1. Periodontology 2000, Vol. 59, 2012, 185–202 Ó 2012 John Wiley & Sons A/S
Printed in Singapore. All rights reserved PERIODONTOLOGY 2000
Advanced reconstructive
technologies for periodontal
tissue repair
C H R I S T O P H A. R A M S E I E R , G I U L I O R A S P E R I N I , S A L V A T O R E B A T I A &
W I L L I A M V. G I A N N O B I L E
Regenerative periodontal therapy uses specific tech- identify clinical procedures that are predictably suc-
niques designed to restore those parts of the tooth- cessful in regenerating periodontal tissues. Hence,
supporting structures that have been lost as a result the extent to which various methods, in combination
of periodontitis or gingival trauma. The term Ôregen- with regenerative biomaterials, such as hard- and
erationÕ is defined as the reconstruction of lost or soft-tissue grafts, or cell-occlusive barrier mem-
injured tissues in such a way that both the original branes used in guided tissue-regeneration proce-
structures and their function are completely restored. dures, are able to regenerate lost tooth support has
Procedures aimed at restoring lost periodontal tissues been investigated (162).
favor the creation of new attachment, including the Periodontal regeneration is assessed using probing
formation of a new periodontal ligament with its measures, radiographic analysis, direct measure-
fibers inserting in newly formed cementum and ments of new bone and histology (133). Many cases
alveolar bone. that are considered clinically successful, including
Deep infrabony defects associated with periodontal those in which significant regrowth of alveolar bone
pockets are the classic indication for periodontal- occurs, may histologically still show an epithelial
regenerative therapy. Different degrees of furcation lining along the treated root surface, instead of newly
involvement in molars and upper first premolars are a formed periodontal ligament and cementum (84). In
further indication for regenerative approaches as the general, however, the clinical outcome of periodon-
furcation area remains difficult to maintain through tal-regenerative techniques is shown to depend on:
instrumentation and oral hygiene. A third group of (i) patient-associated factors, such as plaque control,
indications for regenerative periodontal therapy are smoking habits, residual periodontal infection, or
localized gingival recession and root exposure be- membrane exposure in guided tissue-regeneration
cause they may cause significant esthetic concern for procedures, (ii) effects of occlusal forces that deliver
the patient. The denuding of a root surface with intermittent loads in axial and transverse dimensions,
resultant root sensitivity represents a further indica- as well as (iii) factors associated with the clinical skills
tion for regenerative periodontal therapy in order to of the operator, such as lack of primary closure of the
reduce root sensitivity and to improve esthetics. surgical wound (93). Even though modified flap de-
Professional periodontal therapy and maintenance, signs and microsurgical approaches are shown to
combined with risk-factor control, are shown to positively affect the outcome of both soft- and hard-
effectively reduce periodontal disease progression (7, tissue regeneration, the clinical success for peri-
128). In contrast to the conventional approaches of odontal regeneration still remains limited in many
anti-inflammatory periodontal therapy, however, the cases. Moreover, the surgical protocols for regenera-
regenerative procedures aimed at repairing lost tive procedures are skill-demanding and may there-
periodontal tissues, including alveolar bone, peri- fore lack practicability for a number of clinicians.
odontal ligament and root cementum, remain more Consequently, both clinical and preclinical research
challenging (24). During the last few decades, peri- continues to evaluate advanced regenerative
odontal research has systematically attempted to approaches using new barrier-membrane techniques
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2. Ramseier et al.
(69), cell-growth-stimulating proteins (28, 44, 70) or sue growth factors, which recruit further inflamma-
gene-delivery applications (125) in order to simplify tory cells as well as fibroblastic and endothelial cells,
and enhance the rebuilding of missing periodontal thus playing an essential role in the transition of the
support. The aim of our review was to compare these wound from the inflammatory stage to the granula-
advanced regenerative concepts for periodontal tion tissue-formation stage. The influx of fibroblasts
hard- and soft-tissue repair with conventional and budding capillaries from the gingival connective
regenerative techniques (Table 1). While the focus tissue and the periodontal ligament connective tissue
will be on clinical applications for the delivery of initiate the phase of granulation-tissue formation in
growth factors, the applications for gene delivery of the periodontal wound approximately 2 days after
tissue growth factors are also reviewed. incision. At this stage, fibroblasts are responsible for
the formation of a loose new matrix of collagen,
fibronectin and proteoglycans (12). Eventually, cells
Periodontal wound healing and matrix form cell-to-cell and cell-to-matrix links
that generate a concerted tension, resulting in tissue
Previous research on periodontal wound healing has contraction. The phase of granulation-tissue forma-
provided a basic understanding of the mechanisms tion gradually develops into the final phase of heal-
favoring periodontal tissue regeneration. A number of ing, in which the reformed, more cell-rich tissue,
valuable findings at both the cellular and molecular undergoes maturation and sequenced remodeling to
levels was revealed and subsequently used to engi- meet functional needs (22, 150).
neer the regenerative biomaterials currently available The morphology of a periodontal wound comprises
in periodontal medicine. In order to provide an the gingival epithelium, the gingival connective tis-
overview of the cellular and molecular events and sue, the periodontal ligament and the hard-tissue
their association with periodontal tissue regenera- components, such as alveolar bone and cementum or
tion, the course of periodontal wound healing is dentin on the dental root surface (Fig. 1). This par-
briefly reviewed in this article. ticular composition ultimately affects the healing
The biology and principles of periodontal wound events in each tissue component as well as those in
healing have previously been reviewed (123). Based the entire periodontal site. While the healing of gin-
on observations following experimental incisions in gival epithelia and their underlying connective
periodontal soft tissues, the sequence of healing after tissues concludes in a number of weeks, the regen-
blood-clot formation is commonly divided into the eration of periodontal ligament, root cementum and
following phases: (i) soft-tissue inflammation, (ii) alveolar bone generally takes longer, occurring within
granulation-tissue formation, and (iii) intercellular a number of weeks or months. Aiming for wound
matrix formation and remodeling (22, 150). Plasma closure, the final outcome of wound healing in the
proteins, mainly fibrinogen, accumulate rapidly in epithelium is the formation of the junctional epi-
the bleeding wound and provide the initial basis for thelium surrounding the dentition (16). On the other
the adherence of a fibrin clot (167). The inflammatory hand, the healing of gingival connective tissue results
phase of healing in the soft-tissue wound is initiated in a significant reduction of its volume, thus clinically
by polymorphonuclear leukocytes infiltrating the fi- creating both gingival recession and a reduction of
brin clot from the wound margins, followed shortly the periodontal pocket. Periodontal ligament is
afterwards by macrophages (114). The major function shown to regenerate on newly formed cementum
of the polymorphonuclear leukocytes is to debride created by cementoblasts that have originated from
the wound by removing bacterial cells and injured periodontal ligament granulation tissue (73). Fur-
tissue particles through phagocytosis. The macro- thermore, alveolar bone modeling occurs following
phages, in addition, have an important role to play in the stimulation of mesenchymal cells from the
the initiation of tissue repair. The inflammatory gingival connective tissue that are transformed into
phase progresses into its later stage as the amount of osteoprogenitor cells by locally expressed bone
polymorphonuclear leukocyte infiltrate gradually morphogenetic proteins (78, 154).
decreases while the macrophage influx continues. A series of classical animal studies demonstrated
These macrophages contribute to the cleansing pro- that the tissue derived from alveolar bone or gingival
cess through the phagocytosis of used polymorpho- connective tissue lacks cells with the potential to
nuclear leukocytes and erythrocytes. Additionally, produce a new attachment between the periodontal
macrophages release a number of biologically active ligament and newly formed cementum (74, 112).
molecules, such as inflammatory cytokines and tis- Moreover, granulation tissue derived from the gingi-
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3. Periodontal tissue-engineering technologies
Table 1. Regenerative biomaterials currently available for use in periodontology
Regenerative biomaterials Trade name(s) References
Bone autogenous grafts (autografts)
Intra-oral autografts n⁄a Renvert et al. (134)
¨
Ellegaard & Loe (31)
Extra-oral autografts n⁄a Froum et al. (39)
Bone allogenic grafts (allografts)
Freeze-dried bone allograft GraftonÒ (Osteotech, Eatontown, NJ, USA), Mellonig et al. (96)
LifenetÒ (LifeNet Health Inc., Virginia Beach,
VA, USA)
Demineralized freeze-dried bone Transplant FoundationÒ (Transplant Gurinsky et al. (52)
allograft Foundation Inc., Miami, FL, USA) Kimble et al. (76)
Trejo et al. (156)
Bone xenogenic grafts (xenografts)
Bovine mineral matrix Bio-OssÒ (Geistlich Pharma AG, Wolhusen, Hartman et al. (55)
Switzerland), OsteoGrafÒ (Dentsply, Tulsa, OK, Camelo et al. (13)
USA), Pep-Gen P-15Ò (Dentsply GmbH, Mellonig (97)
Mannheim, Germany) Nevins et al. (108)
Richardson et al. (136)
Bone alloplastic grafts (alloplasts)
Hydroxyapatite (dense, porous, OsteogenÒ (Impladent Ltd, Meffert et al. (95)
resorbable) Holliswood, NY, USA) Galgut et al. (41)
Beta tricalcium phosphate SynthographÒ (Bicon, Boston, MA, USA), Palti & Hoch (117)
alpha-BSMÒ (Etex Corp., Cambridge, MA, Scher et al. (143)
USA) Nery et al. (107)
Hard-tissue replacement polymers BioplantÒ (Kerr Corp., Orange, CA, USA) Dryankova et al. (29)
Bioactive glass (SiO2, CaO, Na2O, PerioGlasÒ (Novabone, Jacksonville, FL, USA), Sculean et al. (146)
P2O2) BioGranÒ (Biomet 3i, Palm Beach Gardens, FL, Reynolds et al. (135)
USA) Trombelli et al. (158)
Fetner et al. (35)
Coral-derived calcium carbonate BiocoralÒ (Biocoral Inc., La Garenne Colombes, Polimeni et al. (122)
France)
Polymer and collagen sponges
Collagen HelistatÒ (Dental Implant Technologies Inc.,
Scottsdale, AZ, USA), CollacoteÒ (Carlsbad, CA,
USA), Colla-TecÒ (Colla-Tec Inc., Plainsboro,
NJ, USA), GelfoamÒ (Baxter, Deerfield, IL, USA)
Polylactide-copolyglycolide barrier membranes
Methylcellulose n⁄a Lioubavina-Hack et al. (83)
Hyaluronic acid ester n⁄a ¨
Wikesjo et al. (163)
Chitosan n⁄a Yeo et al. (171)
Synthetic hydrogel
Polyethylene glycol n⁄a Jung et al. (69)
Nonresorbable cell-occlusive barrier membranes
Polytetrafluorethylene Gore-TexÒ (W. L. Gore & Associates Inc., New- Trombelli et al. (159)
ark, DE, USA) Moses et al. (100)
Murphy & Gunsolley (102)
Needleman et al. (105)
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4. Ramseier et al.
Table 1. Continued
Regenerative biomaterials Trade name(s) References
Resorbable cell-occlusive barrier membranes
Polyglycolide ⁄ Polylactide (synthetic) OssixÒ (ColBar LifeScience Ltd., Rehovot, Israel) Minenna et al. (98)
Stavropoulos et al. (153)
Parashis et al. (118)
Collagen membrane (xenogen) Bio-GideÒ (Geistlich Pharma AG, Wolhusen, Sculean et al. (144)
Switzerland) Owczarek et al. (116)
Camelo et al. (15)
Growth factors
Enamel matrix derivative EmdogainÒ (Straumann AG, Basel, Switzerland) Rasperini et al. (130)
Rosing et al. (139)
Sanz et al. (142)
Francetti et al. (38)
Tonetti et al. (155)
Esposito et al. (32)
Esposito et al. (33)
Esposito et al. (34)
Platelet-derived growth factor Gem 21SÒ (Osteohealth, Shirley, NY, USA) Nevins et al. (110)
Ò
Bone morphogenetic protein Infuse (Medtronic Inc., Minneapolis, MN, Fiorellini et al. (36)
USA)
surgical intervention, the dento–gingival epithelium
migrates apically along the root surface, forming a
protective barrier towards the root surface (11, 75).
The findings from these animal experiments revealed
that ultimately the periodontal ligament tissue con-
tains cells with the potential to form a new connec-
tive tissue attachment (73).
Typically, the down-growth of the epithelium along
the tooth-root surface reaches the level of the peri-
odontal 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
Fig. 1. Periodontal wound following flap surgery: (1) junctional epithelium along the root surface down to
gingival epithelium, (2) gingival connective tissue, (3) the former level of the periodontal ligament (Fig. 2).
alveolar bone, (4) periodontal ligament and (5) cementum
This basic acquisition of knowledge has been the key
or dentin on the dental root surface.
for the engineering of standard clinical procedures
for the placement of a fabricated membrane in gui-
val connective tissue or alveolar bone results in root ded tissue regeneration.
resorption or ankylosis when placed in contact with In summary, the principles of periodontal wound
the root surface. Therefore, it should be expected that healing presented provide a basic understanding of
these complications would occur more frequently the events following wounding in surgical interven-
following regenerative periodontal surgery, particu- tions. In order to obtain new connective tissue
larly following those procedures that include the attachment, the granulation tissue derived from
placement of grafting materials to stimulate bone periodontal ligament cells has to be given both space
formation. The reason for root resorption (which is and time to produce and mature new cementum and
rarely observed), however, may be that following the periodontal ligament. The conventional guided tis-
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5. Periodontal tissue-engineering technologies
attachment (124). Goldman & Cohen (50) originally
proposed a classification for infrabony defects that
referred to the number of osseous walls surrounding
the defect: one-wall, two-wall or three-wall.
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 stimulating
periodontal regeneration. The various graft and im-
plant materials evaluated to date are: (i) autogenous
graft: a graft transferred from one location to another
within the same organism; (ii) allogenic graft: a graft
transferred from one organism to another organism
of the same species; (iii) xenogenic graft: a graft taken
from an organism of a different species; and (iv)
alloplastic material: synthetic or inorganic implant
material used instead of the previously mentioned
Fig. 2. (A) Normal healing process following adaptation of
the periodontal flap with significant reduction of the graft material.
attachment apparatus. (B) In order to enable and promote The biologic rationale behind the use of bone grafts
healing towards the rebuilding of cementum and peri- or alloplastic materials for regenerative approaches is
odontal ligament, the gingival epithelium must be pre- the assumption that these materials may serve as a
vented from forming a long junctional epithelium along
scaffold for bone formation (osteoconduction) and
the root surface down to the former level of the peri-
odontal ligament (e.g., by placement of a bioresorbable contain the bone-forming cells (osteogenesis) or
membrane). bone-inductive substances (osteoinduction).
Histological studies in both humans and animals
have demonstrated that grafting procedures often
sue-regeneration techniques in periodontal practice result in healing with a long junctional epithelium
have shown their predictable, albeit limited, potential rather than a new connective tissue attachment (17,
to regenerate lost periodontal support. Consequently, 84). Therefore, multiple studies have evaluated the
advanced regenerative technologies for periodontal use of hard-tissue graft materials for periodontal
tissue repair aim to increase the current gold stan- regeneration in infrabony defects when compared
dards for success of periodontal regeneration. In with the periodontal flap approach alone.
order to identify appropriate advanced repair tech-
niques for tooth-supporting periodontal tissues, a
number of combinations of conventional regenera- Biomodification of the tooth-root surface
tive techniques have been evaluated: guided tissue A number of studies have focused on the modifica-
regeneration and application of tissue growth fac- tion of the periodontitis-involved root surface in or-
tor(s); guided tissue regeneration and hard-tissue der to advance the formation of a new connective
graft and application of tissue growth factor(s); hard- tissue attachment. However, despite histological
tissue graft and biomodification of the tooth-root evidence of regeneration following root-surface
surface; and hard-tissue graft and application of tis- biomodification with citric acid, the outcomes of
sue growth factors. controlled clinical trials have failed to show any
improvements in clinical conditions compared with
nonacid-treated controls (40, 91, 99).
Advanced repair of alveolar bone In recent years, biomodification of the root surface
defects with enamel matrix proteins during periodontal sur-
gery and following demineralization with EDTA has
The morphology of the alveolar infrabony defect was been introduced to promote periodontal regenera-
shown to play a significant role in the establishment of tion. Based on the understanding of the biological
a predictable outcome of regeneration of periodontal model, the application of enamel matrix proteins
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6. Ramseier et al.
(amelogenins) is seen to promote periodontal factors (44, 46, 58, 87), fibroblast growth factors (49,
regeneration as it initiates events that occur during 101, 149, 77, 151) and bone morphogenetic proteins
the growth of periodontal tissues (43, 54). The com- (42, 59, 152, 164, 165), have been used in preclinical
mercially available product EmdogainÒ, a purified and clinical trials for the treatment of large peri-
acid extract of porcine origin containing enamel odontal or infrabony defects, as well as around dental
matrix derivates, is reported to be able to enhance implants (36, 68, 110). The combined use of re-
periodontal regeneration (Fig. 3). More basic re- combinant human platelet-derived growth factor-BB
search, in addition to the clinical findings, indicates and peptide P-15 with a graft biomaterial has shown
that enamel matrix derivates have a key role in peri- beneficial effects in intraosseous defects (157). How-
odontal wound healing (26, 32). Histological results ever, contrasting results were reported for growth
from both animal and human studies have shown factors such as platelet-rich plasma and graft combi-
that the application of enamel matrix derivates pro- nations, or the use of bioactive agents either alone or
motes periodontal regeneration and confidently in association with graft or guided tissue regeneration
influences periodontal wound healing (147). Thus far, for the treatment of furcation defects (157).
enamel matrix derivates, either alone or in combi-
nation with grafts, have demonstrated their potential
to effectively treat intraosseous defects and the clin- Biological effects of growth factors:
ical results appear to be stable long term (157). platelet-derived growth factor
Periodontal tissue growth factors Platelet-derived growth factor is a member of a
multifunctional polypeptide family that binds to two
Wound-healing approaches using growth factors to cell-membrane tyrosine kinase receptors (platelet-
target restoration of tooth-supporting bone, peri- derived growth factor-Ra and platelet-derived growth
odontal ligament and cementum have been shown to factor-Rb) and subsequently exerts its biological ef-
significantly advance the field of periodontal-regen- fects on cell proliferation, migration, extracellular
erative medicine. A major focus of periodontal re- matrix synthesis and anti-apoptosis (56, 71, 138, 148).
search has studied the impact of tissue growth factors Platelet-derived growth factor-a and -b receptors are
on periodontal tissue regeneration (Table 2) (3, 44, expressed in regenerating periodontal soft and hard
104, 126). Advances in molecular cloning have made tissues (119). In addition, platelet-derived growth
available unlimited quantities of recombinant growth factor initiates tooth-supporting periodontal liga-
factors for applications in tissue engineering. Re- ment cell chemotaxis (111), mitogenesis (113), matrix
combinant growth factors known to promote skin and synthesis (53) and attachment to tooth dentinal sur-
bone wound healing, such as platelet-derived growth faces (172). More importantly, in vivo application of
factors (14, 46, 67, 110, 115, 140), insulin-like growth platelet-derived growth factor alone or in combina-
A B C D E
Fig. 3. Periodontal regeneration of a three-wall infrabony frabony defect is classified and measured: the predomi-
defect using Emdogain. (A) A 32-year-old male patient nant component is a 7-mm-deep three-wall defect. (D)
(nonsmoker with severe periodontitis). Tooth 13 shows a One year after surgical intervention the distal site of tooth
probing pocket depth of 10 mm disto-buccally and clinical 13 shows a probing pocket depth of 2 mm and clinical
attachment loss of 14 mm. (B) Pretreatment radiograph attachment loss of 7 mm. Comparison with the initial
shows the infrabony defect distal to tooth 13. (C) After the measurements indicates that a probing pocket depth gain
buccal incision of the papilla, the interdental tissue is of 8 mm and a clinical attachment gain of 7 mm have
preserved attached to the palatal flap. After debridement been achieved. (E) Radiograph 1 year postsurgery showing
of the granulation tissue and the root surface, the in- filling of the defect.
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7. Periodontal tissue-engineering technologies
Table 2. Effects of growth factors used for periodontal tissue engineering
Growth factor Effects
Platelet-derived growth factor Migration, proliferation and noncollagenous matrix synthesis of mesenchymal
cells
Bone morphogenetic protein Proliferation, differentiation of osteoblasts and differentiation of periodontal lig-
ament cells into osteoblasts
Enamel matrix derivative Proliferation, protein synthesis and mineral nodule formation in periodontal lig-
ament 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
tion with insulin-like growth factor-1 results in the threonine kinases. The type I receptor protein kinase
partial repair of periodontal tissues (46, 47, 87, 88, phosphorylates intracellular signaling substrates
140). Platelet-derived growth factor has been shown called Smads (the sma gene in Caenorhabditis elegans
to have a significant regenerative impact on peri- and the Mad gene in Drosophila). The phosphory-
odontal ligament cells, as well as on osteoblasts (90, lated bone morphogenetic protein-signaling Smads
92, 113, 115). enter the nucleus and initiate the production of bone
The clinical application of platelet-derived growth matrix proteins, leading to bone morphogenesis. The
factor was shown to successfully advance alveolar most remarkable feature of bone morphogenetic
bone repair and clinical attachment level gain. A first proteins is their ability to induce ectopic bone for-
clinical study reported the successful repair of class II mation (160). Bone morphogenetic proteins are not
furcations using demineralized freeze-dried bone only powerful regulators of cartilage and bone for-
allograft saturated with recombinant human platelet- mation during embryonic development and regen-
derived growth factor-BB (109). In a second study, eration in postnatal life, but they also participate in
recombinant human platelet-derived growth factor- the development and repair of other organs such as
BB mixed with a synthetic beta-tricalcium phosphate the brain, kidney and nerves (132).
matrix was shown to advance the repair of deep in- Sigurdsson et al. (149) evaluated bone and
frabony pockets in a large multicenter randomized cementum formation following regenerative peri-
controlled trial (110). Both studies demonstrated that odontal surgery by the use of recombinant human
the use of recombinant human platelet-derived bone morphogenetic protein in surgically created
growth factor-BB was safe and effective in the treat- supra-alveolar defects in dogs (168). Histologic
ment of periodontal osseous defects. In a follow-up analysis showed significantly more cementum for-
trial, the same sample of patients was assessed 18 or mation and regrowth of alveolar bone on bone
24 months following periodontal surgery. Substantial morphogenetic protein-treated sites compared with
radiographic changes in the appearance of the defect the controls.
fill were observed for patients treated with re- Studies have demonstrated the expression of bone
combinant human platelet-derived growth factor-BB morphogenetic proteins during tooth development
(94). and periodontal repair, including alveolar bone (1, 2).
Investigations in animal models have shown the po-
tential repair of alveolar bony defects using re-
Biological effects of growth factors: combinant human bone morphogenetic protein-12
bone morphogenetic proteins (165) or recombinant human bone morphogenetic
protein-2 (86, 166). In a clinical trial by Fiorellini
Bone morphogenetic proteins are multifunctional et al. (36), recombinant human bone morphogenetic
polypeptides belonging to the transforming growth protein-2, delivered by a bioabsorbable collagen
factor-beta superfamily of proteins (169). The human sponge, revealed significant bone formation in a
genome encodes at least 20 bone morphogenetic human buccal wall defect model following tooth
proteins (131). Bone morphogenetic proteins bind to extraction when compared with collagen sponge
type I and type II receptors that function as serine- alone. Furthermore, bone morphogenetic protein-7,
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8. Ramseier et al.
also known as osteogenic protein-1, stimulates bone ably because of proteolytic breakdown, receptor-
regeneration around teeth, endosseous dental im- mediated endocytosis and solubility of the delivery
plants and in maxillary sinus floor-augmentation vehicle (3). Because their half-lives are significantly
procedures (49, 141, 161). reduced, the period of exposure may not be suf-
ficient to act on osteoblasts, cementoblasts or
periodontal ligament cells. Therefore, different
Clinical application of growth factors for
methods of growth-factor delivery need to be
use in periodontal regeneration
considered (4).
In general, the impact of topical delivery of growth Investigations for periodontal bioengineering have
factors to periodontal wounds has been promising, examined a variety of methods that combine delivery
yet insufficient to promote predictable periodontal vehicles, such as scaffolds, with growth factors to
tissue engineering (14, 23) (Fig. 4). Growth factor target the defect site in order to optimize bioavail-
proteins, once delivered to the target site, tend to ability (85). The scaffolds are designed to optimize
suffer from instability and quick dilution, presum- the dosage of the growth factor and to control its
A B C
D E F
G H I
Fig. 4. Periodontal regeneration using platelet-derived together with the graft to rebuild the lost bone. (F) A
growth factor and bone-graft materials. (A) A 27-year-old second internal mattress suture is performed with a 7-0
patient at the re-evaluation visit after the initial nonsur- Gore-TexÒ suture, to allow for optimal adaptation of the
gical therapy; three sites with a probing pocket depth of flap margin without the interference of the epithelium.
>6 mm were identified. One of those sites, distal to tooth The two internal mattress sutures are tied and the knots
44, shows a probing pocket depth of 7 mm and no gingival are performed only after a perfect tension-free closure of
recession. (B) The periapical radiograph shows a deep, the wound. Two additional interrupted 7-0 sutures are
one-wall defect distal to tooth 44 and a lesion between placed to ensure stable contact between the connective
teeth 45 and 46. (C) Measurement of the one-wall defect tissues of the edges of the flaps. The mesial and distal
shows an infrabony component of 6 mm. (D) The grafting papillae are stabilized with additional simple interrupted
material (GEM 21SÒ) is mixed with particles of autoge- sutures. (G) Nine months after surgery, the probing
nous bone chips collected in the surgical area with a pocket depth is 2 mm. (H) Nine months after surgery, the
Rhodes instrument and with the liquid component of the periapical radiograph shows good bone fill of the one-
GEM 21SÒ (platelet-derived growth factor). (E) The liquid wall bony defect. (I) Nine months after surgery, the sur-
platelet-derived growth factor is placed in the defect gical re-entry shows new bone formation.
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9. Periodontal tissue-engineering technologies
release pattern, which may be pulsatile, constant or membranes, as well as barrier materials of polylactic
time-programmed (8). The kinetics of the release and acid, or copolymers of polylactic acid and poly-
the duration of the exposure of the growth factor may glycolic acid, have been tested in animal and human
also be controlled (61). studies.
A new polymeric system, permitting the tissue- Following therapy, guided tissue regeneration has a
specific delivery (at a controlled dose and delivery greater effect on the probing measures of periodontal
rate) of two or more growth factors, was reported in an treatment than periodontal flap surgery alone,
animal study carried out by Richardson et al. (137). including increased attachment gain, reduction of
The dual delivery of vascular endothelial growth fac- probing depth, less gingival recession and more gain
tor with platelet-derived growth factor from a single, in hard-tissue probing at surgical re-entry. Referring
structural polymer scaffold results in the rapid for- to the best evidence currently available, however, it is
mation of a mature vascular network (137). difficult to draw general conclusions about the
clinical benefit of guided tissue regeneration. Al-
though there is evidence demonstrating that guided
Guided tissue regeneration
tissue regeneration has significant benefits over
Histological findings from periodontal-regeneration conventional open-flap surgery, the factors affecting
studies reveal that a new connective tissue attach- outcomes are unclear from the present literature
ment could be predicted if the cells from the peri- because they might be influenced by study conduct
odontal ligament settle on the root surface during issues, such as bias (106).
healing. Hence, the clinical applications of guided In summary, guided tissue regeneration is
tissue regeneration in periodontics involve the currently a very well-documented regenerative
placement of a physical barrier membrane to enable procedure used to achieve periodontal regeneration
the previous periodontitis-affected tooth root surface in infrabony defects and in class II furcations. Further
to be repopulated with cells from the periodontal benefit may be achieved by the additional use of
ligament. In the last few decades, guided tissue grafting materials (155).
regeneration has been applied in many clinical trials
for the treatment of various periodontal defects, such
as infrabony defects (25), furcation involvement (72, Gene therapeutics for periodontal
89) and localized gingival recession (121). In a recent tissue repair
systematic review, the combinations of barrier
membranes and grafting materials used in preclinical Although encouraging results for periodontal regen-
models have been summarized. The analysis of 10 eration have been found in various clinical investi-
papers revealed that the combination of barrier gations using recombinant tissue growth factors,
membranes and grafting materials may result in there are limitations for topical protein delivery, such
histological evidence of periodontal regeneration, as transient biological activity, protease inactivation
predominantly bone repair. No additional histologi- and poor bioavailability from existing delivery vehi-
cal benefits of combination treatments were found in cles. Therefore, newer approaches seek to develop
animal models of three-wall intrabony, class II fur- methodologies that optimize growth-factor targeting
cation, or fenestration defects. In supra-alveolar and to maximize the therapeutic outcome of periodontal-
two-wall intrabony defect models of periodontal regenerative procedures. Genetic approaches in
regeneration, the additional use of a grafting material periodontal tissue engineering show early progress in
gave superior histological results of bone repair achieving delivery of growth-factor genes, such as
compared with the use of barrier membranes alone platelet-derived growth factor or bone morphogenetic
(145). protein, to periodontal lesions (Fig. 5). Gene-transfer
The types of barrier membranes evaluated in clin- methods may circumvent many of the limitations
ical studies vary in design, configuration and com- with protein delivery to soft-tissue wounds (10, 45). It
position. Nonresorbable membranes of expanded has been shown that the application of growth factors
polytetrafluoroethylene have been used successfully (37, 63, 64, 78) or soluble forms of cytokine receptors
in both animal experiments and human clinical trials. (21) by gene transfer provides greater sustainability
In recent years, natural or synthetic bio-absorbable than the application of a single protein. Thus, gene
barrier membranes have been used for guided tissue therapy may achieve greater bioavailability of growth
regeneration in order to eliminate the need for fol- factors within periodontal wounds and hence provide
low-up surgery for membrane removal. Collagen greater regenerative potential.
193

10. Ramseier et al.
A
B
Fig. 5. Advanced approaches for re-
generating tooth-supporting struc-
tures. (A) Application of a graft
material (e.g. bone ceramic) and
growth factor into an infrabony de-
fect covered by a bioresorbable
membrane. (B) Application of gene
vectors for the transduction of
growth factors producing target
cells.
strated to promote bone allograft turnover and
Methods for gene delivery in periodontal
osteogenesis as a mode to enrich human bone allo-
applications
grafts (62). To date, combinations of vascular endo-
Various gene-delivery methods are available to thelial growth factor ⁄ bone morphogenetic protein
administer growth factors to periodontal defects, (120) and platelet-derived growth factor ⁄ vascular
offering great flexibility for tissue engineering. The endothelial growth factor (137) have had highly po-
delivery method can be tailored to the specific sitive synergistic responses in bone repair.
characteristics of the wound site. For example, a Promising preliminary results from preclinical stud-
horizontal one- or two-walled defect may require the ies reveal that host modulation achieved through gene
use of a supportive carrier, such as a scaffold. Other delivery of soluble proteins, such as tumor necrosis
defect sites may be conducive to the use of an ade- factor receptor 1 (TNFR1:Fc), reduces tumor necrosis
novirus vector embedded in a collagen matrix. factor activity and therefore inhibits alveolar bone loss
More importantly from a clinical point of view is (21). These results are comparable to the findings in the
the risk associated with the use of gene therapy in research on rheumatoid arthritis where pathogenesis
periodontal tissue engineering (51). As with maxi- includes high tumor necrosis factor activity and the
mizing growth-factor sustainability and accounting pathways for bone resorption are similar (127).
for specific characteristics of the wound site, both the
DNA vector and delivery method need to be consid-
Preclinical studies evaluating growth
ered when assessing patient safety. In summary,
factor gene therapy for periodontal tissue
studies examining the use of specific delivery meth-
engineering
ods and DNA vectors in periodontal tissue engi-
neering aim to maximize the duration of growth In order to overcome the short half-lives of growth
factor expression, optimize the method of delivery to factor peptides in vivo, gene therapy using a vector
the periodontal defect and minimize patient risk. encoding the growth factor is advocated to stimulate
A combination of an Adeno-Associated Virus- tissue regeneration. So far, two main strategies of
delivered angiogenic molecule, such as vascular gene vector delivery have been applied to peri-
endothelial growth factor, bone morphogenetic pro- odontal tissue engineering. Gene vectors can be
tein signaling receptor (caALK2) and receptor acti- introduced directly to the target site (in vivo tech-
vator of nuclear factor-kappa B ligand, was demon- nique) (63) or selected cells can be harvested, ex-
194

11. Periodontal tissue-engineering technologies
panded, genetically transduced and then re-im- tor signaling. Gene delivery of platelet-derived
planted (ex vivo technique) (64). In vivo gene growth factor-B generally displays higher sustained
transfer involves the insertion of the gene of interest signal-transduction effects in human gingival fibro-
directly into the body anticipating the genetic blasts compared to cells treated with recombinant
modification of the target cell. Ex vivo gene transfer human platelet-derived growth factor-BB protein
includes the incorporation of genetic material into alone. Their data on platelet-derived growth factor
cells exposed from a tissue biopsy with subsequent gene delivery may contribute to an improved
re-implantation into the recipient. Using the in vivo understanding of the pathways that are likely to play
technique, the potential inhibition of alveolar bone a role in the control of clinical outcomes of peri-
loss has been studied in an experimental periodon- odontal-regenerative therapy.
titis model evaluating the inhibition of osteoclasto- In an ex vivo investigation by Anusaksathien et al.
genesis by administering human osteoprotegerin, a (6), it was shown that the expression of platelet-de-
competitive inhibitor of the receptor activator of rived growth factor genes was prolonged for up to
nuclear factor-kappa B ligand-derived osteoclast 10 days in gingival wounds. Adenovirus encoding
activation. Significant preservation of alveolar bone platelet-derived growth factor-B (adenovirus ⁄ plate-
volume was observed among osteoprotegerin:Fc- let-derived growth factor-B) transduced gingival
treated animals compared with controls. Systemic fibroblasts and enhanced defect fill by inducing
delivery of osteoprotegerin:Fc inhibits alveolar bone human gingival fibroblast migration and proliferation
resorption in experimental periodontitis, suggesting (6). On the other hand, continuous exposure of
that inhibition of receptor activator of nuclear fac- cementoblasts to platelet-derived growth factor-A
tor-kappa B ligand may represent an important had an inhibitory effect on cementum mineraliza-
therapeutic strategy for the prevention of progres- tion, possibly via the upregulation of osteopontin and
sive alveolar bone loss (65). the subsequent enhancement of multinucleated giant
cells in cementum-engineered scaffolds. Moreover,
adenovirus ⁄ platelet-derived growth factor-1308 (a
Platelet-derived growth factor gene dominant-negative mutant of platelet-derived growth
delivery factor) inhibited mineralization of tissue-engineered
cementum, possibly owing to the downregulation of
Platelet-derived growth factor-gene transfer strate- bone sialoprotein and osteocalcin and the persis-
gies were originally used in tissue engineering to tence of stimulation with multinucleated giant cells.
improve healing in soft-tissue wounds such as skin These findings suggest that continuous exogenous
lesions (27). Both plasmid (57) and adenovirus ⁄ delivery of platelet-derived growth factor-A may de-
platelet-derived growth factor (125) gene delivery lay mineral formation induced by cementoblasts,
have been evaluated in preclinical and human trials. while platelet-derived growth factor is clearly re-
However, the latter exhibits greater safety in clinical quired for mineral neogenesis (5).
use (51). In a recent animal study reporting on safety Jin et al. (63) demonstrated that direct in vivo gene
and distribution profiles, adenovirus ⁄ platelet-de- transfer of platelet-derived growth factor-B was able to
rived growth factor-B applied for tissue engineering stimulate tissue regeneration in large periodontal de-
of tooth-supporting alveolar bone defects was well fects. Descriptive histology and histomorphometry
contained within the localized osseous defect area revealed that delivery of the human platelet-derived
without viremia or distant organ involvement (18). growth factor-B gene promotes the regeneration of
Early studies in dental applications using re- both cementum and alveolar bone, while delivery of
combinant adenoviral vectors encoding platelet-de- platelet-derived growth factor-1308, a dominant-neg-
rived growth factor demonstrated the ability of these ative mutant of platelet-derived growth factor-A, has
vector constructs to potently transduce cells isolated minimal effects on periodontal tissue regeneration.
from the periodontium (osteoblasts, cementoblasts,
periodontal ligament cells and gingival fibroblasts)
(48, 173). These studies revealed the extensive and Delivery of the bone
prolonged transduction of periodontal-derived cells. morphogenetic protein gene
Both Chen & Giannobile (19) and Lin et al. (82) were
able to demonstrate the effects of adenoviral delivery An experimental study in rodents by Lieberman et
of platelet-derived growth factor to understand, in al. (81) advanced gene therapy for bone regenera-
greater detail, sustained platelet-derived growth fac- tion, with the results revealing that the transduction
195

12. Ramseier et al.
of bone marrow stromal cells with recombinant differentiation of human periodontal ligament cells
human bone morphogenetic protein 2 led to bone (170).
formation within an experimental defect comparable These experiments provide promising evidence
to skeletal bone. Another group was similarly able to showing the feasibility of both in vivo and ex vivo
regenerate skeletal bone by directly administering gene therapy for periodontal tissue regeneration and
adenovirus5 ⁄ bone morphogenetic protein 2 into a peri-implant osseointegration.
bony segmental defect in rabbits (9). Further ad-
vances in the area of orthopedic gene therapy using
Future perspectives: targeted gene
viral delivery of bone morphogenetic protein 2 have
therapy in vivo
provided further evidence for the ability of both in
vivo and ex vivo bone engineering (20, 79, 80, 103). Major advances have been made over the past decade
Franceschi et al. (37) investigated in vitro and in vivo in the reconstruction of complex periodontal and
adenovirus gene transfer of bone morphogenetic alveolar bone wounds that have resulted from disease
protein 7 for bone formation. Adenovirus-trans- or injury. Developments in scaffolding matrices for
duced nonosteogenic cells were also found to dif- cell, protein and gene delivery have demonstrated
ferentiate into bone-forming cells and to produce significant potential to provide ÔsmartÕ biomaterials
bone morphogenetic protein 7 (78) or bone mor- that can interact with the matrix, cells and bioactive
phogenetic protein 2 (20) both in vitro and in vivo. factors. The targeting of signaling molecules or growth
In another study by Huang et al. (60), plasmid DNA factors (via proteins or genes) to periodontal tissue
encoding bone morphogenetic protein 4 adminis- components has led to significant new knowledge
tered using a scaffold-delivery system was found to generation using factors that promote cell replication,
enhance bone formation when compared with blank differentiation, matrix biosynthesis and angiogenesis.
scaffolds. A major challenge that has been studied less is the
In an early approach to regenerate alveolar bone in modulation of the exuberant host response to micro-
an animal model, it was demonstrated that the bial contamination that plagues the periodontal
ex vivo delivery of an adenovirus encoding murine wound microenvironment. To achieve improvements
bone morphogenetic protein 7 was found to promote in the outcome of periodontal-regenerative medicine,
periodontal tissue regeneration in large mandibular scientists will need to examine the dual delivery of
periodontal bone defects (64). Transfer of the bone host modifiers or anti-infective agents to optimize the
morphogenetic protein 7 gene enhanced alveolar results of therapy. Further advancements in the field
bone repair and also stimulated cementogenesis and will continue to rely heavily on multidisciplinary ap-
periodontal ligament fiber formation. Of interest, proaches, combining engineering, dentistry, medicine
alveolar bone formation was found to occur via a and infectious disease specialists in repairing the
cartilage intermediate. However, when genes encod- complex periodontal wound environment.
ing the bone morphogenetic protein antagonist
noggin were delivered, inhibition of periodontal tis-
sue formation resulted (66). In a study by Dunn et al. Acknowledgments
(30), it was shown that direct in vivo gene delivery of
adenovirus ⁄ bone morphogenetic protein 7 in a col- This work was supported by NIH ⁄ NIDCR DE13397
lagen gel carrier promoted successful regeneration of and NIH ⁄ NCRR UL1RR-024986. The authors thank
alveolar bone defects around dental implants. Fur- Mr Chris Jung for his assistance with the figures.
thermore, an in vivo synergism was found of aden-
oviral-mediated coexpression of bone morphogenetic
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