchymal interactions.
The creation and delivery of new tissues to replace diseased, missing, or traumatized pulp is referred to as 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. This approach provides an innovative and novel range of biologically-based clinical treatments for endodontic disease. Regenerative dental procedures have a long history, originating around 1952, when Dr. B. W. Hermann reported on the application of Ca(OH)2 in a case report of vital pulp amputation . Subsequent regenerative dental procedures include the development of guided tissue or bone regeneration (GTR, GBR) procedures and distraction osteogenesis .The application of platelet rich plasma (PRP) for bone augmentation , Emdogain for periodontal tissue regeneration , and recombinant human bone morphogenic protein (rhBMP) for bone augmentation and preclinical trials on the use of fibroblast growth factor 2 (FGF2) for periodontal tissue regeneration . Despite these applications and the considerable evolution of certain medical procedures of tissue regeneration, particularly bone marrow transplants, there has not been significant translation of any of these therapies into clinical endodontic practice Page 7
Tissue Engineering Tissue engineering is an emerging multidisciplinary field that applies the principles of engineering and life sciences for the development of biological substitutes that can restore, maintain, or improve tissue function. The tissues of interest in regenerative endodontics include dentin, pulp, cementum and periodontal tissues . The key elements of tissue engineering are stem cells, morphogens or growth factors, and an extracellular matrix scaffold.
Key Elements for Tissue Engineering
Stem cells Stem cells are generally defined as clonogenic cells capable of both self-renewal and multilineage differentiation. Stem cells are considered to be the most valuable cells for regenerative medicine. Research on stem cells is providing advanced knowledge about how an organism develops from a single cell, and how healthy cells replace damaged ones in adult organisms. Stem cells have the ability to continuously divide to either replicate themselves (self-replication), or produce specialized cells that can differentiate into various other types of cells or tissues (multilineage differentiation). Page 8
Types of 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 an embryo. These early stem cells are totipotent, i.e. possess the ability to become any kind of cell in the body. Blastocyst embryonic stem cells Five days after fertilization, the embryo forms a hollow ball-like structure known as a blastocyst. Embryos at the blastocyst stage contain two types of cellsa) an outer layer of trophoblasts that eventually form the placenta, and b) an inner cluster of cells known as the inner cell mass that becomes the embryo and then develops into a mature organism. The embryonic stem cells in the blastocyst are pluripotent, i.e. having the ability to become almost any kind of cell in the body. Scientists can induce these cells to replicate themselves in an undifferentiated state for very long periods before stimulating them with appropriate signaling molecules to create specialized cells. However, the sourcing of embryonic stem cells is controversial and associated with ethical and legal issues, thus reducing their appeal for the development of new therapies.
Foetal stem cells After 8 weeks of development, the embryo is referred to as a fetus. By this time it has developed a human-like form. Stem cells in the fetus are responsible for the initial development of all tissues before birth. Like embryonic stem cells, fetal stem cells are pluripotent.
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Umbilical cord stem cells The umbilical cord is the lifeline that transports nutrients and oxygen-rich blood from the placenta to the fetus. Blood from the umbilical cord contains stem cells that are genetically identical to the newborn baby. Umbilical cord stem cells are multipotent, i.e. they can differentiate into a limited range of cell types. Umbilical cord stem cells can be stored cryogenically after birth for use in future medical therapy. Here are some advantages and disadvantages of cord stem cells in detail: 1) Harvesting stem cells from the umbilical cord blood is not risky for the child or the mother. However, a donor of bone marrow can get an infection and must be anesthetized. 2) Harvesting of umbilical cord stem cells is a quick process. Umbilical blood is collected away in cryogenic freezers. However, the process of getting in contact with marrow donors and getting them tested is a process that usually takes weeks to complete. You will need substantial time to find a bone marrow donor but units for umbilical blood are ready to be used. In addition, the umbilical blood unit can be taken to the transplant hospital in less than a weeks time. The doctor selects the cord blood to use and can make the transplant urgently. 3) Umbilical cord stem cells have a low incidence of graft vs. host disease, or GVHD (Graft-versus-host disease), as they are more archaic than the stem cells in the bone marrow. This is why a perfect match is not required between the patient and the donor. It is common to have GVHD after allogeneic transplants and it can be life threatening as well as mild. You have less chances of getting GVHD, if you have had a cord blood transplant. Page 10
4) It was earlier believed that one person could not be treated with stem cells obtained from two different umbilical cords. However, this outlook has changed after stem cells from two donors were successfully used in a stem cell transplant. This proves the adaptability of cord stem cells. 5) In cord blood transplant, a matching cord blood unit is not mandatory. The doctor may be able to conduct a transplant even if there is partial match between donors and recipients cord blood cells. The recipient in such transplants has less risk of complications, which is an added advantage. Now that you know the advantages of cord blood stem cells, let us know about its disadvantages: 1) You can be at risk during the stem cell engraftment process as cord stem cells are older than the stem cells found in bone marrow. This engraftment process takes a long time and patient can get infection during this time. 2) Stem cells available in cord blood are not sufficient for treatment in adults. Stem cells in a typical umbilical cord blood are just enough to be transplanted in a smallsized adult or a small child. Advances in medical sciences are bringing new advantages of cord stem cell therapies to light. Scientist are conducting extensive test to see if any advantages offered have any side effects on human body. Adult stem cells The most valuable cells for regenerative medicine are stem cells, with a translational emphasis on the use of postnatal or adult stem cells. Stem cells hold great promise in regenerative medicine, but there are still many unanswered questions that will have to be addressed before these cells can be routinely used in patients, especially in regard to the Page 11
safety of the procedure. The potential for pulp-tissue regeneration from implanted stem cells has yet to be tested in animals and clinical trials. Extensive clinical trials to evaluate efficacy and safety lie ahead before it is likely the Food and Drug Administration (FDA) will approve regenerative endodontic procedures using stem cells. All tissues originate from stem cells. A stem cell is commonly defined as a cell that has the ability to continuously divide and produce progeny cells that differentiate (develop) into various other types of cells or tissues. Stem cells are commonly defined as- either embryonic/ foetal or adult/postnatal .The term embryonic, rather than foetal is preferred, because the majority of these cells are embryonic. The term postnatal is preferred rather than adult, because these same cells are present in babies, infants, and children. The reason why it is important to distinguish between embryonic and postnatal stem cells is because these cells have a different potential for developing into various specialized cells (i.e. plasticity). 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. Adult stem cells typically generate the cell types of the tissue in which they reside. However, some experiments over the last few years have raised the possibility 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. Postnatal stem cells have been found in almost all body tissues, including dental tissues. Stem cells are also commonly subdivided into totipotent, pluripotent, and multipotent categories according to their plasticity. The greater plasticity of the embryonic stem cells makes these cells more valuable among researchers for developing new therapies. However, the sourcing of embryonic stem cells is controversial and is surrounded by ethical and legal issues, which reduces the attractiveness of these cells for developing new therapies. This explains why many researchers are now focusing attention on Page 12
developing stem cell therapies using postnatal stem cells donated by the patients themselves or their close relatives. The application of postnatal stem cell therapy was launched in 1968, when the first allogenic bone marrow transplant was successfully used in the treatment of severe combined immunodeficiency. Since the 1970s, bone marrow transplants have been used to successfully treat leukaemia, lymphoma, various anaemia, and genetic disorders. Postnatal stem cells have been sourced from umbilical cord blood, umbilical cord, bone marrow, peripheral blood, body fat, and almost all body tissues, including the pulp tissue of teeth. One of the first stem cell researchers was Dr. John Enders, who received the 1954 Nobel Prize in medicine for growing polio virus in human embryonic kidney cells. In 1998, Dr. James Thomson, isolated cells from the inner cell mass of the early embryo and developed the first human embryonic stem cell lines. In 1998, Dr. John Gearhart derived human embryonic germ cells from cells in fetal gonadal tissue (primordial germ cells). Pluripotent stem cell lines were developed from donated embryonic cells. In 2001, the president of the United States, George W. Bush, restricted federal funding to pre-existing embryonic cell lines. Of these 78 preexisting embryonic cells lines, 7 were duplicates, 31 were not available, 16 died after thawing, and 2 were withdrawn or are still in development, and the remaining 22 available cell lines did not prove to be very useful to many scientists . This restricted most U.S.-based researchers from working on embryonic stem cells. The legal limitations and the great ethical debate related to the use of embryonic stem cells must be resolved before the great potential of donated embryonic stem cells can be used to regenerate diseased, damaged, and missing tissues as part of future medical treatments. Accordingly, there is increased interest in autogenous postnatal stem cells as an alternative source for clinical applications, because these cells are readily available and have no immunogenicity issues, even though they may have reduced plasticity. From a medical perspective, among the most valuable stem cells are those capable of neuronal differentiation, because these cells Page 13
have the potential to be transformed into different cell morphologies in vitro, using lineagespecific induction factors; these include neuronal, adipogenic, chondrogenic, myogenic, and osteogenic cells . It may be possible to use neuronal stem cells from adipose fat as part of regenerative medicine instead of bone marrow cells, possibly providing a less painful and less threatening alternative collection method. A company called MacroPore
Biosurgery/Cytori Therapeutics Inc. is commercializing this approach, using a 1-hour process for human stem cell purification.
Stem cells are often categorized by their source: a) Autologous stem cells - The most practical clinical application of a stem cell therapy would be to use a patients own donor cells. Autologous stem cells are obtained from the same individual to whom they will be implanted. Bone marrow harvesting of a patients own stem cells and their reimplantation back to the same patient represents one clinical application of autogenous postnatal stem cells. Stem cells could be taken from the bone marrow , peripheral blood , fat removed by liposuction , the periodontal ligament , oral mucosa, or skin. An example of an autologous cell bank is one that stores umbilical cord stem cells . Autologous stem cells have the fewest problems with immune rejection and pathogen transmission . Harvesting the patients own cells makes them the least expensive to obtain and avoids legal and ethical concerns . However, in some cases suitable donor cells may not be available. This concern applies to very ill or elderly persons. One potential disadvantage of harvesting cells from patients is that surgical operations might lead to postoperative sequelae, such as donor site infection . Autologous postnatal stem cells also must be isolated from mixed tissues and possibly expanded in number before they can be used. This takes time, so certain autologous regenerative medicine solutions may not be very quick. To accomplish endodontic regeneration, the most promising cells are autologous Page 14
postnatal stem cells , because these appear to have the fewest disadvantages that would prevent them from being used clinically.
b) Allogenic cells - originate from a donor of the same species . Examples of donor allogenic cells include blood cells used for a blood transfusion , bone marrow cells used for a bone marrow transplant , and donated egg cells used for in vitro transplantation . These donated cells are often stored in a cell bank, to be used by patients requiring them. In contrast to the application of donated cells, there are some ethical and legal constraints to the use of human cell lines to accomplish regenerative medicine . The use of preexisting cell lines and cell organ cultures removes the problems of harvesting cells from the patient and waiting weeks for replacement tissues to form incell organ-tissue cultures . However, the most serious disadvantages of using preexisting cell lines from donors to treat patients are the risks of immune rejection and pathogen transmission . The use of donated allogenic cells, such as dermal fibroblasts from human foreskin, has been demonstrated to be immunologically safe and thus a viable choice for tissue engineering of skin for burn victims. The FDA has approved several companies producing skin for burn victims using donated dermal fibroblasts. The same technology may be applied to replace pulp tissues after root canal therapy, but it has not yet been evaluated and published. c) Xenogenic cells are those isolated from individuals of another species. Pig tooth pulp cells have been transplanted into mice, and these have formed tooth crown structures. This suggests it is feasible to accomplish the reverse therapy, eventually using donated animal pulp stem cells to create tooth tissues in humans. In particular, animal cells have been used quite extensively in experiments aimed at the construction of cardiovascular implants. The harvesting of cells from donor animals removes most of the legal and ethical issues associated with sourcing cells from other humans. However, many problems remain, such as Page 15
the high potential for immune rejection and pathogen transmission from the donor animal to the human recipient. The future use of xenogenic stem cells is uncertain, and largely depends on the success of the other available stem cell therapies. If the results of allogenic and autologous pulp stem cell tissue regeneration are disappointing, then the use of xenogenic endodontic cells remains a viable option for developing an endodontic regeneration therapy.
To date, four types of human dental stem cells have been isolated and characterized: i) ii) iii) iv) Dental pulp stem cells (DPSCs) Stem cells from human exfoliated deciduous teeth (SHED) Stem cells from apical papillae (SCAP) Periodontal ligament stem cells (PDLSCs). Among them, all except SHED are from permanent teeth. The identification of these dental stem cells provides better understanding of the biology of the pulp and periodontal ligament tissues, and their regenerative potential after tissue damage.
Progenitor cells Stem cells generate intermediate cell types before they achieve their fully differentiated state. The intermediate cell is known as a 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 multitissue differentiation and self-renewal properties are demonstrated and they become designated as stem cells. The objectives of regenerative endodontic procedures are to regenerate pulp-like tissue, ideally, the pulp-dentin complex; regenerate damaged coronal dentin, such as following a Page 16
carious exposure; and regenerate resorbed root, cervical or apical dentin. The importance of the endodontic aspect of tissue engineering has been highlighted by the National Institute for Dental and Craniofacial Research. The principles of regenerative medicine can be applied to endodontic tissue engineering. Regenerative endodontics comprises research in adult stem cells, growth factors, organ-tissue culture, and tissue engineering materials. Often these disciplines are combined, rather than used individually to create regenerative therapies. Many aspects of regenerative endodontics are thought to be recent inventions; however, the long history of research in these fields may be surprising. A brief overview of the potential types of regenerative endodontic therapies is provided in this review.
Pulp Stem Cells The dental pulp contains a population of stem cells, called pulp stem cells or, in the case of immature teeth, stem cells from human exfoliated deciduous teeth (SHED) . Sometimes pulp stem cells are called odontoblastoid cells, because these cells appear to synthesize and secrete dentin matrix like the odontoblast cells they replace. After severe pulp damage or mechanical or caries exposure, the odontoblasts are often irreversibly injured beneath the wound site. Odontoblasts are post mitotic terminally differentiated cells, and cannot proliferate to replace subjacent irreversibly injured odontoblasts .The source of the odontoblastoid cells that replace the odontoblasts and secrete reparative dentin bridges has proven to be controversial. Initially, the replacement of irreversibly injured odontoblasts by predetermined odontoblastoid cells that do not replicate their DNA after induction was suggested. It was proposed that the cells within the subodontoblast cellrich layer or zone of Hohl adjacent to the odontoblasts differentiate into odontoblastoids. However, the purpose of these cells appears to be limited to an odontoblast-supporting role, as the survival of these cells was linked to the survival of the odontoblasts and no proliferative or regenerative activity was Page 17
observed. The use of tritiated thymidine to study cellular division in the pulp by autoradiography after damage revealed a peak in fibroblast activity close to the exposure site about 4 days after successful pulp capping of monkey teeth. An additional autoradiographic study of dentin bridge formation in monkey teeth, after calcium hydroxide direct pulp capping for up to 12 days , has revealed differences in the cellular labeling depending on the location of the wound site. Labeling of specific cells among the fibroblasts and perivascular cells shifted from low to high over time if the exposure was limited to the odontoblastic layer and the cell-free zone, whereas labeling changed from high to low if the exposure was deep into the pulpal tissue. More cells were labeled close to the reparative dentin bridge than in the pulp core. The autoradiographic findings did not show any labeling in the existing odontoblast layer, or in a specific pulp location. This provided support for the theory that the progenitor stem cells for the odontoblastoid cells are resident undifferentiated mesenchymal cells. The origins of these cells may be related to the primary odontoblasts, because during tooth development, only the neural crest derived cell population of the dental papilla is able to specifically respond to the basement membrane mediated inductive signal for odontoblast differentiation. The ability of both young and old teeth to respond to injury by induction of reparative dentinogenesis suggests that a small population of competent progenitor pulp stem cells may exist within the dental pulp throughout life. However, the debate on the nature of the precursor pulp stem cells giving rise to the odontoblastoid cells, as well as questions concerning the heterogeneity of the dental pulp population in adult teeth, remain to be resolved . Information on the mechanisms by which these cells are able to detect and respond to tooth injury is scarce, but this information will be valuable for use in developing tissue engineering and regenerative endodontic therapies. One of the most significant obstacles to overcome in creating replacement pulp tissue for use in regenerative endodontics is to obtain progenitor pulp cells that will continually divide and produce cells or pulp tissues that can be Page 18
implanted into root canal systems. Possibilities are the development of an autogenous human pulp stem cell line that is disease- and pathogen-free, and/or the development of a tissue biopsy transplantation technique using cells from the oral mucosa, as examples. The use of a human pulp stem cell line has the advantage that patients do not need to provide their own cells through a biopsy, and that pulp tissue constructs can be premade for quick implantation when they are needed. If a patient provides their own tissue to be used to create a pulp tissue construct, it is possible that the patient will have to wait some time until the cells have been purified and/or expanded in number. This latter point is based on the finding that many adult tissues contain only 1 to 4% stem cells, so purification is needed, and expansion of cell numbers would permit collection of smaller tissue biopsies. Alternatively, larger sources of autologous tissue might be required. The sourcing of stem cells to be used in endodontic, dental, and medical therapies is a significant limiting factor in the development of new therapies and should be a major research priority.
Dental pulp stem cells (DPSCs) DPSCs were isolated for the first time in 2000 by Gronthos et al. based on their striking ability to regenerate a dentin-pulp-like complex composed of a mineralized matrix of tubules lined with odontoblasts, and fibrous tissue containing blood vessels in an arrangement similar to the dentin-pulp complex found in normal human teeth. Then, in a later study , the same group demonstrated that these cells had a high proliferative capacity, a self renewal property and a multi-lineage differentiation potential. Reparative dentin-like structure is deposited on the dentin surface if DPSCs are seeded onto a human dentin surface and implanted into immunocompromised mice, suggesting the possibility of forming additional new dentin on existing dentin. Laino et al. isolated a selected subpopulation of DPSCs known as Stromal Bone-producing Dental Pulp Stem Cells Page 19
(SBP-DPSCs). These were described as multipotential cells that were able to give rise to a variety of cell types and tissues including osteoblasts, adipocytes, myoblasts, endotheliocytes, and melanocytes, as well as neural cell progenitors (neurons and glia), being of neural crest origin . Several studies of DPSCs have shown that they are multipotent stromal cells that proliferate extensively, can be safely cryopreserved, are applicable with several scaffolds, have a long lifespan, posses immunosuppressive properties, and are capable of forming mineralized tissues similar to dentin. Paakkonen et al. demonstrated that DPSCs have a general gene expression pattern similar to that of mature native odontoblasts, and are therefore a valuable human derived cell line for in vitro studies of odontoblasts. However, definitive proof of their ability to produce dentin has not yet been obtained. Recently, Takeda et al. characterized hDPSCs isolated from tooth germs at the crown-completed stage and found that these cells were highly proliferative and had the potential to generate a dentin-like matrix in vivo. However, these characteristics were lost in long-term culture, with a change in their gene expression profile. Meanwhile, Abe et al. have described apical pulp derived cells (APDCs) present in human teeth with immature apices, and suggested that they are an effective source of cells for regeneration of hard tissue. SHED SHED were isolated for the first time in 2003 by Miura et al., who confirmed that they were able to differentiate into a variety of cell types to a greater extent than DPSCs, including neural cells, adipocytes, osteoblast-like and odontoblast-like cells. The main task of these cells seems to be the formation of mineralized tissue, which can be used to enhance orofacial bone regeneration . The ethical constraints associated with the use of embryonic stem cells, together with the limitations of readily accessible sources of autologous postnatal stem cells with multipotentiality, have made SHED an attractive alternative for dental tissue engineering. The use of SHED for tissue engineering might be more advantageous than that Page 20
of stem cells from adult human teeth. They were reported to have a higher proliferation rate than stem cells from permanent teeth, and can also be retrieved from a tissue that is disposable and readily accessible. Thus, they are ideally suited for young patients at the mixed dentition stage who have suffered pulp necrosis in immature permanent teeth as a consequence of trauma. Nr has shown that by seeding SHED onto synthetic scaffolds seated in pulp chamber of a thin tooth slice, odontoblast-like cells can rise from the stem cells and localize against existing dentin surface after implanting the tooth slice construct into immunocompromised mice.
SCAP It is well known that dental papilla is derived from the ectomesenchyme induced by the overlaying dental lamina during tooth development. This developing organ evolves into dental pulp after being encased by the dentin tissue produced by odontoblasts that come from this organ. The apical portion of the dental papilla during the stage of root development has not been described much in the literature. Most information regarding tooth development comes from studies using animal models. Recently, scientists described the physical and histologic characteristics of the dental papilla located at the apex of developing human permanent teeth and termed this tissue apical papilla . The tissue is loosely attached to the apex of the developing root and can be easily detached with a pair of tweezers. Apical papilla is apical to the epithelial diaphragm, and there is an apical cell-rich zone lying between the apical papilla and the pulp. Importantly, there are stem/progenitor cells located in both dental pulp and the apical papilla, but they have somewhat different characteristics. Because of the apical location of the apical papilla, this tissue may be benefited by its collateral circulation, which enables it to survive during the process of pulp necrosis. In general, stem cells are defined by having two major properties. First, they are capable of self-renewal. Second, when Page 21
they divide, some daughter cells give rise to cells that either maintain stem cell character or give rise to differentiated cells. Mesenchymal stem cells (MSCs) have been identified in many tissues and are capable of differentiating into many lineages of cells when grown in defined conditions including osteogenic, chondrogenic, adipogenic, myogenic, and neurogenic lineages The first type of human dental stem cells was isolated from dental pulp tissue of extracted third molars These dental pulp derived cells were named DPSCs for their clonogenic properties (ie, exhibiting CFU-Fs in cultures) and, more importantly, their ability to differentiate into odontoblast- like cells and form dentin/pulp-like complex when implanted into the subcutaneous space of immunocompromised mice . Subsequently, a variety of dental MSCs have been isolated including stem cells from human exfoliated deciduous teeth (SHED), periodontalligament stem cells (PDLSCs), and SCAP. A new unique population of mesenchymal stem cells (MSCs) residing in the apical papilla of permanent immature teeth, known as stem cells from the apical papilla (SCAP), were recently discovered by Sonoyama et al., who reported that these cells express various mesenchymal stem cell markers. Isolated SCAP grown in cultures can undergo dentinogenic differentiation when stimulated with dexamethasone supplemented with L-ascorbate-2phosphate and inorganic phosphate. Alizarin Red (a dye that binds calcium salts)positive nodules form in the SCAP cultures after 4 weeks of induction, indicating a significant calcium accumulation in vitro. Furthermore, cultured SCAP were found to express dentinogenic markers dentin sialophosphoprotein and CBFA1/Runx2. In addition to their dentinogenic potential, SCAP also exhibit adipogenic and neurogenic differentiation capabilities when treated with respective stimuli. SCAP are capable of forming odontoblastlike cells, producing dentin in vivo, and are likely to be the cell source of primary odontoblasts for formation of root dentin. The discovery of stem cells in the apical papilla may also explain a clinical phenomenon described in a number of recent clinical case reports Page 22
showing that apexogenesis can occur in infected immature permanent teeth with periradicular periodontitis or abscess. It is likely that the SCAP residing in the apical papilla survive such pulp necrosis because of their proximity to the vasculature of the periapical tissues. Therefore, after endodontic disinfection, and under the influence of the surviving epithelial root sheath of Hertwig, these cells can generate primary odontoblasts that complete root formation.
SCAP Are a Unique Population of Postnatal Stem Cells It was found that SCAP showed a significantly greater bromodeoxyuridine uptake rate, number of population doublings, tissue regeneration capacity, and number of STRO-1 positive cells when compared with DPSCs. In addition, SCAP express a higher level of survivin (antiapoptotic protein) than DPSCs and are positive for hTERT (human telomerase Page 23
reverse transcriptase that maintains the telomere length) activity, which is usually negative in MSCs. These lines of evidence suggest that SCAP derived from a developing tissue may represent a population of early stem/progenitor cells, which may be a superior cell source for tissue regeneration. Additionally, these cells also highlight an important fact that developing tissues may contain stem cells distinctive from that of mature tissues. The neurogenic potential of SCAP could be because of the fact that SCAP are derived from neural crest cells or at least associated with neural crest cells analogous to other dental stem cells such as DPSCs and SHED that have been shown previously to possess a neurogenic potential .
The Potential Role of SCAP in Continued Root Formation The role of apical papilla in root formation may be observed in clinical cases. A human immature incisor was injured and the crown fractured with pulp exposure. During the treatment, the apical papilla was retained while the pulp was extirpated. Continued root-tip formation was observed after root canal treatment. Further investigation is needed to verify whether the radiographic evidence of continued apical development is because of dentin formation from the apical papilla or if it is merely cementum formation. Using minipigs as a model, a pilot experiment was conducted. Surgically removing the apical papilla at an early stage of the root development halted the root development despite the pulp tissue being intact. In contrast, other roots of the tooth containing apical papilla showed normal growth and development. Although the finding suggests that root apical papilla is likely to play a pivotal role in root formation, further research is needed to verify that this halted root development in the minipig was not due to damage of Hertwigs epithelial root sheath (HERS) during the removal of the apical papilla of that particular root apex.
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The Potential Role of SCAP in Pulp Healing and Regeneration Immature Teeth with Periradicular Periodontitis or Abscess Undergo Apexogenesis. There have been sporadic case reports in the literature showing the potential of root maturation even with the presence of periradicular pathoses of endodontic origin . More recently, several clinical reports with careful documentation and follow-ups have further shown that immature permanent teeth diagnosed with nonvital pulp and periradicular periodontitis or abscess can undergo apexogenesis . These recent reports challenge the traditional approach in managing immature teeth by applying apexification treatment, where there is little to no expectation of continued root development. Instead, it is possible that alternative biologically based treatments may promote apexogenesis/maturogenesis. A common aspect of many of these reported cases is the preoperative presentation of apical periodontitis with sinus tract formation, a condition normally associated with total pulpal necrosis and infection that requires apexification. Although Iwaya et al. and Banchs and Trope applied the term revascularization to describe this phenomenon, what actually occurred was physiological tissue formation and regeneration. The clinical presentation of a negative response to pulpal testing with the presence of apical periodontitis is generally interpreted to indicate pulpal necrosis and infection. Lin et al., however, found that vital tissues can even be present in pulp chambers in mature permanent teeth associated with periapical radiolucencies. The size of the lesions varies, and some may be described as moderately extensive. In the case of immature teeth, this situation could be more common, although there is a lack of histologic studies. The recent case reports of immature teeth that presented with radiolucent lesions and underwent remarkable apexogenesis after conservative treatment suggest that vitalpulp tissue must have remained in the canals . Prolonged infection may eventually lead to a total necrosis of the pulp and apical papilla.Under these conditions, apexogenesis or maturogenesis, a term Page 25
that encompasses not just the completion of root-tip formation but also the dentin of the root , would then be unlikely.
The Potential Role of SCAP in Replantation and Transplantation The fate of human pulp space after dental trauma has been observed in clinical radiographs. Andreasen et al. and Kling et al. showed excellent radiographic images of the ingrowth of bone and periodontal ligament (PDL) (next to the inner dentinal wall) into the canal space with arrested root formation after the replantation of avulsed maxillary incisors, suggesting a complete loss of the viability of pulp, apical papilla, and/or HERS. Some cases showed partial formation of the root accompanied with ingrowth of bone and PDL into the canal space, and in some cases the teeth continued to develop roots to their completion, suggesting that there was partial or total pulp survival after the replantation. It is noted, however, that a pronounced narrowing of canal space is usually associated with a surviving pulp. Skoglund et al. observed revascularization of the pulp of replanted and autotransplanted teeth with incomplete root development in dogs. Ingrowth of new vessels occurs during the first few postoperative days. After 10 days, new vessels are formed in the apical half of the pulp and, after 30 days, in the whole pulp. In some instances, anastomoses seemed to form with preexisting vessels in the pulp. Although revascularization occurs, the pulp space is eventually filled with hard tissue. Other animal studies focusing on the changes in pulp tissue after replantation showed that various hard tissues including dentin, cementum, and bone may form in the pulp space depending on the level of pulp recovery. If pulp and apical papilla are totally lost, then the root canal space may be occupied by cementum, PDL, and bone. By tracing the migration of periodontal cells after pulpectomy in immature teeth, Vojinovic and Vojinovic found that periodontal cells migrate into the apical pulp space during the repair process. Therefore, one may assume that when there is a total loss of pulp Page 26
tissue but the canal space remains in a sterile condition, the outcome is the ingrowth of periodontal tissues. One of the clinical treatment options for missing teeth is autotransplantation. The process often involves extraction of a supernumerary tooth or third molar and implantation into a recipient site. With regard to the status of pulp survival and root formation of the transplanted immature teeth, the clinical observations nicely shown by Tsukiboshi have been that a) if the transplanted tooth has minimal root formation, there will be minimal, if any, further root development after transplantation b) if there is some root formation, it will continue to develop to some extent or to completion after transplantation; and c) pulp tissue will be eventually replaced by hard tissue. It has been considered that as long as the HERS remains viable, it stimulates the undifferentiated mesenchymal cells in periradicular tissues to differentiate into odontoblasts that contribute to the formation of new dentin and root maturation. However, the current understanding is that pulp cells are different from periodontal cells. Based on current available information, it is likely that odontoblast lineages are derived from stem cells in pulp tissue or apical papilla. Both SCAP and HERS appear to be important for the continued root development after transplantation. SCAP are also highly probable to survive after transplantation because minimal vascularity is found in apical papilla based on preliminary findings. The reason that transplanting a tooth with little or no root formation results in almost no further root development is unclear. One may speculate that the integrity of the entire tooth organ at that stage is critical for the root development to continue. During the transplantation, any disruption of the structure such as the follicle, HERS, and apical papilla will prevent further root development . Page 27
These findings provide new light on the possibility of generating pulp and dentin in pulpless canals. However, when implanting cells/scaffolds into root canals that have blood supply only from the apical end, enhanced vascularization is needed in order to support the vitality of the implanted cells in the scaffolds. Recent efforts in developing scaffold systems for tissue engineering have been focusing on creating a system that promotes angiogenesis for the formation of a vascular network. These scaffolds are impregnated with growth factors such as VEGF and/or platelet-derived growth factor or, further, with the addition of endothelial cells. These approaches are particularly important for pulp tissue engineering for the aforementioned reason that blood supply is only from the apical end. Collagen has been considered as a convenient pulp cell carrier and could conveniently be injected into canal space to regenerate pulp clinically, yet collagen as the matrix has been found to contract significantly when carrying pulp cells, which may considerably affect pulp tissue regeneration. Contraction-resistant scaffolds such as PLG (D,Llactide and glycolide) appear to be a more suitable carrier for pulp cells The utilization of stem cells and an optimal scaffold system may one day be used clinically in which engineered constructs may be inserted into canals of immature teeth to allow regeneration of pulp and dentin. The assumption for the multistep engineered tissue installments is based on the concern that blood vessel ingrowth can only occur from the apical end. A single installment, although it is more ideal and will avoid chances of introducing infection, may lead to the cell death in the coronal third region because of a lack of nutrients. Whether impregnation of VEGF and other angiogenic factors would make single installment a possibility awaits experimentation. If the proposed clinical approach can be successful, it implies a fundamental breakthrough in clinical endodontic treatments using cell and tissue engineering therapy.
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SCAP for Bioroot Engineering Dental implants have recently gained momentum as a preferred option for replacing missing teeth instead of bridges or removable dentures. However, although dental implants have had great improvements over the past decades, the fundamental pitfall is the lack of a natural structural relationship with the alveolar bone (ie, the absence of PDL). In fact, it requires a direct integration with bone onto its surface as the prerequisite for success, an unnatural relation with bone as compared with a natural tooth. The lack of natural contours and its structural interaction with the alveolar bone make dental implants a temporary option until a better alternative is available. This alternative may be tooth regeneration. Using animal study models, cells isolated from tooth buds can be seeded onto scaffolds and form ectopic teeth in vivo. Nakao et al. recently engineered teeth ectopically followed by transplantation into an othrotopic site in the mouse jaw. Tooth regeneration at orthotopic sites using larger animals such as dogs and swine has also been tested. The study in dogs failed to show root formation , whereas the swine model was able to show root formation with a 33.3% success rate . The other approach is to use SCAP and PDLSCs to form a bio root. Using a minipig model, autologous SCAP and PDLSCs were loaded onto HA/TCP and gelfoam scaffolds, respectively, and implanted into sockets of the lower jaw. A post channel was precreated to leave space for post insertion. Three months later, the bio root was exposed, and a porcelain crown was inserted. This approach is relatively a quick way of creating a root onto which an artificial crown can be installed. The bio root is different from a natural root in that the root structure is developed by SCAP in a random manner. Nevertheless, the bio root is encircled with periodontal ligament tissue and appears to have a natural relationship with the surrounding bone. What remains to be improved is the mechanical strength of the bio root, which is approximately two thirds of a natural tooth. Page 29
There are also many other challenges- for example, a common question that has been frequently raised in the field of tissue engineering and regenerative medicine is the source of stem cells. Where shall we obtain the stem cells? Autologous stem cells are the best source because allogenic or xenogenic are likely to have immunorejection issues. Banking teeth, tissues, or stem cells for autologous use is a viable option. The recent revelation of the immunosuppression properties of stem cells shed some light on the possibility of allogenic use of stem cells. Many in vitro and in vivo studies have confirmed the immunosuppressive effects of MSCs. The potential mechanisms underlying this immunosuppression can be explained by downregulation of T, dendritic, NK, and B cells .
Periodontal ligament stem cells (PDLSCs) Using a methodology similar to that utilized for isolation of MSCs from deciduous and adult pulp, Seo et al. described the presence of multipotent postnatal stem cells in the human PDL (PDLSCs). Under defined culture conditions, PDLSCs differentiated into cementoblast-like cells, adipocytes, and collagen-forming cells. When transplanted into immunocompromised rodents, PDLSCs showed the capacity to generate a cementum/PDL-like structure and contributed to periodontal tissue repair. The presence of MSCs in the periodontal ligament is also supported by the findings of Trubiani et al., who isolated and characterized a population of MSCs from the periodontal ligament which expressed a variety of stromal cell markers, and Shi et al. , who demonstrated the generation of cementum-like structures associated with PDL-like connective tissue after transplanting PDLSCs with hydroxyapatite/tricalcium phosphate particles into immunocompromised mice. The clinical potential for the use of PDLSCs has been further enhanced by the demonstration that these cells can be isolated from cryopreserved periodontal ligaments while maintaining their stem cell characteristics, including the expression of MSC surface markers, single-colony strain generation, Page 30
multipotential differentiation and cementum/periodontal-ligament-like tissue regeneration, thus providing a ready source of MSCs. Using a minipig model, autologous SCAP and PDLSCs were loaded onto hydroxyapatite/tricalcium phosphate and gelfoam scaffolds, and implanted into sockets in the lower jaw, where they formed a bioroot encircled with periodontal ligament tissue and in a natural relationship with the surrounding bone. Recently, Trubiani et al. suggested that PDLSCs had regenerative potential when seeded onto a three dimensional biocompatible scaffold, thus encouraging their use in graft biomaterials for bone tissue engineering in regenerative dentistry, whereas Li et al. have reported cementum and periodontal ligament-like tissue formation when PDLSCs are seeded on bioengineered dentin. Culturing of stem cells Cell culture is a term that refers to the growth and maintenance of cells in a controlled environment outside an organism. A successful stem cell culture is one that keeps the cells healthy, dividing, and unspecialized. Dental pulp stem cells can be cultured by two methodsa) the first is the enzyme-digestion method in which the pulp tissue is collected under sterile conditions, digested with appropriate enzymes, and then the resulting cell suspensions are seeded in culture dishes containing a special medium supplemented with necessary additives and incubated. Finally, the resulting colonies are subcultured before confluence and the cells are stimulated to differentiate. b) The second method for isolating dental pulp stem cells is the explant outgrowth method in which the extruded pulp tissues are cut into 2-mm3 cubes, anchored via microcarriers onto a suitable substrate, and directly incubated in culture dishes containing the essential medium with supplements. Ample time (up to 2 weeks) is needed to allow a sufficient number of cells to migrate out of the tissues.
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Haung et al. compared both methods and found that cells isolated by enzyme-digestion had a higher proliferation rate than those isolated by outgrowth.
Differentiation of stem cells Generation of specialized cells from unspecialized stem cells is a process known as differentiation, and is triggered by signals inside and outside the cells. The internal signals are controlled by the genes of one cell, which are interspersed across long strands of DNA, and carry coded instructions for all the structures and functions of a cell. The external signals for cell differentiation include chemicals secreted by other cells, physical contact with neighboring cells, and certain molecules in the microenvironment. Cultured dental pulp stem cells can be stimulated to differentiate to more than one cell type according to the contents of the culture medium. a) Osteo/dentinogenic medium contains dexamethasone, glycerophosphate, ascorbate phosphate and 1,25 dihydroxy vitamin D in addition to the basic elements. b) Adipogenic medium whereas c) For neurogenic induction cells are cultured in the presence of B27 supplement, basic fibroblast growth factor, and epidermal growth factor. Cell lines Culturing of stem cells is the first step in establishing a stem cell line, which is a propagating collection of genetically identical cells that can be used for research and therapy development. Once a stable stem cell line has been established, stem cells can be triggered to differentiate into specialized cell types. Odontoblasts are postmitotic terminally differentiated cells, and thus cannot be induced to Page 32 contains dexamethasone, insulin and isobutyl methylxanthine,
undergo further differentiation. The secretion product of differentiated odontoblasts are type I collagen, which forms the scaffold for mineral deposition and provides strength to the mineralized dentin, and two major noncollagenous proteins (NCPs) considered to have mineralization-regulatory capacities , namely dentin phosphophoryn (DPP; or DMP-2) and dentin sialoprotein(DSP) . DPP and DSP are encoded by a single gene, DSPP or DMP-3, which specifically defines the phenotypic characteristics of dentin . Another important noncollagenous protein is dentin matrix protein-1 (DMP-1), which is found primarily in dentin and bone and has been implicated in the regulation of mineralization, being considered to act as a growth factor to induce the differentiation of DPSCs. In order to explore the pulp wound-healing mechanism and to develop a therapeutic strategy for pulp regeneration, development of an odontoblast cell line is very important. Up to now, however, odontogenic differentiation has not been well characterized due to two major limitations: A) The first is the paucity of differentiation markers, which is now being overcome by the characterization of odontoblastspecific markers (DMP-1, DMP-2, and DMP-3) that can indicate the presence of a true odontoblastic cell line. B) The second is the limited life span of the primary cells, which is being addressed by trials of several methodologies including cell cloning and immortalization. Stem Cell Identification Stem cells can be identified and isolated from mixed cell populations by four commonly used techniques: (a) staining the cells with specific antibody markers and using a flow cytometer, in a process called fluorescent antibody cell sorting (FACS) (b) Immunomagnetic bead selection (c) Immunohistochemical staining
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(d) Physiological and histological criteria, including phenotype (appearance) chemotaxis, proliferation, differentiation, and mineralizing activity. FACS together with the protein marker CD34 is widely used to separate human stem cells expressing CD34 from peripheral blood, umbilical cord blood, and cell cultures. Different types of stem cells often express different proteins on their membranes and are therefore not identified by the same stem cell protein marker. The most studied dental stem cells are those of the dental pulp. Human pulp stem cells express von Willebrand factor CD146, alpha-smooth muscle actin, and 3G5 proteins. Human pulp stem cells also have a fibroblast phenoptype, with specific proliferation, differentiation, and mineralizing activity patterns
Growth factors: Growth factors are proteins that bind to receptors on the cell and induce cellular proliferation and/or differentiation. Many growth factors are quite versatile, stimulating cellular division in numerous cell types, while others are more cell specific. The names of individual growth factors often have little to do with their most important functions and exist because of the historical circumstances under which they arose. Growth factors are extracellularly secreted signals governing morphogenesis /organogenesis during epithelial mesenchymal interactions. They regulate the division or specialization of stem cells to the desirable cell type, and mediate key cellular events in tissue regeneration including cell proliferation, chemotaxis, differentiation, and matrix synthesis. Many growth factors are quite versatile, stimulating cellular division in numerous cell types, while others are more cell-specific. Some growth factors are used to increase stem cell numbers, as is the case for platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) , insulin-like growth factor (IGF), colonystimulating factor (CSF) and epidermal growth factor (EGF). Others modulate the humoral and cellular immune responses (interleukins 1- 13) while others are important regulators of Page 34
angiogenesis, such as vascular endothelial growth factor (VEGF), or are important for wound healing and tissue regeneration/ engineering, such as transforming growth factor alpha and beta For example, fibroblast growth factor (FGF) was found in a cow brain extract by Gospadarowicz and colleagues and tested in a bioassay which caused fibroblasts to proliferate. Currently, a variety of growth factors have been identified, with specific functions that can be used as part of stem cell and tissue engineering therapies. Many growth factors can be used to control stem cell activity, such as by increasing the rate of proliferation, inducing differentiation of the cells into another tissue type, or stimulating stem cells to synthesize and secrete mineralized matrix. If regenerative endodontics is to have a significant effect on clinical practice, it must primarily focus on providing effective therapies for regenerating functioning pulp tissue and, ideally, restoring lost dentinal structure. Toward this aim, increased understanding of the biological processes mediating tissue repair has allowed some investigators to mimic or supplement tooth reparative responses. Dentin contains many proteins capable of stimulating tissue responses. Demineralization of the dental tissues can lead to the release of growth factors following the application of cavity etching agents, restorative materials, and even caries. Indeed, it is likely that much of the therapeutic effect of calcium hydroxide may be because of its extraction of growth factors from the dentin matrix. Once released, these growth factors may play key roles in signaling many of the events of tertiary dentinogenesis, a response of pulp-dentin repair. Growth factors, especially those of the transforming growth factor beta (TGF) family, are important in cellular signaling for odontoblasts differentiation and stimulation of dentin matrix secretion. These growth factors are secreted by odontoblasts and deposited within the dentin matrix, where they remain protected in an active form through interaction with other components of the dentin matrix. The addition of purified dentin protein fractions has stimulated an increase in tertiary dentin matrix secretion . Another important family of Page 35
growth factors in tooth development and regeneration consists of the bone morphogenic proteins (BMPs). Bone morphogenetic proteins are multi-functional growth factors belonging to the transforming growth factor super family. The first BMPs were originally identified by their ability to induce ectopic bone formation when implanted under the skin of rodents . To date, about 20 BMP family members have been identified and characterized. They have different profiles of expression, different affinities for receptors and therefore unique biological activities in vivo. During the formation of teeth, BMPs dictate when initiation, morphogenesis, cytodifferentiation, and matrix secretion will occur. Without the BMP family of growth factors, the enamel knot would not be formed, and teeth would be unlikely to develop Recombinant human BMP2 stimulates differentiation of adult pulp stem cells into an odontoblastoid morphology in culture . The similar effects of TGF B1-3 and BMP7 have been demonstrated in cultured tooth slices . Recombinant BMP-2, -4, and -7 induce formation of reparative dentin in vivo . The application of recombinant human insulin-like growth factor-1 together with collagen has been found to induce complete dentin bridging and tubular dentin formation. This indicates the potential of adding growth factors before pulp capping, or incorporating them into restorative and endodontic materials to stimulate dentin and pulp regeneration. In the longer term, growth factors will likely be used in conjunction with postnatal stem cells to accomplish the tissue engineering replacement of diseased tooth pulp.BMPs , as well as other growth factors , have been successfully used for direct pulp capping. This has encouraged the addition of growth factors to stem cells to accomplish tissue engineering replacement of diseased tooth tissues. There are two strategies for the use of BMPs for dentin regenerationa) The first is in vivo therapy, where BMPs or BMP genes are directly applied to the exposed or amputated pulp. b) The second is ex vivo therapy, which consists of isolation of DPSCs, their differentiation Page 36
into odontoblasts with recombinant BMPs or BMP genes, and finally their autogenous transplantation to regenerate dentin . The role played by BMP-2 is reportedly crucial as a biological tool for dentin regeneration. Recombinant human BMP-2 stimulates the differentiation of adult pulp stem cells into odontoblast-like cells in culture, increases their alkaline phosphatase activity and accelerates expression of the dentin sialophosphoprotein (DSPP) gene in vitro , and enhances hard tissue formation in vivo . Also, autogenous transplantation of BMP-2-treated pellet culture onto amputated pulp stimulates reparative dentin formation . Similar effects have been demonstrated for BMP-7, also known as osteogenic protein-1, which promotes reparative dentinogenesis and pulp mineralization in several animal models . Recently, Lin et al. generated a BMP-7-expressing adenoviral vector that induced the expression of BMP-7 in primarily cultured human dental pulp cells. This expression led to a significant increase of alkaline phosphatase activity and induced the expression of DSPP, suggesting that BMP-7 can promote the differentiation of human pulp cells into odontoblast-like cells and promote mineralization in vitro. However, a novel role has been suggested for BMP-4, which is secreted by mesenchymal cells, in the regulation of Hertwigs epithelial root sheath (HERS) during root development by preventing elongation and maintaining cellular proliferation. Therefore it has been utilized as an agent for regulating root formation in a variety of tissue engineering applications.
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THE SOURCE, ACTIVITY AND USEFULNESS OF COMMON GROWTH FACTORS

Abbreviation Factor Primary Source Activity Usefulness
BMP
Bone morphogenetic proteins Colony stimulating factor
Bone matrix
CSF
A wide range of cells
EGF
Epidermal growth factor Fibroblast growth factor Insulin-like growth factor-I or II Interleukins IL1 to IL-13 Plateletderived growth factor
Submaxillary glands
FGF
A wide range of cells
BMP induces differentiation of osteoblasts and mineralization of bone CSFs are cytokines that stimulate the proliferation of specific pluripotent bone stem cells EGF promotes proliferation of mesenchymal, glial and epithelial cells FGF promotes proliferation of many cells
BMP is used to make stemncells synthesize and secrete mineral matrix CSF can be used to increase stem cell numbers
EGF can be used to increase stem cell numbers FGF can be used to increase stem cell numbers IGF can be used to increase stem cell numbers Promotes inflammatory cell Activity PDGF can be used to increase stem cell Numbers Induces epithelial and tissue structure Development TGF-_1 is present in dentin matrix and has been used to promote mineralization of pulp tissue Promotes neuron outgrowth and neural cell survival
IGF
I - liver IIvariety of cells IGF promotes proliferation of many cell types Leukocytes IL are cytokines which stimulate the humoral and cellular immune responses PDGF promotes proliferation of connective tissue, glial and smooth muscle cells TGF-_ may be important for normal wound healing TGF-_ is antiinflammatory, promotes wound healing, inhibits macrophage and lymphocyte proliferation NGF is critical for the survival and maintenance of sympathetic and sensory neurons.
IL
PDGF
Platelets, endothelial cells,placenta
TGF-
Transforming Macrophages, brain growth factoralpha cells,and keratinocytes Transforming Dentin matrix, growth factor-beta activated TH1 cells (T-helper) and natural killer (NK) cells Nerve growth factor A protein secreted by a neurons target tissue
TGF-
NGF
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Scaffolds A scaffold can be implanted alone or in combination with stem cells and growth factors to provide a physicochemical and biological three-dimensional microenvironment or tissue construct for cell growth and differentiation. Tissue engineering using scaffolds reproduces the proper anatomical arrangements for both undifferentiated cells and committed odontoforming or enamel-forming progenitor cells based upon maturation events. Ideal requirements of a scaffold (a) Should be porous to allow placement of cells and growth factors. (b) Should allow effective transport of nutrients, oxygen, and waste. (c) Should be biodegradable, leaving no toxic byproducts. (d) Should be replaced by regenerative tissue while retaining the shape and form of the final tissue structure. (e) Should be biocompatible. (f) Should have adequate physical and mechanical strength. Types of scaffold a) Biological/natural scaffolds These consist of natural polymers such as collagen and glycosaminoglycan, which offer good biocompatibility and bioactivity. Collagen is the major component of the extracellular matrix and provides great tensile strength to tissues. As a scaffold, collagen allows easy placement of cells and growth factors and allows replacement with natural tissues after undergoing degradation. However, it has been reported that pulp cells in collagen matrices undergo marked contraction, which might affect pulp tissue regeneration . b) Artificial scaffolds These are synthetic polymers with controlled physicochemical features such as degradation Page 39
rate, microstructure, and mechanical strength for example: Polylactic acid (PLA), polyglycolic acid (PGA), and their copolymers, poly lactic-coglycolic acid (PLGA). Synthetic hydrogels include polyethylene glycol (PEG)- based polymers. Scaffolds modified with cell surface adhesion peptides, such as arginine, glycine, and aspartic acid (RGD) toimprove cell adhesion and matrix synthesis within the threedimensional network Scaffolds containing inorganic compounds such as hydroxyapatite (HA), tricalcium phosphate (TCP) and calcium polyphosphate (CPP), which are used to enhance bone conductivity , and have proved to be very effective for tissue engineering of DPSCs Micro-cavity-filled scaffolds to enhance cell adhesion Scaffolds for tissue engineering Cumulative reports have shown that pulp cells can be isolated, multiplied in culture, and seeded onto a matrix scaffold where the cultured cells form a new tissue similar to that of the native pulp. These findings have suggested the possibility of generating pulp and dentin in pulpless canals. However, when implanting cells/scaffolds into root canals that have a blood supply only from the apical end, enhanced vascularization is needed in order to support the vitality of the implanted cells in the scaffold. This can be optimized with the addition of growth factors such as VEGF and/or platelet-derived growth factor or, further, with the addition of endothelial cells . Through the use of computer-aided design and threedimensional printing technologies, scaffolds can be fabricated into precise geometries with a wide range of bioactive surfaces. Such scaffolds have the potential to provide environments conducive to the growth of specific cell types. Page 40
Clinical applications of tissue engineering concepts A number of recent clinical case reports have suggested that many teeth that would
traditionally have undergone apexification may be treated by apexogenesis. These reports challenge the traditional approach for managing immature teeth by apexification, where there is little or even no expectation of continued root development. Instead, it is possible that alternative biologically based treatments may promote apexogenesis/maturogenesis, a term that encompasses not just the completion of root-tip formation but also the dentin of the root . Although Iwaya et al. and Banchs and Trope applied the term revascularization to describe this phenomenon, what actually occurred was physiological tissue formation and regeneration. This may be attributed to SCAP surviving the infection and contributing to this phenomenon . It is also possible that the radiographic presentation of increased dentinal wall thickness might be due to ingrowth of cementum, bone, or a dentin-like material . This diversity in cellular response is not surprising, given that DPSCs can develop odontogenic/osteogenic, chondrogenic, or adipogenic phenotypes, depending on their exposure to different cocktails of growth factors and morphogens . The key procedures of the new protocol suggested for treating non-vital immature permanent teeth are- (1) minimal or no instrumentation of the canal while relying on gentle but thorough irrigation of the canal system with sodium hypochlorite and chlorohexidine, (2) augmented disinfection by intra-canal medication with a triple-antibiotic paste (containing equal proportions of ciprofloxacin, metronidazol, and minocycline in a paste form at a concentration of 20 mg/ml) between appointments , and (3) sealing of the treated tooth with mineral trioxide aggregate (MTA) and glass ionomer/resin cement upon completion of the treatment. (4) Finally, periodical follow-ups are made to observe any continued maturation of the root. Page 41
Some investigators have induced haemorrhage in the root canal system by overinstrumentation, allowing a blood clot to form in the canal. Then MTA was placed over the blood clot. They considered that the initiation of a blood clot would provide a fibrin scaffold containing platelet-derived growth factors that would promote the regeneration of tissue within the root canal system. The induction of bleeding to facilitate healing is a common surgical procedure. It had been proposed earlier by Ostby and Myers and Fountain to guide tissue repair in the canal. However, there is a lack of histological evidence that a blood clot is required for the formation of repaired tissues in the canal space. Moreover, there have been no systematic clinical studies to indicate that application of this approach gives significantly better results than procedures that lack it. There is no current evidence-based guideline to help clinicians determine the types of cases that can be treated with this conservative approach. As mentioned above, the presence of radiolucency in the periradicular region can no longer be used as a determining factor, nor can the vitality test be used. In both situations, vital pulp tissue or an apical papilla may still be present in the canal and at the apex. Clinicians are urged to consider choosing a conservative approach first, while apexification can be performed in cases of failure. An Overview of Potential Technologies for Regenerative Endodontics The major areas of research that might have application in the development of regenerative endodontic techniques are: (a) root canal revascularization via blood clotting, (b) postnatal stem cell therapy (c) pulp implantation (d) scaffold implantation (e) injectable scaffold delivery Page 42
(f) three-dimensional cell printing (g) gene delivery. Root Canal Revascularization Via Blood Clotting These regenerative endodontic techniques are based on the basic tissue engineering principles already described and include specific consideration of cells, growth factors, and scaffolds. Root Canal Revascularization via Blood Clotting Several case reports have documented revascularization of necrotic root canal systems by disinfection followed by establishing bleeding into the canal system via overinstrumentation . An important aspect of these cases is the use of intracanal irrigants (NaOCl and chlorhexdine) with placement of antibiotics (e.g. a mixture of ciprofloxacin, metronidazole, and minocycline paste) for several weeks. This particular combination of antibiotics effectively disinfects root canal systems and increases revascularization of avulsed and necrotic teeth , suggesting that this is a critical step in revascularization. The selection of various irrigants and medicaments is worthy of additional research, because these materials may confer several important effects for regeneration in addition to their antimicrobial properties. For example, tetracycline enhances the growth of host cells on dentin, not by an antimicrobial action, but via exposure of embedded collagen fibers or growth factors. However, it is not yet know if minocycline shares this effect and whether these additional properties might contribute to successful revascularization. Although these case reports are largely from teeth with incomplete apical closures, it has been noted that reimplantation of avulsed teeth with an apical opening of approximately 1.1 mm demonstrate a greater likelihood of revascularization . This finding suggests that revascularization of necrotic pulps with fully formed (closed) apices might require instrumentation of the tooth apex to approximately 1 to 2mmin apical diameter to allow systemic bleeding into root canal systems. Clearly, the development of regenerative endodontic procedures may require re-examination of many of the closely held precepts of Page 43
traditional endodontic procedures. The revascularization method assumes that the root canal space has been disinfected and that the formation of a blood clot yields a matrix (e.g., fibrin) that traps cells capable of initiating new tissue formation. It is not clear that the regenerated tissues phenotype resembles dental pulp; however, case reports published to date do demonstrate continued root formation and the restoration of a positive response to thermal pulp testing . Another important point is that younger adult patients generally have a greater capacity for healing There are several advantages to a revascularization approach. First, this approach is technically simple and can be completed using currently available instruments and medicaments without expensive biotechnology. Second, the regeneration of tissue in root canal systems by a patients own blood cells avoids the possibility of immune rejection and pathogen transmission from replacing the pulp with a tissue engineered construct. However, several concerns need to be addressed in prospective research. First, the case reports of a blood clot having the capacity to regenerate pulp tissue are exciting, but caution is required, because the source of the regenerated tissue has not been identified. Animal studies and more clinical studies are required to investigate the potential of this technique before it can be recommended for general use in patients. Generally, tissue engineering does not rely on blood clot formation, because the concentration and composition of cells trapped in the fibrin clot is unpredictable. This is a critical limitation to a blood clot revascularization approach, because tissue engineering is founded on the delivery of effective concentrations and compositions of cells to restore function. It is very possible that variations in cell concentration and composition, particularly in older patients (where circulating stem cell concentrations may be lower) may lead to variations in treatment outcome. On the other hand, some aspects of this approach may be useful; plasma-derived fibrin clots are being used for development as scaffolds in several studies. Second, enlargement of the apical foramen is necessary to promote vascularizaton and to maintain initial cell viability via Page 44
nutrient diffusion. Related to this point, cells must have an available supply of oxygen; therefore, it is likely that cells in the coronal portion of the root canal system either would not survive or would survive under hypoxic conditions before angiogenesis. Interestingly, endothelial cells release soluble factors under hypoxic conditions that promote cell survival and angiogenesis, whereas other cell types demonstrate similar responses to low oxygen availability. Several groups recently have published preclinical research or case reports that offer a biologically based alternative to conventional endodontic treatment of these complex clinical cases. In general, these studies have evolved from the trauma literature, where the following precepts have been established: 1. Revascularization occurs most predictably in teeth with open apices . 2. Instrumentation with NaOCl irrigation is not sufficient to reliably create the conditions necessary for revascularization of the infected necrotic tooth . 3. Placement of Ca(OH)2 in root canal systems prevents revascularization coronal to the location of the Ca(OH)2 . 4. The use of the 3 mix-MP triple antibiotic paste, developed by Hoshino and colleagues and consisting of ciprofloxacin, metronidazole, and minocycline, is effective for disinfection of the infected necrotic tooth, setting the conditions for subsequent revascularization . This triple antibiotic mixture has high efficacy. In a recent preclinical study ondogs, the intracanal delivery of a 20-mg/mL solution of these 3 antibiotics via a Lentulo spiral resulted in a greater than 99% reduction in mean colony-forming unit (CFU) levels, with approximately 75% of the root canal systems having no cultivable microorganisms present . Taken together, these studies provide a strong foundation level of knowledge from the trauma literature that permits subsequent research to focus on developing clinical methods for regeneration of a functional pulp-dentin complex. Although the trauma literature has used the Page 45
term revascularization to describe this treatments outcome, the goal from an endodontic perspective is to regenerate a pulp-dentin complex that restores functional properties of this tissue, fosters continued root development for immature teeth, and prevents or resolves apical periodontitis. Thus, using the term revascularization for regenerative endodontic procedures has been questioned
Postnatal Stem Cell Therapy The simplest method to administer cells of appropriate regenerative potential is to inject postnatal stem cells into disinfected root canal systems after the apex is opened. Postnatal stem cells can be derived from multiple tissues, including skin, buccal mucosa, fat, and bone . A major research obstacle is identification of a postnatal stem cell source capable of differentiating into the diverse cell population found in adult pulp (e.g., fibroblasts, endothelial cells, odontoblasts). Technical obstacles include the development of methods for harvesting and any necessary ex vivo methods required to purify and/or expand cell numbers sufficiently for regenerative endodontic applications. One possible approach would be to use dental pulp stem cells derived from autologous (patients own) cells that have been taken from a buccal mucosal biopsy, or umbilical cord stem cells that have been cryogenically stored after birth; an allogenic purified pulp stem cell line that is disease- and pathogen-free, or xenogneic (animal) pulp stem cells that have been grown in the laboratory. It is important to note that no purified pulp stem cell lines are presently available, and that the mucosal tissues have not yet been evaluated for stem cell therapy. Although umbilical cord stem cell collection is advertised primarily to be used as part of a future medical therapy, these cells have yet to be used to engineer any tissue constructs for regenerative medical therapies. There are several advantages to an approach using postnatal stem cells. First, autogenous stem cells are relatively easy to harvest and to deliver by syringe, and the cells have the Page 46
potential to induce new pulp regeneration. Second, this approach is already used in regenerative medical applications, including bone marrow replacement, and a recent review has described several potential endodontic applications . However, there are several disadvantages to a delivery method of injecting cells. First, the cells may have low survival rates. Second, the cells might migrate to different locations within the body , possibly leading to aberrant patterns of mineralization. A solution for this latter issue may be to apply the cells together with a fibrin clot or other scaffold material. This would help to position and maintain cell localization. In general, scaffolds, cells, and bioactive signaling molecules are needed to induce stem cell differentiation into a dental tissue type . Therefore, the probability of producing new functioning pulp tissue by injecting only stem cells into the pulp chamber, without a scaffold or signaling molecules, may be very low. Instead, pulp regeneration must consider all three elements (cells, growth factors, and scaffold) to maximize potential for success. Pulp Implantation The majority of in vitro cell cultures grow as a single monolayer attached to the base of culture flasks. However, some stem cells do not survive unless they are grown on top of a layer of feeder cells . In all of these cases, the stem cells are grown in two dimensions. In theory, to take two-dimensional cell cultures and make them three-dimensional, the pulp cells can be grown on biodegradable membrane filters. Many filters will be required to be rolled together to form a three dimensional pulp tissue, which can be implanted into disinfected root canal systems. The advantages of this delivery system are that the cells are relatively easy to grow on filters in the laboratory. The growth of cells on filters has been accomplished for several decades, as this is how the cytotoxicity of many test materials is evaluated . Moreover, aggregated sheets of cells are more stable than dissociated cells administered by injection into empty root canal systems. The potential problems associated with the Page 47
implantation of sheets of cultured pulp tissue is that specialized procedures may be required to ensure that the cells properly adhere to root canal walls. Sheets of cells lack vascularity, so only the apical portion of the canal systems would receive these cellular constructs, with coronal canal systems filled with scaffolds capable of supporting cellular proliferation . Because the filters are very thin layers of cells, they are extremely fragile, and this could make them difficult to place in root canal systems without breakage. In pulp implantation, replacement pulp tissue is transplanted into cleaned and shaped root canal systems. The source of pulp tissue may be a purified pulp stem cell line that is disease or pathogen-free, or is created from cells taken from a biopsy, that has been grown in the laboratory. The cultured pulp tissue is grown in sheets in vitro on biodegradable polymer nanofibers or on sheets of extracellular matrix proteins such as collagen I or fibronectin . So far, growing dental pulp cells on collagens I and III has not proved to be successful , but other matrices, including vitronectin and laminin, require investigation. The advantage of having the cells aggregated together is that it localizes the postnatal stem cells in the root canal system. The disadvantage of this technique is that implantation of sheets of cells may be technically difficult. The sheets are very thin and fragile, so research is needed to develop reliable implantation techniques. The sheets of cells also lack vascularity, so they would be implanted into the apical portion of the root canal system with a requirement for coronal delivery of a scaffold capable of supporting cellular proliferation. Cells located more than 200 m from the maximum oxygen diffusion distance from a capillary blood supply are at risk of anoxia and necrosis . The development of this endodontic tissue engineering therapy appears to present low health hazards to patients, although concerns over immune responses and the possible failure to form functioning pulp tissue must be addressed through careful in vivo research and controlled clinical trials.
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Scaffold Implantation To create a more practical endodontic tissue engineering therapy, pulp stem cells must be organized into a three-dimensional structure that can support cell organization and vascularization. This can be accomplished using a porous polymer scaffold seeded with pulp stem cells . A scaffold should contain growth factors to aid stem cell proliferation and differentiation, leading to improved and faster tissue development . Growth factors were described in the previous section. The scaffold may also contain nutrients promoting cell survival and growth , and possibly antibiotics to prevent any bacterial in-growth in the canal systems. The engineering of nanoscaffolds may be useful in the delivery of pharmaceutical drugs to specific tissues . In addition, the scaffold may exert essential mechanical and biological functions needed by replacement tissue . In pulp-exposed teeth, dentin chips have been found to stimulate reparative dentin bridge formation. Dentin chips may provide a matrix for pulp stem cell attachment and also be a reservoir of growth factors . The natural reparative activity of pulp stem cells in response to dentin chips provides some support for the use of scaffolds to regenerate the pulp-dentin complex. To achieve the goal of pulp tissue reconstruction, scaffolds must meet some specific requirements: Biodegradability is essential, since scaffolds need to be absorbed by the surrounding tissues without the necessity of surgical removal . A high porosity and an adequate pore size are necessary to facilitate cell seeding and diffusion throughout the whole structure of both cells and nutrients . The rate at which degradation occurs has to coincide as much as possible with the rate of tissue formation.This means that while cells are fabricating their own natural matrix structure around themselves, the scaffold is able to provide structural integrity within the body, and it will eventually break down, leaving the newly formed tissue that will take over the Page 49
mechanical load. Most of the scaffold materials used in tissue engineering have had a long history of use in medicine as bioresorbable sutures and as meshes used in wound dressings . The types of scaffold materials available are natural or synthetic, biodegradable or permanent. The synthetic materials include polylactic acid (PLA), polyglycolic acid (PGA), and polycaprolactone (PCL), which are all common polyester materials that degrade within the human body. These scaffolds have all been successfully used for tissue engineering applications because they are degradable fibrous structures with the capability to support the growth of various different stem cell types. The principal drawbacks are related to the difficulties of obtaining high porosity and regular pore size. This has led researchers to concentrate efforts to engineer scaffolds at the nanostructural level to modify cellular interactions with the scaffold . Scaffolds may also be constructed from natural materials, in particular, different derivatives of the extracellular matrix have been studied to evaluate their ability to support cell growth . Several proteic materials, such as collagen or fibrin, and polysaccharidic materials, like chitosan or glycosaminoglycans (GAGs), have not been well studied. However, early results are promising in terms of supporting cell survival and function , although some immune reactions to these types of materials may threaten their future use as part of regenerative medicine.
Injectable Scaffold Delivery Rigid tissue engineered scaffold structures provide excellent support for cells used in bone and other body areas where the engineered tissue is required to provide physical support . However, in root canal systems a tissue engineered pulp is not required to provide structural support of the tooth. This will allow tissue engineered pulp tissue to be administered in a soft three-dimensional scaffold matrix, such as a polymer hydrogel. Hydrogels are injectable Page 50
scaffolds that can be delivered by syringe. Hydrogels have the potential to be noninvasive and easy to deliver into root canal systems. In theory, the hydrogel may promote pulp regeneration by providing a substrate for cell proliferation and differentiation into an organized tissue structure. Past problems with hydrogels included limited control over tissue formation and development, but advances in formulation have dramatically improved their ability to support cell survival. Despite these advances, hydrogels at are at an early stage of research, and this type of delivery system, although promising, has yet to be proven to be functional in vivo. To make hydrogels more practical, research is focusing on making them photopolymerizable to form rigid structures once they are implanted into the tissue site . Three-Dimensional Cell Printing The final approach for creating replacement pulp tissue may be to create it using a threedimensional cell printing technique . In theory, an ink-jet-like device is used to dispense layers of cells suspended in a hydrogel to recreate the structure of the tooth pulp tissue. The three-dimensional cell printing technique can be used to precisely position cells , and this method has the potential to create tissue constructs that mimic the natural tooth pulp tissue structure. The ideal positioning of cells in a tissue engineering construct would include placing odontoblastoid cells around the periphery to maintain and repair dentin, with fibroblasts in the pulp core supporting a network of vascular and nerve cells. Theoretically, the disadvantage of using the three-dimensional cell printing technique is that careful orientation of the pulp tissue construct according to its apical and coronal asymmetry would be required during placement into cleaned and shaped root canal systems. However, early research has yet to show that three-dimensional cell printing can create functional tissue in vivo. Gene Therapy The year 2003 marked a major milestone in the realm of genetics and molecular biology. Page 51
That year marked the 50th anniversary of the discovery of the double-helical structure of DNA by Watson and Crick. On April 14, 2003, 20 sequencing centers in five different countries declared the human genome project complete. This milestone will make possible new medical treatments involving gene therapy. All human cells contain a 1-m strand of DNA containing 3 billion base pairs, with the sole exception of nonnucleated cells, such as red blood cells. The DNA contains genetic sequences (genes) that control cell activity and function; one of the most well known genes is p53 . New techniques involving viral or nonviral vectors can deliver genes for growth factors, morphogens, transcription factors, and extracellular matrix molecules into target cell populations, such as the salivary gland. Viral vectors are modified to avoid the possibility of causing disease, but still retain the capacity for infection. Several viruses have been genetically modified to deliver genes, including retroviruses, adenovirus, adenoassociated virus, herpes simplex virus, and lentivirus . Nonviral gene delivery systems include plasmids, peptides, gene guns, DNA-ligand complexes, electroporation, sonoporation, and cationic liposomes . The choice of gene delivery system depends on the accessibility and physiological characteristics of the target cell population. One use of gene delivery in endodontics would be to deliver mineralizing genes into pulp tissue to promote tissue mineralization. However, a literature search indicates there has been little or no research in this field, except for the work of Rutherford . He transfected ferret pulps with cDNA-transfected mouse BMP-7 that failed to produce a reparative response, suggesting that further research is needed to optimize the potential of pulp gene therapy. Moreover, potentially serious health hazards exist with the use of gene therapy; these arise from the use of the vector (gene transfer) system, rather than the genes expressed . The FDA did approve research into gene therapy involving terminally ill humans, but approval was withdrawn in 2003 after a 9-year-old boy receiving gene therapy was found to have developed tumors in different parts of his body . Researchers must learn how to Page 52
accurately control gene therapy and make it very cell specific to develop a gene therapy that is safe to be used clinically. Because of the apparent high risk of health hazards, the development of a gene therapy to accomplish endodontic treatment seems very unlikely in the near future. Gene therapy is a relatively new field, and evidence is lacking to demonstrate that this therapy has the potential to rescue necrotic pulp. At this time, the potential benefits and disadvantages are largely theoretical.
Gene therapy by BMPs - In vivo Half life of BMPs as recombinant proteins is limiting. Therefore gene therapy is a potential alternative to conquer disadvantages of protein therapy. Recombinant adenovirus containing BMP 7 gene induced only a small amount of poorly organized dentin after direct transduction in experimentally inflamed pulp. In vivo gene therapy does not have much effect on reparative dentin formation in case of severe inflammation - Ex-vivo The transplantation of cultured dermal fibroblasts transduced with BMP-7 using a recombinant adenovirus, induced reparative dentin formation in the exposed pulp with reversible pulpitis. The great potency of BMP genes to provoke differentiation of pulp stem cells, even if in reversible pulpitis, demonstrates the utility of ex vivo gene therapy in reparative / regenerative dentin formation for clinical endodontic treatment.
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Tooth regeneration by single cell manipulation Reconstitution of bioengineered tooth germ, like that for other organs, requires that single cells are completely dissociated from both the epithelium and the mesenchyme, and the correct placement of cells is a major issue. The question of how to reconstitute organs from completely dissociated single cells has focused attention upon basic techniques, such as those used in other fields for the ex vivo generation of organs using cell engineering methods, as well as for organ replacement therapy. Bioengineering techniques have been awaited that facilitate high frequencies of reconstitution, such that effective replacement with functional cells occurs in a manner that generally mimics embryonic development. Currently, two approaches are being investigated for reconstructing teeth using cell culture procedures. One approach is to seed tooth germ cells into tooth-shaped scaffolds made of bio degradable materials. The other approach is to reconstitute teeth by reaggregating tooth germ from dissociated single epithelial cells and mesenchymal cells. Bioengineered tooth germ by tissue engineering method using scaffolds One of the three-dimensional tissue engineering methods is scaffolding, in which a complex cell mixture and a prefabricated biodegradable scaffold are employed to grow a tissue of a desired form. This has been used for clinical repairs of various complex, reconstituted tissues, such as cartilage, and bone. Feasibility studies for the production of bioengineered tooth tissues using a cellscaffold composite have been made . Yelicks group examined explants containing dissociated cells isolated from porcine unerupted third molars or rat molar tooth buds. These contained both epithelial cells and mesenchymal cells that were plated onto a tooth-shaped scaffold made of polyglycolate (PGA) and poly- L -lactate-co-glycolate (PLGA). The explants were capable of generating a tooth crown containing both dentin and enamel. This suggests that a tooth-tissue engineering method, with a prefabricated biodegradable scaffold, could be used to produce a bioengineered tooth using tooth tissuePage 55
derived cells. Honda et al. reported the effectiveness of using a collagen sponge as a scaffold, and sequentially seeding epithelial cells and mesenchymal cells. However, it remains to be seen whether tooth formation will be sufficiently efficient and whether the structure of the regenerated teeth will be satisfactory using this method. Bioengineered tooth germ by cell aggregation The other approach for the reconstitution of a bioengineered tooth germ is the cell reaggregation method. The first step in multi-cellular aggregation of epithelial cells and mesenchymal cells is multi-cellular assembly by self-reorganization of each cell type. This occurs through cell migration and selective cell adhesion until cells reach an equilibrium arrangement. Next, reciprocal interactions among epithelial cell layers and mesenchymal cell layers initiate organogenesis that regulates differentiation and morphogenesis. The potentials of cells to self-reorganize and to induce organogenesis are thought to differ greatly among the various cell types of different organs. To create a bioengineered tooth germ, dental epithelium is reassociated with a cell pellet of dissociated mesenchymal cells; the resulting artificial germ is then used to grow a tooth with a proper structure bytransplantation in vivo. A bioengineered tooth germ reconstituted from pellets of dissociated dental epithelium cells and mesenchymal cells, isolated from ED14.5 mouse tooth germ, successfully yielded a complete tooth surrounded by periodontal-like tissue. It has also been reported that a cell aggregate of mixed epithelial and mesenchymal cells, isolated from ED13.5 mouse-derived molar tooth germ, was capable of generating a complete tooth without cell compartmentalization at high cell density. Interestingly, auto-cell sorting and inductive potentials were found in molar-derived epithelial cells. This agrees with Steinbergs theory concerning early organogenesis. In contrast, incisor epithelial cells did not exhibit inductive potential after autologous cell sorting. This suggests that auto-cell sorting and inductive potentials to organize into organ germs differ among the types of organ germs. Page 56
Although these reports indicated that the cell aggregation method is useful for creating bio engineered tooth germ for dental regenerative therapy, a bioengineering method that can replicate the organogenesis of the embryo and that is adaptable to a wide variety of organ germ types is desired. Comparison of cell processing methods During tooth development, the properties of cells vary, depending upon the developmental stage. Different cell manipulation techniques are thus required according to the target stage of the tooth germ. In the early developmental stages of the tooth germ, in which tissues have not yet mineralized, it is essential that reciprocal interactions between epithelial cells and mesenchymal cells regulate the differentiation of odonto-forming or enamel-forming progenitor cells through direct cell-to-cell interactions and cytokine production, and that they promote spontaneous organ development in which cells are properly arranged so as to function normally. In later stages of development such as the late bell stage, in which tissues have mineralized, tooth-specific patterning of the epithelialmesenchymal junction subsequently forms a dentinenamel junction. Ameloblasts and odontoblasts, which are differentiated from epithelial cells and mesenchymal cells, respectively, each have a cell polarity of cell-to-mineralized tissue, and a gradient during the differentiation stage of each cell lineage from the tip of the cusp to the apical part. The cell aggregation method and the tissue engineering method using scaffolds may be useful for regeneration of tooth germ in the early developmental stages (induction phase) and in the late developmental stages (differentiation phase), respectively. Using the cell reaggregation method, epithelial cells and mesenchymal cells isolated from tooth germ at an early developmental stage but not at a late developmental stage, could be induced to reproduce in such a manner so as to direct cell-tocell interactions between each of the cells. The reaggregated cells may self-renew or proliferate, and promote spatial sorting and self-organization resulting in proper cell Page 57
arrangements. The ideal placement of cells may be realized by the scaffolding technique, resulting in full polarization and proper cell morphology. Using scaffolds and seeding the proper anatomical arrangement of both immature cells and committed progenitor cells for each cell lineage maintains both spontaneous cell polarization toward the enamel-dentin junction and the gradient for each cell lineage. Furthermore, using the scaffolding technique, tooth germ in more advanced developmental stages than the induction stage can be reconstituted, and the total time required to develop into a functional tooth from reconstituted tooth germ engrafted into the oral cavity may be shortened. Development of a novel bioengineering method Recently, we developed a bioengineering method for forming a three-dimensional organ germ in the early developmental stages, termed the bioengineered organ germ method [53] . To precisely replicate tooth organogenesis in the early developmental stages, a cell aggregation method using cap-stage tooth-germ-derived epithelial cells and mesenchymal cells was chosen. This method has the distinctive feature that the bioengineered tooth germ is reconstituted between epithelial cells and mesenchymal cells using cell compartmentalization at a high cell density in a collagen gel solution. Both incisor and molar bioengineered tooth germs successfully developed into teeth with the correct tooth structures with a high rate of success in vitro and in vivo; this tooth germ model reproduces the interactions between epithelial cells and mesenchymal cells in early tooth development. Direct cell-to-cell interactions induced by high cell density and cell compartmentalization are essential in tooth organogenesis, and possibly for organogenesis of other organs. Indeed, this method could be applicable to the reconstitution of whisker follicles [53] . Thus, cell compartmentalization, which mimics multicellular assembly and the equilibrium configuration between epithelial cells and mesenchymal cells, is effective for initiating organogenesis in a bio-engineered organ primordium. Page 58
Development of a bio-engineered tooth in the adult oral environment The adult oral environment differs significantly from the embryonic environment in which induction and early development of tooth germ normally occur. It has been reported that murine embryonic tooth primordium can develop in a toothless oral region (diastema) of an adult mouse. This suggests the potential for a dissociated tooth primordium to develop in an adult oral environment. It is also important to determine if a bio-engineered tooth germ can develop in an adult oral environment into a fully functioning tooth that has both a correct tooth structure and a complex network of blood vessels and nerve fibers. Recently, our group reported that a bioengineered tooth primordium, which was isolated from bio-engineered tooth germ, could develop in a tooth cavity formed by the extraction of a mandibular incisor into a tooth with correct tooth structure comprising enamel, dentin, root, dental pulp, periodontal ligament, blood vessels and alveolar bone. This strongly suggested that the replacement of biologically functional teeth is possible by reconstitution in the tooth cavity in cooperation with the surrounding tissues of an adult. Studies of the long-term efficacy of bioengineered tooth germ transplantation should now be undertaken. Identification of cell sources for tooth regeneration Recent studies have demonstrated the potential for adult-tissue stem cells to differentiate into cells arising from all three germ layers, and that stem cells may be candidate sources for tissue engineering, including tooth regeneration. Stem cells that can differentiate into dental cell lineages will be useful for transplantation therapy in tooth decay, bone remodeling and periodontal diseases. The practical application of dental regenerative medicine may be optimized using the patients own cells but not embryo-derived cells. Candidate stem/progenitor cells isolated from various dental tissues have been reported. Human dental pulp tissues contain stem cells called dental pulp stem cells (DPSC). Stem cells from human exfoliated deciduous teeth (SHED) and side-population cells isolated from porcine Page 59
dental pulp cells have also been reported. These stem cells differentiate into odontoblasts and secrete dentin in vivo. One of the first reports showed that periodontal ligament Stem cells (PDLSC), isolated from human periodontal tissue, could regenerate the cementum and periodontal ligament. Recently, a unique approach for tooth root regeneration used the combination of PDLSC and human stem cells from the apical papilla (SCAP). Using a minipig model, a root-shaped hydroxyapatite/tricalcium phosphate (HA/TCP) carrier, which was loaded with SCAP that covered gelfoam/PDLSC, formed a root-like structure to which a porcelain crown was attached, resulting in normal tooth function. Our group reported that tooth germ progenitor cells (TGPCs), which were isolated from human unerupted third molars and single cell-derived clones, were multi-potent and differentiated into cells of the three germ layers, including bone, neural cells and hepatocytes. The dental stem/progenitor cells may also contribute to advances in a wide variety of dental regenerative therapies. Investigations of dental differentiation potentials for various non-dental tissue-derived cells should also offer opportunities to advance tooth regeneration for clinical applications. Cultured, non-dental tissue-derived cells, such as embryonic stem cells, neural stem cells or bone marrow stromal cells, have indicators for early odontogenesis, as measured by gene expression during initiation of odontogenesis in an animal model. Also, a reported cell aggregation method showed that bone marrow cells differentiated into ameloblast-like cells and odontoblast-like cells. Tomookas group, using an organ-germ method, demonstrated that an oral mucosaderived epithelial cell line differentiated into ameloblasts and formed a regenerated tooth in combination with toothgerm- derived mesenchyme. Although these reports have not yet shown that regeneration of a whole tooth is imminently achievable, these findings strongly suggest the presence of cell sources in humans that may be of use for dental regenerative therapy.
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Regulation of tooth size and morphology Regulations of tooth sizes and shapes for the crown and the root are important matters to consider in generating an entirely bio-engineered tooth. Teeth have unique morphological features for incisors, canines, premolars and molars. These features are programmed at predetermined sites in the oral cavity during natural tooth development, and the resulting morphology is controlled so that functional occlusion, or bite, is maintained that involves the teeth, muscles and the temporo-mandibular joints. Tooth morphology is also important for dental esthetics to achieve the beautiful smile, one purpose of dental treatment. To date, many researchers have conducted numerous trials to clarify the molecular mechanisms involved in the regulation of tooth morphology. Spatial distributions of gene expressions provide coordinated signals for positional and morphological control, such as for crowns and roots. The dental mesenchyme controls crown morphologies and epithelial histogenesis, including enamel knots, and regulates the patterning of the cusps and the shapes of tooth crowns. In experiments on reassociations between dental epithelial and mesenchymal cells, the patterning, size, number and cusps of teeth were analyzed. In order to develop a system to control tooth size and morphology of bio-engineered teeth, it is necessary to identify the key molecules regulating tooth patterning and morphogenesis. In the near future, this will be achieved by identifying regulatory systems of gene expression in combination with cell processing methods. Delivery of Regenerative Endodontic procedures Ideally, the delivery of regenerative endodontic procedures must be more clinically effective than current treatments. The method of delivery must also be efficient, cost-effective, and free of health hazards or side-effects to patients. A promising cellular source for regenerative endodontic procedures is autogenous; stem cells from oral mucosa. The oral mucosa cells are readily accessible as a source of oral cells, which avoids the problem of patients being Page 61
required to store umbilical cord blood or third molars immediately after extraction. It also avoids the needs for bone biopsies. The oral mucosa cells may be maintained using in vitro cell culture with antibiotics to remove infection. The cells may then be seeded in the apical 1 to 3 mm of a tissue engineering scaffold with the remaining coronal 15+ mm containing an acellular scaffold that supports cell growth and vascularization. This tissue construct may involve an injectable slurry of [hydrogel + cells + X (growth factors, etc] or [hydrogel + X (growth factors, etc)], then this two layer method would be fairly easy to accomplish. Moreover, by seeding cells only in the apical region, there is reduced demand for large numbers of cells derived from the host. Instead, most of the cellular proliferation would occur naturally in the patient. This would reduce the need to grow large quantities of cells in the laboratory. Both these delivery methods reduce the need for an autogenous pulp stem cell population that will not; be readily available to endodontists, because the teeth requiring treatment are presumably infected and necrotic. This proposed delivery method would help avoid the potential for immune and infection issues surrounding the use of an allogenic pulp stem cell line. Of course these alternative methods must be investigated using preclinical In vitro studies, usage studies in animals, and, eventually, clinical trials.
schematic illustration of proposed stepwise insertion of engineered pulp tissue in the clinical setting.
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Barriers to be addressed to permit introduction of regenerative endodontics: Disinfection & shaping of root canals in a fashion to permit regenerative endodontics Chemomechanical debridement cleaning and shaping root canals Irrigants 6% sodium hypochlorite and 2% chlorhexidine gluconate and alternatives Medicaments Ca(OH)2, triple antibiotics, MTAD and alternatives
Improved Methods to Disinfect and Shape Root Canal Systems The simplest approach to pulp tissue regeneration would be to regrow pulp over remaining pulp tissue. However, attempts to regenerate pulp tissue under conditions of inflammation or partial necrosis have proved unsuccessful , and it is generally recognized that the long-term prognosis of direct pulp capping infected tissue is poor and not recommended . In the presence of infection, the pulp stem cells that survive appear to be incapable of mineralization and deposition of a tertiary dentin bridge. Therefore, the majority of the available evidence suggests that necrotic and infected tooth pulp does not heal. Therefore, in the foreseeable future, it will be necessary to disinfect the root canal systems and remove infected hard and soft tissues before using regenerative endodontic treatments. The literature contains no or few reports of pulp stem cell attachment and adherence to root canal dentin. One of us (P.M.) has completed several unpublished investigations of the interactions between periodontal stem cells and the dentin surface. Our initial unpublished results show that relatively few periodontal stem cells will naturally attach and grow on cleaned and shaped root canal systems, and even fewer cells attach to a dentin smear layer. To successfully attach and adhere to root canal dentin, the stem cells must be supported within a Page 63
polymer or hydrogel scaffold. Furthermore, it has been observed that pulp stem cells, periodontal stem cells, and fibroblasts do not adhere and grow in infected root canal systems; the presence of infection renders the treatment unsuccessful. This indicates that for regenerative endodontics to be successful, the disinfection of necrotic root canal systems must be accomplished in a fashion that does not impede the healing and integration of tissue engineered pulp with the root canal walls. Moreover, the inclusion of a small local amount of antibiotics may need to be considered in developing these biodegradable scaffolds. Substantial numbers of bacterial species have been identified as inhabitants of the oral cavity. However, because of bacterial interactions, nutrient availability and low oxygen potentials in root canal systems, the numbers of bacterial species present in endodontic infections are restricted. These selective conditions lead to the predominance of facultative and strictly anaerobic microorganisms that survive and multiply, causing infections that stimulate local bone resorption. Disinfection is one of the main objectives of root canal preparation. Thorough disinfection removes microorganisms, permits better adaptation of filling materials, and enhances the action of the intracanal medicaments. The choice of an irrigant is of great importance, because the irrigant acts as a lubricant during instrumentation, flushes debris and microorganisms out of the canal, and reacts with pulp, necrotic tissues, and microorganisms and their subproducts. Sodium hypochlorite has been extensively used for several decades for this purpose . Its excellent properties of tissue dissolution and antimicrobial activity make it the irrigant of choice for the treatment of teeth with pulp necrosis, even though it has several undesirable characteristics, such as tissue toxicity at high concentrations and so forth . Moreover, sodium hypochlorite does not totally clean the surfaces of the root canal systems. Chlorhexidine gluconate has been studied for its various properties, antimicrobial activity, and biocompatibility, with the objective of evaluating it as an alternative to sodium hypochlorite. Disinfection of bacteria is clinically important, Page 64
particularly Enterococcus faecalis, because it has been isolated from infected root canal systems and appears more frequently in cases of revisional endodontic treatment . Regenerative endodontics would benefit from a new generation of irrigants that are as effective as current irrigants, but are nonhazardous to patient tissues. This is an important area of research, because development of the ideal disinfectant, irrigant,and chelating agent would benefit patients and the profession.
Smear Layer Removal The presence of a smear layer on root canal walls may inhibit the adherence of implanted pulp stem cells, potentially causing the regenerative endodontic treatment to fail. Improved methods to remove the smear layer from the root canal walls appear to be necessary to help promote the success of regenerative endodontics. The smear layer is a1 to 5 m-thick layer of denatured cutting debris produced on instrumented cavity surfaces, and is composed of dentin, odontoblastic processes, nonspecific inorganic contaminants, and microorganisms . The removal of the smear layer from the instrumented root canal walls is becoming less controversial in clinical practice . Its removal provides better sealing of the endodontic filling material to dentin, and avoids the leakage of microorganisms into oral tissues. Chemical chelating agents are used to remove the smear layer from root canal walls, most commonly a 17% solution of ethylenediaminetetraacetic acid (EDTA) that is applied as a final flush. Several other solutions have been investigated for removing smear layers, including doxycycline, a tetracycline congener , citric acid , and, most recently, MTAD . MTAD is an aqueous solution of 3% doxycycline, 4.25% citric acid, and 0.5% polysorbate 80 detergent. This biocompatible intracanal irrigant is commercially available as a two-part set that is mixed on demand (BioPure MTAD, DentsplyTulsa, Tulsa, OK). In this product, doxycycline hyclate is used instead of its free base, doxycycline monohydrate, to increase the water Page 65
solubility of this broad spectrum antibiotic. MTAD has been reported to be effective in removing endodontic smear layers, eliminating microbes that are resistant to conventional endodontic irrigants and dressings , and providing sustained antimicrobial activity through the affinity of doxycycline to bind to dental hard tissues . However, its interaction with regenerating pulpal tissue is unknown.
Engineering a Functional Pulp Tissue The success of regenerative endodontic therapy is dependent on the ability of researchers to create a technique that will allow clinicians to create a functional pulp tissue within cleaned and shaped root canal systems. The source of pulp tissue may be from root canal revascularization, which involves enlarging the tooth apex to approximately 1 to 2 mm to allow bleeding into root canals and generation of vital tissue that appears capable of forming hard tissue under certain conditions; stem cell therapy, involving the delivery of autologous or allogenic stem cells into root canals; or pulp implantation, involving the surgical implantation of synthetic pulp tissue grown in the laboratory. Each of these techniques to regenerate pulp tissue will have advantages and limitations that still have to be defined through basic science and clinical research.
Creation of replacement pulp-dentin tissues Pulp revascularization by apex instrumentation Stem cells; allogenic, autologous, xenogenic, umbilical cord sources Growth factors; BMP-2, -4, -7; TGF-1, 2, 3 among others Gene therapy; identification of mineralizing genes Tissue engineering; cell culture, scaffolds, hydrogels Page 66
Delivery of replacement pulp-dentin tissues Surgical implantation methods Injection site
Dental restorative materials Improve the quality of sealing between restorative materials and dentin Ensure long-term sealing to prevent recurrent pulpitis
Measuring appropriate clinical outcomes Vascular blood flow Mineralizing odontoblastoid cells Intact afferent innervations Lack of signs or symptoms
Measuring Appropriate Clinical Outcomes Once a tissue engineered pulp has been implanted, it is not ethical to remove functioning tissues to conduct a histological analysis. Therefore, it will not be possible to histologically investigate mineralizing odontoblastoid cell functioning or nerve innervation. Clinicians will have to rely on the noninvasive tests in use today, such as laser Doppler blood flowmetry in teeth, pulp testing involving heat, cold, and electricity, and lack of signs or symptoms. Magnetic resonance imaging (MRI) has shown the potential to distinguish between vital and nonvital tooth pulps, but MRI machines are very expensive and must be greatly reduced in price to become widespread. The ideal clinical outcome is a nonsymptomatic tooth that never needs retreatment, but nonsubjective vitality assessment methods are essential to validate that Page 67
regenerative endodontic techniques are truly effective Regenerative Endodontic Procedures.
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CHALLENGES AND FUTURE DIRECTION
The pulp tissue repair/regeneration recapitulates tooth development. Despite the impressive progress in tissue engineering approaches to regenerative pulp therapy, numerous challenges remain. The associated broad spectrum of responses in pulp includes neural and vascular regeneration. Nerve regeneration Dental pulp is richly innervated. The main nerve supply enters the pulp through the apical foramen along with the vascular elements. Nerves proceed to the coronal area and form a plexus in proximity to the odontoblasts and finally enter the dentinal tubules. They include both sensory and sympathetic nerves. Their functions, locations and interactions with pulp, dentin, vasculature and immune cells are different. In general the A type fibers are myelinated and C fibers are non myelinated. The cytochemical localization of neuropeptides, calcitonin-gene-related peptide (CGRP), nerve growth factor (NGF), glial cell-derived neurotrophic factor (GDNF), and neurofilaments vary with the type of nerve fiber. The temporal sequence of dental pulp innervation is dependent on gradients of neurotrophic growth factors emanating from the pulp cells, NGF, brain-derived neurotrophic factor (BDNF) and GDNF are expressed in dental pulp. Pulpal nerves play a key role in regulation of blood flow, dentinal fluid flow, and pressure. In addition there is evidence for neural regulation of pulpal fibroblasts, inflammation and immunity. The innervation of the pulp has a critical role in the homeostasis of the dental pulp. Invasion of immune and inflammatory cells into sites of injury in the pulp is stimulated by sensory nerves. Sensory denervation results in rapid necrosis of the exposed pulp because of impaired blood flow, extravasation of immune cells. Reinnervation leads to recovery in the coronal dentin. Schwann cells appear to release neurotrophic growth factors and play a role in recruitment of sensory and sympathetic nerves during reinnervation. Thus, the pulpal nerve
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fibers contribute to angiogenesis, extravasation of immune cells and regulate inflammation to minimize initial damage, maintain pulp tissue, and strengthen pulpal defense mechanisms. BMPs have a role in reparative/regenerative dentin formation. It is noteworthy that members of the BMP family have pronounced effects on neurogenesis. Thus, it is likely BMPs can be used for regenerative pulpal therapy and dentinogenesis may have concurrent beneficial effects on nerve regeneration. The increasing interest in tissue engineering of tooth must take in to account neuropulpal interactions and nerve regeneration. The challenges include, but not limited to nociceptive mechanisms, altered thresholds to pain in inflamed teeth and dental pain. Thus, the life of teeth can be possibly prolonged by preservation of pulp and odontoblasts and promoting repair and regeneration by the study of neuropulpal interactions. The recent progress in dental stem/progenitor cells and mechanism of dental pulp cells, assure advances in regeneration of nerves based on neuropulpal interactions.
Vascular regeneration The vascular system in the dental pulp plays a role in nutrition and oxygen supply and as a conduit for removal of metabolite waste. The cellular elements of the blood vessels such as endothelial cells, pericytes and associated cells contribute to pulpal homeostasis along with the nerves. Thus, the vascular contribution to regeneration of dentin-pulp complex is immense. Arterioles enter the pulp chamber through the apical foramen along with the nerve supply. The branching arterioles form a capillary plexus under the odontoblast layer. During development and regeneration there is increased vascular activity and blood flow. There is a common pathway of vascular reaction to varied stimuli such as chemical, physical including mechanical and thermal. This reaction includes a local inflammation and attendant dilation of blood vessels and increased blood flow. Extravasation of leukocytes and increased vascular Page 70
permeability is a hallmark of early vascular response. Pulp vasculature plays an important role in regulating inflammation and subsequent repair and regeneration of dentin. There is an intimate association of the neural elements with vascular supply of the dental pulp, suggesting the interplay of neural and vascular elements and involvement in pulp homeostasis. The critical importance of vasculature in tissue repair and regeneration is well known. Vascular endothelial growth factor (VEGF) is an excellent regulator of angiogenesis and is known to increase vascular permeability. VGEF induced chemotaxis, proliferation and differentiation of human dental pulp cells. In addition human dentin matrix contains VGEF. The presence of VGEF in dentin and response of dental pulp cells to VGEF raises the possibility of the presence of endothelial progenitor cells in dental pulp alongside progenitors for odontoblasts and neuronal cells. In view of the role of endothelial progenitor cells in vascularization during tissue regeneration, it is likely VGEF and vascular endothelial cells are critical for dentin regeneration. The utility of gene therapy in stimulation of vascular growth permits local stimulation of vascularization during regeneration. In fact gene therapy using members of BMP family including BMP7 and GDF11 successfully induced dentinpulp regeneration. Thus, the recent advances in vascular biology and VEGF and techniques of gene therapy will be of potential clinical utility in dentistry especially in endodontics.
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CONCLUSION The counterargument to the development of regenerative endodontic procedures is that the pulp in a fully developed tooth plays no major role in form, function, or esthetics, and thus its replacement by a filling material in conventional root canal therapy is the most practical treatment. However, in terms of esthetics, there is a potential risk that endodontic filling materials and sealers may discolor the tooth crown . In addition, an in vitro study of endodontically treated human teeth found the long-term intracanal placement of calcium hydroxide may reduce the fracture resistance of root dentin. A retrospective study of tooth survival times following root canal filling versus tooth restoration found that although root canal therapy prolonged tooth survival, the removal of pulp in a compromised tooth may still lead to tooth loss in comparison with teeth with normal tissues . On the other hand, although the replacement pulp has the potential to revitalize teeth, it may also become susceptible to further pulp disease and may require retreatment. The implantation of engineered tissues also requires enhanced microbiological control methods required for adequate tissue regeneration. Thus, additional research in regenerative therapies must be conducted to establish reliable and safe methods for all teeth requiring root canal treatment. 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.
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Tissue engineering is the science of design and manufacture of new tissues to replace impaired or damaged ones. The key ingredients for tissue engineering are stem cells, the morphogenes or growth factors that regulate their differentiation and a scaffold of etracellular matrix that constitutes the microenvironment for their growth. Recently there has been increasing interest in applying the concept of tissue engineering to endodontics. The aim of this study was to review the body of knowledge related to dental pulp stem cells, the most common growth factors and the scaffold used to control their differentiation, and a clinical technique for management of immature non vital teeth based on this novel concept.1 The aim of this review is to introduce the background of the recently described stem cells, their isolation, and characterization. The potential role of these stem cells in the contribution of the continued root maturation in endodontically treated immature teeth with periradicular periodontitis or abscess and in autotransplanted teeth are discussed. The possibility of using SCAP and other types of pulp stem cells for pulp/dentin regeneration and the combination of SCAP and periodontal ligament stem cells (PDLSCs) for bioroot engineering shown by Sonoyama et al. in a swine model as a potential future clinical approach to replace dental implants has been thoroughly described and analyzed.2 The purpose of this review was to summarize the findings and illustrate a path forward for the development and evaluation of regenerative endodontic therapies. During the last 1015 years, there has been a tremendous increase in our clinical tools (ie, materials, instruments, and medications) and knowledge from the trauma and tissue engineering fields that can be applied to regeneration of a functional pulpdentin complex. In addition, recent case reports indicate that biologically based endodontic therapies can result in continued root development, increased dentinal Page 73
wall thickness, and apical closure when treating cases of necrotic immature permanent teeth.3 This review provides an overview of regenerative endodontics and its goals, and describes possible techniques that will allow regenerative endodontics to become a reality. These potential approaches include root-canal revascularization, postnatal (adult) stem cell therapy, pulp implant, scaffold implant, three-dimensional cell printing, injectable scaffolds, and gene therapy. These regenerative endodontic techniques will possibly involve some combination of disinfection or debridement of infected root canal systems with apical enlargement to permit revascularization and use of adult stem cells, scaffolds, and growth factors. Although the challenges of introducing endodontic tissue engineering therapies are substantial, the potential benefits to patients and the profession are equally ground breaking. Patient demand is staggering both in scope and cost, because tissue engineering therapy offers the possibility of restoring natural function instead of surgical placement of an artificial prosthesis.4 The aim of this article was to present a case in which extensive decay necessitated endodontic intervention of an inflamed and exposed dental pulp in an incompletely developed permanent molar tooth. The radicular pulp tissues were maintained in a vital state by performing a calcium hydroxide pulpotomy, which allowed complete root formation to take place (apexogenesis). Once apical closure was judged to be complete, the root canal therapy was performed under more optimal conditions and long term retention of the tooth was anticipated.5 The aim of this article was to review the new concept of regenerative endodontics in the management of immature permanent teeth. Two sets of data source were focused in this review: (i) the characterization of various dental stem cells (ii) recent clinical Page 74
case reports showing that after conservative treatment, severely infected immature teeth can undergo healing and apexogenesis or maturogenesis. A new protocol of treating endodontically involved immature permanent teeth has been summarized in the review. The key procedures of the protocol are (1) minimal or no instrumentation of the canal while relying on a gentle but thorough irrigation of the canal system, (2) the disinfection is augmented with intra-canal medication of a triple-antibiotic paste between appointments, and (3) the treated tooth is sealed with mineral trioxide aggregate (MTA) and glass ionomer/resin cement at the completion of the treatment. And periodical follow-ups.6 The aim of this article was to present a new technique to revascularize immature permanent teeth with apical periodontitis. The canal is disinfected with copious irrigation and a combination of three antibiotics. After the disinfection protocol is complete, the apex is mechanically irritated to initiate bleeding into the canal to produce a blood clot to the level of the cementoenamel junction. The double seal of the coronal access is then made. In this case, the combination of a disinfected canal, a matrix into which new tissue could grow, and an effective coronal seal appears to have produced the environment necessary for successful revascularization.7 The aim of this study was to present a case report in which the pulp of two bilateral mandibular premolars with dens evaginatus were revascularized using a modified novel technique to avoid undesired crown discolouration. Recently, regeneration of necrotic pulps has become an alternative conservative treatment option for young permanent teeth with immature roots and is a subject of great interest in the field of endodontics. This novel procedure exploits the full potential of the pulp for dentine deposition and produces a stronger mature root that is better able to withstand the forces than can result in fracture. However, the current protocol has potential clinical Page 75
and biological complications. Amongst them, crown discolouration, development of resistant bacterial strains and allergic reaction to the intracanal medication. In the case presented, a modified technique to avoid undesired crown discolouration was applied sealing the dentinal tubules of the chamber, thus avoiding any contact between the triantibiotic paste and the dentinal walls.8 The present investigation demonstrated larger amounts of reparative dentin formation on the amputated pulp with a BMP2-supplemented pellet compared with a control pellet. The extracellular matrix of the pellet functions as a natural scaffold, which retains and releases BMPs. Techniques to isolate human pulp stem cells and manipulate their growth under defined in vitro conditions have to be established and optimized before cell therapy with BMP2 can become a clinical reality for caries and endodontic therapy. This investigation is a first step toward that long-term goal of biological regenerative endodontic therapy.9 This review provides insight into how teeth are made in nature and how we might make them using our accumulating knowledge and technology. There are also species variations in the extent of enamel coverage on teeth. Despite such differences, most recent teeth, including those of humans, originate from a common precursor and develop under similar molecular instruction. Evolutionary and developmental biologists, as well as tissue engineers, are working together to investigate and compare the tissue origin, patterning and growth of various teeth parts in an effort to restore healthy and/or repair defective tissue. Our understanding of these biological processes may serve as a foundation for the future design and fabrication of regenerated teeth. Research continues with the goal of being able exploit natural processes to generate new therapies. 10
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The purpose of this article was to summarise about stem cells that have the ability to regenerate after trauma, disease or aging. Recently, there has been great interest in mesenchymal stem cells and their roles in maintaining the physiological structure of tissues. The studies on stem cells are thought to be very important and, in fact, it has been shown that this cell population can be expanded ex vivo to regenerate tissues not only of the mesenchymal lineage, such as intervertebral disc cartilage, bone and toothassociated tissues, but also other types of tissues. Several studies have focused on the identification of odontogenic progenitors from oral tissues, and it has been shown that the mesenchymal stem cells obtained from periodontal ligament and dental pulp could have similar morphological and phenotypical features of the bone marrow mesenchymal cells. In fact a population of homogeneous human mesenchymal stem cells derived from periodontal ligament and dental pulp, and proliferating in culture with a well-spread morphology, can be recovered and characterized. Since these cells are considered as candidates for regenerative medicine, the knowledge of the cell differentiation mechanisms is imperative for the development of predictable techniques in implant dentistry, oral surgery and maxillo-facial reconstruction.11 This article will review the isolation and characterization of the properties of different dental MSC-like populations in comparison with those of other MSCs, such as BMMSCs. Different human dental stem/progenitor cells which have been isolated and characterized: dental pulp stem cells (DPSCs), stem cells from exfoliated deciduous teeth (SHED), periodontal ligament stem cells (PDLSCs), stem cells from apical papilla (SCAP), and dental follicle progenitor cells (DFPCs). These postnatal populations have mesenchymal-stem-cell-like (MSC) qualities, including the capacity for self-renewal and multilineage differentiation potential. MSCs derived from bone marrow (BMMSCs) are capable of giving rise to various lineages of cells, such as Page 77
osteogenic, chondrogenic, adipogenic, myogenic, and neurogenic cells. The dentaltissue-derived stem cells are isolated from specialized tissue with potent capacities to differentiate into odontogenic cells.12 This review, the general landscape and current status of the MSC tool as well as their wide potential in tissue engineering, from neuronal to tooth replacement has been discussed. Highlights and pitfalls for further clinical applications have been discussed. Adult mesenchymal stem cells (MSCs) are adherent stromal cells able to self-renew and differentiate into a wide variety of cells and tissues. MSCs can be obtained from distinct tissue sources and have turned out to be successfully manipulated in vitro. As adult stem cells, MSCs are less tumorigenic than their embryonic correlatives and posses another unique characteristic which is their almost null immunogenicity. Moreover, these cells seem to be immunosuppressive in vitro. These facts together with others became MSCs a promising subject of study for future approaches in bioengineering and cell-based therapy. On the other hand, new strategies to achieve long-term integration as well as efficient differentiation of these cells at the area of the lesion are still challenging, and the signalling pathways ruling these processes are not completely well characterized.13 This study conducted an ultrastructural scanning electron microscopic (SEM) investigation of tissue-engineered pulp constructs implanted within endodontically treated teeth. Stem cells from human exfoliated deciduous teeth were seeded on a synthetic open-cell D,D-L,L-polylactic acid scaffold with or without the addition of bone morphogenic protein-2 and transforming growth factor 1 to create pulp tissue constructs. The pulp constructs were implanted into 105 extracted human premolar teeth with a single root canal that had been cleaned and shaped by using rotary Page 78
instrumentation in a crown-down manner to ISO size no. 35. An ultrastructural examination of the SEM micrographs at x2,000 magnification revealed cell adherence within all of the pulp constructs, with little difference between the scaffold types or with the addition of growth factors. These results support the proof-of-concept that it is possible to implant tissue-engineered pulp constructs into teeth after cleaning and shaping.14 The aim of this paper was to review current progress and feasible strategies for dental pulp regeneration and revascularization. Pulp vitality is extremely important for the tooth viability, since it provides nutrition and acts as biosensor to detect pathogenic stimuli. In the dental clinic, most dental pulp infections are irreversible due to its anatomical position and organization. It is difficult for the body to eliminate the infection, which subsequently persists and worsens. The widely used strategy currently in the clinic is to partly or fully remove the contaminated pulp tissue, and fill and seal the void space with synthetic material. Over time, the pulpless tooth, now lacking proper blood supply and nervous system, becomes more vulnerable to injury. Recently, potential for successful pulp regeneration and revascularization therapies is increasing due to accumulated knowledge of stem cells, especially dental pulp stem cells.15 The purpose of this review was to illustrate a path forward for the development and evaluation of regenerative endodontic therapies. During the last 10-15 years, there has been a tremendous increase in our clinical "tools" (ie, materials, instruments, and medications) and knowledge from the trauma and tissue engineering fields that can be applied to regeneration of a functional pulp-dentin complex. In addition, recent case reports indicate that biologically based endodontic therapies can result in continued Page 79
root development, increased dentinal wall thickness, and apical closure when treating cases of necrotic immature permanent teeth.16 This retrospective study included 23 necrotic immature permanent teeth treated for either short-term (treatment period <3 months) or long-term (treatment period >3 months) using conservative endodontic procedures with 2.5% NaOCl irrigations without instrumentation but with Ca(OH)(2) paste medication. For seven teeth treated short-term, the gutta-percha points were filled onto an artificial barrier of mineral trioxide aggregate (MTA). For 16 teeth treated long-term, the gutta-percha points, amalgam, or MTA were filled onto the Ca(OH)2-induced hard tissue barrier in the root canal. We found that all apical lesions showed complete regression in 3 to 21 (mean, 8) months after initial treatment. All necrotic immature permanent teeth achieved a nearly normal root development 10 to 29 months after initial treatment. We conclude that immature permanent teeth with pulp necrosis and apical pathosis can still achieve continued root development after proper short-term or long-term regenerative endodontic treatment procedures.17 The objective of this study was to evaluate in vivo the revascularization and the apical and periapical repair after endodontic treatment using 2 techniques for root canal disinfection (apical negative pressure irrigation versus apical positive pressure irrigation plus triantibiotic intracanal dressing) in immature dogs' teeth with apical periodontitis. Two test groups of canals with experimentally induced apical periodontitis were evaluated according to the disinfection technique: Group 1, apical negative pressure irrigation (EndoVac system), and Group 2, apical positive pressure irrigation (conventional irrigation) plus triantibiotic intracanal dressing. In Group 3 (positive control), periapical lesions were induced, but no endodontic treatment was done. Group 4 (negative control) was composed of sound teeth. A description of the Page 80
apical and periapical features was done and scores were attributed to the following histopathological parameters: newly formed mineralized apical tissue, periapical inflammatory infiltrate, apical periodontal ligament thickness, dentin resorption, and bone tissue resorption. It was concluded that sodium hypochlorite irrigation with the EndoVac system can be considered as a promising disinfection protocol in immature teeth with apical periodontitis, suggesting that the use of intracanal antibiotics might not be necessary.18 The aim of this study was to ivestigate histologically the types of tissues that had grown into the canal space.The canal dentinal walls were thickened by the apposition of newly generated cementum-like tissue termed herein "intracanal cementum (IC)." One case showed partial survival of pulp tissue juxtaposed with fibrous connective tissue that formed IC on canal dentin walls. The IC may also form a bridge at the apex, in the apical third or midthird of the canal. The root length in many cases was increased by the growth of cementum. The generation of apical cementum or IC may occur despite the presence of inflammatory infiltration at the apex or in the canal. These cementum or cementum-like tissues were similar to cellular cementum. Bone or bone-like tissue was observed in the canal space in many cases and is termed intracanal bone (IB). Connective tissue similar to periodontal ligament was also present in the canal space surrounding the IC and/or IB. the study explained in part why many clinical cases of immature teeth with apical periodontitis or abscess may gain root thickness and apical length after conservative treatment with the revitalization procedure.19 In this study, we characterized the self-renewal capability, multi-lineage differentiation capacity, and clonogenic efficiency of human dental pulp stem cells (DPSCs). DPSCs were capable of forming ectopic dentin and associated pulp tissue in Page 81
vivo. Stromal-like cells were reestablished in culture from primary DPSC transplants and re-transplanted into immunocompromised mice to generate a dentin-pulp-like tissue, demonstrating their self-renewal capability. DPSCs were also found to be capable of differentiating into adipocytes and neural-like cells. The odontogenic potential of 12 individual single-colony-derived DPSC strains was determined. Twothirds of the single-colony-derived DPSC strains generated abundant ectopic dentin in vivo, while only a limited amount of dentin was detected in the remaining one-third. These results indicate that single-colony-derived DPSC strains differ from each other with respect to their rate of odontogenesis. Taken together, these results demonstrate that DPSCs possess stem-cell-like qualities, including self-renewal capability and multi-lineage differentiation.20
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References:
1. Shehab el din m Saber. Tissue engineering in endodontics. J Oral Sci 2009; 51:4:495-507. 2. George T.-J. Huang, Wataru Sonoyama,Yi Liu,He Liu, Songlin Wang, Songtao Shi.The Hidden Treasure in Apical Papilla: The Potential Role in Pulp/Dentin Regeneration and BioRoot Engineering. J Endod 2006; 34:6. 3. Kenneth M. Hargreaves, Todd Geisler, Michael Henry,Yan Wang Regeneration Potential of the Young Permanent Tooth: What Does the Future Hold? J Endod 2008; 34:7;S51-58. 4. Peter E. Murray, Franklin Garcia-Godoy, Kenneth M. Hargreaves
Regenerative Endodontics: A Review of Current Status and a Call for Action. J Endod 2007; 33:4:377-390. 5. Craig Barrington,Frederic Barnett. Apexogenesis in an Incompletely Developed Permanent Tooth with Pulpal Exposure. Oral Health 2003:49-54. 6. George T.-J. HuangA paradigm shift in endodontic management of immature teeth: Conservation of stem cells for regeneration. J Dent 2008; 1-8. 7. Francisco Banchs, Martin Trope. Revascularization of Immature Permanent Teeth With Apical Periodontitis: New Treatment Protocol? J Endod 2004; 30:4:196-200. 8. K. Reynolds, J. D. Johnson & N. Cohenca. Pulp revascularization of necrotic bilateral bicuspids using a modified novel technique to eliminate potential coronal discolouration: a case report. Int Endod J 2008; 2-9. Page 83
9. K. Iohara , M. Nakashima1, M. Ito, M. Ishikawa, A. Nakasima and A. Akamine. Dentin Regeneration by Dental Pulp Stem Cell Therapy with Recombinant Human Bone Morphogenetic Protein 2. J Dent Res 2004.83:8:590-595. 10. Despina S. Koussoulakou, Lukas H. Margaritis, Stauros L. Koussoulakos. A Curriculum Vitae of Teeth: Evolution, Generation, Regeneration. Int J Biol Sci 2009; 5:226-243. 11. 11.Trubiani O, Orsini G, Caputis, Piatelli A. Adult mesenchymal stem cells in dental research: a new approach for tissue engineering.Int J Immunopathol Pharmacol- 2006 ;19:451-60. 12. Huang GT, Gronthos S, Shi S. Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine. J Dent Res. 2009; 88:9:792-806. 13. Zeidn-Chuli F, Noda M. "Opening" the mesenchymal stem cell tool box. Eur J Dent. 2009;3:3:240-9.
14. Eric L. Gotlieb, DDS, Peter E. Murray, PhD, Kenneth N. Namerow, DDS,
Sergio Kuttler,
DDS and Franklin Garcia-Godoy, DDS, MS. An
Ultrastructural Investigation of Tissue-Engineered Pulp Constructs Implanted Within Endodontically Treated Teeth. J Am Dent Assoc, 2008; 139:4:457465.
15. Zhang W, Yelick PC. Vital pulp therapy-current progress of dental pulp
regeneration and revascularization. Int J Dent. 2010; 28. 16. Villa P, Fernandez R. Apexification of a replanted tooth using mineral Page 84
trioxide aggregate. Dent Traumatol 2005; 21: 306308.
17. Chueh LH, Ho YC, Kuo TC, Lai WH, Chen YH, Chiang CP. Regenerative
endodontic treatment for necrotic immature permanent teeth. J Endod. 2009 ; 35:2:160-4.
18. da Silva LA, Nelson-Filho P, da Silva RA, Flores DS, Heilborn C, Johnson
JD, Cohenca N. Revascularization and periapical repair after endodontic treatment using apical negative pressure irrigation versus conventional irrigation plus triantibiotic intracanal dressing in dogs' teeth with apical periodontitis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2010 ; 109:5:779-87
19. Wang
X, Thibodeau of
B, Trope regenerated
M, Lin tissues
LM, Huang in canal
GT. space
Histologic after the
characterization
revitalization/revascularization procedure of immature dog teeth with apical periodontitis. J Endod. 2010;36:1:56-63.
20. Gronthos S, Brahim J, Li W, Fisher LW, Cherman N, Boyde A, DenBesten
P, Robey PG, Shi S. Stem cell properties of human dental pulp stem cells. J Dent Res. 2002;81:8:531-5. 21. Hockin H.K. Xu, Michael D. Weir and Carl G. Simon. Injectable and strong nano-apatite scaffolds for cell/growth factor delivery and bone regeneration. Dent Materials 2008; 24:9:1212-22. 22. Thibodeau B.Case report: pulp revascularization of a necrotic, infected, immature, permanent tooth. Pediatr Dent 2009; 31:2:145-8. 23. N.Tamaoki, K.Takahashi, T.Tanaka, T.Ichisaka, H.Aoki, K,Tezuka.Dental Page 85
Pulp Cells for Induced Pluripotent Stem Cell Banking. Int & Am Ass For Dental Research. 24. 16.Cordeiro MM, Dong Z, Kaneko T, Zhang Z, Miyazawa M, Shi S, Smith AJ, Nr JE. Dental pulp tissue engineering with stem cells from exfoliated deciduous teeth. J Endod 2008; 34:8:962-9. 25. Dentistry Of Future? Gene Responsible For Formation Of Enamel Discovered. ScienceDaily; Mar. 3, 2009. 26. Tziafas D, Kodonas K. Differentiation potential of dental papilla, dental pulp, and apical papilla progenitor cells. J Endod 2010; 36:5:781-9. 27. George T.J. Huang, Wataru Sonoyama, Yi Liu, He Liu, Songlin Wang, Songtao Shi. The Hidden Treasure in Apical Papilla: The Potential Role in Pulp/Dentin Regeneration and BioRoot Engineering. J Endod 2008; 34:645 651. 28. Kim JH, Kim Y, Shin SJ, Park JW, Jung IY. Tooth discoloration of immature permanent incisor associated with triple antibiotic therapy: a case report. J Endod 2010; 36:6:1086-91. 29. Witherspoon DE.Vital pulp therapy with new materials: new directions and treatment perspectives--permanent teeth. Pediatr Dent.2008; 30:3:220-4. 30. Trope M. Treatment of the immature tooth with a non-vital pulp and apical periodontitis. Dent Clin North Am 2010; 54:2:313-24. 31. Saber SE. Tissue engineering in endodontics J Oral Sci. 2009 ; 51:4:495-507. 32. Shi S, Bartold PM, Miura M, Seo BM, Robey PG, Gronthos S. The efficacy of Page 86
mesenchymal stem cells to regenerate and repair dental structures. Orthod Craniofac Res.2005; 8:3:191-9. 33. Stem cells sourced in dental pulp. British Dental Journal 2010; 209:5. 34. D. M. Ranlyand F. Garcia-Godoy Current and potential pulp therapies for primary and young permanent teeth. Journal of Dentistry. March 2000; 28:3:153-161.
35. Mullane EM, Dong Z, Sedgley CM, Hu JC, Botero TM, Holland GR, Nr
JE.Effects of VEGF and FGF2 on the revascularization of severed human dental pulps.J Dent Res. 2008;87:12:1144-8. 36. Riccardo d'Aquino , Alfredo De Rosa , Gregorio Laino , Filippo Caruso, Luigi Guida, Rosario Rullo, Vittorio Checchi , Luigi Laino, Virginia Tirino, Gianpaolo Papaccio. Human dental pulp stem cells: from biology to clinical applications. J Experimental Zoology part B: Molecular and development evolution.312B:5: 408 415.
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Regenerative Endodontics

Autogenous Allogenous Xenogenous

Key Elements for Tissue Engineering

THE SOURCE, ACTIVITY AND USEFULNESS OF COMMON GROWTH FACTORS

Bone morphogenetic proteins Colony stimulating factor

A wide range of cells

A wide range of cells

Platelets, endothelial cells,placenta

Delivery of replacement pulp-dentin tissues Surgical implantation methods Injection site

DDS and Franklin Garcia-Godoy, DDS, MS. An

LM, Huang in canal

Histologic after the

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