7.kottoor-biomimetics

Jojo Kottoor Technology - Biomimetic endodontics: barriers and strategies

TECHNOLOGY

BIOMIMETIC ENDODONTICS : BARRIERS AND STRATEGIESJOJO KOTTOORDepartment of Conservative Dentistry and Endodontics,Mar Baselios Dental College, Kothamangalam, Kerala, India

Correspondence to: [email protected]

Abstract

Biomaterials used in the medical field lack the ability to integrate with biological systems through a cellular pathway. Conversely, biomimetic materials transcend the regular biomaterial in utility and will suitably perform the functions of the biological molecule that needs to be replaced. However, certain practical obstacles are yet to be overcome before biomimetic approaches can be applied as evidence-based approach in clinics. The article highlights on the past achievements, current developments and future prospects of tissue engineering and regenerative therapy in the field of endodontics and bioengineered teeth.

Introduction

Biomimetics is defined as the study of the formation, structure, or function of biologically produced substances and materials and biological mechanisms and processes especially for the purpose of synthesizing similar products by artificial mechanisms which mimic natural ones. A material fabricated by biomimetic technique

based on natural process found in biological systems is called a biomimetic material.1,2

Biomimicry or biomimetics (from bios, meaning life, and mimesis, meaning to imitate) involves studying natures most successful developments and then imitating these designs to create new materials. The main disadvantage with traditional biomaterials used in the medical field is that they lack the ability to integrate with

Health Sciences 2013;2(1):JS007 1 An Open Access Peer Reviewed E-Journal


biological systems through a cellular pathway which can lead to failure of the material. However, biomimetic materials transcend the regular biomaterial in utility and will suitably perform the functions of the biological molecule that needs to be replaced.3 A biomimetic approach to restore

tooth structure is based on regenerative endodontic procedures by application of tissue engineering which opens up a whole new arena for the practioner. The key elements of tissue engineering are stem cells, morphogen, and a scaffold of extracellular matrix (Figure 1).4

F igure 1 . Ti s sue eng inee r ing t r i ad

Stem cells are defined as cells that have the ability to continuously divide and produce progeny cells that develop into various other cells or tissues.5 There are two major types of stem cells, Embryonic and Adult stem cells their most important properties being their ability of self renewal and their ability to grow in-vitro. Comparing the different stem cell types, adult stem cells, which have the least amount of ethical concerns, are presently being used in medical therapies and are readily accessible.6 Postnatal stem cells have been found in almost all body tissues, including dental tissues. To date, eight types of human dental stem cells have been isolated and characterized: i) Dental

pulp stem cells (DPSCs), ii) Stem cells from human exfoliated deciduous teeth (SHED), iii) Stem cells from apical papillae (SCAP), iv) Periodontal ligament stem cells (PDLSCs), v) Epithelium-originated dental stem cells (EpSC), vi) Mesenchymal stem cells (BMSC), vii) Stem cells from the dental follicle (DFSC), and viii) Endothelial progenitor cells (EPCs).

Signaling molecules or morphogens are extracellular secreted signalling molecules that play a key role in signaling many of the events of repair and regeneration including tertiary dentinogenesis, a response of pulp-dentin repair. These signalling networks can



be generally classified into growth factors and inflammatory cytokines.7 Growth factors are soluble proteins that act as signaling agents for cells, and influence critical functions, such as cell division, matrix synthesis and tissue differentiation. Primarily, five eminent families of growth factors appear to regualate the process of odontogenesis: Fibroblast growth factor, Bone morphogenic protein (BMP), Hedgehog, Wingless (WNT) and Transforming growth factor. Inflammatory cytokines (Interleukin and Tumor necrosis factor) are molecules that regulate cellular behaviour of bone under inflammation, infection and wound healing.8

The scaffold or the extracellular matrix is a mixture of proteins including collagen, fibronectin, polysaccharide hyaluronic acid, proteoglycans and laminins forming an elastic network surrounding most cells and tissue structures.9 Current scaffolds used in tissue engineering can be grouped into three main categories. Natural scaffolds like Collagen, lyophilized bone and coral are the most commonly used natural scaffold. The main disadvantage of natural scaffolds is that they often lack the desired structural integrity for its independent use in load bearing areas. Mineral based scaffolds usually are made of calcium phosphates in the form of hydroxyapatite or beta Tricalcium phosphate and by varying the content of calcium the rate of degradation of these scaffolds can be controlled. They lack the strength of natural scaffolds and are

brittle making it susceptible to fracture10 and hence were introduced the synthetic scaffolds. These include the porous ceramics, spongiosus collagen, fibrous titanium mesh, poly lactic acid (PLA), poly glycolic acid (PGA), and their copolymers, poly lactic-co-glycolic acid (PLGA) which are all polyester material that degrade within the human body. They have the advantage of being able to function in load bearing; but have the disadvantage of lacking osteoinductiveness and an inherent difficulty in obtaining high porosity and regular pore size.11 This has led researchers to concentrate efforts to engineer scaffolds at the nanostructural level to modify cellular interactions with the scaffold.12

Biomimet ic approaches fo r r egenera t ion

The creation and delivery of new tissues to replace diseased, missing, or traumatized pulp is referred to as regenerative endodontics. 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. However, the challenge lies in designing and fabricating biomimetic materials like enamel, dentin, cementum, pulp, bone and periodontal ligament and focus should be toward regenerating the diseased and necrotic tissues rather than replacing them



with some conventional replacement materials. This article reviews current biomimetic approaches for regeneration tooth and its associated structures.

a. Root canal revascularization

Treatment of the young permanent tooth with a necrotic root canal system and an incompletely developed root is fraught with difficulty. Not only is the root canal system often difficult to fully debride, but the thin dentinal walls increase the risk of a subsequent fracture. Other than the procedure like maturogenesis or apexogenisis, root canal revascularization is a procedure to establish the vitality in a nonvital tooth to allow repair and regeneration of tissues. The typical revascularization protocol advocates that the immature tooth, diagnosed with apical periodontitis, should be accessed and irrigated with either 5% NaOCl _ 3% H2O2 or 5.25% NaOCl and Peridex TM (Procter & Gamble, Cincinnati, OH). An antimicrobial agent (either an antibiotic such as metronidazole, ciprofloxacin or ciprofloxacin, metronidazole, minocycline or Ca (OH)2 should be then applied into the root canal system, and the access cavity is sealed. After an average of 3 weeks, in the absence of symptoms, the tooth is re-entered, the tissue is irritated until bleeding is started and a blood clot produced, and then MTA is placed over the blood clot, and the access is sealed. Within the next 2 years a gradual increase in root development can

be observed.13 However, revascularization procedures lack standardization of treatment protocols with a myriad of reported techniques, intracanal medicaments and irrigants.

b. Stem cell therapy

The simplest method to administer cells of appropriate regenerative potential is to inject the postnatal stem cells into the disinfected root canal system. Autologous dental stem cells are the most accessible stem cells for this therapy. Among the eight different post natal dental stem cells Dental pulp stem cells (DPSCs), Stem cells from human exfoliated deciduous teeth (SHED), and Stem cells from the apical papilla (SCAP) were more commonly used in the field of regenerative endodontics.14

DPSCs are the stem cells isolated from human dental pulp. The most striking feature of DPSCs is their ability to regenerate a dentin-pulp-like complex that is composed of mineralized matrix with tubules lined with odontoblasts, and fibrous tissue containing blood vessels in an arrangement similar to the dentin-pulp complex found in normal human teeth.15

Stem cells from human exfoliated deciduous teeth (SHED) have become an attractive alternative for dental tissue engineering. The use of SHED might bring advantages for tissue engineering over the use of stem cells from adult human teeth as follows: (a)



SHED were reported to have higher proliferation rate compared with stem cells from permanent teeth, which might facilitate the expansion of these cells in vitro before replantation. (b) SHED cells are retrieved from a tissue that is "disposable" and readily accessible in young patients. ie, exfoliated deciduous teeth. It also has an added advantage of abundant cell supply, and painless stem cell collection with minimal invasion.16

A recent finding is the presence of a new population of a mesenchymal stem cells residing in the apical papilla of incompletely developed teeth. They are termed stem cells from the apical papilla (SCAP). It is hypothesised that SCAP appear to be the source of primary odontoblast that are responsible for the formation of root dentine, whereas DPSCs are likely the source of replacement odontoblast.17 Since these stem cells are in the apical papilla, they are benefited by its collateral circulation, which enables it to survive during the process of pulp necrosis.

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 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.18 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.

c. Pulp implantation

Dental pulp tissue is vulnerable to infection. Currently, entire pulp amputation followed by pulp-space disinfection and filling with an artificial rubber-like material is employed to treat the infection - commonly known as root-canal therapy. In pulp implantation, replacement pulp tissue is produced by tissue engineering triad and is transplanted into cleaned and shaped root canal systems. Rebecca et al had generated Dental pulp like tissue by using the tissue engineering triad, the Dental Pulp Stem Cells (DPSCs), a Collagen Scaffold, and Dentin Matrix protein 1 after subcutaneous transplantation in mice. Collagen served as the scaffold, and dentin matrix protein 1 (DMP1) was the growth factor. The result concluded that the triad of DPSCs, a collagen scaffold, and DMP1 can induce an organized matrix formation similar to that of pulpal tissue, which might lead to hard tissue formation.19

Similarly pulp tissue has also been successfully regenerated in-vitro using the tissue engineering triad, but by using a different stem cell, scaffold and morphogens.20



One of the potential problems associated with the implantation of cultured pulp tissue is that specialized procedures may be required to ensure that the cells properly adhere to root canal walls. When implanting pulp into the root canals that have blood supply only from the apical end, enhanced vascularization is needed in order to support its vitality. As a result, microscale technologies that provide open channels or the ability to guide vascular ingress from the apex through the pulp may be of particular benefit.21 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.22 These scaffolds are impregnated with growth factors such as VEGF (vascular endothelial growth factor) and/or platelet derived growth factor or further, with the addition of endothelial cells.

d. 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.23 This will allow tissue engineered pulp tissue to be administered in a soft three-dimensional scaffold matrix. Among the injectable biomaterials investigated so far, hydrogels are more and more attractive

in the field of tissue engineering. Hydrogels are injectable scaffolds that can be delivered by syringe and have the potential to be non-invasive 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.24

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.52 To make hydrogels more practical, research is focusing on making them photopolymerizable to form rigid structures once they are implanted into the tissue site.25

e. Three-dimensional cell printing

One of the most promising approaches in tissue engineering is the application of 3D scaffolds, which provide cell support and guidance in the initial tissue formation stage. The porosity of the scaffold and internal pore organization influence cell migration and play a major role in its biodegradation dynamics, nutrient diffusion and mechanical stability. In order to control cell migration and cellular interactions within the scaffold, novel technologies capable of producing 3D structures in accordance with predefined design are



required. 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.26,27 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. This may involve positioning of odontoblasts around the periphery, with fibroblasts in the core. The major challenge involved is the precise orientation of cellular suspensions according to the apical and coronal asymmetry of pulp.28 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.

f. Gene Therapy

Gene therapy is a method of delivering genes with the help of viral or non-viral vectors. The gene delivery in endodontics would be to deliver mineralizing genes into pulp tissue to promote tissue mineralization. Viral vectors are genetically altered to eliminate ability of causing disease, without losing infectious capacity to the cell. At present adenoviral, retroviral, adenoassociated virus, herpes simplex virus, lentivirus are being developed. Nonviral delivery systems uses plasmids, peptides, cationic liposomes, DNA-ligand complex,

gene guns, electroporation, and sonoporation to address safety concerns such as immunogenicity and mutagenesis.29

Most of the risks of gene therapy may arise from the vector system rather than the gene expressed. Widespread clinical application still awaits the development of vectors that are safe, affordable, efficient, simple for application, and that have ability to express the required level of transgene for the sufficient long term.30

Both in vivo and ex vivo approaches can be used for gene therapy. In the in vivo approach, the gene is delivered systemically into the blood stream or locally to target tissues by injection or inhalation. In the field of conservative dentistry, BMPs or BMP genes are directly applied to the exposed pulp. The ex vivo approach involves genetic manipulation of cells in vitro, which are subsequently transplanted to the regeneration site. In conservative dentistry, DPSCs were isolated, differentiated into odontoblasts with recombinant BMPs or BMP genes, and finally autogenously transplanted to regenerate dentin.32

The main challenges for gene therapy in the next decade will be the requirements to demonstrate that gene therapy can provide cost-effective and safe long-term treatments for conditions that would otherwise lead to significant pulp necrosis. 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.

g. Bioengineered tooth

The ultimate goal of regenerative therapy is to develop fully functioning bioengineered organs that can replace lost or damaged organs following disease, injury, or aging. Research on whole tooth regeneration is also advancing using a strategy of transplanting artificial tooth germ and allowing it to develop in the adult aral environment. Tissue engineering builds a tissue such as skin, bone and cartilage, by seeding cells into a scaffold.33 Research on the fabrication of teeth from dissociated cells was first performed using tooth germ cells.34 When explanted, seeded with porcine third impacted tooth bud cells, were implanted for 20-30 weeks into omentum, bioengineered teeth were visible within the explants.35 However, the regenerated teeth were not identical to their naturally formed counterparts.

Ikeda et al reported a fully functioning tooth replacement achieved by transplantation of a bioengineered tooth germ into the alveolar bone of a lost tooth region in an adult mouse.36 Bioengineered tooth, which was erupted and occluded, had the correct tooth structure, hardness of mineralized tissues for mastication. However, the bioengineered tooth was smaller than the other normal teeth. In addition, the authors couldnt regulate the crown width, cusp position, and

tooth patterning including anterior/posterior and buccal/lingual structures. However, in a more recent study Oshima et al showed that the crown widths and the cusp numbers of bioengineered molar could be regulated by cell manipulation method.37 Tooth regeneration is an important stepping stone in the establishment of engineered organ transplantation, which is one of the ultimate goals of regenerative therapies.

Conc lus ions

The practice of endodontics has grown by leap and bounds in the past few decades. Replacement of diseased or lost tooth structure with biocompatible restorative materials is currently the order of today but each of these procedures does have their own limitations and drawbacks. Regeneration of the lost tooth structure rather than replacement during treatment will ensure better prognosis and higher rate of success. Hence the future of endodontics would involve the use of such biomimetic materials which could successfully replace lost enamel, dentine, cementum and even the pulp tissue.

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7.kottoor-biomimetics

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