Biomimetika nastavak

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3283928/
Enamel matrix derivative

The rationale for the clinical use of enamel matrix derivative is the observation that enamel
matrix proteins are deposited onto the surfaces of developing tooth roots before cementum
formation.[58]
Enamel Matrix Protein (EMPs) are commercially available as Emdogain which have been known
to effect periodontal regeneration. Recent data from a systematic review indicates that
biologically EMPs cause an increase in cell attachment of epithelial cells, gingival fibroblasts,
and PDL fibroblasts. They increase the expression of transcription factors that are related to
chondroblast and osteoblasts/cementoblast differentiation. Stimulation in the synthesis of total
protein and extracellular matrix molecules has also been documented.[59] Use of Enamel matrix
derivative (EMD) and a demineralized freeze dried bone allograft (DFDBA) have been
demonstrated to be osteopromotive in nature; thus, resulting in an additional increase in bone
formation.[60] Studies enumerating the effects of EMD in obtaining periodontal regeneration are
given in Table 4. The only concern with the use of EMD has been related to its application and
its related viscous nature, which may not provide sufficient soft tissue/flap support; thus,
potentially limiting the space available for the regeneration process.

Fajl CCIDEN (bitno za literaturu)

In a series of laboratory studies, the effect of EMD
on migration of mineralized nodules was examined.
Immunoassays
were performed to determine the possible presence
of existing polypeptide factors.16,25

((6)
X Hoang AM, Oates TW, Cochran DL. In vitro wound healing responses
to enamel matrix derivative. J Periodontol. 2000;71:12701277.)

The results showed

that, under in vitro conditions, EMD promotes the proliferation
of periodontal ligament fibroblasts but not epithelial cells,
and increases total protein synthesis of periodontal ligament
fibroblasts as well as formation of mineralized nodules by
periodontal ligament fibroblasts.19 (19. Lyngs tadaas SP, Lundberg E, Ekdahl H, Ander s son C,
Gestrelius S. Autocrine growth factors in human periodontal ligament
cells cultured on enamel matrix derivative. J Clin Periodontol. 2001;28: 181188.)

EMD has
no appreciable effect on osteoclastic differentiation, although
it stimulates cell growth and IGF-1 and transforming growth

factor 1 production in periodontal ligament cells.26

Other data indicate that EMD may contain additional
mitogenic factors, such as transforming growth factor-and
BMP-like growth factors that stimulate fibroblastic proliferation
and contribute to the induction of biomineralization
during periodontal regeneration.28

2.6 Effects of Emdogain on mesenchymal cells

Emdogain and bone-derived cells
Alveolar bone cells, e.g. osteoblasts, osteoclasts and their precursors, are crucial in periodontal
regeneration. During the development of the periodontium, dental follicle cells can differentiate into
periodontal ligament fibroblasts, cementoblasts and osteoblasts (Slavkin et al 1989, Hammarstrm
et al 1997, Spahr et al 1999, Hakki et al 2001, Zeichner- David 2001). EMD has been shown to be
capable of stimulating the proliferation of follicle cells (Hakki et al 2001). In osteoblastogenesis,
EMD has been shown to promote osteogenic differentiation of pluripotential mesenchymal cell
lines into the osteoblast and/or chondroblast lineage and to stimulate transcription factors related to 20
osteogenesis and chondrogenesis (Ohyama et al 2002, Narukawa et al 2007). Furthermore, EMD
enhances the proliferation and osteogenic potential of bone marrow stromal cells (BMSCs) (Keila et
al 2004, Guida et al 2007). In addition, EMD has been observed to increase the ability of BMSCs
to differentiate into cementoblasts (Song et al 2007). In contrast, EMD has been observed to have
no effect on cell growth and differentiation of rat bone marrow cells (Gurpinar et al 2003, van den
Dolder et al 2006).
The studies have concluded that EMD stimulates growth, proliferation and mobility of mature
osteoblastic cell lines and cementoblasts and regulates their gene expression (Tokiyasu et al 2000,
Jiang et al 2001, He et al 2004, Schwarz et al 2004, Shimizu et al 2004, Galli et al 2006, Carinci et
al 2006, Klein et al 2006, Jiang et al 2006). EMD enhances the differentiation of the osteoblasts and
the expression of phenotype markers by increasing the production of BSP, cellular alkaline
phosphatase (ALP) and osteocalcin (Tokiyasu et al 2000, Schwartz et al 2000, Hgewald et al 2004,
Shimizu et al 2004, Galli et al 2006). These observations suggest that EMD stimulates proliferation
at an early stage of osteoblastic maturation and enhances differentiation in later stages of cell

development.
EMD also has effects on cytokines produced by osteoblasts. EMD has been observed to stimulate
TGF-1, connective tissue growth factor (CTGF) and fibroblast growth factor (FGF-2) expression
in human osteoblastic cells (Schwartz et al 2000, Mizutani et al 2003, Heng et al 2007). In addition,
increased COX-2 and decreased MMP-1 expression have been demonstrated (Mizutani et al 2003).
Both EMD and TGF-1 can protect osteoblasts from inflammation-induced apoptosis (He et al
2005). In the endochondral pathway, EMD can stimulate chondrocyte proliferation in addition to
chondrogenic differentiation (Dean et al 2002, Narukawa et al 2007).
EMD has partly contrasting effects on bone resorption. The receptor activator of NF-kB ligand
(RANKL) is expressed on the surface of osteoblasts. RANKLs binding to RANK-receptor on
osteoclast progenitor cells leads to osteoclast differentiation. EMD can reduce the RANKL release
by osteoblasts and enhance the production of osteoblastic osteoprotegerin (OPG), thus inhibiting the
formation and functions of osteoclasts (He et al 2004, Galli et al 2006). Furthermore, the RANKL
mRNA levels of periodontal ligament fibroblast are significantly down-regulated by EMD
(Takayanagi et al 2006). However, it has also been shown that EMD induces osteoclast formation
by interacting with and stimulating the RANKL expressed by osteoblastic cells (Otsuka et al 2005, 21
Itoh et al 2006). In conclusion, EMD may be capable of creating favourable circumstances for bone
regeneration, since it has effects on both bone formation and resorption. Depending on the cell
maturation, EMD can enhance the differentiation of mesenchymal cells into hard tissue-forming
cells and promote their proliferation and growth.
Emdogain and fibroblasts
Several studies have confirmed that EMD enhances the proliferation, migration and in vitro wound
healing of periodontal ligament fibroblasts and gingival fibroblasts (Gestrelius et al 1997,
Lyngstadaas et al 2001, Rincon et al 2003, Cattaneo et al 2003, Davenport et al 2003, Okubo et al
2003, Palioto et al 2004, Keila et al 2004, Rodrigues et al 2007, Zeldich et al 2007). The mitogenic
signalling pathway in which EMD affects PLFs is associated with rapid phosphorylation of the

MAP kinase family and activation of EMD-specific receptor tyrosine kinase (RTK) and
extracellular signal-regulated kinase (ERK) (Kawase et al 2001, Matsuda et al 2002). In addition,
EMD can inhibit gingival fibroblast caspase activation and thereby inhibit TNF--induced
apoptosis and enhance survival (Zeldich et al 2007). EMD induces matrix and total protein
synthesis (Gestrelius et al 1997, Haase & Bartold et al 2001, Rodrigues et al 2007). Furthermore,
EMD stimulates insulin-like growth factor (IGF-I), transforming growth factor-1 (TGF-1),
platelet-derived growth factor (PDGF) and interleukin-6 (IL-6) production in PLFs (Lyngstadaas et
al 2001, Okubo et al 2003). EMD can increase the production of MMP-2 by PLFs more than
threefold (Mirastschijski et al 2004). In angiogenesis, EMD enhances VEGF release by PLFs
(Mirastschijski et al 2004, Schlueter et al 2007). EMD also has profound effects at the gene level.
EMD can down-regulate genes involved in the early inflammatory phase of wound healing, while
simultaneously up-regulating growth and repair related genes (Parkar et al 2004).
The results from studies on the effects of EMD on the differentiation of fibroblasts are somewhat
contradictory. EMD has been observed to both decrease and increase the ALP activity of PLFs
(Okubo et al 2003, Cattaneo et al 2003, Nagano et al 2004, Rodrigues et al 2007, Lossdrfer et al
2007). In addition, EMD can promote bone-like nodule formation but does not induce osteoblastic
differentiation of PLFs or gingival fibroblasts (Gestrelius et al 1997, Okubo et al 2003, Keila et al
2004, Rodrigues et al 2007). EMD can stimulate differentiation, mineralised tissue formation and
bone metabolism by increasing and modulating OPG and RANKL expression in fibroblasts 22
(Takayanagi et al 2006, Lossdrfer et al 2007). Furthermore, EMD alters phenotype markers of
PLFs when cultured on dental materials (Inoue et al 2004).
2.7 Effects of Emdogain on epithelial cells
There have only been a few studies concerning the effects of EMD on epithelial cell. During
periodontal development, molecules from Hertwigs epithelial root sheath can induce differentiation
of mesenchymal precursors to form periodontal tissues. It is assumed that EMD can mimic this
process (Slavkin et al 1989, Hammarstrm et al 1997, Spahr et al 1999, Hakki et al 2001, ZeichnerDavid

2001, Gestrelius et al 2004). The proliferation of epithelial cell rests of Malassez (ERM) is
enhanced by EMD (Rincon et al 2005). In addition, EMD can stimulate the expression of
osteopontin by ERM (Rincon et al 2005). In contrast, EMD has no effect on the proliferation of rat
tongue epithelial cells (Gestrelius et al 1997). In vivo, EMD can enhance wound epithelialisation in
rabbits (Mirastschijski et al 2004).
Evidence on the effect of EMD on endothelial cell proliferation is scarce and somewhat
inconsistent. EMD has been demonstrated to either stimulate or to have no effects on the
proliferation of endothelial cells (Yuan et al 2003, Schlueter et al 2007). Both of these studies have
confirmed that EMD increases the chemotaxis of endothelial cells (Yuan et al 2003, Schlueter et al
2007). In vivo mice experiment showed significantly more blood vessels surrounding EMD-treated
collagen implants than controls (Yuan et al 2003). In addition, EMD has been shown to stimulate
the production of MMP-2 by endothelial cells more than threefold (Mirastschijski et al 2004).

https://helda.helsinki.fi/bitstream/handle/10138/20262/associat.pdf?sequence=1
EMD
increased the osteogenic capacity of bone marrow and
mineralized nodule formation. The presence of EMD in the
initial stages (first 48 hours) of the culture was crucial for
this effect. In contrast, EMD did not induce osteoblastic
differentiation of gingival fibroblasts but increased both cell
numbers and amount of matrix produced by up to two-fold.
Upon further investigation, it was shown that the attachment,
growth, and metabolic rate of periodontal ligament
fibroblasts increased significantly when EMD was addedto cell cultures and that EMD may convert the
differentiation
pathway of pluripotent C2C12 mesenchymal cells into
an osteoblast and/or chondroblast lineage.16,19Periodontal
ligament
fibroblasts showed significantly increased alkaline phosphatase
activity following application of EMD, which also
enhanced the proliferation of human periodontal ligament
fibroblasts.31,32 In the presence of EMD, human periodontal
ligament fibroblasts showed some morphologic changes that
made them more similar to cementoblasts than to fibroblasts,
suggesting a process of cell differentiation.
A recent study
examined the influence of EMD on the viability, proliferation,
and attachment of periodontal fibroblasts to diseased
root surfaces.33 The results indicated that cell viability was
negatively affected by higher doses over time, while low
doses displayed viability effects similar to those of the
29

control.
Periodontal ligament cell proliferation appeared to
be ameliorated following exposure to EMD, and analysis by
scanning electron microscopy suggested that cellular attachment
to diseased dentin was enhanced following application
of EMD. Further investigations demonstrated that EMD significantly
increased mRNA synthesis of the matrix proteins,
versican, biglycan, and decorin, and increased hyaluronan
synthesis in gingival and desmosomal fibroblasts. It was
also suggested that integrins are involved in the interaction
between periodontal ligament cells, gingival fibroblasts,
and EMD.34
it has to be emphasized that in most
studies, EMD had a stronger effect on desmodontal than on
gingival fibroblasts. Other experimental investigations have
shown that the application of EMD can regulate the expression
of genes associated with cementoblasts which, in turn,
has a crucial effect on the mineralization process.35
uticaj na kost

In an in vitro study, the combination of EMD 4 mg and

active demineralized freeze-dried allogenic bone showed
increased bone induction. It was concluded that EMD possesses
osteopromotive but not osteoinductive characteristics
when applied at certain concentrations.39 Schwarz et al have
shown that EMD stimulates the early stages of osteoblast
maturation by increasing cell proliferation.40 However, when
applied to mature cell lines, the main effect was to influence
cell differentiation. A stimulatory role of EMD on mineralized
tissue formation by modulating regulatory molecules critical to
bone metabolism at the RNA level has also been reported.41
It seems that treatment with EMD may enhance
the cellular activities of osteoblasts and osteoclasts, which
in turn might support the regeneration of periodontal bony
defects.44 Because soluble peptides released from EMD may
contribute to the stimulating effects on cell proliferation,
direct contact between EMD and osteoblasts might not be
required to induce cell proliferation.45
Shimizu et al46 investigated
the ability of EMD to regulate bone sialoprotein gene
transcription in osteoblast-like cells. The findings identified
EMD response elements in the rat bone sialoprotein gene
promoter that may mediate the effects of EMD on bone
sialoprotein gene transcription. EMD enhanced the proliferation
of human mesenchymal stem cells and seemed to
enhance mineralization. The reverse transcriptase polymerase
chain reaction test revealed that EMD promoted early-stageosteoblast
differentiation by enhancing collagen type I alpha 2
expression, but exerted an inhibitory effect on mineralization
by lowering gene expression of bone sialoprotein and
-carboxylglutamate.
Uticaj na inflamaciju i oporavak

Parker and Tonetti evaluated the selective effects of EMD

on the activities of 268 cytokine, growth factor, and receptor
genes in periodontal ligament tissue.48 The results indicated
that EMD downregulates the expression of genes involved
in the early inflammatory phases of wound healing while
simultaneously up regulating genes encoding for molecules
that promote growth and repair.
Antibakterijsko dejstvo
It is important to note that certain antibacterial effects
and impairment of bacterial adherence were also found to
be influenced by EMD. .49The results showed that EMD
in the PGA vehicle had a very strong antibacterial effect. It
was concluded that the antibacterial effect of EMD is mainly
due to the effect of the PGA carrier. These findings were
later confirmed in an observer-blind, randomized, five-cell
crossover study, demonstrating for the first time a direct influence
of EMD on the vitality of supragingival dental plaque
in vivo.50 In a further investigation, it was shown that EMD
inhibits the growth of the periodontal pathogenic bacteria
Actinobacillus actinomycetemcomitans, Porphyromonas
gingivalis, and Prevotella intermedia. Twenty-four hours
following the application of EMD, no living colonies of these
pathogenic bacteria could be observed. EMD demonstrated
no negative effect on Gram positive bacteria.51
zarastanje rane

Effect of EMD on early

wound healing
A quantitative study by Lafzi et al146 sought to illustrate the
ultrastructural changes associated with a human gingival
wound 10 days after application of EMD as an adjunct to
a laterally positioned flap in a patient with gingival recession.
Ten days after the operation, a gingival biopsy specimen
was obtained from the dentogingival region and examined
using a transmission electron microscope. A considerable
difference
was found in both the cellular and extracellular
phases of the EMD and non-EMD sites. Fibroblasts
at the EMD site were rounded with plump cytoplasm and
euchromatic
nuclei. Well-developed rough endoplasmic
reticulum and numerous mitochondria could be detected.
In contrast, the fibroblasts at the non-EMD site were offlattened spindle-like morphology. It seems that EMD may
enhance certain features of gingival wound healing, which
may be attributable to its anti-apoptotic, antibacterial, or
anti-inflammatory properties.146
Rincon et al evaluated
the influence of EMD on cultured gingival, periodontal
ligament, and dermal fibroblasts, using an in vitro model of
wound healing. This study demonstrated that cells in vitro
fill an empty space by a combination of proliferation and cell

migration, indicating that EMD may exert an influence on

cells involved in wound healing.55
In a study in rabbits, Mirastschijski et al56 primarily
investigated the in vivo effects of EMD on skin wound
healing. Secondly, they examined the in vitro effects of
EMD on dermal fibroblasts and microvascular endothelial
cells. Treatment with EMD increased the
amount of granulation tissue and accelerated the time taken
to complete epithelialization by 3 days compared with
the controls. Vascular endothelial growth factor levels in
conditioned media were increased by more than five-fold
with EMD treatment compared with the control treatment
in cultured fibroblasts, as measured by enzyme-linked
immunosorbent assay. EMD also increased the release of
MMP-2 from fibroblasts and endothelial cells by more
than three-fold. It was concluded that EMD significantly
accelerated wound closure in rabbits, possibly by increasing
the levels of growth factors and proteinases important
for granulation tissue formation and granulation.56 It
was shown that EMD may express some angiogenetic
effects, which could play an important role in early wound
healing.57
antiinflamatorno dejstvo

Recent evidence points to EMD having antiinflammatory

properties which attenuate the release of
tumor necrosis factor-and interleukin-8 in whole blood
from healthy donors challenged by lipopolysaccharide
or peptidoglycan.58
uticaj na gingivu

EMD may affect gingival health by ways

other than cell proliferation/survival, ie, by stimulation of
tissue inhibitor of metalloproteinase (TIMP)-3 production,
which could improve the MMP-TIMP balance in gingival
tissue and curb extracellular matrix destruction.60

Clinical safety of EMD

Because the commercial formulation of EMD is a porcinederived
xenograft, the potential for it to stimulate an immune
reaction when used in humans is of extreme importance. The
enamel matrix proteins are highly conserved among mammalian
species, and exposure to these proteins takes place during
tooth development in early childhood. Thus, tolerance should
normally be induced and the proteins recognized by the
immune system as self proteins. Therefore, it is reasonable
to assume that they are less likely to act as antigens.
In vitro
studies show that EMD does not significantly modify cellular
or humoral immune responses. Very high concentrations
of EMD induced only a slight increase in proliferation of

human lymphocytes, restricted to the CD25(interleukin-2

receptor) fraction of CD4T lymphocytes. There was a
concomitant decrease in B lymphocytes,
Moreover, it was shown that EMD attenuated the release
of tumor necrosis factor-and interleukin-8 in whole blood
from healthy donors challenged by lipopolysaccharide or
peptidoglycan, but the release of interleukin-10 remained
unchanged. EMD also produced a four-fold increase in the
cAMP levels of peripheral blood mononuclear cell lysates,
which in turn suggested that EMD has anti-inflammatory
properties.85

Surgical periodontal therapy

Data from controlled clinical studies have demonstrated
that treatment of intrabony defects with EMD results in a
significant reduction in probing depths and gain of clinical
attachment. study showed that treatment with open flap surgery EMD
resulted in a three times greater defect fill than treatment with
flap surgery alone (74% defect fill after flap surgery EMD
versus 23% defect fill after flap surgery alone).91 In a further
prospective, controlled clinical study, 40 patients were treated
by surgical therapy with two bioabsorbable barriers and the
outcome compared with that of open flap surgery.92
Generally, most of the data from controlled
clinical studies indicate that the additional application of
EMD in the context of surgical treatment of deep intrabony
periodontal defects may lead to significantly higher gains in
clinical attachment and defect fill compared with open flap
debridement.95100
Surgical treatment with EMD was also
demonstrated to improve supracrestal soft tissue density significantly
compared with open flap debridement alone.101,10However, neither postoperative administration of amoxicillin
and metronidazole nor selective cyclo-oxygenase 2 inhibitors
appeared to enhance the clinical results.103,104 Furthermore,
two studies have suggested that the clinical outcomes of
intrabony defects treated with EMD do not depend on the
use of EDTA root conditioning.105,106Clinical results with EMD are comparable with those
after GTR therapy. A recent prospective multicenter, randomized,
controlled clinical trial compared the clinical outcomes
of EMD and GTR with a bioabsorbable membrane. 108 The
data indicate that the clinical outcomes after treatment
of intrabony defects with EMD can be maintained over a
longer time period.109,110,114 In a case cohort study, Cortellini
and Tonetti indicated that a minimally invasive surgical
technique combined with EMD in the regenerative treatment
of isolated intrabony defects resulted in excellent clinical
improvements while limiting patient morbidity.111 The use
of a minimally invasive surgical technique with EMD promoted
significant improvements in clinical parameters, withminimal
pain/discomfort and maximum esthetic satisfaction
in the treatment of intrabony defects.112

Combination therapy
for intrabony defects
Experimental and clinical studies have indicated that the
extent of regeneration is determined by the available space
under the mucoperiosteal flap.115,116In a
prospective, controlled, clinical study, intrabony defects
were evaluated following treatment with EMD, GTR,
EMD GTR, and open flap surgery.103 It was shown that
all three regenerative treatment procedures resulted in a
significantly greater improvement in clinical parameters compared
with conventional flap surgery; however, combination
therapy of EMD GTR led to no additional improvement
Human histologic studies indicate that a combination
of EMD and a natural bone mineral or bioactive glass may
indeed result in formation of root cementum and mineralization
around the graft particles.118,119 Controlled clinical studies
comparing treatment of intrabony defects with EMD and
different types of bone graft/bone substitute seem to indicate
that combination of EMD and demineralized freeze-dried
allogenic bone or a natural bone mineral may enhance the
clinical outcome.120,121,125,126 Sculean et al reported positive
results from EMD in guided tissue regeneration in a 5-year
and 10-year study.123,124A combination
of EMD autogenous bone resulted in statistically significant
greater soft and hard tissue improvements compared with
treatment with EMD.128 Baltacioglu evaluated the clinical and
radiographic results of intentional replantation of periodontally
hopeless teeth with combined EMD and demineralized
freeze-dried bone allograft therapy.129 Eleven patients with 12
periodontally hopeless teeth resulting from extensive alveolar
bone loss and vertical defects extending to the apices were
studied. At the 12-month clinical and radiographic followup,
significant improvement was observed for all parameters
except gingival recession (

Controlled clinical studies

in recession defects
In two controlled clinical studies, treatment of buccal
Miller class I and II gingival recessions with a coronally
positioned flap alone was examined using the split-mouth
procedure.132,133 The clinical outcome did not show any differences
between the therapies in terms of root coverage over
a short time period of 1 year. However, additional application
of EMD induced statistically significant greater formation
of keratinized tissue, compared with that using a coronally
positioned flap alone.133Kuru
et al demonstrated in two cases the possibility of treating
human buccal recessions with EMD plus a laterally sliding
flap, with predictable root coverage and clinical attachment

gain.140 Aroca et al showed that EMD did not enhance clinical

outcome in the treatment of a class III recession-type defect
when used with a modified tunnel/connective tissue graft
technique compared with the modified tunnel/connective
tissue graft technique alone.141 The additional use of EMD
combined with a subepithelial connective tissue graft procedure
does not produce a beneficial clinical outcome in terms
of root coverage.142

Controlled clinical studies

in furcation defects
Defects were randomly assigned to treatment
with EMD or GTR using a bioresorbable membrane. It was
concluded that there was a significantly greater reduction
in horizontal furcation depth and a comparatively lower
incidence of postoperative pain/swelling following EMD
than with GTR therapy.

Conclusion
Application of enamel matrix proteins in the form of Emdogain
has set a modern standard for periodontal regeneration
therapy. Surgical periodontal treatment of deep intrabony
defects with EMD promotes periodontal regeneration.
Surgical
periodontal treatment of deep intrabony defects
using EMD may lead to significantly greater improvements
in clinical parameters compared with open flap debridement
alone. The effect of treatment with EMD is comparable with
that for GTR and can be maintained over a 10-year period.
The combination of EMD and some types of bone graft/bone
substitute may result in the soft and hard tissue parameters
compared with treatment with EMD alone. Further studies are
needed in order to clarify definitively the possible advantage
of combination therapy using EMD and bone grafts/bone
substitutes in relation to single therapies. Application of
EMD seems to provide better long-term results than coronally
repositioned flaps alone. Application of EMD may enhance
periodontal regeneration in mandibular class II furcations,
comparable with that obtained using GTR.
The indications
Emdogain is intended as an adjunct to
periodontal surgery as a topical
application onto exposed root surfaces
to treat intrabony defects due to
moderate or severe periodontitis. It has
been proven to be effective in:

 one-wall, two-wall, and three-wall

intrabony defects
class II mandibular furcation defects
with minimal interproximal bone loss
recession defects
-minimaly invasive surgery technique in esthetic zones

Contraindications
Emdogain should not be used in patients with disorders or conditions :unconntroled diabetes and other uncontrolled
systemic diseases,disorders or trweatments that compromise wound healing,chronic high dose steroid therapy,bone
metabolic diseases,radiation and other immuno oppresive therapy and infections or vascular impairment at
sutrgicval site. Kontrindikovana je primena Emdogaina kod pacijenata sa premalignim, ili
malignim lezijama usne duplje zbog toga to neke od aktivnih komponenti EMD-a u
tumorski izmenjenom tkivu mogu poveati produkciju odreenih citokina povezanih sa
karcinogenezom (tabela 6.- in vitro studije) [84].
Laaksonen, Matti, Timo Sorsa, and Tuula Salo. "Emdogain in carcinogenesis: a systematic
review of in vitro studies." Journal of oral science 52.1 (2010): 1-11.

Hirurska tehnika

Tkivni ininjering http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3283928/

Tissue engineering, aimed at developing techniques
for the fabrication of new tissues to replace damaged
or diseased tissues, is based on principles of cell biology,
developmental biology and biomate6.
Bartold PM, Narayanan AS. Periodontal regeneration. In:
Biology of the periodontal connective tissues, Chapter 11.
Chicago: Quintessence Publishing, 1998.rials (6).

Tissue engineering is a highly promising field of reconstructive biology that draws on recent
advances in medicine, surgery, molecular and cellular biology, polymer chemistry, and
physiology. The objective of using tissue engineering as therapeutic application has been to
harness its ability to exploit selected and primed cells together with an appropriate mix of
regulatory factors, to allow growth and specialization of cells and matrix. The tissue engineering
approach to bone and periodontal regeneration combines three key elements to enhance
regeneration.

1. Conductive scaffolds/Extracellular matrix.

2. Signalling molecules.
3. Stem/Progenitor cells.[7]
This concept is often represented as a triangle, indicating that by combining the three key
elements tissue regeneration can often be accomplished [Figure 1].

Figure 1
The tissue engineering triad
Scaffolds

The scaffold provides a 3D substratum on to which the cells can proliferate and migrate, produce
a matrix and form a functional tissue with a desired shape. A suitable bioactive threedimensional scaffold for the promotion of cellular proliferation and differentiation is critical in
periodontal tissue engineering.
A scaffold plays many roles in tissue regeneration process:[8]

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

It serves as a delivery vehicle for exogenous cells, growth factors, and genes.
It may structurally reinforce the defect to maintain the shape of the defect.
It serves as a barrier to prevent infiltration of surrounding tissue that may impede the process of
regeneration.
Before its absorption, a scaffold can serve as a matrix for exogenous and endogenous cell
adhesion and thus facilitates and regulates certain cellular processes, including mitosis,
synthesis and migration.

Biomaterials used as scaffolds

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

Table 1
Scaffolds used for the purpose of periodontal regeneration

Naturally derived.
Synthetic.

Liao et al. in a study compared porous beta-tricalcium phosphate/chitosan composite scaffolds

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

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

Figure 2
Methods of fabrication of scaffolds

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

Cell source is an important parameter to consider when applying tissue engineering strategies to
restore lost tissues and functions.
Stem cells are immature progenitor cells capable of self renewal and multi-lineage differentiation
through a process of asymmetric mitosis that leads to two daughter cells, one identical to the
stem cell (daughter stem cell) and one capable of differentiation into more mature cells
(progenitor cells).[29]
Stem cells may be:

1. Totipotent, i.e. early embryonic cells (one to three days from oocyte fertilization), which can
give rise to all the embryonic tissues and placenta.
2. Pluripotent, i.e. embryonic cells from blastocystis (4-14 days after oocyte fertilization), which
can differentiate only into embryonic tissues belonging to the inner cell mass (ectoderm,
mesoderm, and endoderm).
3. Multipotent, i.e. embryonic cells from the 14th day onwards, which can give rise to tissues
belonging to only one embryonic germ layer (ectoderm or mesoderm or endoderm).[30]

Depending on the development stage of the tissues from which the stem cells are isolated, stem
cells can be broadly divided into two categories: Adult stem cells and embryonic stem cells.[31
33]
Embryonic stem cells are derived from embryos that are 2 11 days old called blastocysts. They
are totipotent cells. Due to ethical concerns and the risk of tumorogenicity and teratoma
formation, its use has been restricted to the research field.
Adult stem cells are multipotent stem cells, and depending upon their origin, they can be further
classified into hemopoetic stem cells and mesenchymal stem cells. Friedenstein and colleagues
first identified mesenchymal stem cells in aspirates of adult bone marrow.[34] Among the adults
stem cells, bone marrowderived stem cells or mesenchymal stem cells (MSCs) are adherent,
proliferating, and capable of multi-lineage differentiation having the capability of differentiating
into multiple tissue types, including bone, cartilage, muscle, tendon, etc., and hold great potential
for autologous cell-based therapy.[33]
Another important characteristic of MSCs for regenerative medicine is their potential allogenic
use without immunosuppressive therapy.[35] Within the sphere of periodontal tissue
engineering, mesenchymal derived cells have been applied for simultaneous regeneration of the
attachment apparatus components.
Signals

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

Mitogenic (proliferative);
Chemotactic (stimulate directed migration of cells); and
Angiogenic (stimulate new blood vessel formation) effects.[36]

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

Table 2
Various growth factors and their sources
Platelet-derived growth factor

Platelet-derived growth factor (PDGF) is the natural wound healing hormone. It is naturally
produced by the body at sites of soft tissue and bone injury. It was discovered by Lynch and
coworkers in the late 1980s.[37]
While PDGF secreted from platelets play an important role in initial wound healing, its
subsequent secretion from macrophages continues the events of wound healing through upregulation of other growth factors and cells that ultimately promote fibroblastic and osteoblastic
functions.[38]
Moon et al. applied PDGF-BB to promote migration and proliferation of periodontal ligament
fibroblasts. They demonstrated that PDGF has the capacity to stimulate bone formation and
periodontal regeneration in vivo and indicate that it holds promise as an important adjuvant to
periodontal surgery.[39]
Insulin like growth factor

Insulin like growth factor (IGF) is a potent chemotactic agent for vascular endothelial cells
resulting in increased neovascularization. It also stimulates mitosis of many cells in vitro such as
fibroblasts, osteocytes, and chondrocytes.[40] Insulin like growth factor-I is found in substantial
levels in platelets and is released during clotting along with the other growth factors.
Han and Amar demonstrated that in vitro IGF-I substantially enhanced cell survival in
periodontal ligament fibroblast compared to gingival fibroblasts by the up-regulation of antiapoptic molecules and down-regulation of pro-apoptotic molecules.[41]
Transforming growth factor family

The two best characterized polypeptides from this group of growth factors are Transforming
growth factor family (TGF)- and TGF-. TGF- appears to be a major regulator of cell
replication and differentiation. Three forms of TGF- have been identified namely TGF-1,
TGF-2, and TGF-3. TGF- isoforms have multiple regulatory roles in the synthesis,
maintenance and turnover of the extracellular matrix. TGF- is chemotactic for fibroblasts and
cementoblasts, and promotes fibroblast accumulation and fibrosis in the healing process. It can
also modulate other growth factors such as PDGF, TGF-, and EGF and fibroblast growth factor
(FGF) possibly by altering their cellular response or by inducing their expression.[42]
Oates et al. compared the mitogenic activity of TGF- with interleukin-1 and PDGF in fibroblast
cells derived from periodontal ligament explants. TGF- was relatively a weak mitogen for
Periodontal (PDL) cells compared to PDGF, suggesting that TGF- may indirectly stimulate
DNA synthesis.[43]
Fibroblast growth factor family

Fibroblast growth factors are the members of heparin binding growth factor family. The two
most thoroughly characterized forms are: Basic FGF (bFGF) and acidic FGF (aFGF). Both aFGF
and bFGF are single chain proteins that are proteolytically derived from different precursor
molecules to generate biologically active proteins of 15,000 molecular weight. They promote
proliferation and attachment of endothelial cells and PDL cells in wound healing process. FGF-2
is known to attract epithelial cells more effectively than FGF-1.[44]
Kitamura et al. did a recent randomized clinical trial trying to evaluate the therapeutic response
to varying doses of FGF-2 (bFGF). They demonstrated a significant increase in the alveolar bone
height on using 0.3% FGF-2.[45]
Takayama et al. examined the efficiency of topical application of FGF-2 with periodontal
regeneration in the bony defects by surgically creating furcation class II bone defects in nonhuman primates and concluded that a topical application of FGF-2 can enhance considerable
periodontal regeneration.[46]
Hepatocyte growth factor

Hepatocyte growth factor (HGF) is a secreted, heparin sulfate glycosaminoglycan-binding

protein. HGF has been shown to have mitogenic effects on osteoblasts; thus, participating in the
bone remodeling process.
Yamada et al. cultured fibroblasts in a culture medium containing HGF and concluded that they
produced good cell proliferation and vascular endothelial growth factor (VEGF) release. The
results suggest that it may provide a new tool for the treatment of gingival recession.[47]
Bone morphogenetic proteins

Bone morphogenetic proteins (BMPs) are the members of transforming growth factor- (TGF-)
superfamily, which play a crucial role in cell growth and differentiation. They are a group of
related proteins that are known to possess the unique ability to induce cartilage and bone
formation.[48] They trigger cellular effects by way of heterotetrameric serine/threonine kinase
receptors and intracellular signaling proteins known as small mothers against decapentaplegic
(Smads).[49] BMPs, like PDGF, play a role in the blood vessel formation. They play an
important role in the angiogenetic activity by up-regulating the angiogenetic peptides like VEGF,
may bind to endothelial cells and stimulate the migration and promote blood vessel
formation.[50]
The hallmark property of BMP is the differentiation factor. BMP will differentiate an
undifferentiated mesenchymal cell into an osteoblast. In contrast, PDGF is a chemotactic and
mitogenic factor for osteoblast like precursors.[50]
Go to:

CLINICAL APPLICATIONS OF TISSUE ENGINEERING FOR PERIODONTAL

TISSUE REGENERATION
Guided tissue regeneration

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

Table 3
Data extraction from studies done on membranes to treat infrabony defects
Protein based approaches[56,57]

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

Transforming growth factor ;

Bone morphogenetic proteins (super family members);

Basic fibroblast growth factor; and

Platelet derived growth factor.

Enamel matrix derivative

The rationale for the clinical use of enamel matrix derivative is the observation that enamel
matrix proteins are deposited onto the surfaces of developing tooth roots before cementum
formation.[58]
Enamel Matrix Protein (EMPs) are commercially available as Emdogain which have been known
to effect periodontal regeneration. Recent data from a systematic review indicates that
biologically EMPs cause an increase in cell attachment of epithelial cells, gingival fibroblasts,
and PDL fibroblasts. They increase the expression of transcription factors that are related to
chondroblast and osteoblasts/cementoblast differentiation. Stimulation in the synthesis of total
protein and extracellular matrix molecules has also been documented.[59] Use of Enamel matrix
derivative (EMD) and a demineralized freeze dried bone allograft (DFDBA) have been
demonstrated to be osteopromotive in nature; thus, resulting in an additional increase in bone
formation.[60] Studies enumerating the effects of EMD in obtaining periodontal regeneration are
given in Table 4. The only concern with the use of EMD has been related to its application and
its related viscous nature, which may not provide sufficient soft tissue/flap support; thus,
potentially limiting the space available for the regeneration process.

Table 4
Data extraction from studies done on enamel matrix derivative used to treat infrabony defects
Platelet rich plasma

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

Table 5
Data extraction from studies done on platelet rich plasma used to treat infrabony defects
Recombinant protein therapeutics

With advances in recombinant technology, the development and commercialization of pure

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

binding and release profile for these bioactive proteins that actively recruit healing cells to the
treatment site and expand their cell numbers, in order to achieve the greatest regeneration.
To date, only three recombinant growth factor products have been widely used

rh PDGF-BB (gel).[73]

rhPDGF-BB (with tricalcium phosphate).*74]

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

Recombinant platelet derived growth factor (rh PDGF BB) is more than 98% pure recombinant
protein developed using conventional recombinant expression techniques under highly controlled
conditions. They are first produced by removing the specific DNA sequences from a human cell
and transfecting it into a bacterial plasmid. The bacterial plasmid is then transfected into the host
cells capable of large scale growth. These are essentially protein factories that synthesize and
secrete many proteins. The rh PDGF BB is then separated using sophisticated analytical protein
chemistry techniques, sterile filtered and formulated into dose specified for clinical use.[76]
Use of rh PDGF has been one of the options to regenerate the periodontium and has received
FDA clearance for use.
The mitogenic responsiveness of periodontal cells to local application of PDGF-BB was
confirmed in a dog model by Wang and Castelli. Its levels were raised in cases of periodontitis,
but not in diabetic cases; thus, suggesting that PDGF-BB driven repair process is suppressed
under diabetic conditions.[77]
The concept of the use of recombinant protein therapeutics delivered in an allograft matrix has
provided significant clinical results. The efficacy of GEM 21S (growth enhanced matrix
TCP+PDGF), biomimmetic therapeutics were recently reported by Nevins and co workers.[57]
Studies have also suggested that the use of rh PDGF+ TCP and a collagen membrane may
represent an acceptable alternative to connective tissue graft for covering gingival recession
defects.[74]
Simion M conducted a study using rh PDGF in conjunction with anorganic bone block for
vertical ridge augmentation. It resulted in better healing and increased amount of regenerated
bone[78] [Table 6].

Table 6
Data extraction from studies done on recombinant platelet derived growth factor used to treat
infrabony defects and furcation defects
Role of rhBMP-2 in periodontal regeneration

The identification and development of recombinant human bone morphogenetic protein-2

(rhBMP-2) has lead to the commercial availability for the first time of an osteoinductive
autograft replacement (INFUSE Bone Graft). rhBMP-2 is a homodimeric protein consisting of
two BMP-2 protein subunits.
Studies provide an important insight that space provision appears critical to draw clinically
significant benefits from a BMP construct.

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

rhBMP2 has also been used in a DFDBA/fibrin clot carrier.[85]
rhBMP2 and calcium phosphate cement matrix.[86]

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

Table 7
Data extraction from studies done on recombinant bone morphogenetic protein-2 used to treat
infrabony defects and furcation defects

Tissue engineering using PRP or recombinant protein therapeutics is a clinical reality in

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

Cell based approaches

Cell transplantation using autologous cells is expected to play a central clinical role in the future.
Dental cell seeding attempts have attempted to regenerate the periodontal tissues since 1990s.
Attempts have been made to create the target tissue in the laboratory by culturing and
proliferating mesenchymal cells together with scaffolds, before transplanting them into the body.
Typical cell harvesting methods using enzymatic dispersion might destroy critical cell surface
proteins such as ion channels, while growth factor receptors and cell to cell junctions remain
intact. Okano et al. developed temperature responsive culture dishes (commercially available
under the name of UpCell, CellSeed Inc., Tokyo, Japan) by grafting a polymer poly N
isopropylacylamide (PIPAAm) onto tissue culture graded polystyrene dishes by irradiation with
an electron beam. Cells generally adhere to hydrophobic surfaces, but not to hydrophillic
surfaces. At temparatures lower than 32C, PIPAAm is fully hydrated. This dish allowed intact
cells with preserved extracellular matrix proteins and normal cell functions to be harvested with
just low temperature treatment.[50] This has evolved into a novel strategy called Cell sheet
engineering which produces tissues without a specific scaffold. Transplanted cell sheets can be
grafted to the recipient tissues without suturing.
Akizuki et al. investigated periodontal healing after the application of periodontal ligament cell
sheet in beagle dogs. These results demonstrated that, in the experimental group, periodontal
tissue healing with the formation of bone, periodontal ligament and cementum occurred in three
out of the five defects.[91]
Hasegawa et al. assessed the ability of periodontal ligament cell sheets to regenerate the
periodontal ligament tissue and demonstrated its usefulness in periodontal tissue
regeneration.[92] Flores et al. evaluated whether human PDL cell sheet could reconstruct
periodontal tissue and found that transplanted PDL cell sheet cultured with osteogenic
differentiation medium induced periodontal tissue regeneration containing an obvious cementum
layer and Sharpey's fiber.[93]
Huang and Zhang have set forward a hypothesis of transplanting PDL cell obtained from the
periodontium of autogenous extracted teeth, such as the third molar and premolar for orthodontic
purposes sheets when cultured using the cell sheet engineering approach into the implant beds
before inserting the implants.[94]
Gene delivery based approaches

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

Comparative evaluation

Comparison of different agents and techniques used to treat infrabony and furcation defects have
been tabulated as in Tables Tables337 and a brief conclusion has been drawn on them:

Clinical outcome obtained in the EMD patients are similar to those with the use of bioresorbable membranes. The advantage of using EMD is that they are technically more simple
with less risk of exposure, less invasive and resulting in lesser recession after surgery. However,

the adjunctive use of EMD with GTR doesnt seem to enhance the outcome of GTR.
PRP which provides similar results has the advantage of having hemostatic activity, giving a user
friendly environment and acts as a stabilizing agent, immobilizing the blood clot and bone graft
from the area.

The clinical results as well as histologically evaluated periodontal regeneration obtained using rh
PDGF and rh BMP produce much superior, but patient centered outcomes, including adverse
effects, cost effectiveness, and risk benefit have been evaluated in a very limited number of
studies.
Other bioactive agents are also being experimentally tested to treat periodontal defects
including OP-1, transforming growth factor , b FGF, IGF-1, cementum derived growth factor,
vascular endothelial growth factor and many more. More clinical studies need to prove their
effectiveness in treating periodontal defects.

Challenges ahead

1. Structural and functional complexity of the periodontium The fact that more than one tissue
must be reconstructed, namely alveolar bone, periodontal ligament, root cementum, and
gingiva, makes it much more difficult to find both the right combination and the doses of growth
factors.
2. To overcome the rapid clearance of growth factors, a carrier system must be found that stores
and releases the growth factors over a longer period of time so that their resident time is
prolonged. Although many carrier systems have been tested, none of them appears to be ideal.
3. While high developmental and therapeutic costs appear justified for severe skeletal conditions
such as non-unions, open fractures, spinal fusion, and large bone defects, for example in the
mandible, the same cannot necessarily be said for relatively small and non-life-threatening
periodontal defects where preventive and maintenance measures are still mandatory.
Go to:

CONCLUSION
We need to look beyond before we can achieve the dream. Tissue engineering has enlarged our
vision and thus made the fascination of being able to achieve regeneration of periodontal
complex in its entirety a reality.
Though the task has been arduous, but the promise still remains

Tissue engineering, according to National Institute of

Health definition, is an emerging multidisciplinary field
involving biology, medicine, and engineering that is
likely to revolutionize the way we improve the health
and quality of life for millions of people worldwide by
restoring, maintaining, or enhancing tissue and organ
function (1). For periodontal tissue engineering, this
specifically relates to repair of alveolar bone, toothassociated
cementum and periodontal ligament (PDL) (2).
1. Sipe JD, Kelley CA, McNichol LA. Reparative medicine: growing
tissues and organs. Ann N Y Acad Sci 2002;961:1389.
2. Giannobile WV. Periodontal tissue engineering by growth factors.
Bone 1996;19(Suppl. 1):23S37S.

The regeneration of the periodontal tissues is dependent

on four basic components. The appropriate signals,
cells, blood supply and scaffold need to target the tissue
defect (Fig. 1). Each of these elements plays a fundamental
role on the healing process in a simultaneous
and temporal timeframe and is interconnected into the
generation of new tissues. Cells provide the machinery
for new tissue growth and differentiation. Growth factors
or morphogens modulate the cellular activity and
provide stimuli to cells to differentiate and produce
matrix toward the developing tissue. New vascular
networks promoted by angiogenic signals provide the
nutritional base for tissue growth and homeostasis.
Finally, scaffolds guide and create a template structure
three-dimensionally to facilitate the above processes
critical for tissue regeneration. A major complication
and limiting factor in the achievement of periodontal
regeneration is the presence of microbial pathogens
that contaminate periodontal wounds and reside on
tooth surfaces as plaque-associated biofilms (11).
Appropriate strategies in controlling infection at the
reparative wound site are required to optimize periodontal
regeneration.
11. Slots J, MacDonald ES, Nowzari H. Infectious aspects of periodontal
regeneration. Periodontology 2000 1999;19:16472.

Cells contributing to periodontal repair

Specialized tissues such as the periodontium include

epithelialmesenchymal interactions and junctional
complexes (to the tooth and connective tissues).
Based on its embryonic origin, the periodontal
tissues form by the interaction of mesenchymal and
epithelium cells that respond differently to a variety of
stimuli (12). 12. Giannobile WV, Somerman MJ. Growth and amelogenin-like
factors in periodontal wound healing. A systematic review. Ann
Periodontol 2003;8:193204.

The cellular constitution of the gingival connective

tissue and alveolar bone include: fibroblasts, macrophages,
mast cells, osteoblasts and osteoblast precursor
cells, cementoblasts and cementoblast precursor
cells, osteoclasts and odontoclasts, assorted inflammatory
cells, and cells that make up vascular channels and
nerves. Inflammatory cells include polymorphonuclear
leucocytes, lymphocytes, and plasma cells (13). The
connective tissue also contains undifferentiatedectomesenchymal cells that serve as a replacement
source for more differentiated cells, primarily fibroblasts.
Fibroblasts are irregularly shaped cells responsible
for the maintenance and remodeling of
the connective tissue, synthesizing and removing
connective tissue fibers and the ground substance in
which they are embedded (14).
The PDL contains fibroblasts, macrophages, undifferentiated
ectomesenchymal cells, cementoblasts and
cementoclasts, osteoblasts and osteoclasts, cell rests of
Malassez and vascular and neural elements that are
capable of generating and maintaining three distinct
tissues, PDL, and the mineralized tissues: cementum
and alveolar bone (13,15). As specific cell lineages
retain the potential of regeneration (12,16), stimulatory
(9) and selective (10) approaches have been attempted
to re-engineer lost periodontal supporting tissues.
9. Urist MR. Bone: formation by autoinduction. Science
1965;150:8939.
10. Melcher AH. On the repair potential of periodontal tissues. J
Periodontol 1976;47:25660. 13. Schroeder HE, Listgarten MA. The gingival tissues: the architecture
of periodontal protection. Periodontology 2000 1997;13:91
120.
14. Howard PS, Kucich U, Taliwal R, Korostoff JM. Mechanical forces
alter extracellular matrix synthesis by human periodontal ligament
fibroblasts. J Periodontal Res 1998;33:5008.
15. Aukhil I. The potential contributions of cell and molecular biology
to periodontal tissue regeneration. Curr Opin Dent 1992;2:916.
16. Saygin NE, Giannobile WV, Somerman MJ. Molecular and cell
biology of cementum. Periodontology 2000 2000;24:7398.

Somerman et al. have also demonstrated the potential

use of cell therapy with cloned cementoblasts (25).
In a series of reports, the group has cloned and characterized
a cementoblast cell line, which possesses
many of the phenotypic characteristics of tooth-lining
cells in vivo. More recently, it has been demonstrated
that transplantation of cloned cementoblasts into polylactidepolylactide-

co-glycolide acid (PLGA) carriers leads to the

repair of large periodontal alveolar bone defects in
rodents (26). The achieved regenerationwas only limited
by the size of the transplanted PLGAcarrier. Jin et al. have
also shown that skin fibroblasts transduced by the
BMP-7 gene promoted the tissue engineering of periodontal
bone defects including new bone, functional
PDL and tooth root cementum (27).
25. Somerman MJ, Ouyang HJ, Berry JE, Saygin NE, Strayhorn CL,
DErrico JA et al. Evolution of periodontal regeneration: from the
roots point of view. J Periodontal Res 1999;34:4204.
26. Zhao M, Jin Q, Berry JE, Nociti FH Jr, Giannobile WV, Somerman
MJ. Cementoblast delivery for periodontal tissue engineering.
J Periodontol 2004;75:15461.
27. Jin QM, Anusaksathien O, Webb SA, Rutherford RB, Giannobile
WV. Gene therapy of bone morphogenetic protein for periodontal
tissue engineering. J Periodontol 2003;74:20213.

In order to enhance the in vivo efficacy, incorporation

of bioactive molecules into scaffolding materials may
Table 1. Cell-occlusive barriers used for periodontal regeneration
Cell-occlusive barrier Trade name
Non-resorbable
Cellulose, ePTFE Millipore filter_,
Gore-Tex_
Resorbable
Polylactic acid and poly-glycolic
acid, Polyglactin-910, poly(L-lactide)
Resolut_, Atrisorb_,
Vicryl-Netz_
Collagen
Bovine tendon type I,
Porcine dermis type I + III
Biomend_, BioGide_,
Ossix_
Plaster of Paris
Calcium sulfate Cap-Set_, Hap-Set_

A
B
C
Fig. 2. Cell therapy for periodontal tissue engineering: (A) SEM image
of poly-lactide-co-glycolide acid (PLGA) scaffold and seeded cells into
scaffolds; (B) tissue engineering approach for ectopic mineralization
in mice, cells are expanded in culture prior to being seeded into the
scaffolds for implantation; (C) periodontal ligament cells (PDL) do
not promote mineralization in vivo, while cementoblasts show mineralization,
20 magnification.
Orthod Craniofacial Res 8, 2005/292302 295
Taba et al. Current concepts in periodontal bioengineering

Growth factors for periodontal

Regeneration
facilitate sustained factor release for periods of time.
There are two basic modes of incorporating bioactive
molecules into the scaffolds at the time of fabrication
(28) or after the fabrication (29). Bioactive molecules
incorporated directly into a bioresorbable scaffold are
generally released by a diffusion-controlled mechanism
that is regulated by the pore sizes such that different
pore sizes affect the tortuosity of the scaffold and
thereby control the release of protein (30). The rate

of growth factor release depends on the type and rate of

degradation of the delivery device, and the rate of
growth factor diffusion through pores of the scaffolds
28. Whang K, Tsai DC, Nam EK, Aitken M, Sprague SM, Patel PK
et al. Ectopic bone formation via rhBMP-2 delivery from porous
bioabsorbable polymer scaffolds. J Biomed Mater Res
1998;42:4919.
29. Fournier N, Doillon CJ. Biological molecule-impregnated polyester:
an in vivo angiogenesis study. Biomaterials 1996;17:1659
65.
30. Babensee JE, McIntire LV, Mikos AG. Growth factor delivery for
tissue engineering. Pharm Res 2000;17:497504.

Several bioactive molecules have demonstrated

strong effects in promoting periodontal wound repair in
preclinical and clinical studies. These bioactive moleculesmolecules
include PDGF (36), IGF-I (37), basic fibroblast
growth factor (FGF-2) (38), TGF-1 (39), BMP-2 (40), -4
(41), -7 (42) and -12 (43), and enamel matrix derivative
(EMD) (44) that have shown positive results in stimulating
periodontal regeneration. In addition, PDGF,
BMP-2, and BMP-7 have been shown to promote periimplant
bone regeneration (2). Recently, a comparative
study in periodontal defects found that rhBMP-2 in
association with collagen sponges promoted superior
bone regeneration than rhBMP-12. Cementum formation
is similar for both BMPs, however, only the rhBMP12 treatment induces to functionally oriented PDL
bridging newly formed bone and new cementum (43).
The details of preclinical and clinical studies have
been reviewed in several articles (2,5,12,4547). In
general, the results from the in vivo studies have shown
that all of the above-mentioned bioactive molecules,
with the exception of TGF-1, exhibited an ability to
promote periodontal tissue regeneration. BMPs
showed the greatest potential in critical-size defectsBMPs
showed the greatest potential in critical-size defects, as
they are potent osteoinductive mediators. A phase I/II
human trial using a combination of PDGF and IGF-I
demonstrated the stimulation of alveolar bone repair in
patients with severe periodontal disease (48).
2. Giannobile WV. Periodontal tissue engineering by growth factors.
Bone 1996;19(Suppl. 1):23S375. Nakashima M, Reddi AH. The application of bone morphogenetic
proteins to dental tissue engineering. Nat Biotechnol
2003;21:102532.S.
12. Giannobile WV, Somerman MJ. Growth and amelogenin-like
factors in periodontal wound healing. A systematic review. Ann
Periodontol 2003;8:193204.
37. Giannobile WV, Hernandez RA, Finkelman RD, Ryan S, Kiritsy CP,
DAndrea M et al. Comparative effects of platelet-derived growth
factor-BB and insulin-like growth factor-I, individually and in
combination, on periodontal regeneration in Macaca fascicularis.
J Periodontal Res 1996;31:30112.
38. Murakami S, Takayama S, Ikezawa K, Shimabukuro Y,
Kitamura M, Nozaki T et al. Regeneration of periodontal tissues
by basic fibroblast growth factor. J Periodontal Res 1999;34:425
30.

39. Saygin NE, Tokiyasu Y, Giannobile WV, Somerman MJ. Growth

factors regulate expression of mineral associated genes in
cementoblasts. J Periodontol 2000;71:1591600.
40. Zhao M, Xiao G, Berry JE, Franceschi RT, Reddi A, Somerman MJ.
Bone morphogenetic protein 2 induces dental follicle cells to
differentiate toward a cementoblast/osteoblast phenotype. J Bone
Miner Res 2002;17:144151.
41. Ahn SH, Kim CS, Suk HJ, Lee YJ, Choi SH, Chai JK et al. Effect of
recombinant human bone morphogenetic protein-4 with carriers
in rat calvarial defects. J Periodontol 2003;74:78797.
42. Jin QM, Zhao M, Economides AN, Somerman MJ, Giannobile WV.
Noggin gene delivery inhibits cementoblast-induced mineralization.
Connect Tissue Res 2004;45:509.
43. Wikesjo UM, Sorensen RG, Kinoshita A, Jian Li X, Wozney JM.
Periodontal repair in dogs: effect of recombinant human bone
morphogenetic protein-12 (rhBMP-12) on regeneration of alveolar
bone and periodontal attachment. J Clin Periodontol
2004;31:66270.
44. Lyngstadaas SP, Lundberg E, Ekdahl H, Andersson C, Gestrelius S.
Autocrine growth factors in human periodontal ligament cells
cultured on enamel matrix derivative. J Clin Periodontol
2001;28:1818.
45. Anusaksathien O, Giannobile WV. Growth factor delivery to
re-engineer periodontal tissues. Curr Pharm Biotechnol
2002;3:12939.
46. Giannobile WV, Al-Shammari KF, Sarment DP. Matrix molecules
and growth factors as indicators of periodontal disease activity.
Periodontology 2000 2003;31:12534.
47. Terranova VP, Wikesjo UM. Extracellular matrices and polypeptide
growth factors as mediators of functions of cells of the
periodontium. A review. J Periodontol 1987;58:37180.

Scaffold and extracellular matrix

The extracellular matrix is a biologically active tissue
composed of a complex mixture of macromolecules,
that in addition to serving a structural function, also
profoundly affect the cellular physiology of an organism
(47). Cell adhesion, migration, proliferation and
differentiation are examples of biological processes
influenced by the composition and structural organization
of surrounding extracellular matrices (49). extracellular
matrix components combine active morphogens to
confer the optimal conformation, which then can initiate
contact-mediated interactions (51). Considering that scaffolds simulate the extracellular
matrix, the rate of material degradation plays a critical
role on the initiation of tissue replacement for engineered
structures. A list of commercially available
biomaterials for tissue repair applications is shown in
Table 3. It was demonstrated by Lu et al. that the release
rate of growth factor from the scaffold can profoundly
affect the results (52). Furthermore, the pharmacokinetics
of the BMPs incorporated on biomaterials, such
as collagen and hydroxyapatite (53), may be different
from the kinetics in the bone, periodontium and teeth
because the retention of BMPs at the site of implantation
is dependent on the charge characteristics and
isoelectric point of the morphogens (54).
49. Kleinman HK, Philp D, Hoffman MP. Role of the extracellular

matrix in morphogenesis. Curr Opin Biotechnol 2003;14:52632.

51. Reddi AH. Interplay between bone morphogenetic proteins and
cognate binding proteins in bone and cartilage development:
noggin, chordin and DAN. Arthritis Res 2001;3:15.
52. Lu L, Zhu X, Valenzuela RG, Currier BL, Yaszemski MJ. Biodegradable
polymer scaffolds for cartilage tissue engineering. Clin
Orthop 2001;391(Suppl.):S25170.
53. Brandao AC, Brentegani LG, Novaes AB Jr, Grisi MF, Souza SL,
Taba M Jr et al. Histomorphometric analysis of rat alveolar
wound healing with hydroxyapatite alone or associated to BMPs.
Braz Dent J 2002;13:14754.
54. Uludag H, Gao T, Porter TJ, Friess W, Wozney JM. Delivery systems
for BMPs: factors contributing to protein retention at an
application site. J Bone Joint Surg Am 2001;83-A(Suppl. 1):S12835.

Angiogenic factors for periodontal repair

It is not only important to maintain
the local homeostasis and adequate host defense by
transporting cells and defensins to the gingival crevice
(56), the blood supply has a key role on the nutrition of
newly engineered tissues. However, a major challenge
in periodontal regeneration is the targeting of angiogenesis
to an avascular tooth root surface.
Subsequent to an injury, capillaries invading a fibrin
clot will deliver nutrients, inflammatory cells, and
oxygen to the wound site for early granulation tissue
formation (57). Additionally, new blood vessels will
contribute to provide an adequate environment for cell
migration, proliferation, differentiation and extracellular
matrix synthesis; all essential features noted during
the initial phases of periodontal healing (58).
Basic fibroblast growth factor (bFGF or FGF-2) has
been demonstrated to have potent angiogenic activity
(59) and potential to induce the growth of immature
PDL cells (38). The mRNA level of laminin in PDL cells,
which plays an important role in angiogenesis, is
upregulated by FGF-2 stimulation (38).
Also, a recent
report addressed the effect of the EMD on the stimulation
of angiogenesis of the periodonAlthough EMD has angiogenic effects both in vitro
and in vivo (60), the more rapid initial healing may not
be directly influenced by the angiogenic effect of EMD
alone. At least two other mechanisms probably contribute
to the acceleration of wound healing. First, PDL
cells can secrete growth factors, including TGF-b1, IL-6,
and PDGF-AB after exposure to EMD (44,61).tal wounds (60).
The technical challenges confronting the tissue
engineering of vasculature are many (65). First is the
selection of appropriate vascular cells and scaffold
materials. The majority of studies to date typically
involve in vitro culturing of bone marrow cells or

smooth muscle cells in combination with a collagenbased

matrix until a tubular structure is formed, subsequently
allowing endothelial cells to attach to the
vessel wall (66). Scaffolds need to be designed to
support the proper formation of vascular tissue and
possess the mechanical properties that can match
those of native arteries. The synthetic vessel must
withstand the fluid shear stress and strain from
blood flow and have an adequate burst strength to
withstand physiological blood pressures (65). Finally,
incompatibilities between synthetic engineered grafts
and native blood vessels must be quantified and
evaluated. Present and future
studies involving the incorporation of angiogenic
growth factors (most notably VEGF) directly into scaffolds
(67), or using gene therapy to deliver growth
factors to these sites may prove to be an important key
for stimulating new vessel formation (68). In a dual
growth factor delivery model targeting rapid formation
of mature vascular networks, it was demonstrated that
both PDGF + VEGF, each with distinct kinetics,
improve angiogenesis by leading the formation of a
significant number of blood vessels and inducing their
maturation (69)
58. Okuda K, Murata M, Sugimoto M, Saito Y, Kabasawa L, Yoshie H
et al. TGF-beta1 influences early gingival wound healing in rats:
an immunohistochemical evaluation of stromal remodelling by
extracellular matrix molecules and PCNA. J Oral Pathol Med
1998;27:4639.
59. Folkman J, Klagsbrun M. Angiogenic factors. Science
1987;235:4427.
60. Yuan K, Chen CL, Lin MT. Enamel matrix derivative exhibits
angiogenic effect in vitro and in a murine model. J Clin Periodontol
2003;30:7328.
61. van der Pauw MT, Everts V, BeertsenW. Expression of integrins by
human periodontal ligament and gingival fibroblasts and their
involvement in fibroblast adhesion to enamel matrix-derived
proteins. J Periodontal Res 2002;37:31723.
62. Dennison DK, Vallone DR, Pinero GJ, Rittman B, Caffesse RG.
Differential effect of TGF-beta 1 and PDGF on proliferation of
periodontal ligament cells and gingival fibroblasts. J Periodontol
1994;65:6418.
63. Bartold PM, Raben A. Growth factor modulation of fibroblasts in
simulated wound healing. J Periodontal Res 1996;31:20516.
64. Sculean A, Auschill TM, Donos N, Brecx M, Arweiler NB. Effect of
an enamel matrix protein derivative (Emdogain) on ex vivo dental
plaque vitality. J Clin Periodontol 2001;28:10748.
65. Zisch AH, Lutolf MP, Ehrbar M, Raeber GB, Rizzi SC, Davies N
et al. Cell-demanded release of VEGF from synthetic, biointeractive
cell ingrowth matrices for vascularized tissue growth.
FASEB J 2003;17:22602.
66. Shum-Tim D, Stock U, Hrkach J, Shinoka T, Lien J, Moses MA
et al. Tissue engineering of autologous aorta using a new biodegradable
polymer. Ann Thorac Surg 1999;68:2298304 (discussion
305).
67. Zisch AH, Lutolf MP, Hubbell JA. Biopolymeric delivery matrices
for angiogenic growth factors. Cardiovasc Pathol 2003;12:295310.
68. Liu PY, Tong W, Liu K, Han SH, Wang XT, Badiavas E et al.
Liposome-mediated transfer of vascular endothelial growth factor
cDNA augments survival of random-pattern skin flaps in the rat.
Wound Repair Regen 2004;12:805.
69. Richardson TP, Peters MC, Ennett AB, Mooney DJ. Polymeric
system for dual growth factor delivery. Nat Biotechnol

2001;19:102934.
38. Murakami S, Takayama S, Ikezawa K, Shimabukuro Y,
Kitamura M, Nozaki T et al. Regeneration of periodontal tissues
by basic fibroblast growth factor. J Periodontal Res 1999;34:42530.
44. Lyngstadaas SP, Lundberg E, Ekdahl H, Andersson C, Gestrelius S.
Autocrine growth factors in human periodontal ligament cells
cultured on enamel matrix derivative. J Clin Periodontol
2001;28:1818.

Gene therapy for periodontal engineering

As the half-life of growth factors in vivo is transient (on
the order of minutes to hours), periodontal regeneration
is not a common outcome following conventional
surgical therapy. Typically, high concentrations of
growth factors are required to promote tissue regeneration
(70). Therefore, supplemental local growth
factor production via gene transfer could be superior to
bolus delivery methods.
Simply stated, gene therapy consists of the insertion
of genes into an individuals cells either directly or
indirectly with a matrix to promote a specific biological
effect. Gene therapy typically aims to supplement a
defective mutant allele with a functional one in a
therapeutic approach but can also be used to induce
a more favorable host response. Targeting cells for gene
therapy requires the use of vectors or direct delivery
methods to transfect them. Using an ex vivo approach for periodontal repair, Jin
et al. used BMP-7 gene transfer to stimulate new
alveolar bone, tooth root cementum, and PDL (27).
Ad-BMP-7 or its antagonist, Ad-noggin transduced
syngeneic dermal fibroblast that were seeded onto
gelatin carriers and transplanted into large alveolar
bone defects. The repair of periodontal defects was
observed through a rapid chondrogenesis, followed by
osteogenesis, cementogenesis and predictable bridging
of the bone defect (Fig. 3) (27).
PDGF has demonstrated strong potential in promoting
gingival (71), alveolar bone (72), and cementum
(73) regeneration in a variety of wound healing models
(Fig. 4) (72). When periodontal defects were treated
with adenovirus encoding PDGF-B, strong evidence of
bone and cementum regeneration beyond that of
control vectors, consisting of nearly fourfold increases
in bridging bone and sixfold increases in tooth-lining
cemental repair was seen (72).
27. Jin QM, Anusaksathien O, Webb SA, Rutherford RB, Giannobile
WV. Gene therapy of bone morphogenetic protein for periodontal
tissue engineering. J Periodontol 2003;74:20213
70. Fang J, Zhu YY, Smiley E, Bonadio J, Rouleau JP, Goldstein SA
et al. Stimulation of new bone formation by direct transfer of
osteogenic plasmid genes. Proc Natl Acad Sci U S A 1996;93:5753

8.
71. Anusaksathien O, Webb SA, Jin QM, Giannobile WV. Plateletderived
growth factor gene delivery stimulates ex vivo gingival
repair. Tissue Eng 2003;9:74556.
72. Jin Q, Anusaksathien O, Webb SA, Printz MA, Giannobile WV.
Engineering of tooth-supporting structures by delivery of PDGF
gene therapy vectors. Mol Ther 2004;9:51926.
73. Anusaksathien O, Jin Q, Zhao M, Somerman MJ, Giannobile WV.
Effect of sustained gene delivery of platelet-derived growth factor
or its antagonist (PDGF-1308) on tissue-engineered cementum.
J Periodontol 2004;75:42940.

Future perspectives
Many advances have been made over the past decade
in the reconstruction of complex periodontal and
alveolar bone wounds. Developments in polymeric and
ceramic scaffolding systems for cell, protein and gene
delivery have undergone significant growth. The targeting
of signaling molecules or growth factors (via
proteins or genes) to the periodontium has lead to
significant new knowledge generation using bioactive
molecules that promote cell proliferation, differentiation,
matrix biosynthesis, and angiogenesis. A major
challenge that has been overlooked has been the
modulation of the exuberant host response to microbial
contamination that plagues the periodontal wound
environment. For improvements in the outcomes in
periodontal regenerative medicine, scientists will needto examine dual delivery of host modifiers or antiinfective
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.

Advanced reconstructive
technologies for periodontal
tissue repair
Biomodification 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
biomodification with citric acid, the outcomes of
controlled clinical trials have failed to show any
improvements in clinical conditions compared with
nonacid-treated controls (40, 91, 99).
In recent years, biomodification of the root surface
with enamel matrix proteins during periodontal surgery
and following demineralization with 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 as it initiates events that occur during

the growth of periodontal tissues (43, 54). The commercially

available product Emdogain_, a purified
acid extract of porcine origin containing enamel
matrix derivates, is reported to be able to enhance
periodontal regeneration (Fig. 3).

Periodontal tissue growth factors

Periodontal tissue growth factors
Wound-healing approaches using growth factors to
target restoration of tooth-supporting bone, periodontal
ligament and cementum have been shown to
significantly advance the field of periodontal-regenerative
medicine. 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 (14, 46, 67, 110, 115, 140), insulin-like growth
factors (44, 46, 58, 87), fibroblast growth factors (49,
101, 149, 77, 151) and bone morphogenetic proteins
(42, 59, 152, 164, 165), have been used in preclinical
and clinical trials for the treatment of large periodontal
or infrabony defects, as well as around dental
implants (36, 68, 110). The combined use of recombinant
human platelet-derived growth factor-BB
and peptide P-15 with a graft biomaterial has shown
beneficial effects in intraosseous defects (157).

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 (plateletderived
growth factor-Ra and platelet-derived growth
factor-Rb) and subsequently exerts its biological effects
on cell proliferation, migration, extracellular
matrix synthesis and anti-apoptosis (56, 71, 138, 148).
Platelet-derived growth factor-a and -b receptors are
expressed in regenerating periodontal soft and hard
tissues (119). In addition, platelet-derived growth
factor initiates tooth-supporting periodontal ligament
cell chemotaxis (111), mitogenesis (113), matrix
synthesis (53) and attachment to tooth dentinal surfaces
(172). More importantly, in vivo application of
platelet-derived growth factor alone or in combination with insulin-like growth factor-1 results in the
partial repair of periodontal tissues (46, 47, 87, 88,
140). Platelet-derived growth factor has been shown
to have a significant regenerative impact on periodontal
ligament cells, as well as on osteoblasts (90,
92, 113, 115).

Biological effects of growth factors:

bone morphogenetic proteins

Bone morphogenetic proteins are multifunctional
polypeptides belonging to the transforming growth
factor-beta superfamily of proteins (169). The human
genome encodes at least 20 bone morphogenetic
proteins (131). Bone morphogenetic proteins bind to
type I and type II receptors that function as serinethreonine
kinases. The type I receptor protein kinase
phosphorylates intracellular signaling substrates
called Smads (the sma gene in Caenorhabditis elegans
and the 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 their ability to induce ectopic bone formation
(160). Bone morphogenetic proteins are not
only powerful regulators of cartilage and bone formation
during embryonic development and regeneration
in postnatal life, but they also participate in
the development and repair of other organs such as
the brain, kidney and nerves (132).
Sigurdsson et al. (149) evaluated bone and
cementum formation following regenerative periodontal
surgery by the use of recombinant human
bone morphogenetic protein in surgically created
supra-alveolar defects in dogs (168). Histologic
analysis showed significantly more cementum formation
and regrowth of alveolar bone on bone
morphogenetic protein-treated sites compared with
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
(165) or recombinant human bone morphogenetic
protein-2 (86, 166).

Clinical application of growth factors for

use in periodontal regeneration
In general, the impact of topical delivery of growth
factors to periodontal wounds has been promising,
yet insufficient to promote predictable periodontal
tissue engineering (14, 23) (Fig. 4). Growth factor
proteins, once delivered to the target site, tend to
suffer from instability and quick dilution, presumably
because of proteolytic breakdown, receptormediated
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).
Investigations for periodontal bioengineering have
examined a variety of methods that combine delivery
vehicles, such as scaffolds, with growth factors to
target the defect site in order to optimize bioavailability
(85). The scaffolds are designed to optimize
the dosage of the growth factor and to control itsrelease pattern, which may be pulsatile, constant or
time-programmed (8). The kinetics of the release and
the duration of the exposure of the growth factor may
also be controlled (61). A new polymeric system, permitting the tissuespecific
delivery (at a controlled dose and delivery
rate) of two or more growth factors, was reported in an
animal study carried out by Richardson et al. (137).
The dual delivery of vascular endothelial growth factor
with platelet-derived growth factor from a single,
structural polymer scaffold results in the rapid formation
of a mature vascular network (137)

Gene therapeutics for periodontal

tissue repair
Although encouraging results for periodontal regeneration
have been found in various clinical investigations
using recombinant tissue growth factors,
there are limitations for 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 periodontalregenerative
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 (10, 45). It
has been shown that the application of growth factors
(37, 63, 64, 78) or soluble forms of cytokine receptors
(21) by gene transfer provides greater sustainability
than the application of a single protein. Thus, gene
therapy may achieve greater bioavailability of growth
factors within periodontal wounds and hence provide
greater regenerative potential.

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, such as a scaffold. Other
defect sites may be conducive to the use of an adenovirus
vector embedded in a collagen matrix.

A combination of an Adeno-Associated Virusdelivered

angiogenic molecule, such as vascular
endothelial growth factor, bone morphogenetic protein
signaling receptor (caALK2) and receptor activator
of nuclear factor-kappa B ligand, was demonstrated
to promote bone allograft turnover and
osteogenesis as a mode to enrich human bone allografts
(62). To date, combinations of vascular endothelial
growth factor bone morphogenetic protein
(120) and platelet-derived growth factor vascular
endothelial growth factor (137) have had 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
(21).

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)
(63) or selected cells can be harvested, ex-panded, genetically transduced and then re-implanted
(ex vivo technique) (64). 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 receptor activator of
nuclear factor-kappa B ligand-derived osteoclast
activation.

Platelet-derived growth factor gene

Delivery
Both plasmid (57) and adenovirus
platelet-derived growth factor (125) gene delivery
have been evaluated in preclinical and human trials
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)

(48, 173). These studies revealed the extensive and
prolonged transduction of periodontal-derived cells
In an ex vivo investigation by Anusaksathien et al.
(6), 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 plateletderived growth factor-B) transduced gingival
fibroblasts and enhanced defect fill by inducing
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
the subsequent enhancement of multinucleated giant
cells in cementum-engineered scaffolds. 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. (63) 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 delivery of the human platelet-derived
growth factor-B gene promotes the regeneration of
both cementum and alveolar bone

Delivery of the bone

morphogenetic protein gene
An experimental study in rodents by Lieberman et
al. (81) advanced gene therapy for bone regeneration,
with the results revealing that the transductionof bone marrow stromal cells with recombinant
human bone morphogenetic protein 2 led to bone
formation within an experimental defect comparable
to skeletal bone. Another group was similarly able to
regenerate skeletal bone by directly administering
adenovirus5 bone morphogenetic protein 2 into a
bony segmental defect in rabbits (9).
Adenovirus-transduced
nonosteogenic cells were also found to differentiate
into bone-forming cells and to produce
bone morphogenetic protein 7 (78) or bone morphogenetic
protein 2 (20) both in vitro and in vivo.
In another study by Huang et al. (60), plasmid DNA
encoding bone morphogenetic protein 4 administered
using a scaffold-delivery system was found to
enhance bone formation when compared with blank
scaffolds.
Transfer of the bone
morphogenetic protein 7 gene enhanced alveolar
bone repair and also stimulated cementogenesis and
periodontal ligament fiber formation. Of interest,

alveolar bone formation was found to occur via a

cartilage intermediate. In a study by Dunn et al.
(30), 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.

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 led to significant new knowledge
generation using factors that promote cell replication,
differentiation, matrix biosynthesis and angiogenesis.
A major challenge that has been studied less is the
modulation of the exuberant host response to microbial
contamination that plagues the periodontal
wound microenvironment. To achieve improvements
in the outcome of periodontal-regenerative medicine,
scientists will need to examine the 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.

Regeneracija i uvod
The complex series of events
associated with periodontal regeneration involves
recruitment of locally derived progenitor cells to
the site which can subsequently differentiate into
periodontal ligament-forming cells, mineral-forming
cementoblasts, or bone-forming osteoblasts (6).
To date, restoration of damaged or diseased periodontal
tissues has relied almost entirely on the use of
implantation of structural substitutes, often with little
or no reparative potential. In general, these efforts
have focused almost exclusively on regenerating
lost alveolar bone and have included the use of
autografts (cortical cancellous bone, bone marrow),
allografts (demineralized freeze-dried freeze-dried
bone) and alloplastic materials (ceramics, hydroxyapatite,
polymers and bioglass). Due to issues such as

the variability in safety, clinical effectiveness and

stability over time of these agents, their use for periodontal
regeneration has been questioned (5, 8, 37).
More recently, biological approaches based on the
principles of tissue-engineering have emerged as
prospective alternatives to conventional treatments
Such approaches have included gene therapy and the
local administration of biocompatible scaffolds with
or without the presence of selected growth factors (21,
31, 34, 60). These new approaches, based on an
understanding of the cell and molecular biology of the
developing and regenerating periodontium, offer
interesting alternatives to existing therapies for the
repair and regeneration of the periodontium.

5. Future perspectives of cell-based therapy for periodontal and bone

regeneration
5.1. PROGRESS IN REGENERATIVE THERAPY FOR PERIODONTIUM AND MAXILLOFACIAL
BONES
First, material-and scaffold-based therapies have been developed using bone replacement graft and GTR
membranes. Second, growth factor application has increased for mixed components and recombinant proteins.
Several approaches involving enamel-matrix derivatives and PRP/PRF have been reported, and recombinant
growth factors (BMP, FGF-2, PDGF, and GDF-5) have been introduced for periodontal and bone regenerative
therapy. Third, cell-based therapy has recently emerged using bone marrow cells, periosteal cells, and
periodontal ligament cells. Finally, combination therapy with scaffolds, growth factors, and cells appears to
present a promising strategy to achieve the regeneration of large defects and to enlarge the indication of
therapy [Egusa et al., 2012].
Egusa H, Sonoyama W, Nishimura M, Atsuta I, & Akiyama K. (2012) Stem cells in dentistry-Part II: Clinical applications. J Prosthodont Res56: 229-248.

5.2. THREE CHALLENGES FOR COMBINED CELL-BASED THERAPY

5.2.1. BIOLOGICAL AND CLINICAL SAFETY CHALLENGES

Prevention of tumor formation following stem cell implantation is a major safety consideration from the
biological point of view. It is critical to have an understanding of the cell genetic instability, culture medium
and conditions. In addition, practical errors and accidents involving contamination and infection with virus and
mycoplasma should be monitored and avoided.

5.2.3. CHALLENGES OF CELL DELIVERY SYSTEM AND CELL BANKING

Cell processing center/room (CPC) is required for cell-based therapy, and the refinement of techniques to
facilitate laboratory handing of cells is also crucial for biological and clinical applications. CPC should be
established at hospital, however, it is impossible to establish CPC. Thus, a well-controlled cell delivery system
is necessary for wide area of several prefectures. Furthermore, cell banking may be one strategy to realize the
potential of cell-based regenerative therapy. Several types of cells can be cryopreserved to retain regenerative
potential. Dental cells can be isolated from the cryopreserved tissue whenever required for future regenerative
therapies [Chen et al., 2012].
Chen FM, Sun HH, Lu H, & Yu Q. (2012) Stem cell-delivery therapeutics for periodontal tissue
regeneration. Biomaterials 33: 6320-6344.

5.2.2. BENEFICIAL TECHNICAL CHALLENGES

It is definitely possible to achieve remarkable regeneration using cell-based therapy compared with
conventional regenerative treatment for satisfaction of patients and cost benefit aspects. The matrix and
scaffold should have good biocompatibility in terms of the cellular and molecular components during the
process of developing and regenerating tissues. The synergistic effects of cell-based therapy with suitable
scaffolds and growth factors are anticipated and should be substantiated in the near future.

In our basic studies using in vitro cell culture systems and in vivo animal implantation systems, we have
demonstrated several remarkable characteristics of the periosteal sheet, which are summarized below:

Tissue-like thickness: The periosteal sheet displays a unique structure that is composed of cell-multilayers
and abundantly deposited extracellular matrices (ECM).
Osteogenicity: Periosteal cells within the periosteal sheet differentiate to osteogenic cells slowly but
spontaneously with time of expansion using a conventional medium.
Osteoinduction: The periosteal sheet produces the major growth factors involved in bone metabolism.

Stem celijeStem cells and periodontal

regeneration

The concept that stem cells may reside in the

periodontal tissues was first proposed almost
20 years ago by Melcher, who queried whether
the three cell populations of the periodontium
(cementoblasts, alveolar bone cells and periodontal
ligament fibroblasts) were ultimately derived from
a single population of ancestral cells or _stem cells
The most compelling evidence that these cells
are present within the periodontal tissues has been
provided by the in vivo and histological studies of
McCulloch and coworkers (42, 4547).
Studies to date
have shown that periodontal ligament cells can be
transplanted into periodontal defects with no adverse
immunologic or inflammatory consequences (40, 44,
70)
2. Cell Sheet Engineering (CSE)
The cell delivery for periodontal regeneration is usually performed with the combination
use of cells and scaffolds, although the location and the differentiation of transplanted is dif
ficult to control. In contrast to approaches that utilize scaffolds, we have developed an alter
native technology for cell transplantation using temperature responsive culture dishes,
which we call Cell Sheet Engineering.
2.1. Intelligent surface of N-isopropylacrylamide (PIPAAm) and fabrication of cell sheets
Poly(N-isopropylacrylamide) (PIPAAm) is a temperature responsive polymer that has been
widely utilized for novel biomedical applications. We have developed a PIPAAm-grafted

surface as a smart biointerface wherein cell attachment/detachment can be easily controlled

by simply changing the temperature (Okano et al., 1995; Yamada et al., 1990). This surface is
slightly hydrophobic under cell culture conditions of 37 C, but readily becomes hydrated
and hydrophilic below its lower critical solution temperature (LCST) of 32 C. Cells can ad
here, spread, and proliferate similarly to that on ungrafted tissue culture grade polystyrene
surfaces at 37 C (Figure 1A), and cells detach from the surface by reducing temperature be
low LCST, making it possible to harvest the cells from the culture surfaces without the use
of proteolytic enzymes (Figure 1B).
were limited and associated with poor clinical predictability (Esposito et al., 2009). Therefore, stem
cellbased
approaches for periodontal regeneration have been studied and translated into clinical settings as

the third generation. In this chapter, we would like to describe the principles of Cell Sheet Engineering
and its application of clinical settings, featuring our recent translational research for periodontal
regeneration.
(14pt)
2. Cell Sheet Engineering (CSE)
(6pt)
The cell delivery for periodontal regeneration is usually performed with the combination use of cells and
scaffolds, although the location and the differentiation of transplanted is difficult to control. In contrast
to
approaches that utilize scaffolds, we have developed an alternative technology for cell transplantation
using temperature responsive culture dishes, which we call Cell Sheet Engineering.
(14pt)
2.1 Intelligent Surface of N-isopropylacrylamide (PIPAAm) and Fabrication of Cell Sheets
Poly(N-isopropylacrylamide) (PIPAAm) is a temperature responsive polymer that has been widely
utilized for novel biomedical applications. We have developed a PIPAAm-grafted surface as a smart
biointerface wherein cell attachment/detachment can be easily controlled by simply changing the
temperature (Okano et al., 1995; Yamada et al., 1990). This surface is slightly hydrophobic under cell
culture conditions of 37 C, but readily becomes hydrated and hydrophilic below its lower critical
solution
temperature (LCST) of 32 C. Cells can adhere, spread, and proliferate similarly to that on ungrafted
tissue culture grade polystyrene surfaces at 37 C (Figure 1A), and cells detach from the surface by
reducing temperature below LCST, making it possible to harvest the cells from the culture surfaces
without the use of proteolytic enzymes (Figure 1B).
(9pt)
AB
(4pt)
Fig. 1. The principle of Cell Sheet Engineering.
A: Cells can attach and proliferate on grafted surface of the temperature responsive polymer (poly
(Nisopropylacrylamide:

PIPAAm) at 37 C, wherein PIPAAm is extensively dehydrated and compact. B: At

Figure 1. The principle of Cell Sheet Engineering.
A: Cells can attach and proliferate on grafted surface of the temperature responsive polymer
(poly (N-isopropylacrylamide: PIPAAm) at 37 C, wherein PIPAAm is extensively dehy
drated and compact. B: At temperatures below 32 C, cells with extracellular matrix proteins
spontaneously detach from the temperature responsive culture dishes, wherein PIPAAm is
fully hydrated with an extended-chain conformation. A simple temperature change can con
trol cell attachment/detachment without any damages. Modified and reprint from Iwata et
al., 2013.