Tissue engineering in periodontics

Regeneration of the lost periodontal tissues is the ultimate goal of a successful periodontal therapy. A lot of techniques have been used to achieve this goal so far. More recent introduction in this context is the concept of tissue engineering. Tissue engineering is defined as the science of fabrication of new tissues for replacement and regeneration of lost tissues or defined tissues 1. This approach for regeneration of the lost tissues was proposed by Langer and Vacanti in 1993 2. The primary aim of this therapy is to……………..

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Key elements in tissue engineering

To achieve complete regeneration of the lost tissue, we need to have progenitor cells which can differentiate into desired cell types and give rise to desired structural components of extracellular matrix (ECM) and signaling molecules which initiate desired cellular activities, a scaffold which is able to carry these components in the active form and a conducive environment (Figure 69.1). Presently, there are two approaches used to regenerate tissues,

Figure 69.1 Key elements in periodontal regeneration

Key elements in periodontal regeneration

Ex vivo approach:

In this approach, the tissue is created in a laboratory by culturing the cells on a biodegradable scaffold in the presence of molecular factors required for growth and then it is transferred into the body.

In-vivo approach:

In this technique all the components which are required for regeneration are placed in the tissue defect and an environment which is conducive to maximum regeneration is created to achieve favorable regeneration.

To achieve any kind of tissue regeneration, some basic components are required, which participate at different levels in the formation of desired tissue. These can be called as key elements of tissue engineering. These include,

  • Progenitor cells
  • Scaffold or supporting matrix
  • Signaling molecules

Progenitor cells

There has been a lot or research on the regenerative capacity of post-natal progenitor cells. These cells can differentiate into different types of end cells and can form the desired structural components of the lost tissue. However, these cells should satisfy the following criteria to achieve effective, long-lasting repair of damaged tissues 4,

  1. An adequate number of cells must be produced to fill the defect.
  2. Cells must be………………….

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The source of these progenitor cells may be autologous, allogeneic or xenogeneic. Autologous or autogenic source (derived from the patient) is an ideal source of these cells because of low association with immune complications and almost complete acceptability. Allogeneic source (derived from another member of the same species) of these cells is also commonly used and has an advantage of uniformity, standardization of procedure, quality control and cost-effectiveness over the autogenous source. Xenogeneic (derived from other species) sources have an advantage of unlimited supply but carry a greater risk of immunological reaction.

Progenitor cell population in periodontal tissues:

The first evidence for the presence of undifferentiated mesenchymal within periodontal tissues was provided by McCulloch and co-workers (1987) 5. Most of the present literature on periodontal regeneration has discussed periodontal ligament-derived cells for their potential to differentiate into various different types of cells required for the regeneration of lost tissues. However, other sources including periodontal ligament-derived mesenchymal stromal cells, periosteal cells, bone marrow-derived mesenchymal stem cells (BM-MSCs), adipose-derived stem cells (ADSCs), ES/iPS cells and gingival fibroblasts have also been investigated recently.

Periodontal ligament (PDL) derived progenitor cells:

The ability of PDL-derived progenitor cells in periodontal regeneration was discovered way back when research in this field had just started. The PDL stem cells have the capability to produce cementum and periodontal ligament-like structure and contribute to periodontal tissue repair. It has been shown that PDL stem cells can differentiate into cementoblast-like cells, adipocytes and collagen-forming cells 6.  Procedures like guided tissue regeneration are based on the fact that these cells should be allowed to proliferate in the area of the periodontal defect to differentiate into different cells required for regeneration.

Periodontal ligament (PDL) derived mesenchymal stromal cells:

The mesenchymal stem cells were initially identified in aspirates of adult bone marrow. Later on, Friedenstein et al. (1970, 1976, 1987) 7-9 in their experiments developed………….

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Periosteal cells:

Along with differentiation into an osteoblastic lineage, periosteal cells also have the capability to express PDL related genes. In an experiment, it was observed that these cells were clonogenic, displayed long telomeres and expressed markers of mesenchymal stem cells (MSCs), regardless of donor age 11. In another experiment Mizuno et al. (2006) 12 repaired Class III furcation defects in Beagle dogs by grafting autologous periosteal cells, which was cultured from membrane derived from the periosteum. In another experiment, Yamamiya et al. (2008) 13 showed that when combined with platelet-rich plasma and hydroxyapatite, cultured periosteum demonstrated clinical improvement in human infrabony defects.

Bone marrow-derived mesenchymal stem cells (BM-MSCs):

These cells have a potential to differentiate into various target cells, which are capable of producing bone, cartilage, adipose, muscle, and periodontal tissues etc. 14-17. Recently, it has been demonstrated by in vitro studies that these cells are capable of inducing periodontal regeneration 16, 18. In another experiment, BM-MSCs were used in combination with platelet-rich plasma (PRP). The results of the study demonstrated that this combination was capable of inducing periodontal regeneration 19.

Adipose-derived stem cells (ADSCs):

Recent research has reported the potential of ADSCs to be used in periodontal tissue regeneration. One major advantage of ADSCs is that adipose tissue from where these cells are obtained is abundant and easy to obtain as compared to other sources. These cells have been shown to differentiate into different tissue types 20-23.  In vitro experimental studies have shown that the combination of PRP and ADSCs was capable of inducing periodontal regeneration in bony defects 24, 25. Hence, these cells need to be further investigated for their potential for periodontal regeneration.

Gingival fibroblasts:

Cell transplantation therapy using gingival fibroblasts has been developed for root coverage in areas with recession. The gingival fibroblasts were seeded onto sponges of human type I or III recombinant collagen. After culturing, vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) were released in the culture media. It was observed that there was a significant increase in fibroblast proliferation indicating that the technique may provide a new tool for the treatment of gingival recession 26.  In another study 27, 14 recession sites in 4 patients were transplanted with autologous gingival fibroblast sheets to achieve root coverage. The results of the study demonstrated regeneration of gingival tissue in recession areas. It was concluded that gingival fibroblasts cloud be easily harvested and prepared for transplantation in areas with gingival recession.

Know more………..

Present status of PDL-derived mesenchymal stem cells (PDL-MSCs) in periodontal regeneration:

The present evidence strongly suggests that PDL-MSCs are capable of enhancing regeneration of periodontal tissue, including alveolar bone, cementum and PDL 28. MSCs derived from other sources than PDL may not have similar properties as that of PDL-derived MSCs. For example, MSCs derived from PDL have been shown to exhibit a greater regenerative capacity for PDL tissue as compared to bone marrow derived MSCs 29. PDL-MSCs can be easily obtained during tooth extraction, but cannot be obtained from patients who lack teeth to be extracted. However, recent studies have demonstrated that PDL-MSCs can be obtained from inflamed PDL tissue 30. During periodontal treatment, the inflamed periodontal tissue can be obtained and PDL-MSCs can be extracted. However, the technique of doing this procedure is still under investigation.

 

Scaffold or supporting matrix

Scaffolds are natural or synthetic material used to carry biologically active molecules to the site of regeneration. Tissue engineering scaffolds have been fabricated using several natural and synthetic polymers. The primary requirements for a scaffold are,

  • It must be inherently biocompatible.
  • It should be…………….

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Naturally derived scaffold materials:

The natural polymers used for tissue engineering applications include fibrin, collagen, gelatin, chitosan, alginate, and hyaluronic acid 31-36.

Fibrin: It is an important component of the blood clot and is used in mixtures with thrombin to produce an in-situ forming gel which is used as a scaffold to carry various biologically active molecules.

Collagen: It is one of the most widely used scaffold material. Type I collagen is usually derived from animal tissues and gelatin.

Chitosan: It is a cationic polymer derived from chitin. Its scaffold produces a hydrophilic surface which is osteoconductive which indicates its potential use for bone tissue engineering.

Alginate: It is an anionic polysaccharide derived from brown algae. It forms a gel when complexed with divalent cations such as Ca2+.

Hyaluronic acid: It is a non-sulfated glycosaminoglycan of repeating disaccharide units. It is a major component of connective tissue, forming cross-linkable hydrogels with various modifications.

Synthetically derived scaffold materials:

These are biodegradable synthetic polymers which have matrices in their structure to carry bioactive molecules. The commonly used chemical compounds to fabricate synthetic scaffolds include poly(α-hydroxyester)s, polyanhydrides, and polyorthoesters 37.  Among these polymers, poly(α-hydroxyester)s such as…………….

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These polymers can also be fabricated in the form of microspheres and can be injected at the site of the defect. Furthermore, these can also be used to make scaffolds with nanofibrous structure. The disadvantage of these polymers is that when they degrade, they produce acidic by-products which may hamper the process of regeneration. The solution to this problem is the use of biodegradable hydrogel scaffolds fabricated with hydrophilic polymers such as poly (ethylene glycol), which do not produce acidic by-products on degradation.

Scaffold fabrication:

There are various methods to make scaffolds which are then used to carry bioactive molecules. These include,

  • Fiber bonding
  • Emulsion freeze drying
  • Solvent casting/particulate leaching
  • High-pressure processing
  • Gas foaming/particulate leaching
  • Thermally induced phase separation
  • Electrospinning
  • Rapid prototyping

Fiber bonding:

In this technique, interconnected fiber networks within the matrix are achieved. The PGA fibers are aligned in the shape of the desired scaffold and then embedded in a Poly (L -lactide) /methylene chloride solution. Once the solvent evaporates, Poly (L-lactide)/PGA composite is heated above the melting temperatures of both the polymers. Poly (L-lactide) is then removed by selective dissolution after cooling. PGA fibers are then physically joined at their cross-points, making a cross-linked network. One disadvantage of this technique is that the matrix porosity achieved cannot be precisely controlled.

Emulsion freeze drying:

In this technique, an emulsion solution containing a dispersed water phase and an organic continuous phase is freeze dried. It results in the formation of a porous scaffold with various pore sizes and inter-connectivities. Using this technique, up to 95% porous scaffold with a pore size up to 200 μm has been prepared 38.

Solvent casting/particulate leaching:

Another method to create pores involves the use of a water-soluble porogen, such as salt (NaCl). In this technique, the polymer (PLLA or PLGA) is first dissolved in chloroform or methylene chloride and then is casted onto a petri dish filled with the porogen. Once the solvent evaporates, the polymer/salt composite is leached in water for two days to remove the porogen. The amount of porosity depends on the quantity of salt added and the pore size depends on the crystal size of the salt particles.

High-pressure processing:

In this technique, a gas such as CO2 is applied at high pressure to the dry polymer. It results in the formation of a single phase polymer/gas solution. After the formation of this single phase polymer/gas solution, pressure is reduced, which creates thermodynamic instability of the dissolved CO2 and results in nucleation and growth of gas cells to generate pores within the polymer matrix. This technique has been used to make highly porous sponges of PLGA 39.

Gas foaming/particulate leaching:

This technique was developed by Park et al., where they made a binary solution of PLA-solvent gel containing dispersed ammonium bicarbonate salt particles. The mixture was casted in a…………….

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Thermally induced phase separation:

This procedure involves phase separation by thermodynamic demixing of a homogeneous polymer-solvent solution into a polymer-rich phase and a polymer-poor phase. Liquid-liquid phase separation or emulsification/freeze-drying method is used to separate the two phases. The polymer solution is quenched below the freezing point of the solvent and subsequently freeze dried. It results in the formation of a highly porous structure. The porosity of the structure can be modified by changing the thermodynamic and kinetic parameters.

Electrospinning:

This is the most widely used method for preparation of nanofiber non-woven matrices. In this technique, a polymer solution is pumped at a constant rate through a syringe with a small-diameter needle that is connected to a high-voltage source. When this voltage source is turned on, an electric field is created between the needle and a metallic collecting plate. Under the strong electrical field, electric charge overcomes the surface tension of the polymer solution droplet. Then, a polymer jet is sprouted from the nozzle followed by solvent evaporation which forms the solid nanofibers. With this technique, a highly porous three-dimensional scaffold is formed.

Rapid prototyping:

In this method, a computer aided design (CAD) with pre-decided three-dimensional architecture is formed in a layer-by-layer manner with precise control over its morphological characteristics. This is the most recent introduction in the field of tissue engineering and is being extensively investigated for fabrication of scaffolds for carrying bioactive molecules. The main advantage of this technique is that a scaffold with predetermined size, shape, porosity, chemical composition and desired mechanical properties can be fabricated.

Signaling molecules:

The signaling molecules play a vital role during various biological processes. They are secreted from various cells in response to a stimulus and they act on the same, neighboring or distant cells to cause specific effects. The knowledge of signaling molecules involved in regeneration is primarily derived from wound healing. A detailed description of biological events that happen during wound healing has been discussed in chapter 60 “Biology of periodontal regeneration”.

The application of…………………

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Presently, some recombinant  growth  factors have been made available for commercial use, including platelet-derived  growth  factor  (PDGF;  GEM21®)  and bone  morphogenic protein-2 (BMP-2;  Infuse®) 43. The potential of fibroblast growth factor (FGF)-2 has recently been investigated for its efficacy in periodontal regeneration in large clinical trials 24, 41. These studies warrant further research on this growth factor and its clinical use on a large scale. Table 69.1 describes various studies that have been done to evaluate the efficacy of various scaffolds used with signaling molecules in achieving periodontal regeneration.

Table 69.1 Studies evaluating the efficacy of various scaffolds used with signaling molecules in achieving periodontal regeneration

Scaffold used
Growth factor incorporated
Experimental model
Study
Bone grafts
• Bone allograft

rhPDGF-BB

Human

Nevins et al. (2003) 44
Bioactive ceramics
• Calcium carbonate carrier
• β- tricalcium phosphate

• Hydroxyapatite (HA)

TGF- β 1
rhPDGF-BB
rhGDF-5
rhBMP-2

Dog
Human
Monkey
Dog

Tatakis et al. (2000) 45
Sarment et al. (2006) 46
Emerton KB et al. (2011) 47
Wiskesjo et al. (2002) 48
Naturally occurring polymers
• Porous chitosan/ collagen scaffolds
• Methylcellulose gel

• Hydroxypropyl cellulose

PDGF or BMP-7gene
rhPDGF-BB/rhIGF-1

FGF-2

Dog
Human

Human

Zhang et al. (2009) 49
Lynch et al. (1991) 50

Kitamura et al. (2008, 2011) 51, 41
Synthetic polymers
• PLA-PGLA copolymer

• Pluronic F127

rhBMP-2

TGF- β 1

Mouse

Sheep

Saito et al. (2001) 52

Mohammed et al. (1998) 53
Hydrogels
• Dex-GMA gelatin gel
• Collagen gel
• Gelatinous carrier

rhBMP-2
FGF-2
FGF-2

Dog
Dog
Monkey

Chen et al. (2007) 54
Sato et al. (2004) 55
Takayama et al. (2001) 56

Desired properties of scaffolds used for periodontal regeneration

To achieve regeneration in the periodontal defects, the biomaterial placed in the defect should have some desirable properties. The scaffold material should allow cell-cell and cell-matrix interaction of bioactive molecules (such as growth factors) with the surrounding environment. The scaffold should hold the growth factor in its matrix for a desirable duration of time so that the growth factor can exert its effect on the surrounding cells and matrix for long enough. The scaffold should be completely compatible with the surrounding tissues and should allow cellular proliferation on its surface. Scaffolds designed to carry stem cells should not induce environmental changes (such as pH, pO2 oxidative stress, etc.) which are not in favor of regeneration in the areas, where it is placed.

Current strategies for periodontal regeneration

The current methods to achieve periodontal regeneration are based on using a scaffold which may or may not carry biologically active molecules in periodontal defect (bone grafts or hard tissue replacement polymer) or creating a three-dimensional area secured by a membrane to allow the proliferation of cells from PDL to facilitate regeneration (guided tissue regeneration). Presently, research is being done to incorporate various biologically active molecules (BMP’s and growth factors) in scaffolds which are placed in the periodontal defect to achieve maximum regeneration. The scaffolds used so far in periodontal research include bone grafts, synthetic polyesters such as polyglycolic acid, polylactic acid and polycaprolactone and natural polymers such as collagen fibrin, albumin, hyaluronic acid, cellulose, chitosan, polyhydroxyalkanoates, alginate, agarose and polyamino acids.

Studies evaluating the efficacy of various scaffolds used with signaling molecules in achieving periodontal regeneration

Bone grafts:

Bone grafts can be autogenic, allogeneic or xenogeneic depending on its source from where it is derived from. The biological properties of bone grafts can be described by three interrelated but not identical terminologies: osteogenic (formation of new bone by stem cell lineage derived from graft material); osteoinductive (bone growth by the surrounding immature cells recruited by graft material); and osteoconductive (bone growth on the surface of a material with fabrication) 58. Autogenous bone grafts are considered as gold standard because of their osteogenic and osteoinductive properties and complete acceptance. A detailed description of bone grafts has been given in the chapter 63,“Bone grafts in periodontics”. The tissue engineering approach in using bone scaffold is to incorporate osteogenic factors like bone morphogenetic proteins (BMP’s) and other growth factors which induce bone formation when placed in periodontal defects.

Barrier membranes:

Barrier membranes are used to achieve regeneration by selectively allowing the growth of cells derived from PDL. Both biodegradable and non-degradable membranes have been used extensively to achieve periodontal regeneration. A detailed description of these membranes has been given in chapter 62, “Guided tissue regeneration”.

Gene therapy in periodontal tissue engineering:

The problem associated with the delivery of proteins (growth factors and other bioactive molecules) is that they get easily degraded in the biological environment due to proteolytic breakdown by host enzymes. Secondly, to exert their effect, these factors should attach to their respective ligands on the target cells. The binding of the growth factors on target cells is not predictable. Their inability to bind to their receptors on the cell surface results in no biological activity by the target cell. Finally, lack of stability of scaffold carrying these molecules further jeopardizes the potential for regeneration.

To overcome these problems, gene therapy………….

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Ribonucleic acid mediated silencing:

Fire and Mello were awarded the Nobel Prize in Physiology and Medicine in 2006 for their discovery of RNA interference (RNAi)- gene silencing by double-stranded DNA 63. It is a biological process in which RNA molecules inhibit the expression of certain genes which are detrimental to the tissue regeneration by causing the destruction of specific mRNA molecules. RNA interference is executed by two types of small RNA molecules – microRNA (miRNA) and small interfering RNA (siRNA). RNAs are the direct products of genes, and these small RNAs can bind to other specific messenger RNA (mRNA) molecules. Most of the RNA-based research done presently uses siRNAs because they are safe and cost-effective. This technique is presently a focus of intensive research to make it useful in clinical practice and making it widely available.

Implantation of live cells to achieve regeneration:

Implantation of living cells in the periodontal defect and achieving regeneration is a challenging task. However, attempts have been made in this direction with varying degree of success. In a study McGuire and Scheyer (2007) 64, implanted autologous fibroblast following a minimally invasive papilla priming procedure to augment open interproximal spaces. The interdental papillary height was assessed using subject visual analog scale. The results were found to be significantly better in test sites as compared to placebo sites. In another experiment Bowsma and D’souza (2005) 65, expanded autologous fibroblasts and injected them in the periodontal pocket. The results of the study demonstrated that in periodontal pockets where autologous fibroblasts were placed, pocket depth reduction was significantly more than placebo sites.

Implantation of tissue engineered human fibroblast-derived dermal substitute:

Human fibroblast-derived dermal substitute (HF-DDS) is……….

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Application of bi-layered cell therapy as a substitute to tissue from palate:

Tissue-engineered bi-layered skin substitutes have been used for immediate replacement of both, lost dermis and epidermis in cases of skin burn or other injuries. This tissue is composed of type I bovine collagen and viable allogeneic human fibroblasts and keratinocytes isolated from human foreskin. The tissue behaves in a similar manner as normal human skin. The proliferation of keratinocytes takes place in the basal layer of the epidermis and fibroblast proliferation takes place in the matrix. The keratinocytes produce various growth factors which facilitate wound healing. In an experiment, Momose et al. (2002) 68 estimated the levels of growth factors, including vascular endothelial growth factor (VEGF),  transforming growth factor-α and β1 (TGF-α and β1), and epidermal growth factor (EGF) in tissue culture of human cultured gingival epithelial sheets (HCGES). The gingival tissues were obtained from the patient with generalized chronic periodontitis while performing flap surgery. It was observed that levels of these growth factors were significantly high in test cell culture media as compared to culture media without cells. Hence, these growth factors influence the surrounding environment in favor of healing. These substitutes have a potential application in periodontal mucogingival surgeries, where they can be used for root coverage or for increasing the width of keratinized gingiva.

Critical analysis of present status of tissue engineering in periodontics

Tissue engineering is one of the most extensively researched fields presently. So far we have succeeded in fabricating scaffolds suitable to deliver various bioactive molecules at the sites where tissue regeneration is required. We can isolate the growth factors or synthesize them in vivo. We can expand cell line in tissue cultures and implant them at the site of interest. However, there are many challenges which need to be addressed. These are,

  • Various growth factors act through intracellular signaling mechanism once they attach to their corresponding surface receptors. Our knowledge of these intracellular mechanisms is still incomplete.
  • The exact mechanism by which the growth factors enhance periodontal regeneration yet remains to be proven in vivo. Although tritiated thymidine and proline labeling studies would yield valuable information regarding in vitro effects of PDGF/IGF-1 69, more research is required in this field.
  • Ideally, once delivered at the site of interest, the growth factors…………….

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Conclusion

There are a lot of questions in the field of tissue engineering, which still need to be answered. Delivery systems which are suitable for the placement in periodontal defects need to be evaluated. More research is required to find out the exact molecular mechanism involved in the action of various growth factors and other bioactive molecules. Particular attention is required to authenticate the clinical usage of gene therapy. Because gene therapy involves changes in the genetic material, i.e., DNA, both pros and cons need to be analyzed thoroughly before this therapy is implemented clinically for periodontal regeneration.

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