Tissue engineered periodontal products

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1 J Periodont Res 2016; 51: 1 15 All rights reserved Review Article Tissue engineered periodontal products Bartold PM, Gronthos S, Ivanovski S, Fisher A, Hutmacher DW. Tissue engineered periodontal products. J Periodont Res 2016; 51: John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Attainment of periodontal regeneration is a significant clinical goal in the management of advanced periodontal defects arising from periodontitis. Over the past 30 years numerous techniques and materials have been introduced and evaluated clinically and have included guided tissue regeneration, bone grafting materials, growth and other biological factors and gene therapy. With the exception of gene therapy, all have undergone evaluation in humans. All of the products have shown efficacy in promoting periodontal regeneration in animal models but the results in humans remain variable and equivocal concerning attaining complete biological regeneration of damaged periodontal structures. In the early 2000s, the concept of tissue engineering was proposed as a new paradigm for periodontal regeneration based on molecular and cell biology. At this time, tissue engineering was a new and emerging field. Now, 14 years later we revisit the concept of tissue engineering for the periodontium and assess how far we have come, where we are currently situated and what needs to be done in the future to make this concept a reality. In this review, we cover some of the precursor products, which led to our current position in periodontal tissue engineering. The basic concepts of tissue engineering with special emphasis on periodontal tissue engineering products is discussed including the use of mesenchymal stem cells in bioscaffolds and the emerging field of cell sheet technology. Finally, we look into the future to consider what CAD/CAM technology and nanotechnology will have to offer John Wiley & Sons A/S. Published by John Wiley & Sons Ltd JOURNAL OF PERIODONTAL RESEARCH doi: /jre P. M. Bartold 1, S. Gronthos 2, S. Ivanovski 3, A. Fisher 3, D. W. Hutmacher 4 1 Colgate Australian Clinical Dental Research Centre, Dental School, University of Adelaide, Adelaide, SA, Australia, 2 School of Medical Sciences, University of Adelaide, Adelaide, SA, Australia, 3 Griffith Health Institute, School of Dentistry and Oral Health, Griffith University, Gold Coast, Qld, Australia and 4 Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Qld, Australia P. Mark Bartold, Colgate Australian Clinical Dental Research Centre, Dental School, University of Adelaide, Frome Road, Adelaide, SA 5005, Australia Tel: Fax: mark.bartold@adelaide.edu.au Key words: cell sheets; periodontal ligament; periodontal ligament stem cells; tissue engineering Accepted for publication March 3, 2015 In recent years, a great deal has been learnt from the variety of techniques that have been used to promote periodontal regeneration with varying degrees of success. It has become apparent that for optimal regeneration to occur, the surgical/regenerative site has to be adequately isolated from the oral environment. From this perspective, it is advantageous for a rapid epithelial seal to be formed around the cervical portion of the tooth surface. It is also evident that there are cell types that need to be recruited into the area to promote regeneration and other cells that need to be excluded. As our knowledge of cellular and molecular processes increases, so too does the appreciation of the complexity involved in the formation of the periodontium. To achieve regeneration of periodontal tissues, then we must endeavor to replicate this process in a clinical setting. For periodontal regeneration to occur there is an intricate process involving differentiation of the various cell types involved and the production of these cells in sufficient numbers in the correct location and environment. A detailed understanding of how to manipulate these processes will therefore increase our potential successfully to regenerate damaged periodontal tissues. It is now recognized that molecular signaling systems principally drive the cell selection process (1). These are soluble mediators or growth factors, which interact with the extracellular matrix and receptors on the cell surface. This results in a cascade of intracellular reactions resulting in gene expression and resultant differentiation and proliferation of cell types. Many mediators and growth factors have been implicated in the regenerative/formative processes of the periodontium. Unfortunately, there is no simple association with one single growth factor that can

2 2 Bartold et al. elicit periodontal regeneration. The biology involved is far too complex for all of these interactions to be fully appreciated, but there has been some evidence that certain growth factors can enhance regenerative procedures (1,2). Factors influencing success for periodontal regeneration It is important to understand that not all regenerative procedures are associated with the same level of clinical success. There are several factors that can influence the clinical outcome, which can be broadly divided into patient, tooth, defect and surgical technique factors. As with all periodontal conditions/ procedures, the patient s oral hygiene and health of the periodontal tissues affects the ability to achieve a successful outcome from a surgical procedure (3). A superior result will be obtained from the institution of an adequate oral hygiene regimen (3). Other factors that need to be considered are the patient s overall general health, smoking status and systemic disease status. Factors such as age, genetics and stress have all been postulated to have an effect, but this is not yet supported by the literature (4). Tooth-related factors to be considered include endodontic status and any hypermobility. There are conflicting views regarding the endodontic treatment of teeth undergoing regenerative therapy, but it must be assumed that frank infective processes should be controlled if a regenerative procedure is to be considered an appropriate treatment option (5). Any tooth with horizontal mobility < 1 mm can be successfully treated with regenerative treatment (6,7). Not all types of periodontal defects are amenable to regeneration. Indeed, periodontal regenerative procedures should be limited to intrabony and class II furcation defects. In particular, better results are obtained with narrow intrabony defects with increased number of walls (3,8). Horizontal defects and class III furcations cannot yet be regenerated with current techniques (4,9). Precursor products to periodontal tissue engineering Guided periodontal tissue regeneration membranes First described in the 1980s, guided tissue regeneration (GTR) for the periodontium provided an early biological approach to periodontal tissue engineering (10). Guided periodontal tissue regeneration was based on the principle of using a non-resorbable teflon-based membrane (eptfe) to exclude epithelial and gingival tissue ingrowth into a periodontal defect while allowing proliferation of the periodontal ligament at such a site with the expectation of regrowth of cementum, periodontal ligament and alveolar bone (Fig. 1). While such a process was demonstrated biologically feasible the clinical results were often disappointing due to the technical sensitivity of the surgical procedure and high rate of postoperative complication. The most common cause of failure of this procedure was exposure of the non-resorbable eptfe membranes used at this time. A list of commonly available GTR membranes is shown in Table 1. Subsequent development of resorbable membranes made from a wide variety of biodegradable materials improved the clinical outcomes considerably. However, even with this development the clinical results were unpredictable. Despite the clinical limitations of guided periodontal regeneration, it has been considered a significant development in the field of regenerative periodontal therapy. The recognition that periodontal tissues possess a capacity for regeneration stimulated further studies to understand the molecular and cell biology of periodontal regeneration. The benefit of using GTR over the conventional use of open flap debridement has been reviewed extensively in the literature and the findings support the use of GTR in two clinical situations, furcation involvement and intrabony defects. For furcations, the consensus is that GTR is not effective for regenerating class III furcation defects and has a modest clinical improvement for maxillary class II furcation defects (9). Overall, GTR shows enhanced clinical outcomes over the use of open flap debridement alone for the treatment of mandibular class II furcation defects (9). A Cochrane Review has compared the use of GTR to open flap debridement alone for the treatment of intrabony defects (8). Improvements were noted when GTR was compared to open flap debridement for the rele- Fig. 1. Schematic representation of guided tissue regeneration. The barrier membrane is draped over the periodontal defect to allow sufficient space for cells from the periodontal ligament to repopulate this space. The gingival tissues are physically excluded from access to the wound space by the membrane.

3 Periodontal tissue engineering 3 Table 1. Examples of currently available periodontal regeneration materials Guided tissue regeneration membranes Non-resorbable Expanded PTFE GoreTex Ò High-density PTFE Cytoplast TXT-200 Ò Titanium enforced high-density PTFE Cytoplast Ti-250 Ò Resorbable, synthetic Poly DL-lactic/co-glycolic acid Polyglactin 910 Poly DL-lactide and solvent (N-methylpyrrolidone) Resorbable collagen-based Cadaveric human skin type I collagen Porcine skin collage type I Bovine tendon collagen type I Bovine tendon type I collagen Bone grafting materials Alloplasts Beta tricalcium phosphate Hydroxyapatitie Calcium sulfate Calcium phosphate Bioactive glass Autografts Endogenous bone grafts Allografts Demineralized freeze dried bone allograft Freeze dried bone allograft Xenografts Anorganic bovine bone xenograft Bovine-derived xenograft and type I collagen Growth factors and biologicals studied for periodontal regeneration Bone morphogenetic proteins-2, -3, -4, -6, -7 and -12 Cell-binding peptide P-15 Fibroblast growth factor-2 Growth/differentiation factor-5 Insulin-like growth factor-1 Matrix factors (fibronectin, amelogenins, thrombospondin) Platelet-derived growth factor Platelet-rich plasma Vascular endothelial growth factor Enamel matrix derivative PTFE, polytetrafluorethylene. fibrovascular tissue ingrowth with incorporation of the graft material with the new bone. Additionally, an ideal bone graft should be osteogenic or osteoinductive resulting in the formation of new bone or at a minimum, osteoconductive, promoting direct bone contact and growth along the graft material. Ideal graft materials should also undergo remodeling with a resorption rate similar to the rate of new bone formation resulting in an augmented site consisting of host bone alone. Four different types of bone graft materials have been commonly used and are classified as autografts, allograft, xenografts or alloplasts (Table 1). Autografts Autografts are harvested from the intended graft recipient and are considered the gold standard bone graft material (15). Autografts are osteogenic, due to the presence of viable osteogenic cells, osteoinductive, due to the presence of bone matrix proteins such as bone morphogenetic proteins (BMP) and osteoconductive, due to the porous mineralized component of bone (16). However, the amount of graft material that can be harvested is limited and may be associated with increased morbidity and risk of surgical or postoperative complications. vant clinical parameters of attachment gain, reduced pocket depth, less increase in gingival recession and more gain in hard tissue probing at re-entry. Most recently, similar results have been reported for absorbable collagen GTR membranes (11). From the systematic reviews published to date it is important to note that improvements using GTR are only modest, variable and may not even result in any improvement in the long-term goal of tooth retention (8,12). Although the attachment gains achieved with GTR can be maintained over many years (13), there is no significant difference in attachment gain between GTR and open flap debridement after 12 years (14). GTR therefore is conceptually sound, but the clinical results are somewhat unreliable and predictable regeneration is elusive in most periodontal defects with a possible exception of a few ideal situations. These ideal clinical scenarios are seldom encountered in clinical practice so biologically active agents have been explored to improve treatment outcomes by optimizing the healing dynamics of the periodontal wound. Periodontal bone grafting materials The ideal bone graft material should be biocompatible, with a physicochemical structure similar to natural bone to promote angiogenesis and Allografts Allografts are grafts that have been harvested from one individual and implanted into another individual of the same species (15). Donors can be living related persons, living unrelated persons or, more commonly, from cadavers after the removal of viable cells. Allografts are generally prepared in freeze-dried forms or hydrochloric acid treated to produce demineralized freeze-dried forms to expose growth factors such as the BMP sequestered in the bone matrix. As cells are removed from allografts, they are not osteogenic and the extent of osteoinductive and osteoconductive properties of the allograft may vary depending on graft processing as well

4 4 Bartold et al. as the donor age (17). Although allografts have been used with clinical success in implant dentistry, their application may be limited due to high cost and patient s perceived risks of viral transmission, immunogenicity or other social and religious concerns. Xenografts Xenografts are grafts harvested from different species, commonly of bovine origin consist of hydroxyapatite bone mineral after removal of the organic component. Xenografts are osteoconductive as they have a mineral content and porosity similar to human bone but do not contain osteogenic cells or osteoinductive agents (18). Animal and human studies have demonstrated new bone formation in direct contact with demineralized bovine bone matrix particles when used in bone grafting procedures. Similar to allografts, the use of the demineralized bovine bone matrix may not be acceptable to some patients due to the perceived risk of disease transmission or other social or religious concerns. Alloplasts Alloplasts represent a large group of chemically and structurally diverse materials with varying mechanical and biological properties. These materials include calcium sulfate, composite polymers, bioactive glass ceramics as well as calcium phosphate-based ceramics. As alloplasts do not provide any osteogenic cells or osteoinductive proteins, they are considered osteoconductive only (19). The literature is conflicting on the ability of any of these grafting materials to promote true periodontal regeneration. Indeed the concept of focusing only on bone for regeneration is conceptually flawed if one considers that true periodontal regeneration involves not only bone regeneration but also regeneration of the periodontal ligament and cementum (20,21). It is unlikely that a bone graft can achieve this. A recent review of the use of bone grafting materials in intrabony and furcation defects found that bone grafts increased clinical attachment level and probing depth compared to surgical open flap debridement. For furcation defects, it was concluded that some benefit could be obtained in mandibular class II furcations, but the lack of consistency among the studies meant that no clear consensus was obtained. The different types of grafting materials produced similar types of results (22). This potential improvement in clinical parameters in some situations, however, does not necessarily reflect true regeneration. The only method to assess whether regeneration has occurred is via histological examination and the literature suggests that there are conflicting findings as to whether this actually occurs or not with this treatment approach. Some studies support the generation of new attachment on to the root surface (23), whereas others illustrate repair via a long junctional epithelium. The consensus, however, is that these grafting materials are ineffective at producing complete regeneration (24 26). This is thought to be because they rely on proliferation of the cells that are locally available in the defect site and the amount of viable periodontal ligament present. This is significantly influenced by the size of the defect, disease status and shape/morphology of the defect (27). Guided tissue regeneration in conjunction with grafting materials With the development of non-supporting absorbable GTR membranes the need for some form of support underneath the membrane was seen as a critical factor to successful clinical outcomes. A recent systematic review comparing GTR alone to GTR with grafting materials concluded that the significant gains in clinical outcomes achieved by GTR over open flap debridement alone could be even further enhanced using grafting materials (28). Growth factors for periodontal regeneration With the discovery of growth and differentiation factors is renewed interest in biological approaches to periodontal regeneration. Table 1 lists some of the more comprehensively studied growth factors used for periodontal regeneration. The use of growth factors has presented an important step forward in achieving periodontal regeneration but it has still failed to deliver consistent results (29). The reasons for these limited outcomes relate to our limited understanding of differentiation requirements of the cells responsible for periodontal regeneration, specific target cells within the periodontium, release kinetics of growth factors delivered to the site and stability of the regenerated tissues formed following growth factor delivery. The rationale for the application of growth factors to the root surface is based on their ability to influence key cellular functions as reported by in vivo and in vitro investigations. Some growth factors have been found to promote the growth and differentiation of certain cells within the periodontal ligament (30). This is through the regulation of cell proliferation, cell activity, chemotaxis and/or cell differentiation. This would theoretically result in regeneration of the periodontium by activation and differentiation of the appropriate cell types required. There are several limitations involved in the use of growth factors (31). There is considerable difficulty with determining what function each growth factor performs and its precise influence on different cell types in a complex in vivo environment. Even if the factors can be isolated and their function perfectly understood, targeted delivery of these to the appropriate cells in the right concentrations at the right time during development/regeneration is a problem constrained by numerous practical considerations. There is also the difficulty common to all periodontal regenerative procedures, which is the inability to obtain primary surgical closure necessary to allow the growth factor to have an influence in a sterile, closed environment. The above considerations are particularly relevant for the growth factors that have been investigated in

5 the field of periodontal regeneration. These include platelet-derived growth factor (PDGF), insulin-like growth factor, fibroblast growth factor, vascular endothelial growth factor, BMPs and enamel matrix derivative (EMD). Apart from EMD, the most studied growth factors for periodontal regeneration have been PDGF/insulin-like growth factor (32). BMPs appear to be promising in promoting bone formation, in particular BMP-2 and BMP-7, but even these growth factors do not result in complete periodontal regeneration. More recently, fibroblast growth factor has been studied with encouraging results (33,34). A recent human randomized controlled clinical trial using fibroblast growth factor-2 has demonstrated good gains in alveolar bone height 36 wk after application (35). Of the other growth factors, the outcomes of their use are extremely heterogeneous or the level of evidence is limited to sporadic case reports so they cannot be recommended as a panacea for periodontal regeneration attempts. Further clinical trials are needed to determine the clinical utility of these agents. Enamel matrix derivative Notwithstanding the above, the most widely studied biologically active material for periodontal regeneration is EMD, which is a complex mixture of enamel matrix proteins (EMPs) (36). EMPs are a group of molecules associated with amelogenesis and include amelogenins, ameloblastin, amelotin, tuftelin and enamelin (37,38). In addition to their well-documented role in enamel biomineralization, EMPs are considered involved in epithelial mesenchymal interactions at the developing root surface (39). EMP exposed on the root surface by the breakdown of Hertwig s epithelial root sheath is thought to play a role in the differentiation of mesenchymal tissues and subsequent formation of the attachment apparatus of the periodontium (39 41). By harnessing this process, it was postulated that regeneration of periodontal attachment that has been lost because of periodontal disease could be achieved. The principle behind the use of EMPs in regenerative approaches is therefore that of biomimetics or mimicking natural biology. EMD is available commercially as Emdogain (Straumann, Basel, Switzerland). This is an injectable gel solution comprising EMD, water and a carrier, propylene glycol alginate. The EMD primarily consists of amelogenins but also contains other enamel proteins (42). Histological evidence of regeneration following EMD application has been obtained from animal trials including the creation of surgical buccal dehiscence defects in monkeys (43). In humans, histological evidence of regeneration has also been obtained from case reports (44) and this is corroborated by clinical parameters in other studies (45 47). The combination of research from human and animal trials has led to the use of this product becoming an accepted treatment option in contemporary periodontics. Despite these positive results, it is not clear whether the amelogenin or non-amelogenin components of Emdogain regulate regeneration (48,49). Systematic reviews on the effectiveness of EMD vs. open flap debridement or GTR in randomized clinical trials with a minimum of 1 year follow-up show some additional improvement when using EMD selectively. For example, when EMD is compared with open flap debridement, EMD showed statistically significant attachment gain improvements and probing depth reduction (50). When compared to GTR, there were no statistically significant differences between the two treatment approaches with respect to attachment gain or probing depth reduction. However, GTR was associated with more recession and postoperative complications. The selection criteria for this review were randomized controlled clinical trials on patients affected by periodontitis having intrabony defects of at least 3 mm treated with EMD compared with open flap debridement, GTR and various bone grafting procedures with at least 1 year follow-up. Periodontal tissue engineering 5 Grade II furcations have been shown to have superior regenerative responses when EMD is used compared to GTR (51). Both GTR and EMD have superior responses to open flap debridement alone, but EMD has an additional improvement in horizontal furcation depth reduction compared to GTR. In addition, there is a comparatively lower incidence of postoperative pain and swelling following EMD treatment compared to GTR (51). EMD appears safe to use, with no documentation of adverse side effects, except for isolated case reports of infrequent external root resorption (52). Long-term results also seem to indicate stability, with the outcomes obtained from regenerative surgery using EMD are being maintained over time for up to 5 years (46,53). Gene therapy Gene therapy is not yet a clinical reality for periodontal regeneration, but a theoretical approach would be to drive the differentiation of specific cells involved in periodontal regeneration through the delivery of the appropriate genes (54). Using vectors, it may be possible to insert genetic material into certain cell types of the periodontal ligament, eliciting transcription of these genes and subsequent growth and differentiation of cementoblasts leading to new attachment formation. An example of this is the delivery of PDGF genes into animal cells inducing more cementoblastic activity (55). This process has been studied in an animal model, which demonstrated that gene delivery of PDGF stimulated more cementoblast activity and improved regeneration compared to a single application of recombinant PDGF (56). However, the safety and efficacy of using gene therapy for periodontal regeneration has yet to be fully evaluated. Summary of currently available materials for periodontal regeneration With the exception of gene therapy, all of the above materials and procedures

6 6 Bartold et al. have undergone evaluation in humans (Table 2). All of the products have shown efficacy in promoting periodontal regeneration in animal models (Table 3) but the results in humans are variable and equivocal (57). Periodontal tissue engineering The limitations of the above techniques, currently available procedures and products using membranes, bone grafts, growth factors and gene therapy have led to the consideration of alternatives. These are based on contemporary approaches driven by our increasing knowledge in a variety of relevant fields. Tissue engineering involves using techniques for the fabrication of tissues outside the body for implantation into the body to regenerate the lost biological function of that respective tissue (58). The requirements for tissue engineering are the appropriate levels and sequencing of regulatory signals, the presence and numbers of responsive progenitor cells, an appropriate extracellular matrix, carrier or scaffold and an adequate blood supply (1,59) (Fig. 2). The principle Successful periodontal regeneration via tissue engineering approaches requires a number of important principles to be addressed (60); the engineered tissues have to have sufficient biomechanical strength, architectural properties and space-maintaining ability. The engineered tissue has to maintain space for in-growth of alveolar bone, but it also has to be exclusionary with respect to the epithelial tissues to prevent the formation of a long junctional epithelium. In addition, biological functions have to be appropriate to allow cellular recruitment and proliferation, vascularization and the delivery of the appropriate factors for regeneration (1). Vascularization is an important part of any regeneration to avoid tissue necrosis. Using a tissue engineering construct of human periodontal ligament fibroblasts co-cultured with or without human umbilical vein endothelial cells that were found to form capillary-like structures when co-cultured with the human periodontal ligament fibroblasts (61). These cultures demonstrated longer survival, higher alkaline phosphatase activity and lower osteocalcin production than the human periodontal ligament fibroblast cultures alone. This is all consistent with a greater potential for regeneration and this approach may be beneficial in maintaining adequate vascularization for regeneration to be successful. Recent advances in technology and bioengineering have now opened the scope for the use of stem cells in these tissue engineering constructs. With the recognition of the presence of mesenchymal stem cells (MSCs) within the periodontal ligament there has been considerable research carried out on the isolation, characterization and clinical utilization of these cells (62). Stem cells are divided into two broad categories, embryonic stem cells and adult stem cells. These are then further subclassified according to their origin and differentiation potential (Fig. 3). Human embryonic stem cells, derived from the inner cell mass of blastocysts, are pluripotent cells capable of differentiating into cells of all three germ layers, ectoderm, mesoderm and endoderm (63). Human Table 2. Testing of periodontal regenerative materials in periodontal defects Intrabony defect Class II furcation Class III furcation Recession Animal Human Animal Human Animal Human Animal Human Guided tissue regeneration membranes Yes Yes Yes Yes Yes Yes Yes Yes Bone grafts Yes Yes Yes Yes Yes Yes Yes Yes Growth factors Yes Yes Yes Yes Yes Yes Yes Yes Enamel matrix Yes Yes Yes Yes Yes Yes Yes Yes Gene therapy Yes No Yes No No N0 No No Periodontal ligament stem cells Yes Yes No No No No No No Table 3. Outcomes of periodontal regenerative materials for periodontal defects Intrabony defect Class II furcation Class III furcation Recession Animal Human Animal Human Animal Human Animal Human Guided tissue regeneration membranes Good Good Good Good Good Poor Good Good Bone grafts Good Good Good Good Good Poor Good Questionable Growth factors Yes Yes Yes Yes Yes Unknown Good Good Enamel matrix Good Good Good Good Questionable Poor Yes Yes Gene therapy Good Unknown Good Unknown Good Unknown Unknown Unknown Periodontal ligament stem cells Good Good Good Unknown Good Unknown Unknown Unknown

7 Fig. 2. Requirements for periodontal tissue engineering. The concept of tissue engineering consists of implanted cells, signaling molecules and an appropriate delivery scaffold. This together with an adequate blood supply should be sufficient to allow new tissue regeneration. embryonic stem cells have two unique properties, i.e., (i) virtually unlimited proliferative potential in an undifferentiated state, and (ii) their pluripotency, which is the capability of differentiating into cells from all three germ layers mentioned above (63). However, human embryonic stem cell research has been associated with major ethical concerns (63). Adult stem cells are found in the majority of fetal and adult tissues and are thought to play roles in long-term tissue maintenance and/or repair by replacing cells that are either injured or lost (64). They are generally multipotent stem cells that can form a limited number of cell types. Two common examples are hematopoietic and MSC. As the periodontium is mesenchymal in origin, MSCs have been studied in periodontal regeneration research. In a recent study bone marrow mesenchymal stromal cells on microcarrier gelatin beads were placed into surgically created rat periodontal defects with encouraging results being described for periodontal regeneration outcomes (65). Periodontal tissue engineering By the early 2000s, the concept of tissue engineering was presented and subsequently this has become well accepted within the field of periodontal regenerative medicine (1,66). The concept is based on regenerating damaged tissues through the implantation of cells within a bioscaffold and is based on what we have learnt from cell biology of tissue regeneration, developmental biology and biomaterials science. For tissue engineering the utilization of an appropriate scaffold is central for a successful outcome (60). In recent years, considerable progress has been made in the field of periodontal tissue engineering. However, there are still many issues that need to be addressed for this to become a clinical reality with predictable results. Two particular areas that need to be addressed are (i) fabricating materials, which will maintain an in vitro substratum relevant to the tissues to be regenerated with regards to architectural geometry and space maintaining properties, and (ii) how to produce a substratum that is compatible with cell growth and differentiation, allows neovascularization and incorporates the appropriate growth and differentiation signals needed for tissue regeneration. Scaffold-based tissue engineering The fundamental concept underlying tissue engineering is to combine a scaffold with living cells, and/or biologically active molecules to form a tissue engineering construct, which promotes the repair and/or regeneration of tissues (67,68). The scaffold is expected to perform various functions, including the support of cell colonization, migration, growth and differentiation. The design of these scaffolds also needs to consider physicochemical properties, morphology and degradation kinetics. External size and shape of the construct are of importance, particularly if the construct is customized for an individual patient. Most importantly, clinically successful bone constructs should stimulate and support both the onset and the continuance of bone ingrowth as well as subsequent remodeling and maturation by providing optimal stiffness and external and internal geometrical shapes. Henceforth, scaffolds must provide sufficient Periodontal tissue engineering 7 initial mechanical strength and stiffness to substitute for the loss of mechanical function of the diseased, damaged or missing tissue. Continuous cell and tissue remodeling is important for achieving stable biomechanical conditions and vascularization at the host site. The degree of remodeling depends on the tissue itself (e.g., skin 4 6 wk, bone 4 6 mo), and its host anatomy and physiology. In addition to these essentials of mechanics and geometry, a suitable construct will (i) possess a 3D and highly porous interconnected pore network with surface properties, which are optimized for the attachment, migration, proliferation and differentiation of cell types of interest (depending on the targeted tissue) and enable flow transport of nutrients and metabolic waste, and (ii) be biocompatible and biodegradable with a controllable rate to compliment cell/tissue growth and maturation (69). It cannot be emphasized enough how essential it is to understand and control this scaffold degradation process, for successful tissue formation, remodeling and maturation at the defect site. In the early days of tissue engineering, it was believed that scaffolds should degrade and vanish as the tissue is growing. Yet, tissue ingrowth and maturation differs temporally from tissue to tissue and, furthermore, tissue in-growth does not equate to tissue maturation and remodeling, in other words a defect filled with immature tissue should not be considered regenerated. Hence, many scaffold-based strategies have failed in the past, as the scaffold degradation was more rapid than tissue remodeling and/or maturation. The onset of degradation should only occur after the regenerated tissue within the scaffold has remodeled at least once in the natural remodeling cycle (70). This paradigm shift is particularly relevant for higher load bearing tissues, such as bone. Original hypotheses in the field promoted scaffold degradation to onset immediately as new tissue starts to form. In contrast, work over the last 10 years underline the importance of the scaffold remaining intact as the tissue

8 8 Bartold et al. Fig. 3. Sources and derivation of stem cell populations. Depending on the site, stage of development or cell culture induction environment human stem cells can be classified as being of pluripotent, multipotent, totipotent or inducible pluripotent potential. matures in the scaffold pores with bulk degradation occurring later. Tissue in-growth and maturation differs temporally from tissue to tissue and secondly tissue in-growth does not equate to tissue maturation and remodeling, in other words a defect filled with immature tissue should not be considered regenerated. Hence, many scaffold-based bone engineering strategies have failed in the past, as the scaffold degradation was more rapid than the tissue remodeling and/ or maturation (Fig. 4). A number of small and large animal studies have demonstrated that this rationale leads to structural and functional bone regeneration in a large critical-sized defect model (70). A recent development in scaffold design for periodontal tissue engineering has been the use of multiphasic scaffolds (67). A multiphasic scaffold can be defined as a construct that incorporates variations in architectural organization (porosity, pore organization, etc.) and/or chemical composition, which aims to recapitulate the structural organization of the target tissue. Multiphasic scaffolds may be favorable for facilitating periodontal regeneration, because of the requirement to control temporally and spatially the interaction between multiple soft and hard tissues. Recently, a number of research groups have utilized periodontal tissue engineering approaches using multiphasic scaffolds, with promising results in animal studies (71 76). Stem cells and periodontal tissue engineering Recent advances in technology and bioengineering have now opened the scope for the use of stem cells in tissue engineering constructs. With the recognition of the presence of MSCs within the periodontal ligament there has been considerable research carried out on the isolation, characterization and clinical utilization of these cells (77). Periodontal cells and periodontal regeneration There are many cells present in the periodontal ligament, including cementoblasts, osteoblasts, fibroblasts, myofibroblasts, endothelial cells, nerve cells and epithelial cells. In addition to these, a smaller population of progenitor cells has been identified by in vivo kinetic studies (78). These progenitor cells are enriched in locations adjacent to blood vessels and exhibit some of the classical cytological features of stem cells. MSCs reside within the periodontal ligament and are responsible for tissue homeostasis serving as a source of renewal cells for cementoblasts, osteoblasts and fibroblasts throughout adult life (Fig. 4). Periodontal regeneration is a reenactment of the development process, including morphogenesis, cytodifferentiation, extracellular matrix production and mineralization. In the event of injury to the periodontium, these MSCs

9 Periodontal tissue engineering 9 Stem cell Precursor cells Limited potential precursor cell Multipotent precursor cell Cbfa1 Other mesenchymal lineages (cartilage, muscle, fat, fibrous tissues) could be activated towards terminal differentiation and tissue repair or regeneration. Studies to date have demonstrated that periodontal ligament cells can be transplanted into periodontal defects with no adverse immunologic or inflammatory consequences (79 82). Therefore, a tissue engineering strategy for periodontal regeneration that exploits the regenerative capacity of stem cells residing within the periodontium is an attractive thesis (83). Recently, studies have demonstrated successful regenerative outcomes in experimental periodontal defects implanted with cultured periodontal ligament fibroblasts (57,84 87). Therefore, the literature supports the principle that cultured periodontal ligament cells applied in a scaffold or gel can induce periodontal regeneration around teeth (82). However, it must be noted that complete periodontal regeneration is often unobtainable, and adverse outcomes such as ankylosis have been shown to occur (88). This indicates that although theoretically possible, our approaches to regeneration have so far lacked the sophistication to become sufficiently predictable for mainstream clinical practice. It is possible that the key to regeneration lies in the ability to isolate those cells residing within the periodontal ligament that possess stem cell properties. It is thought that these cells direct the periodontal regeneration process. Periodontal ligament stem cells Commited precursor cells Differentiated cells Osteoblasts Osteocytes lining cells Cementoblasts Fibroblasts A small population of multipotent stem cell populations, termed periodontal ligament stem cells (PDLSCs), have been isolated from the periodontal ligament (62,89,90). When cultured, these cells produce adherent clonogenic clusters that resemble fibroblasts and are capable of differentiating into adipocytes, osteoblast- and cementoblast-like cells in vitro, and demonstrate the capacity to produce cementum- and periodontal ligamentlike tissues in vivo. These cells have been identified within the periodontal ligament using a number of MSC-associated markers including STRO-1, CD146 and CD44 (91). This led to their discovery in healthy and diseased periodontium and showed that they were mainly located in the perivascular regions and more widely distributed in diseased periodontium. Based on the stem cell properties of self-renewal and multilineage differentiation, PDLSCs offer significant potential for periodontal regenerative therapies. Periodontal regeneration using mesenchymal stem cells BONE CEMENTUM Fig. 4. Cellular differentiation in periodontal tissues. Reproduced with permission from Sodek and McKee (119). PDL The concept of using stem cells for periodontal regeneration initially involved the isolation of MSCs from the bone marrow and implantation into the furcation defect in dog molars (92). The results of this study showed a more ordered and enhanced regenerative process on histological examination than control sites, indicating a role for stem cells in regeneration via tissue engineering. Furthermore, periodontal regeneration was accompanied by continuing MSC activity, based on experiments using fluorescence-labeled MSC. This illustrated that this regeneration is likely to be due to the activity of the MSC, rather than natural repair resulting after the creation of an acute surgical defect. The use of MSC to promote regeneration has been used in case studies and early phase human clinical trials (93). MSC extracted from iliac crest, placed in a platelet-rich plasma gel and injected on to the root surface of an isolated site resulted in significant improvement in clinical and radiographic parameters. However, this should not be relied upon as definitive evidence for the clinical effectiveness of this approach, but it illustrates the potential clinical applications of tissue engineering in humans. In addition, a major problem with the use of bone marrow-derived MSC in periodontal regeneration is that their extraction involves an additional traumatic surgical procedure unrelated to the oral cavity. A novel approach using an autologously derived sheet of periosteum placed over artificially created class 3 furcations in adult dogs, has demonstrated complete regeneration in all experimental sites (94). All control sites without the membrane had no evidence of regeneration or repair, in contrast to the excellent results from the experimental sites. This showed that not only bone, but also new cementum was formed in these defects, illustrating an elegant approach for using autologous periosteum in a tissue engineering approach essentially to simulate GTR. The use of intraoral autologous cells, without the addition of exogenous molecules, is attractive. Although this approach does not specifically involve periodontal ligament cells, it does provide evidence that a tissue engineering approach can be used to promote periodontal regeneration. Periodontal regeneration using periodontal ligament stem cells The regenerative capacity of PDLSCs has been studied in considerable detail in animal models (82,95). The large number of animal studies carried out to date have clearly shown that PDLSCs have the potential to form

10 10 Bartold et al. bone, cementum, periodontal ligament-like structures and enhance overall periodontal regeneration. Thus far, PDLSCs appear to have a greater capacity to generate periodontal structures, in comparison to MSC-like cells derived from other tissues, making them highly amenable for use in periodontal regeneration. To date there has been only one human clinical study reporting the potential for autologous PDLSCs for the regeneration of periodontal intrabony defects (96). The literature therefore suggests that PDLSCs can be used for regeneration of the periodontium in surgically created defects in both small and large animal models, albeit with limited success and in only a narrow field of application. One problem encountered with this approach is that very few of these stem cells attach to the surface of the alveolar bone and teeth. This has led to the utilization of cell sheet technology in conjunction with the principles to deliver the regenerative potential of the PDLSC to the appropriate location. Cell sheets and periodontal tissue engineering The principle A novel technique for using biologically driven approaches for regeneration involves the use of cell sheets (97). This requires the identification and isolation of cells required for periodontal regeneration and then growing these cells on a temperaturesensitive sheet in culture plates. Cell sheet construction involves the use of a temperature-sensitive polymer biomaterial, poly-n-isopropylacrylamide (PIPA Am), in the cell culturing process. Once a mature cell sheet is formed, it is harvested by decreasing the temperature, which leads to the detachment from the temperature-sensitive substrate. This allows harvesting of a complete sheet of cellular material with an intact extracellular matrix and cell cell junctions, in an attempt to optimize the regenerative potential of the implanted cells in situ. Cell sheet material This cell sheet technique was first developed using bovine endothelial cells and rat hepatocytes cultured on PIPA Am-grafted surfaces (98). Cell sheets have since been fabricated from various cell types, including fibroblasts, endothelial cells, retinal pigmented cells and gastric cells and have been applied in vivo (97,99). This PIPA Am material is covalently attached to a solid surface by a specific chemical immobilization reaction or electron beam irradiation. The coated culture shows a similar hydrophobicity to a normal cell culture above 32 C. This is because the PIPA Am chains are dehydrated on the surface of the culture. The PIPA Am molecules rapidly hydrate when the temperature is reduced below 32 C. This leads to a situation where this material is hydrophilic below 32 C and hydrophobic above this temperature, giving it the useful property of temperature sensitivity (98,100). The PIPA Am cell sheet material can therefore support monolayers of confluent periodontal ligament cells forming on its surface at 37 C. This cell sheet layer can be harvested by lowering the temperature to 20 C. When the temperature is lowered, the cells detach spontaneously and float free as a thin, viable cell sheet. The advantage of this technique is that the cell sheet can avoid enzymatic degradation that has been historically used to harvest cultured cells, and thus retain the extracellular matrix, cell-to-cell junctions and differentiated features. Traditional methods of culturing periodontal ligament cells relied on recovering them by proteolytic treatment using trypsin- EDTA which has been shown to degrade a5 integrin and damage b1 integrin molecular structure and may hamper cell adhesion. The use of the temperature-sensitive method does not cause any destruction to integrins and it is the maintenance of the integrity of the fibronectin molecules that assists in the adherence of the cell sheets to the denuded root surface (27). Trials on animal models using cell sheet technology In a pilot study autologous periodontal ligament cells from extracted premolars of beagle dogs were implanted into surgically created dehiscence defects on mandibular first molars (101). In all animals, there were no signs of inflammation or recession postoperatively. Histologically, in the experimental group, healing with bone, periodontal ligament and cementum formation was noted in three of five defects. Among the control sites, only one showed such tissue formation. Histometric analysis showed more cementum formation in the experimental group than controls. There was also more bone observed in the experimental group, but this difference was not significant. Since this pilot study, a number of studies have been published describing the use of cell sheet technology for periodontal regeneration (27, ). All of these studies have demonstrated good results with histomorphometric analyses demonstrating that transplanted periodontal ligament cell sheets have the ability to regenerate periodontal tissues. A complementary approach to cell sheet technology is the utilization of decellularization techniques, which could make the clinical application of cell sheets more viable through the use of allogenic material (105). More recently, a clinical study of periodontal regeneration using cell sheet technology in humans has commenced in Japan (106). Following approval by the appropriate government regulatory bodies, autologous cell sheets grown in autologous serum were prepared using a standard operating procedure to ensure the quality of the transplant material. Following in vitro and in vivo testing, the cell sheets have been prepared and approved for human clinical trials in January To date we still await the results of these trials but based on preclinical testing, the potential for this technology for periodontal regeneration is exciting (107).

11 The future CAD/CAM CAD/CAM for tissue engineering was first introduced into the clinic in the 1990s, and enabled surgeons to reconstruct trauma or tumor defects in an accurate manner (108). Medical technologies including procedures such as computed tomography, magnetic resonance imaging and 3D laser scanning has enabled the acquisition of site-specific digital 3D models. An impressive array of CAM techniques to create physical models has been developed and includes selective laser sintering, stereolithography and fused deposition modeling. These models are very useful in the preoperative planning stages of advanced surgical regenerative procedures. In dentistry, CAD/CAM has found applications in dental implant placement performed using customized drill guides and this has significantly improved the accuracy of implant positioning and placement over conventional surgical guides (109). Furthermore, using medical imaging and CAD/CAM technology presurgical planning can be enhanced through the production of moulds and study models, which can be used for real-time implant placement in a controlled environment replicating the surgical site. In addition to the indirect use of such customized models, which permit direct in vivo applications, such CAD/CAM technology is increasingly being utilized to manufacture anatomically precise scaffolds with flexible properties (54,57). In recent years a number of promising studies have been published demonstrating the utility of such advances for periodontal regeneration (59,60,110). Nanotechnology Nanotechnology is defined as the development and use of specific phenomena and direct manipulation of materials at the nanoscale (111). This developing field embraces research and development of materials, devices and systems and biological properties that differ from those present in larger scales. Nanotechnology is a multidisciplinary field and encompasses a diverse range of technologies derived from biological sciences, physics, chemistry, biochemistry, engineering and materials science. To date the use of nanotechnology in periodontics has been limited to the development of drug delivery systems mainly for antibacterial and host modulation devices (112,113). However, these applications are beyond the scope of this review and here we focus on the emerging field of nanotechnology for tissue engineering purposes. The development and introduction of biomaterials with nanoscale features with the ability to form ideal interactions with tissues has been a very significant development in the field of tissue engineering (114). Using biology as a guide, significant technological advances have been achieved in incorporating biology and nanomaterials for tissue engineering purposes (115). These applications are beginning to be adopted in the field of periodontal tissue engineering (71). Nanomaterials for periodontal tissue engineering Current approaches for periodontal tissue engineering are based on the fabrication of bioscaffolds, which are biocompatible and can act as a suitable vehicle for cell delivery (1). While this approach is showing good results, the next development in this rapidly moving field will likely involve nanotechnology solutions. Nanotechnology provides a new frontier to create selfassembling systems that are wholly biocompatible for tissue engineering purposes (116,117). Synthetic biologic systems that are self-assembling are based on known biological processes, which occur in tissues during the development and maintenance of tissue homeostasis. Through such approaches, systems can be constructed at the nano-, micro- or even macro-scale. An important feature of nano-constructed self-assembling materials is their ability to be constructed into nano-scaled domains and blocks to allow purpose built control and delivery systems. In this Periodontal tissue engineering 11 regard, the possibilities of this technology for periodontal regeneration are unlimited. It is well recognized that current production of bioscaffolds for cell seeding, growth factor delivery and tissue engineering purposes is producing encouraging results. However, future developments in this field may well be further enhanced through the development of nanodevices and nanostructures for regeneration of damaged periodontal tissues. For example, nano-sized hydroxyapatite has been developed to for periodontal regeneration and shown to have slightly improved clinical outcomes compared to traditional b-tricalcium phosphate for the management of intrabony periodontal defects (118). While current work is focused on the recent development particularly on nanoparticles and nanotubes for periodontal management, the materials developed from them such as the hollow nanospheres, core shell structures, nanocomposites, nanoporous materials and nanomembranes will play a growing role in materials development for the dental industry. Although many studies have been published concerning nanocomposite and nanoporous materials it will become of increasing importance to develop specifically nanomaterials for the management of periodontal diseases. It is envisaged that this trend will be further improved in the future as more nanotechnologies are being commercially explored. Conclusion Periodontal regeneration remains the ideal goal for clinicians in the treatment of destructive periodontal disease. Various regenerative treatment modalities have shown mixed results and success has been limited to a small range of clinical applications. The discovery of periodontal stem cells and advances in tissue engineering technology has enabled the development of new techniques with promising regenerative potential. The contemporary approach of cell sheet technology allows the delivery of autologous cells to the periodontal