Advanced regenerative technologies for periodontal tissue repair

Regenerative periodontal therapy comprises techniques which are particularly designed to restore those parts of the tooth-supporting structures which have been lost due to periodontitis or gingival trauma. The term ‘regeneration’ is defined as a reconstruction of lost or injured tissues in such a way that both the original structures and their function are completely restored. Procedures aimed at restoring lost periodontal tissues favor the creation of new attachment, including the formation a new periodontal ligament with its fibers inserting in newly formed cementum and alveolar bone.

Deep infra-bony defects associated with periodontal pockets are the classic indication for periodontal regenerative therapy. Additionally, different degrees of furcation involvement in molars and upper first premolars are a further indication for regenerative approaches as the furcation area remains difficult to maintain through instrumentation and oral hygiene. A third group of indications for regenerative periodontal therapy are localized gingival recessions and root exposure since they may cause a significant esthetic concern for the patient. The denuding of a root surface with resultant root sensitivity represents a further indication to apply regenerative periodontal therapy in order to achieve both the reduction of root sensitivity and the improvement of esthetics.

Professional periodontal therapy and maintenance, combined with risk factor control, are shown to effectively reduce periodontal disease progression. In contrast to the conventional approaches of anti-inflammatory periodontal therapy, however, the regenerative procedures aimed at repairing lost periodontal tissues, including alveolar bone, periodontal ligament and root cementum, remain more challenging (22). Periodontal research in the past few decades has attempted to systematically determine predictably successful clinical procedures to regenerate periodontal tissues. Hence, various methods in combination with regenerative biomaterials, such as hard- and soft-tissue grafts, or cell occlusive barrier membranes used in guided tissue regeneration procedures, have been pursued to regenerate lost tooth support (160).

Periodontal regeneration is assessed by probing measures, radiographic analysis, direct measurements of new bone and histology (129). Many cases that are considered clinically successful, including cases with significant re-growth of alveolar bone, may histologically still show an epithelial lining along the treated root surface instead of newly formed periodontal ligament and cementum (81). In general, however, the clinical outcome of periodontal regenerative techniques is shown to depend on 1) patient associated factors such as plaque control, smoking habits, residual periodontal infection, or membrane exposure in guided tissue regeneration procedures, 2) effects of occlusal forces that deliver intermittent loads in axial and transverse dimensions, as well as 3) factors associated with the clinical skills of the operator such as lack of primary closure of the surgical wound (90). Even though modified flap designs and microsurgical approaches are shown to positively affect the outcome of both soft and hard tissue regeneration, the clinical success for periodontal regeneration still remains limited in many cases. Moreover, the surgical protocols for regenerative procedures are skill-demanding and may therefore lack practicability for a number of clinicians. Consequently, both clinical and pre-clinical research continues to evaluate advanced regenerative approaches using either new barrier membrane techniques (67), cell-growth stimulating proteins (26, 42, 68) or gene delivery applications (125), respectively, in order to simplify and enhance the rebuilding of missing periodontal support. The aim of our review is to compare these advanced regenerative concepts for periodontal hard and soft tissue repair with conventional regenerative techniques (Table 1). While a focus will be given on clinical applications with the delivery of growth factors, the applications for gene delivery of tissue growth factors are also reviewed.

Table 1.

Currently available regenerative biomaterials in periodontology

Regenerative biomaterials	Trade name(s)	References
Bone autogenous grafts (Autografts)
Intraoral autografts	n/a	Renvert et al. 1985 (130) Ellegaard & Löe 1971 (29)
Extraoral autografts	n/a	Froum et al. 1975 (37)
Bone allogenic grafts (Allografts)
Freeze dried bone allograft	Grafton®, Lifenet®	Mellonig et al. 1991 (93)
Demineralized freeze dried bone allograft	Transplant Foundation®	Gurinsky et al. 2004 (50) Kimble et al. 2004 (74) Trejo et al. 2000 (154)
Bone xenogenic grafts (Xenografts)
Bovine mineral matrix	Bio-Oss, OsteoGraf®, Pep-Gen P-15®	Hartman et al. 2004 (53) Camelo et al. 2001 (12) Mellonig et al. 2000 (94) Nevins et al. 2000 (107) Richardson et al. 1999 (132)
Bone alloplastic grafts (Alloplasts)
Hydroxyapatite (dense, porous, resorbable)	Osteogen®, Periograf®, ProOsteone®	Meffert et al. 1985 (92) Galgut et al. 1992 (39)
Beta Tricalcium phosphate	Sunthgraft®, alpha-BSM®	Palti et al. 2002 (114) Scher et al. 1999 (139) Nery et al. 1992 (104)
Hard tissue replacement polymers	Bioplant®	Dryankova et al. 2001 (27)
Bioactive glass (SiO2, CaO, Na2O, P2O2)	PerioGlas®, BioGran®	Sculean et al. 2005 (142) Reynolds et al. 2003 (131) Trombelli et al. 2002 (156) Fetner et al. 1994 (33)
Coral-derived calcium carbonate	Biocoral®	Polimeni et al. 2004 (119)
Polymer and Collagen sponges
Collagen	Helistat®, Collacote®, Colla-Tec®, Gelfoam®
Poly lactide-copolyglycolide barrier membranes
Methylcellulose	n/a	Lioubavina-Hack et al. 2005 (80)
Hyaluronic acid ester	Hy®	Wikesjö et al. 2003 (161)
Chitosan	n/a	Yeo et al. 2005 (169)
Synthetic hydrogel
Polyethylene glycol	n/a	Jung et al. 2007 (67)
Non-resorbable cell occlusive barrier membranes
Polytetrafluorethylene	Gore-Tex®	Trombelli et al. 2005 (157) Moses et al. 2005 (97) Murphy et al. 2003 (99) Needleman et al. 2002 (102)
Resorbable cell occlusive barrier membranes
Polyglycolide/Polylactide (synthetic)	Ossix®	Minenna et al. 2005 (95) Stavropoulos et al. 2004 (148) Parashis et al. 2003 (115)
Collagen membrane (xenogen)	Bio-Gide®	Sculean et al. 2005 (140) Owczarek et al. 2003 (113) Camelo et al. 1998 (14)
Growth Factors
Enamel matrix derivative	Emdogain®	Rasperini et al. 2005 (126) Rosing et al. 2005 (135) Sanz et al. 2004 (138) Francetti et al. 2004 (36) Tonetti et al. 2002 (153) Esposito et al. 2003 (31) Esposito et al. 2004 (30) Esposito et al. 2005 (32)
Platelet-derived growth factor	Gem 21S®	Nevins et al. 2005 (106)
Bone morphogenetic protein	Infuse®	Fiorellini et al. 2005 (34)

Periodontal wound healing

Research on periodontal wound healing in the past was able to provide the basic understanding of the mechanisms favoring periodontal tissue regeneration. A number of valuable findings at both the cellular and molecular levels was revealed and subsequently used for the engineering of regenerative biomaterials available in periodontal medicine today. In order to provide an overview of the cellular and molecular events and their relation to periodontal tissue regeneration, the course of periodontal wound healing is briefly reviewed in this article.

The biology and principles of periodontal wound healing have previously been reviewed (120). Based on observations following experimental incisions in periodontal soft tissues, after blood clot formation, the sequence of healing is commonly divided into the following phases: 1) soft tissue inflammation; 2) granulation tissue formation; and 3) intercellular matrix formation and remodeling (20, 146). Plasma proteins, mainly fibrinogen, dominate rapidly in the bleeding wound and provide an initial basis for the adherence of a fibrin clot (165). The inflammatory phase of healing in the soft tissue wound is initiated by polymorphonuclear leukocytes infiltrating the fibrin clot from the wound margins shortly followed by macrophages (111). The major function of the polymorphonuclear leukocytes is to debride the wound by removing bacterial cells and injured tissue particles through phagocytosis. The macrophages, in addition, have an important role to play in the initiation of tissue repair. The inflammatory phase progresses into its later stage as the polymorphonuclear leukocyte infiltrate gradually decreases while the macrophage influx continues. These macrophages contribute to the cleansing process by phagocytosis of used polymorphonuclear leukocytes and erythrocytes. Additionally, macrophages release a number of biologically active molecules such as inflammatory cytokines and tissue growth factors, which recruit further inflammatory cells as well as fibroblastic and endothelial cells, thus playing an essential role in the transition of the wound from the inflammation into the granulation tissue formation. The influx of fibroblasts and budding capillaries from the gingival connective tissue and the periodontal ligament connective tissue initiate the phase of granulation tissue formation in the periodontal wound approximately two days after incision. At this stage, fibroblasts are responsible for the formation of a loose new matrix of collagen, fibronectin, and proteoglycans (11). Eventually, cells and matrix form cell-to-cell and cell-to-matrix links that generate a concerted tension resulting in tissue contraction. The phase of granulation tissue formation gradually develops into the final phase of healing in which the reformed, more cell-rich tissue undergoes maturation and sequenced re-modeling to meet functional needs (20, 146).

The morphology of a periodontal wound comprises 1) the gingival epithelium, 2) the gingival connective tissue, 3) the periodontal ligament, and the hard tissue components such as 4) alveolar bone and 5) cementum or dentin on the dental root surface (Fig. 1). This particular composition ultimately affects both the healing events in each tissue component as well as in the entire periodontal site. While the healing of gingival epithelia and their underlying connective tissues concludes in a number of weeks, the regeneration of periodontal ligament, root cementum and alveolar bone generally only occur within a number of weeks or months. Aiming for wound closure, the final outcome of wound healing in the epithelium is the formation of the junctional epithelium surrounding the dentition (15). The healing of gingival connective tissue on the other hand, results in a significant reduction of its volume, thus clinically creating both gingival recession and a reduction of the periodontal pocket. Periodontal ligament is shown to regenerate on newly formed cementum created by cementoblasts originated from periodontal ligament granulation tissue (71). Furthermore, alveolar bone modeling occurs following the stimulation of mesenchymal cells from the gingival connective tissue that are transformed into osteoprogenitor cells by locally expressed bone morphogenetic proteins (75, 149).

Fig. 1.

Periodontal wound following flap surgery: 1) gingival epithelium, 2) gingival connective tissue, 3) periodontal ligament, 4) alveolar bone and 5) cementum or dentin on the dental root surface

A series of classical animal studies were able to demonstrate that the tissue derived from alveolar bone or gingival connective tissue lack cells with the potential to produce a new attachment between the periodontal ligament and newly formed cementum (72, 109). Moreover, granulation tissue derived from the gingival connective tissue or alveolar bone result in root resorption or ankylosis when placed in contact with the root surface. It should be expected, therefore, that these complications would occur more frequently following regenerative periodontal surgery, particularly following those procedures which include the placement of grafting materials to stimulate bone formation. The reason for root resorption (which is rarely observed), however, may be that following the surgical intervention, the dento-gingival epithelium migrates apically along the root surface, forming a protective barrier towards the root surface (10, 73). The findings from these animal experiments revealed that ultimately the periodontal ligament tissue contains the cells with the potential to form a new connective tissue attachment (71).

Typically, the downgrowth of the epithelium along the tooth root surface reaches the level of the periodontal ligament before the latter has regenerated with new layers of cementum and newly inserting connective tissue fibers. Therefore, in order to enable and promote healing towards the rebuilding of cementum and periodontal ligament, the gingival epithelium must be prevented from forming a long junctional epithelium along the root surface down to the former level of the periodontal ligament (Fig. 2). This basic acquisition of knowledge has been the key for the engineering of standard clinical procedures for the placement of a fabricated membrane in guided tissue regeneration.

Fig. 2.

A) Regular healing process following the periodontal flap adaptation with significant reduction of the attachment apparatus.

B) In order to enable and promote the healing towards the rebuilding of cementum and periodontal ligament, the gingival epithelium must be prevented from forming a long junctional epithelium along the root surface down to the former level of the periodontal ligament (e.g. by placement of a bioresorbable membrane).

In summary, the principles of periodontal wound healing presented provide the basic understanding of the events following wounding in surgical interventions. In order to obtain new connective tissue attachment, the granulation tissue derived from periodontal ligament cells has to be given both space and time to format and mature new cementum and periodontal ligament. The conventional guided tissue regeneration techniques in periodontal practice have shown their predictable, however limited, potential to regenerate lost periodontal support. Consequently, advanced regenerative technologies for periodontal tissue repair aim to increase the current gold standards for success of periodontal regeneration. In order to identify advanced repair of tooth-supporting periodontal tissues a number of combinations of conventional regenerative techniques have been evaluated:

Periodontal and alveolar bone repair

• –Guided tissue regeneration & application of tissue growth factor(s)
• –Guided tissue regeneration & hard tissue graft & application of tissue growth factor(s)
• –Hard tissue graft & bio-modification of the tooth root surface
• –Hard tissue graft & application of tissue growth factors

Advanced repair of alveolar bone defects

The morphology of the alveolar infra-bony defect was shown to play a significant role for the establishment of a predictable outcome of regeneration of periodontal attachment (121). Goldman and Cohen originally proposed a classification for infra-bony defects which referred to the number of osseous walls surrounding the defect, either being one-, two- or three-wall, respectively (48).

Hard tissue grafts

In a number of clinical trials and animal experiments, the periodontal flap approach was combined with the placement of bone grafts or implant materials into the curetted bony defects with the aim of favoring periodontal regeneration. The various graft and implant materials evaluated so far can be listed as follows:

1. Autogenous graft: graft transferred from one location to another within the same organism

2. Allogenic graft: graft transferred from one organism to another organism of the same species

3. Xenogenic graft: graft taken from an organism of a different species

4. Alloplastic material: synthetic or inorganic implant material used instead of the previously mentioned graft material.

The biologic rationale behind the use of bone grafts or alloplastic materials for regenerative approaches is the assumption that these materials may serve as a scaffold for bone formation (osteoconduction), contain the bone forming cells (osteogenesis) or bone inductive substances (osteoinduction).

Histological studies in both humans and animals have demonstrated that grafting procedures often result in healing with a long junctional epithelium rather than a new connective tissue attachment (16, 81). Therefore, multiple studies have evaluated the use of hard tissue graft materials for periodontal regeneration in infra-bony defects when compared to the periodontal flap approach alone.

Bio-modification of the tooth root surface

A number of studies have focused on the modification of the periodontitis-involved root surface in order to advance the formation of a new connective tissue attachment. However, despite histological evidence of regeneration following root surface bio-modification with citric acid, the outcome of controlled clinical trials have failed to show any improvements in clinical conditions compared to non-acid treated controls (38, 88, 96).

In recent years, bio-modification of the root surface with enamel matrix proteins during periodontal surgery and following demineralization with ethylene-diamine-tetra-acetic acid (EDTA) has been introduced to promote periodontal regeneration. Based on the understanding of the biological model, the application of enamel matrix proteins (amelogenins) is seen to promote periodontal regeneration since it initiates events that occur during the growth of periodontal tissues (41, 52). The commercially available product Emdogain®, a purified acid extract of porcine origin contains enamel matrix derivates, is reported to be able to enhance periodontal regeneration (Fig. 3). More basic research, in addition to the clinical findings, indicates that enamel matrix derivates have a key role in periodontal wound healing (24, 30). Histological results from both animal and human studies have shown that the application of enamel matrix derivates promotes periodontal regeneration and confidently influences periodontal wound healing (143). Thus far, enamel matrix derivates either alone or in combination with grafts have demonstrated its potential to effectively treat intra-osseous defects and the clinical results appear to be stable long term (155).

Fig. 3.

Periodontal regeneration of a 3 wall infrabony defect using Emdogain

A: 32 year old patient, male, non smoker with severe periodontitis. Tooth 13 shows a probing pocket depth of 10 mm disto-buccally and clinical attachment loss of 14 mm.

B: Pre-treatment radiograph shows the infra-bony defect distal to the tooth 13.

C: After the buccal incision of the papilla, the interdental tissue is preserved attached to the palatal flap. After the debridement of the granulation tissue and the root surface debridement, the infra-bony defect is classified and measured: the predominant component is a 7 mm deep 3-wall defect.

D: 1-year after surgical intervention the distal site of the tooth #13 shows a probing pocket depth of 2 mm and clinical attachment loss of 7 mm. If compared with the initial measurements a probing pocket depth gain of 8 mm and a clinical attachment loss gain of 7 mm has been achieved.

E: Radiograph 1 year post-surgery showing filling of the defect.

Periodontal tissue growth factors

Wound healing approaches using growth factors to target restoration of tooth-supporting bone, periodontal ligament, and cementum has been shown to significantly advance the field of periodontal regenerative medicine. A major focus of periodontal research has studied the impact of tissue growth factor applications on periodontal tissue regeneration (Table 2) (3, 42, 101, 123). Advances in molecular cloning have made available unlimited quantities of recombinant growth factors for applications in tissue engineering. Recombinant growth factors known to promote skin and bone wound healing, such as platelet-derived growth factors (13, 44, 65, 106, 112, 136), insulin like growth factors (42, 44, 56, 84), fibroblast growth factors) (47, 98, 145, 151, 152) and bone morphogenetic proteins (40, 57, 147, 162, 163) have been used in pre-clinical and clinical trials for the treatment of large periodontal or infra-bony defects, as well as around dental implants (34, 66, 106). The combined use of rhplatelet-derived growth factor-BB and peptide P-15 with a graft biomaterial has shown beneficial effects in intra-osseous defects (155). However, contrasting results were reported for growth factors such as platelet-rich plasma and graft combinations, or the use of BAs either alone or in association with graft or guided tissue regeneration for the treatment of furcation defects (155).

Table 2.

Effects of growth factors used for periodontal tissue engineering

Growth Factor	Effects
Platelet-derived growth factor	Migration, proliferation and non-collagenous matrix synthesis of mesenchymal cells
Bone morphogenetic protein	Proliferation, differentiation of osteoblasts, differentiation of periodontal ligament cells into osteoblasts
Enamel matrix derivative	Proliferation, protein synthesis and mineral nodule formation in periodontal ligament cells, osteoblasts and cementoblasts
Transforming growth factor Beta	Proliferation of cementoblasts and periodontal ligament fibroblasts
Insulin-like growth factor-1	Cell migration, proliferation, differentiation and matrix synthesis
Fibroblast growth factor-2	Proliferation and attachment of endothelial cells and periodontal ligament cells

Biological effects of growth factors: platelet-derived growth factor

Platelet-derived growth factor is a member of a multifunctional polypeptide family that binds to two cell membrane tyrosine kinase receptors (platelet-derived growth factor-Rα and platelet-derived growth factor-Rβ) and subsequently exerts its biological effects on cell proliferation, migration, extracellular matrix synthesis, and anti-apoptosis (54, 69, 134, 144). Platelet-derived growth factor-α and -β receptors are expressed in regenerating periodontal soft and hard tissues (116). In addition, platelet-derived growth factor initiates tooth-supporting periodontal ligament cell chemotaxis (108), mitogenesis (110), matrix synthesis (51), and attachment to tooth dentinal surfaces (170). More importantly, in vivo application of platelet-derived growth factor alone or in combination with insulin-like growth factor-1 results in partial repair of periodontal tissues (44, 45, 84, 85, 136). Platelet-derived growth factor has been shown to have a significant regenerative impact on periodontal ligament cells as well as on osteoblasts (87, 89, 110, 112).

The clinical application of platelet-derived growth factor was shown to successfully advance alveolar bone repair and clinical attachment level gain. A first clinical study reported the successful repair of class II furcations using demineralized freeze-dried bone allograft saturated with rhplatelet-derived growth factor-BB (105). Subsequently, rhplatelet-derived growth factor-BB mixed with a synthetic beta-tricalcium phosphate matrix was shown to advance the repair of deep infra-bony pockets in a large multicenter randomized controlled trial (106). Both studies demonstrated that the use of rhplatelet-derived growth factor-BB was safe and effective in the treatment of periodontal osseous defects. In a follow-up trial, the same sample patients were again assessed 18 or 24 months following periodontal surgery. Substantial radiographic changes in the appearance of the defect fill were observed for patients treated with rhplatelet-derived growth factor-BB (91).

Biological effects of growth factors: bone morphogenetic proteins

Bone morphogenetic proteins are multifunctional polypeptides belonging to the transforming growth factor-β superfamily of proteins (167). The human genome encodes at least twenty bone morphogenetic proteins (128). Bone morphogenetic proteins bind to type I and II receptors that function as serine-threonine kinases. The type I receptor protein kinase phosphorylates intracellular signaling substrates called Smads (Sma gene in Caenorhabditis elegans and Mad gene in Drosophila). The phosphorylated bone morphogenetic protein-signaling Smads enter the nucleus and initiate the production of bone matrix proteins leading to bone morphogenesis. The most remarkable feature of bone morphogenetic proteins is the ability to induce ectopic bone formation (158). Bone morphogenetic proteins are not only powerful regulators of cartilage and bone formation during embryonic development and regeneration in postnatal life, but also participate in the development and repair of other organs such as the brain, kidney, and nerves (127).

Sigurdsson et al. (145) evaluated bone and cementum formation following regenerative periodontal surgery using recombinant human bone morphogenetic protein in surgically-created supra-alveolar defects in dogs (166). Histologic analysis showed significantly more cementum formation and regrowth of alveolar bone on bone morphogenetic protein treated sites as compared to the controls.

Studies have demonstrated the expression of bone morphogenetic proteins during tooth development and periodontal repair including alveolar bone (1, 2). Investigations in animal models have shown the potential repair of alveolar bony defects using recombinant human bone morphogenetic protein-12 (rhBMP-12) (163) or rhBMP-2 (83, 164). In a clinical trial by Fiorellini et al. (34), recombinant human bone morphogenetic protein-2 (rhBMP-2) delivered by a bioabsorbable collagen sponge revealed significant bone formation in a human buccal wall defect model following tooth extraction when compared to collagen sponge alone. Furthermore, bone morphogenetic protein-7, also known as osteogenic protein-1, stimulates bone regeneration around teeth, endosseous dental implants, and in maxillary sinus floor augmentation procedures (47, 137, 159).

Clinical applications of growth factors for use in periodontal regeneration

In general, the impact of a topical delivery of growth factors to periodontal wounds has shown to be promising, yet insufficient for the promotion of predictable periodontal tissue engineering (13, 21) (Fig. 4). Growth factor proteins, once delivered to the target site, tend to suffer from instability and quick dilution, presumably due to proteolytic breakdown, receptor-mediated endocytosis, and solubility of the delivery vehicle (3). Because their half-lives are significantly reduced, the period of exposure may not be sufficient to act on osteoblasts, cementoblasts, or periodontal ligament cells. Therefore different methods of growth factor delivery need to be considered (4).

Fig. 4.

Periodontal regeneration using platelet-derived growth factor and bone graft materials

A: 27-year old patient at the reevaluation visit after the initial non-surgical therapy presented 3 sites with probing pocket depth > 6 mm, one of those distal to the tooth #44 shows a probing pocket depth of 7 mm and no gingival recession.

B: The periapical radiograph shows a deep 1-wall defect distal to tooth #44 and a lesion between the teeth #45 and 46.

C: The measurement of the 1-wall defect shows an infra-bony component of 6 mm

D: The Grafting material (GEM 21S®) is mixed with particles of autogenous bone chips collected in the surgical area with a Rhodes instrument and with the liquid component of the GEM 21S® (platelet-derived growth factor).

E: The liquid platelet-derived growth factor is placed in the defect together with the graft to rebuild the lost bone.

F: A second internal mattress suture in performed with a 7- 0 Gore Tex® suture, to allow for optimal adaptation of the flap margin without the interference of the epithelium. The two internal mattress sutures are tied and the knots are performed only after a perfect free tension closure of the wound. Two additional interrupted 7-0 sutures are placed to ensure stable contact between the connective tissues of the edges of the flaps. The mesial and distal papillae are stabilized with additional simple interrupted sutures

G: 9 months after surgery, the probing pocket depth is 2 mm.

H: 9 months after surgery, the periapical radiograph shows a good bone fill of the 1 wall bony defect.

I: 9 months after surgery, the surgical re-entry shows the new bone formation

Investigations for periodontal bioengineering have examined a variety of methods combining delivery vehicles, such as scaffolds, with growth factors to target the defect site in order to optimize bioavailability (82). The scaffolds are designed to optimize the dosage of the growth factor and to control its release pattern which may be pulsatile, constant or time programmed (7). Additionally, the kinetics of the release and the duration of the exposure of the growth factor may be controlled (59).

A new polymeric system was reported in an animal study by Richardson et al. (133) enabling the tissue-specific delivery of two or more growth factors, with a controlled dose and rate of delivery. The dual delivery of vascular endothelial growth factor together with platelet-derived growth factor from a single, structural polymer scaffold results in the rapid formation of a mature vascular network (133).

Guided tissue regeneration

Histological findings from periodontal regeneration studies reveal that a new connective tissue attachment could be predicted if the cells from the periodontal ligament settle on the root surface during healing. Hence, the clinical applications of guided tissue regeneration in periodontics involve the placement of a physical barrier membrane to enable the previous periodontitis-affected tooth root surface to be repopulated with cells from the periodontal ligament. In the last decades, guided tissue regeneration has been applied in many clinical trials for the treatment of various periodontal defects, such as infra-bony defects (23), furcation involvements (70, 86), and localized gingival recessions (118). In a recent systematic review, the combinations of barrier membranes and grafting materials used in preclinical models have been summarized. The analysis of ten papers revealed that the combination of barrier membranes and grafting materials may result in histological evidence of periodontal regeneration, predominantly bone repair. No additional histological benefits of combination treatments were found in animal models of three wall intrabony, class II furcation, or fenestration defects. In supra-alveolar and two wall intrabony defect models of periodontal regeneration, the additional use of a grafting material gave superior histological results of bone repair to barrier membranes alone (141).

The types of barrier membranes evaluated in clinical studies vary regarding design, configuration, and composition. Non-resorbable membranes of expanded polytetrafluoroethylene have been used successfully in both animal experiments and human clinical trials. In recent years, natural or synthetic bio-absorbable barrier membranes have been used for guided tissue regeneration in order to eliminate the need for a follow-up surgery for membrane removal. Collagen membranes as well as barrier materials of polylactic acid or copolymers of polylactic acid and polyglycolic acid have been tested in animal and human studies.

Following therapy, guided tissue regeneration has a greater effect on probing measures of periodontal treatment than periodontal flap surgery alone, including increased attachment gain, reduction of probing depth, less gingival recession and more gain in hard tissue probing at surgical re-entry. Referring to the best available evidence today, however, it is difficult to draw general conclusions about the clinical benefit of guided tissue regeneration. Even as there is evidence that guided tissue regeneration can demonstrate a significant improvement over conventional open flap surgery, the factors affecting outcomes are unclear from the present literature since they might comprise study conduct issues such as bias (103).

In summary, guided tissue regeneration represents a very well documented regenerative procedure today to achieve periodontal regeneration in infra-bony defects and in degree II furcations. An added benefit may be achieved by the additional use of grafting materials (153).

Gene therapeutics for periodontal tissue repair

Although encouraging results for periodontal regeneration have been found in various clinical investigations using recombinant tissue growth factors, limitations exist with topical protein delivery such as transient biological activity, protease inactivation, and poor bioavailability from existing delivery vehicles. Therefore, newer approaches seek to develop methodologies that optimize growth factor targeting to maximize the therapeutic outcome of periodontal regenerative procedures. Genetic approaches in periodontal tissue engineering show early progress in achieving delivery of growth factor genes such as platelet-derived growth factor or bone morphogenetic protein to periodontal lesions (Fig. 5). Gene transfer methods may circumvent many of the limitations with protein delivery to soft tissue wounds (9, 43). It has been shown that the application of growth factors (35, 61, 63, 75) or soluble forms of cytokine receptors (150) by gene transfer provides a greater sustainability than that of single protein application. Thus, gene therapy may achieve greater bioavailability of growth factors within periodontal wounds and thus providing greater regenerative potential.

Fig. 5.

Advanced approaches for regenerating tooth-supporting structures.

A) Application of a graft material (e.g. bone ceramic) and growth factor into an infra-bony defect covered by a bioresorbable membrane.

B) Application of gene vectors for the transduction of growth factors producing target cells.

Methods for gene delivery in periodontal applications

Various gene delivery methods are available to administer growth factors to periodontal defects offering great flexibility for tissue engineering. The delivery method can be tailored to the specific characteristics of the wound site. For example, a horizontal one or two-walled defect may require the use of a supportive carrier like a scaffold. Other defect sites may be conducive to the use of an adenovirus-vector embedded in a collagen matrix.

More importantly from a clinical point of view is the risk associated with the use of gene therapy in periodontal tissue engineering (49). As with maximizing growth factor sustainability and accounting for specific characteristics of the wound site, both the DNA vector and delivery method need to be considered when assessing patient safety. In summary, studies examining the use of specific delivery methods and DNA vectors in periodontal tissue engineering reflect the aim to maximize the duration of growth factor expression, optimize delivery method to periodontal defect, and minimize patient risk.

A combination of an AAV-delivered angiogenic molecule such as vascular endothelial growth factor, bone morphogenetic protein signaling receptor (caALK2) and receptor activator of nuclear factor-kappa B ligand (RANKL) were demonstrated to promote bone allograft turnover and osteogenesis as a mode to enrich human bone allografts (60). To date, combinations of vascular endothelial growth factor/bone morphogenetic protein (117) and platelet-derived growth factor/vascular endothelial growth factor (133) have been performed with highly positive synergistic responses in bone repair.

Promising preliminary results from preclinical studies reveal that host modulation achieved through gene delivery of soluble proteins such as tumor necrosis factor receptor 1 (TNFR1:Fc) reduces tumor necrosis factor activity and therefore inhibits alveolar bone loss (150). These results are comparable to the findings in the research on rheumatoid arthritis where pathogenesis includes high tumor necrosis factor activity, and pathways for bone resorption are similar (124).

Preclinical studies evaluating growth factor gene therapy for periodontal tissue engineering

In order to overcome the short half-lives of growth factor peptides in vivo, gene therapy using a vector encoding the growth factor is advocated to stimulate tissue regeneration. So far, two main strategies of gene vector delivery have been applied to periodontal tissue engineering. Gene vectors can be introduced directly to the target site (in vivo technique) (61) or selected cells can be harvested, expanded, genetically transduced and then re-implanted (ex vivo technique) (63). In vivo gene transfer involves the insertion of the gene of interest directly into the body anticipating the genetic modification of the target cell. Ex vivo gene transfer includes the incorporation of genetic material into cells exposed from a tissue biopsy with subsequent re-implantation into the recipient. Using the in vivo technique, the potential inhibition of alveolar bone loss has been studied in an experimental periodontitis model evaluating the inhibition of osteoclastogenesis by administering human osteoprotegerin, a competitive inhibitor of the RANKL-derived osteoclast activation. Significant preservation of alveolar bone volume was observed among osteoprotegerin:Fc-treated animals compared to the controls. Systemic delivery of osteoprotegerin:Fc inhibits alveolar bone resorption in experimental periodontitis, suggesting that RANKL inhibition may represent an important therapeutic strategy for the prevention of progressive alveolar bone loss (62).

Platelet-derived growth factor gene delivery

The application of platelet-derived growth factor-gene transfer strategies to tissue engineering has originally been generated to improve healing in soft tissue wounds such as skin lesions (25). Both plasmid- (55) and adenovirus/platelet-derived growth factor gene delivery (122) have been evaluated in preclinical and human trials. However, the latter has been able to exhibit more safety favorable for clinical use (49). In a recent animal study reporting on safety and distribution profiles, adenovirus-platelet-derived growth factor-B applied for tissue engineering of tooth-supporting alveolar bone defects was well contained within the localized osseous defect area without viremia or distant organ involvement (17).

Early studies in dental applications using recombinant adenoviral vectors encoding platelet-derived growth factor demonstrated the ability of these vector constructs to potently transduce cells isolated from the periodontium (osteoblasts, cementoblasts, periodontal ligament cells, and gingival fibroblasts) (46, 171). These studies revealed the extensive and prolonged transduction of periodontal-derived cells. Both Chen & Giannobile (18) and Lin et al. (79) were able to demonstrate the effects of adenoviral delivery of platelet-derived growth factor for the better understanding of sustained platelet-derived growth factor signaling. Gene delivery of platelet-derived growth factor-B generally displays higher sustained signal transduction effects in human gingival fibroblasts when compared to cells treated with recombinant human platelet-derived growth factor-BB protein alone. Their data on platelet-derived growth factor gene delivery may contribute to an improved understanding of these pathways that are likely to play a role in the control of clinical outcomes of periodontal regenerative therapy.

In an ex vivo investigation by Anusaksathien et al. () it was shown that the expression of platelet-derived growth factor genes was prolonged for up to 10 days in gingival wounds. Adenovirus encoding platelet-derived growth factor-B (adenovirus/platelet-derived growth factor-B) transduced gingival fibroblasts and enhanced defect fill by induction of human gingival fibroblast migration and proliferation (6). On the other hand, continuous exposure of cementoblasts to platelet-derived growth factor-A had an inhibitory effect on cementum mineralization, possibly via the upregulation of osteopontin and subsequent enhancement of multinucleated giant cells in cementum engineered scaffolds. Moreover, adenovirus/platelet-derived growth factor-1308 (a dominant-negative mutant of platelet-derived growth factor) inhibited mineralization of tissue-engineered cementum possibly due to downregulation of bone sialoprotein and osteocalcin with a persistence of stimulation of multinucleated giant cells. These findings suggest that continuous exogenous delivery of platelet-derived growth factor-A may delay mineral formation induced by cementoblasts, while platelet-derived growth factor is clearly required for mineral neogenesis (5).

Jin et al. (61) demonstrated that direct in vivo gene transfer of platelet-derived growth factor-B was able to stimulate tissue regeneration in large periodontal defects. Descriptive histology and histomorphometry revealed that human platelet-derived growth factor-B gene delivery promotes the regeneration of both cementum and alveolar bone, while platelet-derived growth factor-1308, a dominant negative mutant of platelet-derived growth factor-A, has minimal effects on periodontal tissue regeneration.

Bone morphogenetic protein gene delivery

An experimental study in rodents by Lieberman et al. (78) advanced gene therapy for bone regeneration with results revealing that the transduction of bone marrow stromal cells with rhBMP-2 lead to bone formation within an experimental defect comparable to skeletal bone. Another group was similarly able to regenerate skeletal bone by directly administering adenovirus5/BMP-2 into a bony segmental defect in rabbits (8). Further advances in the area of orthopedic gene therapy using viral delivery of bone morphogenetic protein-2 have provided further evidence for the ability of both in vivo and ex vivo bone engineering (19, 76, 77, 100). Franceschi et al. (35) investigated in vitro and in vivo adenovirus gene transfer of bone morphogenetic protein-7 for bone formation. Adenovirus transduced nonosteogenic cells were also found to differentiate into bone-forming cells and to produce bone morphogenetic protein-7 (75) or bone morphogenetic protein-2 (19) both in vitro and in vivo. In another study by Huang et al. (58), plasmid DNA encoding for bone morphogenetic protein-4 administered with a scaffold delivery system was found to enhance bone formation when compared with blank scaffolds.

In an early approach to regenerate alveolar bone in an animal model, it was demonstrated that the ex vivo delivery of adenovirus encoding murine bone morphogenetic protein-7 was found to promote periodontal tissue regeneration in large mandibular periodontal bone defects (63). Bone morphogenetic protein-7 gene transfer not only enhanced alveolar bone repair, it also stimulated cementogenesis and periodontal ligament fiber formation. Of interest, the alveolar bone formation was found to occur via a cartilage intermediate. However, when genes encoding the bone morphogenetic protein antagonist noggin were delivered, inhibition of periodontal tissue formation resulted (64). In a study by Dunn et al. (28), it was shown that direct in vivo gene delivery of adenovirus/bone morphogenetic protein-7 in a collagen gel carrier promoted successful regeneration of alveolar bone defects around dental implants. Furthermore, an in vivo synergism of adenoviral-mediated coexpression of bone morphogenetic protein-7 and insulin like growth factor-1 on human periodontal ligament cells in up-regulating alkaline phosphatase activity and mRNA levels of collagen type I and Runx2 was found (168). Implantation with scaffolds illustrated that the transduced cells exhibited osteogenic differentiation and formed bone-like structures. It was concluded that the combined delivery of bone morphogenetic protein-7 and insulin like growth factor-1 genes using an internal ribosome entry site-based strategy synergistically enhanced differentiation of human periodontal ligament cells (168).

These experiments provide promising evidence showing the feasibility of both in vivo and ex vivo gene therapy for periodontal tissue regeneration and peri-implant osseointegration.

Future perspectives: targeted gene therapy in vivo

Major advances have been made over the past decade in the reconstruction of complex periodontal and alveolar bone wounds that have resulted from disease or injury. Developments in scaffolding matrices for cell, protein and gene delivery have demonstrated significant potential to provide “smart” biomaterials that can interact with the matrix, cells and bioactive factors. The targeting of signaling molecules or growth factors (via proteins or genes) to periodontal tissue components has lead to significant new knowledge generation using factors that promote cell replication, differentiation, matrix biosynthesis and angiogenesis. A major challenge that has been less studied is the modulation of the exuberant host response to microbial contamination that plagues the periodontal wound microenvironment. For improvements in the outcomes in periodontal regenerative medicine, scientists will need to examine dual delivery of host modifiers or anti-infective agents to optimize the results of therapy. Further advancements in the field will continue to rely heavily on multidisciplinary approaches combining engineering, dentistry, medicine, and infectious disease specialists in repairing the complex periodontal wound environment.