Tissue Engineering

Chapter outline

- Current Strategies for Treatment of Lost Tissue

- Strategies of Stem Cell Technology - Triad of Tissue Engineering - Dental Pulp Stem Cells (DPSCs) - Stem Cells from Human Exfoliated Deciduous Teeth (SHED) - Periodontal Ligament Stem Cells (PDLSCs)

- Morphogens/Signaling Molecules - Scaffold/Matrix - Revascularization to Induce Apexification/ Apexogenesis in Infected, Nonvital,

Immature Tooth

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- Every year millions of Indians suffer from some type of tissue loss or end-stage organ failure which can be due to inherited disorders, trauma, and neoplastic or infectious diseases.

- Tissueengineering is expected to solve many such problems by the use of stem cells. - Stem cell is a special kind of cell that has a unique capacity to renew itself and to

give rise to specialized cell types.

- Although most cells of the body, such as heart cells or skin cells, are committed to perform a specific function, a stem cell is uncommitted and remains uncommitted, until it receives a signal to develop into a specialized cell.

- One novel approach to restore tooth structure is based on biology: “Regenerative

Endodontics” procedures by application of tissue engineering.

- Regenerative endodontics is a biological procedure designed to replace the diseased, missing, and traumatized tissue including dentin and root structures as well as cells of pulp-dentin complex.

Current strategies for treatment of lost tissue

- Currently, the replacement of lost or deficient tissues involves prosthetic materials, drug therapies, and tissue and organ transplantation.

- However, all of these have limitations, including the inability of synthetic prostheses to replace the structural functions of a tissue.

- It includes: 1. Autografts 2. Allografts 3. Synthetic materials (Xenografts)

- But all these materials have their own limitations. Therefore, the gold standard to replace a lost or damaged tissue is the same natural healthy tissue, which has led to the concept of regenerating new tissue from pre-existing tissue.

Tissue Engineering

- Tissue engineering is a multidisciplinary field. - Tissue engineering is the use of a combination of cells, engineering materials, and

suitable biochemical factors to improve or replace biological functions. - Probably the first definition of tissue engineering was by Langer and Vacanti who

stated it to be “an interdisciplinary field that applies the principles of engineering and life sciences towards the development of biological substitutes that restore, maintain, or improve tissue function or a whole organ”.

- MacArthur and Oreffo defined tissue engineering as “understanding the principles of tissue growth, and applying this to produce functional replacement tissue for clinical use”.

Strategies of stem cell technology

- There are three strategies to stem cell technology. ( figs 37.1A to C ) • Conductive • Inductive • Cell base transplantation

- These approaches all typically utilize a material component, although with different

goals.

Conductive

- Conductive approaches utilize biomaterials in a passive manner to facilitate the growth or regenerative capacity of existing tissue.

- An example of this is guided tissue regeneration in which the appropriate use of barrier membranes promotes predictable bone repair and new attachment with new formation of cementum and periodontal ligament fibers.

- Conductive approach utilizes biomaterial in a passive manner to facilitate the growth or regenerative capacity of existing tissue.

- Example: Application of Ca(OH)2 uses conductive approach.

- Limitation of conductive approach is that it is not predictable.

Inductive

- The second major tissue engineering strategy (induction) involves activating cells in close proximity to the defect site with specific biological signals like BMPs.

- Urist first showed that new bone could be formed at non-mineralizing, or ectopic, sites after implantation of powdered bone (bone demineralized and ground into fine particles).

- Contained within the powdered bone were proteins (BMPs), which turned out to be the key elements for inducing bone formation.

- Limitation of this technique is that the inductive factor for a particular tissue may not be known.

Cell Transplantation

- This approach involves direct transplantation of cells grown in the laboratory. ( fig. 37.2 )

Triad of tissue engineering ( fig. 37.3 )

- 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: - Biological scaffolds (e.g. collagen, glycosaminoglycan) - Artificial scaffolds (e.g. PLA, PGA, PLGA).

Stem Cells/Progenitor Cells

- Adult stem/progenitor cells reside in a variety of tissues. - Tissues are composed of cells, insoluble extracellular matrix (ECM) and soluble

molecules that serve as regulators of cell function. - Stem cells are undifferentiated cells which divide and respond into specialized cell

on response to morphogens. ( fig. 37.4 ) - Stem cells are commonly defined as either embryonic/fetal or adult/postnatal. - The term embryonic is preferred to fetal, because the majority of these cells are

embryonic and the term postnatal is preferred to adult because these same cells are present in babies, infants and children.

- The differentiation between embryonic and postnatal is because these cells have different potential for developing into various specialized cells (i.e. plasticity).

- Studies indicate that postnatal stem cells are more plastic than the embryonic stem cells.

- Unique Characteristics of Stem Cells A. They exist as undifferentiated cells and maintain this phenotype by the

environment and/or the adjacent cell populations until they are exposed to and respond to the appropriate signals.

B. Ability to self replicate for prolonged periods C. Maintain their multiple differentiation potential throughout the life of the

organism.

Stem Cells are Often Categorized by Their Source

1. Autologous cells are obtained from the same individual to whom they will be reimplanted. Advantage of autologous stem cells is that they have minimum problems with rejection and pathogen transmission, however, the disadvantage is limited availability.

2. Allogenic cells are obtained from the body of a donor of the same species. 3. Xenogenic cells are those isolated from individuals of another species. In

particular animal cells have been used quite extensively in experiments aimed at the construction of cardiovascular implants.

4. Syngeneic or isogenic cells are isolated from genetically identical organisms, such as twins, clones, or highly inbred research animal models.

- Primary cells are from an organism. - Secondary cells are from a cell bank.

Progenitor Cells

These cells retain the differentiation potential and high proliferation capability but have lost the self-replication property unlike stem cells. ( fig. 37.5 )

Types of Stem Cells

1. Embryonic stem cells (pluripotent) 2. Fetal stem cells (multipotent) 3. Adult stem cells (multipotent)

- Embryonic stem cells can be isolated from normal blastocyst, the structures formed at about 32 cell stage during embryonic development.

- Adult stem cells can be collected directly from the bone marrow, from umbilical cord blood and from circulating blood of individuals receiving cytokines which mobilizes stem cells.

- Stem cells defined by their capacity for asymmetric division, where in a single cell division results in one cell identical to mother cell and another more differentiated cell, thus maintaining the stem cell population even after differentiation.

Dental pulp stem cells (DPSCS) - Although the regenerative capacity of the human dentin-pulp complex is not well

understood, it is known that, upon injury, reparative dentin is formed as a protective barrier for the pulp.

- Accordingly, one might anticipate that dental pulp contains the dentinogenic progenitors, i.e dental pulp stem cells (DPSCs) that are responsible for dentin repair.

- 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.

- Stem cell properties of human dental pulp stem cells: 1. Self-renewal capability. 2. Multilineage differentiation capacity. 3. Clonogenic efficiency of human dental pulp stem cells (DPSCs). 4. DPSCs were capable of forming ectopic dentin and associated pulp tissue in

vivo.

Stem cells from human Exfoliated deciduous teeth (SHED)

- The exfoliated deciduous tooth contains living pulp remnants consisting of connective tissue, blood vessels, and odontoblasts.

- This tissue contains special kind of cells known as SHED (stem cells from human exfoliated deciduous teeth).

- SHED can differentiated into odontoblast-like cells that form small dentinlike structures.

- SHEDs are distinctive from DPSCs with respect to odontogenic differentiation and osteogenic induction.

Periodontal ligament stem cells (PDLSCS)

- The periodontal ligament (PDL) connects the cementum to alveolar bone, and functions primarily to support the tooth in the alveolar socket.

- A recent report identified stem cells in human PDL (PDLSCs) and found that PDLSCs implanted into nude mice generated cementum/PDL-like structures that resemble the native PDL as a thin layer of cementum that interfaced with dense collagen fibers, similar to Sharpey’s fibers.

- Thus, the PDLSCs have the ability of forming periodontal structures, including the cementum and PDL.

Morphogens/signaling molecules

- Morphogens are extracellularly secreted signals governing morphogenesis during epithelial-mesenchymal interactions.

- These are biological factors that regulate stem cells to form the desirable cell type. - They are injected alone or bound to a biomaterial used as delivery system.

Functions

1. To stimulate division of neighboring cells and those infiltrating the defect (Example: Growth factors - PDGF)

2. To stimulate the differentiation of certain cells along a specified pathway (Example: Differentiation factors - BMP)

3. To stimulate angiogenesis 4. To serve as chemoattractants for specific cell types.

Different types of morphogens are:

1. Bone morphogenic proteins (BMPs) 2 . Insulin-like growth factor (IGF)

Scaffold / matrix

- The scaffold provides a physicochemical and biological threedimensional microenvironment for cell growth and differentiation, promoting cell adhesion and migration.

- The scaffold serves as a carrier for morphogen and for cells. - Natural scaffold are proteic materials such as collagen or fibrin, and polysaccharide

materials, like chitosan or glycosaminoglycans which offer good biocompatibility and bioactivity, and synthetic scaffold can elaborate physicochemical features

such as degradation rate, microstructure, and mechanical strength. - Commonly used synthetic materials are polylactic acid (PLA), polyglycolic acid (PGA),

and their copolymers, polylacticco- glycolic acid (PLGA) and polycaprolactone (PCL).

- Synthetic hydrogels include poly ethylene glycol (PEG) based polymers. - Scaffolds containing inorganic compounds such as hydroxyapatite and calcium

phosphate are used to enhance bone conductivity.

Requirements of a Scaffold

1. It should be effective for transport of nutrients, oxygen, and waste. 2. It should be gradually degraded and replaced by regenerativetissue, retaining the

feature of the final tissue structure. 3. It should be biocompatible, nontoxic, and should have proper physical and

mechanical strength. 4. Easy cell penetration, distribution and proliferation. 5. Permeability of culture medium. 6. In vivo vascularization (once implanted). 7. Maintenance of osteoblastic cell phenotype. 8. Adequate mechanical stiffness. 9. Ease of fabrication.

Calcium Hydroxide

1. As direct capping agent induces formation of reparative dentinal bridge 2. As indirect capping agent, it brings about formation of reactionary dentin.

Biologic properties of Ca(OH)2 ↓

High alkaline pH induces burn of limited amplitude at surface of exposed pulp ↓

Below the scar, once inflammatory process has resolved , reparative cells are recruited in central part of pulp

↓ Migration to the site

↓ Proliferation and differentiation

New Bioactive Molecules

1. Bone sialoprotein 2. BMP-7 3. Amelogenin gene splice products A M + 4, A – 4. 4. Dentin phosphoprotein (DPP) 5. Dentin matrix protein (DMP – 1)

- These stimulate the formation of thick homogenous dentinal bridge in contrast to dentinal bridge after Ca(OH)2 pulp capping. ( fig. 37.12 )

Different Strategies Used for Regenerative Endodontics

Definition - Regenerative endodontic procedures are biologically based procedures designed to

replace damaged structures, including dentin and root structures, as well as cells of the pulp-dentin complex.

- Regenerative dental procedures have a long history, originating around 1952, when Dr BW 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.

- Several major areas of research that might have application in the development of regenerative endodontic techniques.

- These techniques are: a. Root canal revascularization via blood clotting, b. Postnatal stem cell therapy, c. Pulp implantation, d. Scaffold implantation, e. Injectable scaffold delivery, f. Three-dimensional cell printing, and g. Gene delivery.

Revascularization to induce apexification/apexogenesis in infected, nonvital, immature tooth

- Trauma to teeth during development may lead to open apex and blunderbuss

canals. If vital pulp is present, apexogenesis is the best option. - But if teeth are nonvital, several treatment challenges are there:

I. Adequate mechanical cleaning/shaping of tooth II. Thin, fragile lateral dentinal walls may fracture during mechanical filing III. Large amount of necrotic debris in a wide root canal is difficult to disinfect. IV. Obturation is problematic V. Surgical procedures with retrograde filling may leave the tooth with an

unfavorable crown root ratio.

Apexification

- It is the method used to induce a calcified barrier in nonvital immature teeth that serves as a matrix against which the root filling material is compacted.

- Conventional materials used for apexification are Ca(OH)2, Ca(OH)2 in combination with CMCP, cresanol, saline or local anesthetic solution, ZnO, Tricalcium phosphate, collagen, calcium phosphate gel, osteogenic protein I and II, MTA,etc.

Calcium Hydroxide

- It has been the most often advocated material for this purpose.

Disadvantages i. Variability of treatment time ii. Patient compliance for attending the recalls iii. Although the open apex might be “closed” by a calcific barrier, apexification

does not promote the continueddevelopment of the root. iv. Because the pulp canal space is physically occupied by the material, there is no

room for vital tissue to proliferate v. Calcium hydroxide treatment will have short roots with thin dentinal walls and a

high risk of fracture vi. Its high pH may cause necrosis of tissues that can potentially differentiate into

new pulp vii. May make teeth brittle because of its proteolytic and hygroscopic properties viii. Barrier formation is often porous and noncontinuous.

Mineral Trioxide Aggregate

- A new technique has been proposed to decrease the time to create a bridge at the apex.

- After disinfection of the canal, MTA is placed in the apical third of the immature root to create a stop for the filling material. This technique will also not allow new tissue to grow into the root canal, and the root remains thin and weak.

- Hence, apexification would not lead to continued root formation or thickening of the root canal wall, leading to the risk of an undesirable side effect of a short and weakened root that is susceptible to fracture.

- An alternative treatment regime is preferred to overcome these problems, i.e. pulp revascularization.

Pulp Revascularization

Definition - Revascularization is the procedure to re-establish vitality in a nonvital tooth to allow

repair and regeneration of tissue.

Advantages - Revascularization will allow further development of root and dentin structure with a

better long-term prognosis. - It is favored when apical foramen is not completely formed and the apical diameter

exceeds 1 mm.

Pulp Revascularization in Immature Teeth

- Once the canal infection is controlled, it resembles avulsed tooth that has a necrotic but sterile pulp canal space.

- Blood clot is then introduced so as to mimic the scaffold that is in place with ischemic necrotic pulp as in avulsed tooth and access cavity is restored with a bacteria tight seal.

- In teeth with open apices and necrotic pulp, it is possible that some vital pulp tissue and Hertwig’s epithelial root sheath remain.

- When canal is disinfected, inflammatory process reverses and these tissues may proliferate.

- It depends mainly on: i. Disinfection of root canal. ii. Placement of matrix in canal for tissue ingrowth. iii. Bacteria tight seal of access opening.

Indications - The teeth that present with symptoms of acute or chronic apical periodontitis (i.e.

pain, diffuse facial and/or mucosal swelling, tenderness to percussion, or intraoral sinuses).

- Radiographically, the tooth had an immature apex, either blunderbuss or in the form of a wide canal with parallel walls and an open apex.

- On electric pulp test the affected tooth is nonvital.

Techniques - The tooth is anesthetized with a local injection with epinephrine and is isolated with

rubber dam. - The access cavity is prepared with a round diamond and Endo-Z bur - The canal is gently flushed with 20 mL of 5.25% sodium hypochlorite (NaOCl)

solution.

A. For teeth with chronic apical periodontitis (vital pulp) i. The canal is dried with sterile paper points and etching and dentin bonding agent

light curing is done followed by application of a flowable composite. A mixture of ciprofloxacin 250 mg, metronidazole 250 mg, and minocycline 250 mg, is placed in the sterile root canal 2 mm from the working length and left for 7 days.

ii. In the next visit, the antibiotic dressing material is removed by rinsing with 5.25 percent NaOCl, if the tooth is symptom free. The canal is dried with sterile paper points and is confirmed to have no exudate.

iii. A size #40 K-file is introduced into the root canal until vital tissue is felt, and this instrument is used to irritate this tissue to create some bleeding into the root canal.

iv. The bleeding is allowed to reach a level 3 mm below the cementoenamel junction, and tooth is left for 15 minutes so that a blood clot is formed. Then a grey mineral trioxide aggregate is placed over the clot carefully upto the level of CEJ followed by a wet cotton pellet and restored with a temporary dressing material.

Mechanism of Revascularization

i. Few cells remain at the apical end of the root canal. These cells might proliferate into the newly formed matrix and differentiate into odontoblasts under the organizing influence of cells of Hertwig’s epithelial root sheath, which are quite resistant to destruction, even in the presence of inflammation. The newly formed odontoblasts can lay down a tubular dentin at the apical end, causing apexogenesis as well as on lateral aspects of dentinal walls of the root canal, reinforcing and strengthening the root.

ii. Continued root development due to multipotent dental pulp stem cells, which are

present in permanent teeth and might be present in abundance in immature teeth. These cells from the apical end might be seeded onto the existing dentinal walls and might differentiate into odontoblasts and deposit tertiary or atubular dentin.

iii. Stem cells in the periodontal ligament can proliferate, grow into the apical end and within the root canal, and deposit hard tissue both at the apical end and on the lateral root walls.

iv. Root development could be attributed to stem cells from the apical papilla or the

bone marrow. Instrumentation beyond the confines of the root canal to induce bleeding can also transplant mesenchymal cells from the bone into the canal lumen. These cells have excellent proliferative capacity. Transplantation studies have shown that human stem cells from bone marrow can form bone or dentin in vivo.

v. Blood clot itself being a rich source of growth factors could play important role regeneration. These include platelet-derived growth factor, vascular factor, tissue growth factor and could stimulate differentiation, growth and maturation of fibroblasts, odontoblasts, cementoblasts, etc. from the immature undifferentiated mesenchymal cells in the newly formed tissue matrix.

Advantages of Revascularization Procedure

1. Short treatment time. 2. The approach is technically simple and can be completed using currently available

instruments and medicaments without expensive biotechnology. 3. The regeneration of tissue in root canal systems by a patient’s own blood cells

avoids the possibility of immune rejection and pathogen transmission from replacing the pulp with a tissue engineered construct.

4. Cost-effective. 5. Obturation of canal not required. 6. Continued root development and strengthening.