Tissue engineering: A new vista in periodontal regeneration.

Tissue engineering is a highly promising field of reconstructive biology that draws on recent advances in medicine, surgery, molecular and cellular biology, polymer chemistry, and physiology. The objective of using tissue engineering as therapeutic application has been to harness its ability to exploit selected and primed cells together with an appropriate mix of regulatory factors, to allow growth and specialization of cells and matrix. The authors reviewed controlled clinical trials which also included histological studies that evaluated the potential of tissue engineering as a clinical tool in regeneration. PubMed/MEDLINE databases were searched for studies up to and including June 2010 to identify appropriate articles. A comprehensive search was designed, and the articles were independently screened for eligibility. Articles with authentic controls and proper randomization and pertaining specifically to their role in periodontal regeneration were included. Studies demonstrated that the periodontal regeneration with the use of combination of tissue engineered products with an osteoconductive matrix improve the beneficial effect of these materials by accelerating cellular in growth and revascularization of the wound site. Studies have suggested the use of rh Platelet-derived growth factor + beta tricalcium phosphate for regeneration of the periodontal attachment apparatus in combination with collagen membranes as an acceptable alternative to connective tissue graft for covering gingival recession defects. The studies concluded that growth factors promote true regeneration of the periodontal attachment apparatus and the use of combination protein therapeutics which is commercially available can provide more predictable, faster, less invasive, less traumatic, and efficient outcome for the patient.

INTRODUCTION

The ultimate goal of periodontal therapy is to completely restore the periodontal attachment including cementum, periodontal ligament, and alveolar bone lost due to periodontal disease or trauma. In the past few decades, many attempts have been made to unravel the “magic filler” material that could result in new clinical and histological attachment, but have only culminated in healing by repair.

Periodontal repair refers to healing that does not allow the original morphological nor functional restoration of the tissue, considered as non-functional scarring.

Periodontal regeneration attributes to a complete recovery of the periodontal tissues in both height and function, that is, the formation of alveolar bone, a new connective attachment through collagen fibers functionally oriented on the newly formed cementum.[1]

Regeneration of the periodontal tissues is a complex phenomenon requiring interplay between various processes in a timely manner.

Tissue engineering was proposed as a possible technique for regenerating lost periodontal tissues by Langer and colleagues in 1993.[2]

Tissue engineering is an interdisciplinary field that applies principles and methods of engineering and the life sciences towards the development of biological substitutes that restore, maintain, and improve the function of damaged tissues and organs.[3]

The goal of tissue engineering is to promote healing, and ideally, true regeneration of a tissue's structure and function, more predictably, more quickly, less invasively, and more qualitatively than allowed by previous passive techniques.[45]

The purpose behind writing this brief review has been to integrate the evidence of research related to tissue engineering so as to implement them in our daily practice.

From passive to active – The tissue engineering approach

Periodontal healing is the most complex process in the human body. The cells of five or more tissue type i.e. the epithelium, gingival and periodontal connective, cementum, bones are essentially asked to create a new connection to the nonvascular and non vital hard tissues of the root surface. Healing of the periodontal tissues is also rendered more complex because it must occur in an open system permanently contaminated and under a significant bacterial load. Added to this complexity are the occlusal forces on the tooth complex in the transverse and the axial planes which affect the stability of the healing wound.[6]

Various approaches like the use of bone replacement grafts, barrier membranes have been used, but none have proved successful in achieving “Restitutio ad integrum” – A complete functional connection.

In wound healing, the natural healing process usually results in tissue scarring or repair. Using tissue engineering, the wound healing process is manipulated so that tissue regeneration occurs.

Whether the damaged tissues heal by regeneration or repair depends upon two crucial factors.

The availability of cell types needed; and

The presence or absence of cues or signals necessary to recruit and stimulate these cells.

The tissue engineering approach to bone and periodontal regeneration combines three key elements to enhance regeneration.

Conductive scaffolds/Extracellular matrix.

Signalling molecules.

Stem/Progenitor cells.[7]

This concept is often represented as a triangle, indicating that by combining the three key elements tissue regeneration can often be accomplished [Figure 1].

Figure 1

The tissue engineering triad

Scaffolds

The scaffold provides a 3D substratum on to which the cells can proliferate and migrate, produce a matrix and form a functional tissue with a desired shape. A suitable bioactive three-dimensional scaffold for the promotion of cellular proliferation and differentiation is critical in periodontal tissue engineering.

A scaffold plays many roles in tissue regeneration process:[8]

It serves as a framework to support cellular migration into the defect from surrounding tissues.

It serves as a delivery vehicle for exogenous cells, growth factors, and genes.

It may structurally reinforce the defect to maintain the shape of the defect.

It serves as a barrier to prevent infiltration of surrounding tissue that may impede the process of regeneration.

Before its absorption, a scaffold can serve as a matrix for exogenous and endogenous cell adhesion and thus facilitates and regulates certain cellular processes, including mitosis, synthesis and migration.

Biomaterials used as scaffolds

Biomaterials used as scaffolds in tissue engineering are classified into two broad categories [Table 1].

Table 1

Scaffolds used for the purpose of periodontal regeneration

Naturally derived.

Synthetic.

Liao et al. in a study compared porous beta-tricalcium phosphate/chitosan composite scaffolds with pure chitosan scaffolds. Composite scaffolds showed higher proliferation rate of human periodontal ligament cells (HPLCs) and up-regulated the gene expression of bone sialoprotein and cementum attachment protein. In vivo, HPLCs in the composite scaffold not only proliferated, but also recruited vascular tissue ingrowth; thus, suggesting the benefit of using these composite scaffolds.[23]

Fabrication of a scaffold

Many methods have been used to produce porous materials to be used as scaffolds for tissue engineering [Figure 2].

Figure 2

Methods of fabrication of scaffolds

The underlying concepts guiding the development of scaffolds can be predicated on the selected biomaterials and/or on the method of production of the scaffold.[24]

Cells

Cell source is an important parameter to consider when applying tissue engineering strategies to restore lost tissues and functions.

Stem cells are immature progenitor cells capable of self renewal and multi-lineage differentiation through a process of asymmetric mitosis that leads to two daughter cells, one identical to the stem cell (daughter stem cell) and one capable of differentiation into more mature cells (progenitor cells).[29]

Stem cells may be:

Totipotent, i.e. early embryonic cells (one to three days from oocyte fertilization), which can give rise to all the embryonic tissues and placenta.

Pluripotent, i.e. embryonic cells from blastocystis (4-14 days after oocyte fertilization), which can differentiate only into embryonic tissues belonging to the inner cell mass (ectoderm, mesoderm, and endoderm).

Multipotent, i.e. embryonic cells from the 14th day onwards, which can give rise to tissues belonging to only one embryonic germ layer (ectoderm or mesoderm or endoderm).[30]

Depending on the development stage of the tissues from which the stem cells are isolated, stem cells can be broadly divided into two categories: Adult stem cells and embryonic stem cells.[31-33]

Embryonic stem cells are derived from embryos that are 2 – 11 days old called blastocysts. They are totipotent cells. Due to ethical concerns and the risk of tumorogenicity and teratoma formation, its use has been restricted to the research field.

Adult stem cells are multipotent stem cells, and depending upon their origin, they can be further classified into hemopoetic stem cells and mesenchymal stem cells. Friedenstein and colleagues first identified mesenchymal stem cells in aspirates of adult bone marrow.[34] Among the adults stem cells, bone marrow–derived stem cells or mesenchymal stem cells (MSCs) are adherent, proliferating, and capable of multi-lineage differentiation having the capability of differentiating into multiple tissue types, including bone, cartilage, muscle, tendon, etc., and hold great potential for autologous cell-based therapy.[33]

Another important characteristic of MSCs for regenerative medicine is their potential allogenic use without immunosuppressive therapy.[35] Within the sphere of periodontal tissue engineering, mesenchymal derived cells have been applied for simultaneous regeneration of the attachment apparatus components.

Signals

Signaling molecules are proteins that may act locally or systemically to affect the growth and function of cells in various manners. The two types of signaling molecules that have received the greatest attention are growth factors and morphogens that act by altering the cell phenotype i.e. by causing the differentiation of stem cells into bone forming cells - a process commonly known as osteoinduction.

These cytokines have pleotropic effects some of which include

Mitogenic (proliferative);

Chemotactic (stimulate directed migration of cells); and

Angiogenic (stimulate new blood vessel formation) effects.[36]

Growth factors act on the external cell membrane receptors of a target cell, provide the signal to local mesenchymal and epithelial cells to migrate, divide, and increase matrix synthesis. The growth factor that has received the most attention in hard and soft tissue wound healing is platelet derived growth factor. Various potential growth factors and their sources have been summarized in Table 2.

Table 2

Various growth factors and their sources

Platelet-derived growth factor

Platelet-derived growth factor (PDGF) is the natural wound healing “hormone”. It is naturally produced by the body at sites of soft tissue and bone injury. It was discovered by Lynch and coworkers in the late 1980s.[37]

While PDGF secreted from platelets play an important role in initial wound healing, its subsequent secretion from macrophages continues the events of wound healing through up-regulation of other growth factors and cells that ultimately promote fibroblastic and osteoblastic functions.[38]

Moon et al. applied PDGF-BB to promote migration and proliferation of periodontal ligament fibroblasts. They demonstrated that PDGF has the capacity to stimulate bone formation and periodontal regeneration in vivo and indicate that it holds promise as an important adjuvant to periodontal surgery.[39]

Insulin like growth factor

Insulin like growth factor (IGF) is a potent chemotactic agent for vascular endothelial cells resulting in increased neovascularization. It also stimulates mitosis of many cells in vitro such as fibroblasts, osteocytes, and chondrocytes.[40] Insulin like growth factor-I is found in substantial levels in platelets and is released during clotting along with the other growth factors.

Han and Amar demonstrated that in vitro IGF-I substantially enhanced cell survival in periodontal ligament fibroblast compared to gingival fibroblasts by the up-regulation of anti-apoptic molecules and down-regulation of pro-apoptotic molecules.[41]

Transforming growth factor family

The two best characterized polypeptides from this group of growth factors are Transforming growth factor family (TGF)-α and TGF-β. TGF-β appears to be a major regulator of cell replication and differentiation. Three forms of TGF-β have been identified namely TGF-β1, TGF-β2, and TGF-β3. TGF-β isoforms have multiple regulatory roles in the synthesis, maintenance and turnover of the extracellular matrix. TGF-β is chemotactic for fibroblasts and cementoblasts, and promotes fibroblast accumulation and fibrosis in the healing process. It can also modulate other growth factors such as PDGF, TGF-α, and EGF and fibroblast growth factor (FGF) possibly by altering their cellular response or by inducing their expression.[42]

Oates et al. compared the mitogenic activity of TGF-β with interleukin-1 and PDGF in fibroblast cells derived from periodontal ligament explants. TGF-β was relatively a weak mitogen for Periodontal (PDL) cells compared to PDGF, suggesting that TGF-β may indirectly stimulate DNA synthesis.[43]

Fibroblast growth factor family

Fibroblast growth factors are the members of heparin binding growth factor family. The two most thoroughly characterized forms are: Basic FGF (bFGF) and acidic FGF (aFGF). Both aFGF and bFGF are single chain proteins that are proteolytically derived from different precursor molecules to generate biologically active proteins of 15,000 molecular weight. They promote proliferation and attachment of endothelial cells and PDL cells in wound healing process. FGF-2 is known to attract epithelial cells more effectively than FGF-1.[44]

Kitamura et al. did a recent randomized clinical trial trying to evaluate the therapeutic response to varying doses of FGF-2 (bFGF). They demonstrated a significant increase in the alveolar bone height on using 0.3% FGF-2.[45]

Takayama et al. examined the efficiency of topical application of FGF-2 with periodontal regeneration in the bony defects by surgically creating furcation class II bone defects in non-human primates and concluded that a topical application of FGF-2 can enhance considerable periodontal regeneration.[46]

Hepatocyte growth factor

Hepatocyte growth factor (HGF) is a secreted, heparin sulfate glycosaminoglycan-binding protein. HGF has been shown to have mitogenic effects on osteoblasts; thus, participating in the bone remodeling process.

Yamada et al. cultured fibroblasts in a culture medium containing HGF and concluded that they produced good cell proliferation and vascular endothelial growth factor (VEGF) release. The results suggest that it may provide a new tool for the treatment of gingival recession.[47]

Bone morphogenetic proteins

Bone morphogenetic proteins (BMPs) are the members of transforming growth factor-β (TGF-β) superfamily, which play a crucial role in cell growth and differentiation. They are a group of related proteins that are known to possess the unique ability to induce cartilage and bone formation.[48] They trigger cellular effects by way of heterotetrameric serine/threonine kinase receptors and intracellular signaling proteins known as small “mothers against” decapentaplegic (Smads).[49] BMPs, like PDGF, play a role in the blood vessel formation. They play an important role in the angiogenetic activity by up-regulating the angiogenetic peptides like VEGF, may bind to endothelial cells and stimulate the migration and promote blood vessel formation.[50]

The hallmark property of BMP is the differentiation factor. BMP will differentiate an undifferentiated mesenchymal cell into an osteoblast. In contrast, PDGF is a chemotactic and mitogenic factor for osteoblast like precursors.[50]

CLINICAL APPLICATIONS OF TISSUE ENGINEERING FOR PERIODONTAL TISSUE REGENERATION

Guided tissue regeneration

Nyman and Karring in the 1982 were the first ones to have proposed the use of guided tissue regeneration for periodontal regeneration, which marked the evolution of periodontal regeneration technologies using tissue engineering. The placement of barrier membranes over the denuded root surface and the debrided periodontal defect has shown space provision, epithelial cell occlusion, and exclusion of gingival connective tissue from the root surface and selective repopulation of periodontal ligament cells. Various studies demonstrating the clinical benefits of using Guided tissue regeneration (GTR) membrane alone or in combination with other regenerating materials have been specified in Table 3.

Table 3

Data extraction from studies done on membranes to treat infrabony defects

Protein based approaches[5657]

The use of growth and differentiation factors evolved tissue engineering to its next level and has been the most popular tissue engineering approach for regeneration of periodontal tissues.

Several growth factors have been used including

Transforming growth factor β;

Bone morphogenetic proteins (super family members);

Basic fibroblast growth factor; and

Platelet derived growth factor.

Enamel matrix derivative

The rationale for the clinical use of enamel matrix derivative is the observation that enamel matrix proteins are deposited onto the surfaces of developing tooth roots before cementum formation.[58]

Enamel Matrix Protein (EMPs) are commercially available as Emdogain which have been known to effect periodontal regeneration. Recent data from a systematic review indicates that biologically EMPs cause an increase in cell attachment of epithelial cells, gingival fibroblasts, and PDL fibroblasts. They increase the expression of transcription factors that are related to chondroblast and osteoblasts/cementoblast differentiation. Stimulation in the synthesis of total protein and extracellular matrix molecules has also been documented.[59] Use of Enamel matrix derivative (EMD) and a demineralized freeze dried bone allograft (DFDBA) have been demonstrated to be osteopromotive in nature; thus, resulting in an additional increase in bone formation.[60] Studies enumerating the effects of EMD in obtaining periodontal regeneration are given in Table 4. The only concern with the use of EMD has been related to its application and its related viscous nature, which may not provide sufficient soft tissue/flap support; thus, potentially limiting the space available for the regeneration process.

Table 4

Data extraction from studies done on enamel matrix derivative used to treat infrabony defects

Platelet rich plasma

Since physiologic concentrations of growth factors may not be sufficient to stimulate local bone formation, the use of exogenous growth factors to supplement endogenous biological mediators has been explored. Platelet rich plasma (PRP) is a volume of autologous plasma that contains a platelet concentration above baseline values. The development of PRP from autologous blood by simple, sterile (office based and Food and Drug Administration (FDA) cleared devices) by gradient density centrifugation produces a concentration of platelets with enhanced growth factors including PDGF, TGF-β, and insulin growth factor-1. It has been reported that PRP preparations may increase the concentrations of platelets up to 338%.[66] PRP works through transmembrane receptors and intra cytoplasmic signaling pathways, as do all other growth factor preparations. PRP stimulates the proliferation of human osteogenic cells and periodontal ligament cells.[67] Because PRP and all growth factor preparations work through normal regulated genes and are not autogenous, they are safe promoters of biologic healing and there is no risk of promoting neoplasia. The data reflecting the clinical gain with the use of PRP has been given in [Table 5].

Table 5

Data extraction from studies done on platelet rich plasma used to treat infrabony defects

Recombinant protein therapeutics

With advances in recombinant technology, the development and commercialization of pure recombinant human growth factor - matrix combination has been developed. Combination products which represent the next generation of tissue engineering therapeutics, have gained increasing attention from clinicians and researchers as a strategy to optimize tissue regeneration. Proteins may now be synthesized, concentrated, purified, and packaged in large sterile quantities under tightly controlled and regulated conditions. Providing growth modulating molecules in a highly concentrated pure and consistent form and the ability to combine highly concentrated forms of individual signaling proteins with conductive matrices is important in order to increase the predictability of regenerative procedures. This allows clinical researchers to develop improved regenerative products combining the physical and chemical characteristics of tissue specific matrices required for specific cell attachment, growth and differentiation, with optimal binding and release profile for these bioactive proteins that actively recruit healing cells to the treatment site and expand their cell numbers, in order to achieve the greatest regeneration.

To date, only three recombinant growth factor products have been widely used

rh PDGF-BB (gel).[73]

rhPDGF-BB (with β tricalcium phosphate).[74]

rh BMP-2 (with type I collagen sponge).[75]

Recombinant platelet derived growth factor (rh PDGF BB) is more than 98% pure recombinant protein developed using conventional recombinant expression techniques under highly controlled conditions. They are first produced by removing the specific DNA sequences from a human cell and transfecting it into a bacterial plasmid. The bacterial plasmid is then transfected into the host cells capable of large scale growth. These are essentially protein factories that synthesize and secrete many proteins. The rh PDGF BB is then separated using sophisticated analytical protein chemistry techniques, sterile filtered and formulated into dose specified for clinical use.[76]

Use of rh PDGF has been one of the options to regenerate the periodontium and has received FDA clearance for use.

The mitogenic responsiveness of periodontal cells to local application of PDGF-BB was confirmed in a dog model by Wang and Castelli. Its levels were raised in cases of periodontitis, but not in diabetic cases; thus, suggesting that PDGF-BB driven repair process is suppressed under diabetic conditions.[77]

The concept of the use of recombinant protein therapeutics delivered in an allograft matrix has provided significant clinical results. The efficacy of GEM 21S (growth enhanced matrix β TCP+PDGF), biomimmetic therapeutics were recently reported by Nevins and co workers.[57]

Studies have also suggested that the use of rh PDGF+ β TCP and a collagen membrane may represent an acceptable alternative to connective tissue graft for covering gingival recession defects.[74]

Simion M conducted a study using rh PDGF in conjunction with anorganic bone block for vertical ridge augmentation. It resulted in better healing and increased amount of regenerated bone[78] [Table 6].

Table 6

Data extraction from studies done on recombinant platelet derived growth factor used to treat infrabony defects and furcation defects

Role of rhBMP-2 in periodontal regeneration

The identification and development of recombinant human bone morphogenetic protein-2 (rhBMP-2) has lead to the commercial availability for the first time of an osteoinductive autograft replacement (INFUSE® Bone Graft). rhBMP-2 is a homodimeric protein consisting of two BMP-2 protein subunits.

Studies provide an important insight that space provision appears critical to draw clinically significant benefits from a BMP construct.

rhBMP 2 has been combined with ACS atellocollagen sponge (ACS).[84]

rhBMP2 has also been used in a DFDBA/fibrin clot carrier.[85]

rhBMP2 and calcium phosphate cement matrix.[86]

Hanisch O Tatakis reported that rhBMP-2/ACS at 1.5 mg/cc, INFUSE® Bone Graft, induced significant bone formation suitable for implant placement.[87] Additional clinical studies are needed to evaluate rhBMP2 in combination with other materials for further potential applications [Table 7].

Table 7

Data extraction from studies done on recombinant bone morphogenetic protein-2 used to treat infrabony defects and furcation defects

Tissue engineering using PRP or recombinant protein therapeutics is a clinical reality in periodontal, cranio maxillofacial, and orthopedics indications.

Dental surgeons at long last have access to pure recombinant tissue growth factors, allowing us to progress from previously passive therapies to new active treatments, thereby enhancing the opportunity for regeneration of bone and other tissues and providing more predictable, faster, less invasive, less traumatic, and efficient outcome for the patient.[74]

Cell based approaches

Cell transplantation using autologous cells is expected to play a central clinical role in the future. Dental cell seeding attempts have attempted to regenerate the periodontal tissues since 1990s. Attempts have been made to create the target tissue in the laboratory by culturing and proliferating mesenchymal cells together with scaffolds, before transplanting them into the body.

Typical cell harvesting methods using enzymatic dispersion might destroy critical cell surface proteins such as ion channels, while growth factor receptors and cell to cell junctions remain intact. Okano et al. developed temperature responsive culture dishes (commercially available under the name of UpCell™, CellSeed Inc., Tokyo, Japan) by grafting a polymer poly N isopropylacylamide (PIPAAm) onto tissue culture graded polystyrene dishes by irradiation with an electron beam. Cells generally adhere to hydrophobic surfaces, but not to hydrophillic surfaces. At temparatures lower than 32°C, PIPAAm is fully hydrated. This dish allowed intact cells with preserved extracellular matrix proteins and normal cell functions to be harvested with just low temperature treatment.[50] This has evolved into a novel strategy called “Cell sheet engineering” which produces tissues without a specific scaffold. Transplanted cell sheets can be grafted to the recipient tissues without suturing.

Akizuki et al. investigated periodontal healing after the application of periodontal ligament cell sheet in beagle dogs. These results demonstrated that, in the experimental group, periodontal tissue healing with the formation of bone, periodontal ligament and cementum occurred in three out of the five defects.[91]

Hasegawa et al. assessed the ability of periodontal ligament cell sheets to regenerate the periodontal ligament tissue and demonstrated its usefulness in periodontal tissue regeneration.[92] Flores et al. evaluated whether human PDL cell sheet could reconstruct periodontal tissue and found that transplanted PDL cell sheet cultured with osteogenic differentiation medium induced periodontal tissue regeneration containing an obvious cementum layer and Sharpey's fiber.[93]

Huang and Zhang have set forward a hypothesis of transplanting PDL cell obtained from the periodontium of autogenous extracted teeth, such as the third molar and premolar for orthodontic purposes sheets when cultured using the cell sheet engineering approach into the implant beds before inserting the implants.[94]

Gene delivery based approaches

Numerous tissue regeneration studies have investigated various gene delivery techniques. These techniques involve a gene encoding a therapeutic protein being introduced into the cells which can then express the target protein. This technique avoids the problems associated with the protein delivery method by maintaining constant protein levels at the site of the defect.[2]

Comparative evaluation

Comparison of different agents and techniques used to treat infrabony and furcation defects have been tabulated as in Tables 3–7 and a brief conclusion has been drawn on them:

Clinical outcome obtained in the EMD patients are similar to those with the use of bio-resorbable membranes. The advantage of using EMD is that they are technically more simple with less risk of exposure, less invasive and resulting in lesser recession after surgery. However, the adjunctive use of EMD with GTR doesn’t seem to enhance the outcome of GTR.

PRP which provides similar results has the advantage of having hemostatic activity, giving a user friendly environment and acts as a stabilizing agent, immobilizing the blood clot and bone graft from the area.

The clinical results as well as histologically evaluated periodontal regeneration obtained using rh PDGF and rh BMP produce much superior, but patient centered outcomes, including adverse effects, cost effectiveness, and risk benefit have been evaluated in a very limited number of studies.

Other bioactive agents are also being experimentally tested to treat periodontal defects including OP-1, transforming growth factor β, b FGF, IGF-1, cementum derived growth factor, vascular endothelial growth factor and many more. More clinical studies need to prove their effectiveness in treating periodontal defects.

Challenges ahead

Structural and functional complexity of the periodontium The fact that more than one tissue must be reconstructed, namely alveolar bone, periodontal ligament, root cementum, and gingiva, makes it much more difficult to find both the right combination and the doses of growth factors.

To overcome the rapid clearance of growth factors, a carrier system must be found that stores and releases the growth factors over a longer period of time so that their resident time is prolonged. Although many carrier systems have been tested, none of them appears to be ideal.

While high developmental and therapeutic costs appear justified for severe skeletal conditions such as non-unions, open fractures, spinal fusion, and large bone defects, for example in the mandible, the same cannot necessarily be said for relatively small and non-life-threatening periodontal defects where preventive and maintenance measures are still mandatory.

CONCLUSION

We need to look beyond before we can achieve the dream. Tissue engineering has enlarged our vision and thus made the fascination of being able to achieve regeneration of periodontal complex in its entirety a reality.

Though the task has been arduous, but the promise still remains….