Tissue Engineering - A Seminar

•      INTRODUCTION

•      TRIAD OF TISSUE ENGINEERING

−        STEM CELLS

−        SCAFFOLD

−        MORPHOGENES

•      APPLICATIONS IN CONSERVATIVE DENTISTRY & ENDODONTICS

•      REGENERATIVE ENDODONTICS

•      CONCLUSION

INTRODUCTION

“Imagine a world where transplant patients do not wait for a donor or a world where burn victims leave the hospital without disfiguring scars. Imagine implant materials that can "grow", reshape themselves, or change their function as the body requires”

-Professor M.V. Sefton

Pulpal regeneration after tooth injury is difficult to accomplish. This is the reason why infected pulp requires root canal therapy or tooth extraction.

Tissue engineering aims at the regeneration of affected or lost pulp tissue using stem cell therapy. 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.

            The dental pulp contains progenitor/stem cells, which can proliferate and differentiate into dentinforming odontoblasts (Nakashima et al, 1994; Gronthos et al, 2000, 2002). Following physiological stimulation or injury, such as caries and operative procedures, stem cells in pulp may be mobilized to proliferate and differentiate into odontoblasts by morphogens released from the surrounding dentin matrix. Tissue engineering with the triad of dental pulp progenitor/stem cells, morphogens, and scaffolds may provide a useful alternative method for pulp-capping and root canal treatment.

                                                            However, the technique for manipulation of the growth of the isolated pulp progenitor/stem cells and induction of three-dimensional tissue formation invitro needs to be developed.

What is Tissue Engineering?

The use of a combination of cells, engineering and materials methods, and suitable biochemical and physico-chemical factors to improve or replace biological functions.

An interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function or a whole organ.

-Langer and J. Vacanti

In other words Tissue Engineering is using a persons cells to create a new artificial fully alive tissue or organ that can replace or improve/heal the old one in the body.

Triad Of Tissue Engineering

Tissue engineering employs use of three materials:

1.      Stem cells/progenitor cells: These are capable of differentiating into specialized cells and are able to respond to morphogens by dividing or specializing.

2.      Morphogens/signaling molecules: These are the biological factors that regulate stem cells to form desirable cell type,

e.g. BMPs, which are major morphogens family for tooth regeneration

3.      Scaffold/matrix: It provides a biocompatible 3-dimensional structure for cell adhesion and migration. It can be

a.       Biological scaffolds (e.g. collagen, glycosaminoglycan)

b.      Artificial scaffolds (e.g. PLA, PGA, PLGA).

These days some authors consider  DELIVERY SYSTEM  as part of basic triad forming it to be a tetrad.

The mixture must be delivered in a spatially appropriate fashion into the space of root canal system.

Eg: All cells are within 0.1 to 1 mm of a blood vessel in order to maintain adequate diffusion of oxygen and nutrients.

Stem Cells

Stem cells are primal undifferentiated cells that retain the ability to divide and differentiate into other cell types.

            Stem cells differ from other kinds of cells in the body as all stem cells regardless of their source have three general properties: they are capable of dividing and renewing themselves for long periods; they are unspecialized; and they can give rise to specialized cell types.

There are two types of stem cells:

1. Embryonic (Fetal) stem cells, as their name suggests, they are derived from embryos. Specifically, from embryos that develop from eggs that have been fertilized in vitro, in an in-vitro fertilization clinic and then donated for research purposes with informed consent of the donors.

They are not derived from eggs fertilized in a woman's body.

2. Adult (Postnatal) stem cell is an undifferentiated cell found among differentiated cells in a tissue or organ, can renew itself, and can differentiate to yield the major specialized cell types of the tissue or organ.

The primary roles of adult stem cells in a living organism are to maintain and repair the tissue in which they are found.

Depending on their origin, adult stem cells can be further classified as

−        Hemopoetic stem cells (HSCs) and

−        Mesenchymal stem cells (MSCs).

•      HSCs are obtained either from cord blood or peripheral blood.

•      MSCs are those that originate from the mesoderm layer of the fetus and in the adult reside in a variety of tissues such as the bone marrow stem cells (BMSCc), limbal stem cells, hepatic stem cells, dermal stem cells, etc.

VARIOUS SOURCES OF STEM CELLS:

•      Dental pulp stem cells:-

−        Stem cells derived from the dental pulp can form pulp like tissue , in future it is possible to replace infected pulp tissue of a paining tooth with newly generated pulp like tissue instead of doing RCT ,thus preserving the vitality of the tooth.

−        It also has the ability to form bone that is useful for the osseointegration of dental implants, thus increaseing its success rate.

•      Stem cells of  Human Exfoliated Deciduous Teeth

−        have higher rate of proliferation

−        have potential to form bone which is useful during osseointegration of dental implants

−        have the potential to repair calvarial defects in immunocompromised mice.

•      Periodontal Ligament Stem Cells:

−        have potentials of regenerating typical cementum and periodontal ligament like structure.

−        tissue of the periodontium made by stem cell can be used as a treatment modality to replace the diseased periodontium around teeth so as treatment to mobility of teeth .

•      Stem Cells from Apical Papilla:

−        can only be isolated at certain specific stages of the development of tooth.

−        dental papilla contain higher number of adult stem cells than mature dental pulp, thus have a greater potential for regenerating dentin than DPSCs.

•      Dental Follicle Precursor Cells:

−        Dental follicles contain progenitor cells which have the capability of differentiating into cementum forming cells (cementoblasts), osteoblasts of the alveolar bone, and periodontal ligament fibroblasts.

     

Functions of Adult Stem Cell

a. They exist as undifferentiated cells and maintain this phenotype by the environment and/or the adjacent cell populations until they are exposed to and respond to the appropriate signals.

b. They have an ability to self-replicate for prolonged periods.

c. They maintain their multiple differentiation potential throughout the life of the organism.

According to their source, stem cells can be categorized as autologous, allogenic or xenogenic, while according to their plasticity, stem cells can be totipotent, pluripotent and multipotent.

GOALS OF STEM CELL THERAPY IN TISSUE ENGINEERING

• Proliferate extensively and generate sufficient quantities of tissue.

• Differentiate into the desired cell types.

• Survive in the recipient after transplant.

• Integrate into the surrounding tissue after transplant.

• Function appropriately for the duration of the recipient's life.

• Avoid harming the recipient in any way.

SOURCE OF PULP STEM CELLS

The source of odontoblastoid cells that repair dentinal bridges has proved to be controversial. Initially, the replacement of irreversibly injured odontoblasts by predetermined odontoblastoid cells that do not replicate DNA after induction was suggested. Researchers proposed that the  cells within the subodontoblast cell rich layer of Hohl adjacent to odontoblasts differentiate into odontoblastoids. The purpose of these cells seem to be limited to an odontoblast supporting role, as the survival of these cells is linked to the survival of the odontoblasts, and no proliferative or regenerative activity was observed.

Stem Cell Therapy

Stem cell therapy is one of the most promising area s of tissue engineering because the transplantation of the materials that contain pulp stem cells grown in the laboratory provides an an inductive means to regenerate new tooth tissues. The transplantation of odontoblastoid stem cells into teeth to accomplish regeneration removes the problem of delivering growth factors and genes into host target cells and waiting for the target cells to differentiate. Recent studies focus on evaluating the use of human odontoblastoid stem cells transplantation for regeneration of oral tissues in conjunction with in vitro tissue engineering to produce regenerative biomimetic materials.

Scaffold

Scaffold provides a three-dimensional micro environment for cell growth and differentiation, promoting cell adhesion, and migration (Fig. 24.4). The scaffold acts as a carrier for morphogen cells in cell therapy. Scaffold should have transport of nutrients, oxygen, and waste across them.

                        Scaffold should be slowly degraded and replaced by regenerative tissue, retaining the feature of the final tissue structure. They should have biocompatibility, nontoxicity, and good physical properties.

Scaffold materials can be:

1. Natural

a. Collagen

b. Glycosaminoglycans

2. Synthetic

a. Synthetic polymers

• Poly (lactic acid) (PLA)

• Poly (glycolic acid) (PGA)

• Poly (lactic-co-glycolic acid) (PLGA).

b. Synthetic hydrogels

• Poly (ethylene glycol) (PEG) based polymers.

c. Synthetic hydrogels modified with cell surface adhesion peptides

• Arginine.

• Glycine.

• Asparticacid (RGD).

d. Inorganic compounds

• Hydroxyapatite

• Calcium phosphate.

Morphogens

A major focus of contemporary studies in developmental biology has been to delineate the biological cues that drive stem cell proliferation and differentiation.

Four signaling protein families that govern patterning and morphogenesis have been identified:

• Fibroblast growth factors,

• Hedgehog proteins,

• Bone morphogenetic proteins,

• Wingless- and int-related proteins (Wnts).

Proteins from each of these families are now being evaluated for their utility for stem cell based engineering of craniofacial defects. Recently, these applications have been extended to treat the diseases of endodontic origin. Naturally derived collagen or synthetic materials such as polyglycolic acid (PGA) are used as a scaffold for attachment and guidance of cells. The pulp derived fibroblasts adhering to the PGA fibers can proliferate and form a new tissue similar to that of native pulp

            The synthetic matrices, however, must undergo degradation simultaneously with the new tissue formation by the cultured cells.

Bone morphogenetic proteins (BMPs) have been implicated in tooth development, and the expression of BMP2 is increased during the terminal differentiation of odontoblasts. Beads soaked in human recombinant BMP2 induce the mRNA expression of Dspp, the differentiation marker of odontoblasts after implantation onto dental papilla in organ culture.

                                                                                                            BMP2 also induces a large amount of reparative dentin on the amputated pulp in vivo (Nakashima, 1994a). It has been suggested that BMP2 may regulate the differentiation of pulp cells into odontoblastic lineage and stimulate reparative dentin formation (Nakashima and Reddi, 2003).

Applications in Conservative Dentistry & Endodontics

•      Regeneration of

−        Damaged coronal dentin and pulp,

−        Resorbed root, &

−        cervical or apical dentin.

•      Repair perforations

•      Whole tooth regeneration.

Regeneration of damaged coronal dentin and pulp

•      To this date, no restorative material has been able to mimic all physical and mechanical properties of tooth tissue.

•      If the regeneration of tooth tissue is possible in these situations, it facilitates physiologic dentin deposition that forms an integral part of the tooth thereby restoring structural integrity, minimizing interfacial failure, microleakage, and other consequent complications.

•      Similarly, young permanent teeth that require apexogenesis or apexification are the perfect candidates for the regeneration of pulp as they allow completion of both vertical and lateral root development, improving the long-term prognosis.

•      However, pulp regeneration in fully formed teeth may not be of great benefit, although there is sufficient evidence to say that a restored vital tooth serves longer than a root-canal-treated one.

•      Pulp tissue regeneration involves either delivery of autologous/allogenic stem cells into the root canals or implantation of the pulp that is grown in the laboratory using stem cells.

•      A landmark study conducted by Gronthos et al. demonstrated both in vitro and in vivo in animals that dental pulp stem cells (DPSCs) were capable of forming ectopic dentin and associated pulp tissue.

•      Batouli et al. used an in vivo stem cell transplantation system to investigate differential regulation mechanisms of bone marrow stromal stem cells (BMSCs) and DPSCs. 

•      DPSCs were found to be able to generate a reparative dentin-like tissue on the surface of human dentin in vivo.

•      This study provided direct evidence to suggest that osteogenesis and dentinogenesis mediated by BMSCs and DPSCs, respectively, may be regulated by distinct mechanisms, leading to the different organization of the mineralized and nonmineralized tissues.

Whole tooth regeneration

•      A therapeutic option that was unthinkable a few years ago seems an achievable goal today.

•      Even to this day, the replacement of missing teeth has limitations. Although, implants are a significant improvement over dentures and bridges, their fundamental limitation is the lack of natural structural relationship with the alveolar bone (absence of periodontal ligament).

•      Ohazama et al. reported the reconstruction of murine teeth using cultured stem cells which when transferred into renal capsules resulted in the development of tooth structures and associated bone.

•      Nakao et al. recently engineered teeth ectopically and transplanted them into an anthrotopic site in a mouse jaw.

Regenerative Endodontics

•      Regenerative endodontic procedures can be defined as biologically based procedures which are designed to replace damaged structures including dentin and root structures, as well as the cells of the pulp-dentin complex.

•      The factors contributing to the success of regenerative endodontics comprises of the research on adult stem cells, growth factors, organ tissue cultures and tissue engineering materials.

•      The objectives of the regenerative endodontic procedures are to regenerate pulp-like tissues: ideally, the dentin pulp complex; regenerate damaged coronal dentine

Steps in regenerative endodontics:

A. Disinfection and shaping of canals

B. Creation of replacement pulp-dentin tissue

C. Delivery of replacement pulp-dentin tissue

D. Dental restorative materials

E. Measuring appropriate clinical outcomes

Various developemental approaches for regenerative endodontics

There are several techniques for the application of regenerative endodontics.

These techniques are:

1. Root canal revascularization via blood clotting.

2. Post natal stem cell therapy.

3. Pulp implantation.

4. Scaffold implantation.

5. Injectable scaffold delivery.

6. Three dimensional cell printing.

7. Gene therapy.

These regenerative endodontic techniques are based on the basic principles of tissue

engineering.

Root canal revascularization via blood clotting:

The development of regenerative endodontic procedures may require the reexamination of many of the closely held percepts of traditional endodontic procedures.The revascularization method assumes that the root canal space has been disinfected effectively by the use of intracanal irrigants, with the placement of antibiotics for several weeks. Several case reports have documented the revascularization of the necrotic root canal systems by disinfection, followed by establishing bleeding into the canal system via over instrumentation[12].

Post natal stem cell therapy:

The simplest method to administer the cells of appropriate regenerative potential is to inject the post natal stem cells into the disinfected root canal systems after the apex is opened. The post natal stem cells can be derived from multiple tissues including skin, buccal mucosa, fat and bone. One recent approach could be to use the dental pulp stem cells that have been taken from the umbilical cord, which are mostly disease and pathogen free.

Pulp implantation:

In pulp implantation, the cultured pulp tissue is transplanted into cleaned and shaped root canal systems. The 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[13]. The limitation of this technique is that specialized procedures may be required to ensure that the cells properly adhere to the root canal walls.

Scaffold implantation:

Pulp stem cells must be organized into a three-dimensional structure that can support cell organization and vascularization. This can be accomplished by using a porous polymer scaffold which is seeded with pulp stem cells[14]. In pulpexposed teeth, dentin chips have been found to stimulate reparative dentin bridge formation. Dentin chips may provide a matrix for pulp stem cell attachment and they may also be a reservoir of growth factors15. The natural reparative activity of the pulp stem cells in response to the dentin chips provides some support for the use of scaffolds to regenerate the pulp dentin complex.

Injectable scaffold delivery:

Tissue engineered pulp tissue is seeded into the soft three-dimensional scaffold matrix, such as a polymer hydrogel. Hydrogels are injectable scaffolds that can be delivered by syringe16, they have the potential to be noninvasive and are easy to deliver into the 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. 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.

Three dimensional cell printing:

The threedimensional 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 structure17. 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.

Gene therapy:

Gene therapy has been recently used as a means of delivering genes for growth factors, morphogens, transcription factors and extracellular matrix molecules locally to the somatic cells of individuals, with resulting therapeutic effect3. The gene can stimulate or induce a natural biological process by expressing the molecules which are involved in the regenerative response for the tissue of interest. Both an in-vivo and ex-vivo approach can be used for gene therapy. One use of gene delivery in endodontics would be to deliver mineralizing genes into the pulp tissues to promote tissue mineralization. Gene therapy is a relatively a new field and evidence is lacking to demonstrate that this therapy has the potential to rescue the necrotic pulp.

Conclusion

The last decade has proved to be an exciting time for pulp biology and has led to rapid advances in our knowledge of repair in this tissue. At the start of a new millennium, the use of biological molecules for the development of novel restorative treatment modalities in clinical dentistry is in sight. These approaches have potential applications in unexposed cavity preparations for protection of the pulp from harmful effects of dental materials by increasing the residual dentin thickness through reactionary dentinogenesis, as well as in exposed pulp situations for restoration of the structural integrity of the dentin wall by reparative dentinogenesis. In the severely compromised pulp, it may even be possible to use biological approaches in endodontic therapy to seal the root canal.